Method for producing a fermentation broth containing short-chain fatty acids

Abstract

The present invention is a method for producing a fermentation broth containing colloidal particles having a particle size of not more than 50 nm and short-chain fatty acids, with an acidity of pH 3 to 4. The method is a method comprising the steps of: a step of preparing a fermentation device comprising a plurality of sets of fermentation vessels subjected to temperature management in each stage of a multi-stage fermentation process; a step of using soft water as a starter culture, a spore-forming Clostridium genus-containing primary culture solution containing 7 kinds of fermentation bacteria (International Depositary Accession Nos. NITE BP-02945 to NITE BP-02951) managed at a low temperature, and 3 kinds of fermentation media derived from natural raw materials generated by separately fermenting a first culture medium of dried soybeans, a second culture medium of a mixed culture medium of dried plants containing Chinese jujube, Chinese wolfberry fruit, and turmeric, and a third culture medium of a honey raw material; and a step of producing a fermentation broth by a multi-stage fermentation process.

Description

Technical Field

[0001] The present invention relates to a method for producing a fermentation broth containing short-chain fatty acids by fermenting natural raw materials. The fermentation broth produced by the present invention has an acidity of pH 3-4 and contains colloidal particles with a particle size of no more than 50 nm and short-chain fatty acids, including butyric acid, propionic acid, and lactic acid. The colloidal particle content of the fermentation broth is 6.5% to 7.5%. The fermentation broth contains 0.5g to 0.6g of butyric acid per 100mL of the fermentation broth. Background Art

[0002] A large number of fermented foods, for example, vinegar, miso, soy sauce and brewed wine, etc. are sold on the market as daily ingredients. In addition, a large number of liquid fermented foods are sold on the market as functional foods or the stock solution of functional foods. Traditionally, liquid fermentation extracts are made by crushing the raw materials comprising legumes such as fruits, vegetables, soybeans, and nuts such as chestnuts or walnuts, dropping the crushed raw materials into natural water, and fermenting them by the fermentation bacteria of selection. Such methods and processed foods manufactured by this method have long been known to people and have been improved in various ways.

[0003] Regarding short-chain fatty acids such as butyric acid, propionic acid, and lactic acid involved in the present invention, Non-Patent Document 1, for example, states that butyric acid is produced during the anaerobic fermentation of dietary fiber by intestinal bacteria, and that investigations into the effects of butyric acid have confirmed that it promotes the production of regulatory T cells. Non-Patent Document 1 reports that "feeding a butyric acid starch diet to a chronic enteritis model mouse model promoted the production of regulatory T cells derived from the transplanted cells in the large intestine, improving enteritis symptoms."

[0004] Furthermore, non-patent document 2 reports that two types of pTreg, thymus-derived tTreg and peripherally induced pTreg, have been identified among regulatory T cells (Treg). pTreg is associated with RORγt, the master transcription factor of TH17 lymphocytes. Butyric acid produced by Clostridium clusters IV and XIVa is involved in the induction of pTreg. Therefore, short-chain fatty acids, especially butyric acid, are important for the induction of pTreg in the large intestine.

[0005] Furthermore, Non-Patent Document 3 reports that "recent studies have consistently revealed that short-chain fatty acids, in addition to being utilized as a host energy source, also play an essential role in maintaining energy homeostasis, including weight gain suppression, glucose uptake, improved glucose metabolism, and increased insulin sensitivity." This report highlights the importance of consuming plant fiber, including indigestible polysaccharides, as a source of short-chain fatty acids. Non-Patent Document 3 describes various molecular mechanisms of short-chain fatty acids, particularly butyric acid, through the fatty acid receptors GPR41, GPR43, GPR109a, and O1fr78.

[0006] Non-Patent Document 4 reports that mixed cultivation of Shigella dysenteriae and Clostridium butyricum MIYAIRI 588 under anaerobic conditions inhibited Shigella dysenteriae growth even when the bacterial count was low. Non-Patent Document 4 reports that the anti-inflammatory effects of butyric acid and Clostridium butyricum in vivo have been attracting attention in recent years.

[0007] Non-Patent Document 5 reports that butyric acid, a type of short-chain fatty acid produced by intestinal bacteria in the large intestine, is an essential nutrient for the large intestine and is consumed as energy in epithelial cells. Impaired butyric acid metabolism is a cause of ulcerative colitis. Non-Patent Document 5 lists physiological effects of short-chain fatty acids such as enhanced absorption of minerals such as calcium, inhibition of cholesterol synthesis, and inhibition of colorectal cancer development through butyric acid, and also presents analytical results.

[0008] Conventional methods for producing fermented liquids include, for example, Patent Document 1, which describes a fermentation product and its production method. This method involves grinding natural raw materials such as beans and nuts into a lactose solution, then uniformly mixing fresh microorganisms and a fermentation extract using at least two fermentation tanks and a microbial culture propagator to produce an immunologically active fermented product. Patent Document 2 describes a multi-stage fermentation process involving nanofiltration, and Patent Document 3 describes a method for producing a fermented liquid by adding Bacillus natto to ashitaba leaves and soybean powder through a multi-stage fermentation process.

[0009] Patent Document 4 describes a method for producing a lactobacillus production liquid by dividing 16 types of lactobacillus into a plurality of groups, subculturing the groups while maintaining a symbiotic state, and filtering the culture liquid. Patent Document 4 also describes a method for producing a lactobacillus production liquid.

[0010] Regarding the promotion of calcium metabolism in vivo, Patent Document 5 describes a physiologically active agent having calcium absorption promoting activity and antioxidant activity, comprising a peptide or peptide mixture obtained by decomposing casein with a protease produced by lactic acid bacteria. Patent Document 6 describes a health supplement for beverages with calcium absorption promoting function, comprising a soybean fermentation polymer containing folic acid obtained by adding soybeans to purified water for fermentation.

[0011] Regarding fermented foods using soybeans as a raw material, Patent Documents 7 to 9 disclose fermented foods produced by methods using soybeans as a raw material and bifidobacteria or lactic acid strains as a starter.

[0012] Regarding the acidity of the fermented liquid, Patent Document 10 describes a method for producing a fermented soymilk beverage with an acidity adjusted to less than pH 4.5, and a fermented soymilk beverage with an acidity less than pH 4.5 and a viscosity of 5.9 mPa·s or greater. Patent Document 11 describes an enzyme-containing health food with an acidity of approximately pH 3.7 to 3.9, and a method for producing such a health food by adding several fermented liquids to a wild grass extract and fermenting the resulting product.

[0013] Prior art literature

[0014] Patent Literature

[0015] Patent Document 1: Japanese Patent Application No. 2013-524791

[0016] Patent Document 2: Japanese Patent Application Laid-Open No. 2016-000039

[0017] Patent Document 3: Japanese Patent Application Laid-Open No. 2013-132290

[0018] Patent Document 4: Japanese Patent No. 4540376

[0019] Patent Document 5: Japanese Patent Application Laid-Open No. 5-304889

[0020] Patent Document 6: Japanese Patent Application No. 2010-540623

[0021] Patent Document 7: Japanese Patent Application Laid-Open No. 2001-120180

[0022] Patent Document 8: Japanese Patent Application Laid-Open No. 2005-218390

[0023] Patent Document 9: Japanese Patent Application Laid-Open No. 2011-167190

[0024] Patent Document 10: Japanese Patent Application Laid-Open No. 2014-168441

[0025] Patent Document 11: Japanese Patent Application Laid-Open No. 2009-178084

[0026] Non-patent literature

[0027] Non-Patent Document 1: “Intestinal bacterial metabolites that control the production of regulatory T cells”, Morijin Hayakawa, Pharmacia, Vol. 50, No. 8, p. 815 (2014)

[0028] Non-patent Document 2: “Allergic Diseases and Intestinal Bacteria” Shimojo Naoki, Experimental Medicine Supplement Vol. 37, No. 2, pp. 97-103 (2019)

[0029] Non-patent document 3: “Host metabolic control and intestinal bacterial flora”, Ikuo Kimura, Experimental Medicine Supplement Vol. 37, No. 2, pp. 119-126 (2019)

[0030] Non-Patent Document 4: "Inhibitory Effect of Clostridium butyricum MIYAIRI 588 on Intestinal Pathogens" by Kuroiwa Toyoaki, Kobari Ichifeng, and Iwanaga Masaaki, Journal of Infectious Diseases, Vol. 64, No. 3, pp. 257-263 (1990)

[0031] Non-patent document 5: "Physiological effects of short-chain fatty acids produced in the large intestine from prebiotics", Hara Hiroshi, Journal of Enterobacteriacology, Vol. 16, p. 35-42 (2002) Summary of the Invention

[0032] Technical problems to be solved by the present invention

[0033] The present invention provides a method for producing a fermentation broth containing short-chain fatty acids by fermenting natural raw materials. More specifically, the present invention provides a method for producing a fermentation broth containing colloidal particles with a particle size of no more than 50 nm and short-chain fatty acids, with an acidity of pH 3 to 4.

[0034] The fermentation broth produced by the present invention contains colloidal particles with a particle size of no more than 50 nm and short-chain fatty acids, wherein the short-chain fatty acids include butyric acid, propionic acid, and lactic acid, and has an acidity of pH 3 to 4. The content of the colloidal particles in the fermentation broth can be 6.5% to 7.5%. The fermentation broth can contain 0.5g to 0.6g of butyric acid per 100mL of the fermentation broth.

[0035] This fermented liquid can be used as a functional food, and the following functions have been confirmed: while doubling the number of lactic acid bacteria and bifidobacteria that promote gastrointestinal health, it halved the number of Clostridium perfringens, a species harmful to gastrointestinal health, and increased calcium absorption in the body, promoting calcium metabolism and significantly contributing to bone strength and bone metabolism. This is described in detail in Non-Patent Document 3, and is believed to be consistent with the various molecular mechanisms of short-chain fatty acids, particularly butyric acid, through the fatty acid receptors GPR41, GPR43, GPR109a, and O1fr78.

[0036] The present invention, which was completed by the inventors through years of practical research, provides a method for producing a fermentation broth containing colloidal particles having a particle size of no more than 50 nm and short-chain fatty acids, wherein the short-chain fatty acids include butyric acid, propionic acid, and lactic acid, and having an acidity of pH 3 to 4, by fermenting natural raw materials.

[0037] More specifically, the present invention provides a method for producing a fermentation broth containing colloidal particles having a particle size of not more than 50 nm and short-chain fatty acids, wherein the short-chain fatty acids include butyric acid, propionic acid, and lactic acid, and having an acidity of pH 3 to 4, wherein the fermentation broth is produced by a multi-stage fermentation process in which pre-symbiotic and stabilized bacteria, which have been managed under low temperature, function symbiotically, wherein the bacteria are bacteria comprising spore-forming Clostridium bacteria that produce organic acids containing short-chain fatty acids (seven fermentation bacteria deposited with the Japan Patent Microorganisms Depository (NPMD), an independent administrative institution serving as an international depositary institution under the Budapest Treaty, and having international deposit numbers NITE BP-02945, NITE BP-02946, NITE BP-02947, NITE BP-02948, NITE BP-02949, NITE BP-02950, ​​and NITE BP-02951).

[0038] The method of the present invention has the following characteristics:

[0039] A fermentation apparatus is prepared that includes a plurality of sets of temperature-controlled fermentation containers in each stage of a multi-stage fermentation process.

[0040] use:

[0041] Soft water as a starter;

[0042] A bacterial liquid b containing seven fermentation bacteria of the genus Clostridium with international deposit numbers NITE BP-02945 to NITE BP-02951, which is maintained at low temperature; and

[0043] The three fermentation media m1, m2, and m3 derived from natural raw materials are generated by fermenting a first culture medium pm1 of dried soybeans, a second culture medium pm2 of a mixed culture medium containing dried jujubes, wolfberries, and turmeric, and a third culture medium pm3 of honey raw materials.

[0044] Through a multi-stage fermentation process, a fermentation liquid containing colloidal particles with a particle size of no more than 50 nm and short-chain fatty acids with an acidity of pH 3 to 4 is produced.

[0045] The non-patent and patent documents do not disclose or suggest a fermentation broth containing colloidal particles with a particle size of no more than 50 nm and short-chain fatty acids, wherein the short-chain fatty acids include butyric acid, propionic acid, and lactic acid, and having an acidity of pH 3-4. Furthermore, the non-patent and patent documents do not disclose or suggest a method for producing a fermentation broth containing colloidal particles with a particle size of no more than 50 nm and short-chain fatty acids, wherein the short-chain fatty acids include butyric acid, propionic acid, and lactic acid, and having an acidity of pH 3-4 through a multi-stage fermentation process. Furthermore, with respect to any combination of the technical elements described in these non-patent and patent documents, it is clearly understood that none of them suggests that a person skilled in the art could conceive of the method of the present invention for producing a fermentation broth containing colloidal particles with a particle size of no more than 50 nm and three short-chain fatty acids, namely butyric acid, propionic acid, and lactic acid, and having an acidity of pH 3-4.

[0046] Technical means to solve technical problems

[0047] According to an embodiment of the present invention, a first feature of the method for producing a fermentation liquid containing colloidal particles having a particle size of not more than 50 nm and short-chain fatty acids and having an acidity of pH 3 to 4 lies in the first fermentation line f1 in the multi-stage fermentation process.

[0048] The first fermentation line f1 uses a first fermentation system s1 and a second fermentation system s2. The first fermentation system s1 includes Figure 1 The second fermentation system s2 includes a plurality of (four or more) ceramic fermentation bottles 10, a first culture medium tank 20 equipped with a heating kettle 30, and two first fermentation tanks 100. Figure 1 A second fermentation tank 200 shown in [6] to [8], Figure 4 [b] shows the take-out device 110 of the first fermentation tank 100 connected to the second fermentation tank 200.

[0049] The first fermentation production line f1 consists of the following steps: Figure 2 The first step p11 of producing the first preliminary fermentation liquid pn1 and the fermented soybean fs shown in (2) Figure 2 [b]~[d] and Figure 16The second step p12 of generating the first fermentation liquid n1 with an acidity of pH 5.3± shown in the first stage, Figure 1 [8] and Figure 16 The third step p13 of producing the second fermentation liquid n2 with a pH of 5.0± is shown in stage 1.

[0050] In this manual, the symbol ± after the pH value indicates Figure 16 The annual variation in pH value between batches of the fermentation liquid in the fermentation process shown in FIG was within 0.3 above and below the average value.

[0051] More specifically, all the steps of the first fermentation production line f1 are as follows: Figure 1 [1] to [8] are shown. The first fermentation production line f1 includes the first step p11. In the first step p11, each Figure 2 Into the plurality of (4 or more) ceramic fermentation bottles 10 shown in (1), soft water w with an acidity of pH 7.3± as a fermentation seed and dried soybean ds as a raw material of the first culture medium pm1 are put. Figure 2 As shown in pp10 of FIG, in order to promote the fermentation of soybeans soaked in soft water, a part of the original bacterial liquid b and sugar chains are added. The contents of the fermentation bottle 10 are kept as Figure 16 The fermentation was carried out at 37°C to 40°C for 3 days (68 to 74 hours) as shown in stage 1. Figure 2 As shown in (2), a first preliminary fermentation liquid pn1 at pH 4.5± and fermented soybean fs were generated.

[0052] The first step p11 of the first fermentation line f1 is as follows Figure 1 [3]~[4] and Figure 2 As shown in steps pp11 to pp13, further comprising:

[0053] Fermented soybean fs is ground by a pulverizing means (not shown) to produce soybean paste gfs, which is then Figure 2 (3), the steps of transferring the fermented liquid to a plurality of (four or more) containers 11 corresponding to the fermentation bottles 10, mixing the fermented liquid with the first preliminary fermentation liquid pn1, and transferring the fermented liquid from the containers 11 to the heating kettle 30; and

[0054] like Figure 2 As shown in step pp14 of [a], the soybean paste gfs and the first preliminary fermentation liquid pn1 are heated to 55-60°C in a heating kettle 30 and transferred to a first culture medium tank 20 in which soft water w has been previously added for cooling, thereby generating a first fermentation medium m1 having a pH of 4.5±.

[0055] The first fermentation production line f1 includes Figure 1The second step p12 shown in [5] to [6]. Figure 1 The second step p12 shown in [5] includes:

[0056] like Figure 2 (4) As shown in [b], the first fermentation medium m1 is evenly transferred to two first fermentation tanks 100, and soft water w and bacterial solution b are further added to each first fermentation tank 100; and

[0057] like Figure 2 As shown in step pp15 of [c], the first fermentation medium m1, soft water w, and bacterial liquid b in each first fermentation tank 100 are stirred and mixed to produce a second preliminary fermentation liquid pn2 with a pH of 6.4±.

[0058] The second preliminary fermentation liquid pn2, with a pH of 6.4, generated in the second preliminary step, is a turbid liquid pn2 formed by mixing the first fermentation medium m1, soft water w, and the original bacterial solution b. After the second preliminary step in the first fermentation line f1, no new soft water w or original bacterial solution b is added in the multi-stage fermentation step. Therefore, this turbid liquid pn2 becomes the stock solution θ used to produce a fermentation liquid containing colloidal particles with a particle size of no more than 50 nm and short-chain fatty acids, with an acidity of pH 3-4.

[0059] like Figure 1 As shown in [6], the second step p12 further includes a third preparatory step. The third preparatory step is as follows Figure 2 As shown in step pp16 of [d], the second preliminary fermentation liquid pn2 having a pH of 6.4± produced in each first fermentation tank 100 is maintained as follows Figure 16 The fermentation was carried out for 50 to 100 days at 37 to 40° C. in the fermentation state shown in stage 1, thereby producing a first fermentation liquid n1 having a pH of 5.3±.

[0060] like Figure 4 [a], and detailed information is given below. Figure 1 In each first fermentation tank 100 shown in [6], a sponge layer sp containing the supernatant of fermentation gas is formed on the upper layer, a first precipitate layer dep1 containing the fibrous first fermentation medium m1 is accumulated on the lower layer, and a first fermentation liquid n1 with a pH of 5.3± is formed between the sponge layer sp and the first precipitate layer dep1.

[0061] The first fermentation production line f1 may include Figure 1 The third step p13 shown in [7] to [8]. The third step p13 includes:

[0062] like Figure 4As shown in [b], a step of taking out only the first fermentation liquid n1 having a pH of 5.3± produced in the second step p12 from the two first fermentation tanks 100 by the taking-out device 110 of the first fermentation tank 100;

[0063] The first fermentation liquid n1 is transferred to a second fermentation tank 200 in which a fermentation environment is formed without using a fermentation medium, and stirred and mixed; and

[0064] The fermentation bacteria b1 of the original bacterial liquid b contained in the first fermentation liquid n1 keeps the first fermentation liquid n1 as Figure 16 The fermentation is carried out at 37°C to 40°C for 3 to 5 days as shown in stage 1.

[0065] The second fermentation liquid n2 with a pH of 5.0± was generated.

[0066] According to an embodiment of the present invention, a second feature of the method for producing a fermentation liquid containing colloidal particles having a particle size of not more than 50 nm and short-chain fatty acids and having an acidity of pH 3 to 4 lies in the second fermentation line f2 in the multi-stage fermentation process.

[0067] The second fermentation line f2 uses the third fermentation system s3. The third fermentation system s3 is as follows Figure 1 [9]~

[12] and Figure 7 As shown in , including:

[0068] Figure 1 [9] or Figure 7 The second culture medium tank 40 shown in [a];

[0069] Shown in Figure 1

[11] and detailed information is given in Figure 8 and Figure 9 A third fermentation tank 300 equipped with a bag-shaped culture medium filter 320 and a circulation pump device 330 installed in a suspended manner inside; and

[0070] and Figure 1 The extraction device 210 of the second fermentation tank 200 connected to the third fermentation tank 300 shown in

[12] (the operating principle of the extraction device 210 is the same as that of the second fermentation tank 200) Figure 4 [b] is the same as the removal device 110, not shown).

[0071] The second fermentation line f2 is composed of a first step p21 for producing a second fermentation medium m2 having a pH of 4.8, and a second step p22 for producing a third fermentation liquid n3 having a pH of 4.5.

[0072] All the processes of the second fermentation production line f2 are as follows Figure 1The first process p21 of generating the second fermentation medium m2 at pH 4.8 ± is as shown in [9] to

[12] . Figure 1 [9] and

[10] , and more specifically, includes the following first and second preliminary processes pp21 and pp22.

[0073] The first preliminary process pp21 includes:

[0074] As shown in [9] or Figure 1 [9] or Figure 7 As shown in [a] of [9], a step of charging a part of either one of the second fermentation broth n2 at pH 5.0 ± or the first fermentation broth n1 at pH 5.3 ±, which is a fermentation seed, a sterilized mixed medium pm2 of dried plants including jujube, Chinese wolfberry fruit, and turmeric, into the second medium tank 40;

[0075] A step of dipping the sterilized mixed medium pm2 into a part of either one of the second fermentation broth n2 or the first fermentation broth n1 and performing mixed stirring; and

[0076] A step of allowing them to ferment for 2 to 3 days in a fermentation state at 37 to 40 °C as shown in stage 2, Figure 16

[0077] As shown in

[10] or Figure 1

[10] or Figure 7 [b] of [9], a preliminary fermentation mixed medium pfm2 and a third preliminary fermentation broth pn3 are generated.

[0078] In the first preliminary process pp21, the mixed medium pm2 preferably contains jujube, Chinese wolfberry fruit, and turmeric at a ratio of 7 to 4 to 1 by weight. For example, with respect to the total amount of either one of the second fermentation broth n2 or the first fermentation broth n1, 35 liters, 200 to 210 g of dried jujube, 110 to 120 g of dried Chinese wolfberry fruit, and 25 to 30 g of dried turmeric are used in the mixed medium pm2.

[0079] In the second preliminary process pp22, from Figure 1

[10] or Figure 7 The preliminary fermentation mixed medium pfm2 is taken out of the second medium tank 40 as shown in

[10] or[b] of [9], is pulverized to a degree that the seeds of jujube are not ground using a pulverizing means (not shown), and a third preliminary fermentation broth pn3 at pH 4.8 ± and a second fermentation medium m2 are generated.

[0080] More specifically, the second process p22 of generating the third fermentation broth n3 at pH 4.5 ± in the second fermentation line f2 includes the following preliminary process.

[0081] The second process p22 includes:

[0082] As shown in [9] or Figure 1 As shown in

[11] , the second fermentation medium m2 produced in the first step p21 is sealed in a bag-shaped medium filter 320, the bag-shaped medium filter 320 is suspended in a third fermentation tank 300, and the third preliminary fermentation liquid pn3 having a pH of 4.8± is transferred to the third fermentation tank 300;

[0083] By connecting to the third fermentation tank 300 Figure 1 The removal device 210 of the second fermentation tank 200 shown in [8] is a step of transferring the second fermentation liquid n2 having a pH of 5.0± from the second fermentation tank 200 to the third fermentation tank 300;

[0084] start up Figure 9 , wherein the circulation pump device 330 shown in FIG. 1 integrally circulates the third preliminary fermentation liquid pn3 having a pH of 4.8± and the second fermentation liquid n2 having a pH of 5.0± via the bag-shaped medium filter 320 so that the second fermentation medium m2 is not mixed into the fermentation liquid; and

[0085] Keep them as Figure 16 The fermentation is carried out at 37°C to 40°C for 8 to 9 days as shown in stage 2, and finally a third fermentation liquid n3 having a pH of 4.5± is produced.

[0086] According to an embodiment of the present invention, a third feature of the method for producing a fermentation liquid containing colloidal particles having a particle size of not more than 50 nm and short-chain fatty acids and having an acidity of pH 3 to 4 lies in the third fermentation line f3 in the multi-stage fermentation process.

[0087] The third fermentation line f3 uses the fourth fermentation system s4. The fourth fermentation system s4 is as follows Figure 1

[13] ~

[16] and Figure 12 As shown in [a] to [g], including:

[0088] Figure 1

[13] ~

[14] and Figure 12 The third culture medium tank 50 shown in [b],

[0089] Figure 1

[15] and Figure 12 [d] shows a fourth fermentation tank 400,

[0090] and Figure 12 The take-out device 310 of the third fermentation tank 300 connected to the fourth fermentation tank 400 shown in [e].

[0091] The third fermentation line f3 is composed of a first step p31 for producing a third fermentation medium m3 having a pH of 4.4, and a second step p32 for producing a fourth fermentation liquid n4 having a pH of 3.7.

[0092] All the processes of the third fermentation production line f3 are as follows Figure 1

[13] to

[16] More specifically, the first step p31 of producing the third fermentation medium m3 at pH 4.4± includes the following preparatory steps.

[0093] The first step p31 includes:

[0094] like Figure 12 As shown in [b] to [c], a step of adding a honey raw material pm3 equivalent to 3 to 5% of the amount of the third fermentation liquid n3 produced in the third fermentation tank 300, preferably a honey raw material pm3 composed of multi-flower honey and acacia honey in a ratio of 1 to 4, to the third culture medium tank 50, and further adding a portion of the third fermentation liquid n3 with a pH of 4.5± equivalent to 4 times the amount of the honey raw material pm3 as a starter seed;

[0095] A step of stirring the honey raw material pm3 and a portion of the third fermentation liquid n3 in the third culture medium tank 50 to generate a preliminary fermentation medium pfm3;

[0096] like Figure 16 As shown in stage 2, Figure 12 The step of maintaining the preliminary fermentation medium pfm3 shown in step pp31 at 37° C. to 40° C. for 2 to 3 days to finally generate the third fermentation medium m3 having a pH of 4.4±.

[0097] More specifically, the second step p32 of producing the fourth fermentation liquid n4 having a pH of 3.7± in the third fermentation line f3 includes the following preliminary steps.

[0098] The second step p32 includes:

[0099] like Figure 12 [d] shows a step of transferring the third fermentation medium m3 to the fourth fermentation tank 400;

[0100] like Figure 12 As shown in [e] to [f], the third fermentation liquid n3 having a pH of 4.5±, which serves as a starter, is further transferred from the third fermentation tank 300 to the fourth fermentation tank 400 by the extraction device 310 of the third fermentation tank 300 connected to the fourth fermentation tank 400 and stirred; and

[0101] like Figure 12 [g] and Figure 16 Stage 2 shows a step of maintaining the content of the fourth fermentation tank 400 at 37° C. to 40° C. for 30 to 60 days to finally produce a fourth fermentation liquid n4 having a pH of 3.7±.

[0102] According to an embodiment of the present invention, a fourth feature of the method for producing a fermentation liquid containing colloidal particles having a particle size of not more than 50 nm and short-chain fatty acids and having an acidity of pH 3 to 4 lies in the fourth fermentation line f4 in the multi-stage fermentation process.

[0103] The fourth fermentation line f4 uses the fifth fermentation system s5. The fifth fermentation system s5 includes:

[0104] Figure 1 A fifth fermentation tank 500 shown in

[17] ,

[0105] The take-out device 410 of the fourth fermentation tank 400 connected to the fifth fermentation tank 500 (the operating principle of the take-out device 410 is the same as that of the Figure 12 [e] is the same as the removal device 310, not shown).

[0106] The fourth fermentation production line f4 consists of Figure 12 The step p41 shown in [g] is to generate the fifth fermentation liquid n5 at pH 3.6± from the fourth fermentation liquid n4 at pH 3.7±.

[0107] More specifically, the step p41 of producing the fifth fermentation liquid n5 having a pH of 3.6 in the fourth fermentation line f4 includes the following preliminary steps.

[0108] Process p41 as Figure 1 As shown in

[16] to

[17] , it includes:

[0109] a step of transferring a fourth fermentation liquid n4 having a pH of 3.7±, serving as a starter, from the fourth fermentation tank 400 to the fifth fermentation tank 500 via the extraction device 410 of the fourth fermentation tank 400 connected to the fifth fermentation tank 500;

[0110] A step of stirring the fourth fermentation liquid n4 at pH 3.7± in the fifth fermentation tank 500 several times to establish a fermentation environment without using a fermentation medium;

[0111] like Figure 16 As shown in stage 2, the fourth fermentation liquid n4 is kept in a fermentation state at 37°C to 40°C and fermented by the fermentation bacteria b1 for 30 to 60 days to finally generate the fifth fermentation liquid n5 with a pH of 3.6±.

[0112] According to an embodiment of the present invention, a fifth feature of the method for producing a fermentation liquid containing colloidal particles having a particle size of not more than 50 nm and short-chain fatty acids and having an acidity of pH 3 to 4 lies in the fifth fermentation line f5 in the multi-stage fermentation process.

[0113] The 5th fermentation line f5 uses the 6th fermentation system s6. The 6th fermentation system s6 is as follows Figure 1

[18] ~

[19] or Figure 15 As shown in [a] to [c], it has:

[0114] Figure 1

[19] or Figure 15 [c] shows a sixth fermentation tank 600,

[0115] Figure 1

[18] or Figure 15 The cooling device 610 shown in [b],

[0116] Figure 15 [a] shows the preparation tank 620 and the extraction device 510 of the fifth fermentation tank 500 connected to the preparation tank 620.

[0117] The fifth fermentation production line f5 consists of the following steps:

[0118] like Figure 1

[18] or Figure 15 As shown in [b], the first step p51 is to rapidly cool the fifth fermentation liquid n5 at pH 3.6± at 35 to 40°C to a liquid temperature of 4 to 5°C or less;

[0119] The rapidly cooled 5th fermentation liquid n5 of pH 3.6± was restored to room temperature. Figure 1

[19] or Figure 15 As shown in [c], the second step p52 is to generate the sixth fermentation liquid n6 with an acidity of pH 3.3± while maintaining the normal temperature state.

[0120] More specifically, the first step p51 of rapidly cooling the fifth fermentation liquid n5 at pH 3.6 at 35 to 40°C to a liquid temperature of 4 to 5°C or lower in the fifth fermentation line f5 includes the following preliminary steps.

[0121] The first step p51 includes:

[0122] like Figure 15 As shown in [a], a step of transferring the fifth fermentation liquid n5 having a liquid temperature of 35 to 40°C and a pH of 3.6± from the fifth fermentation tank 500 to a preparatory tank 620 connected to the extraction device 510 of the fifth fermentation tank 500; and

[0123] like Figure 15 As shown in [b], the preliminary tank 620 is immersed in the cooling water of the cooling device 610 to rapidly cool the fifth fermentation liquid n5 at 35 to 40°C to a final liquid temperature of 4 to 5°C or less.

[0124] More specifically, if Figure 15As shown in [c], the second step p52 of producing the sixth fermentation liquid n6 having a pH of 3.3± while maintaining the fifth fermentation line f5 at room temperature includes the following preliminary steps.

[0125] The second step p52 includes:

[0126] like Figure 15 As shown in [c], the preparation tank 620 is removed from the cooling device 610, the rapidly cooled fifth fermentation liquid n5 is restored to room temperature, and then the fifth fermentation liquid n5 is transferred to the sixth fermentation tank 600;

[0127] A step of establishing a new fermentation environment without using a fermentation medium; and

[0128] The step of fermenting the fifth fermentation liquid n5 having a pH of 3.6± for 180 to 240 days by fermentation bacteria b1 while maintaining the temperature to finally produce the sixth fermentation liquid n6 having a pH of 3.3±.

[0129] As described above, the method of the present invention is briefly composed of the first fermentation production line f1 to the fifth fermentation production line f5, and is a method for producing a fermentation liquid containing colloidal particles with a particle size of not more than 50 nm and short-chain fatty acids, the short-chain fatty acids containing three types of short-chain fatty acids, namely butyric acid, propionic acid and lactic acid, and having an acidity of pH 3 to 4.

[0130] At once Figure 1 As for the feed amount of the two first fermentation tanks 100 shown in [5], that is, if the amount of the second preliminary fermentation liquid pn2 is expected to be 1800 L, the amount of soft water w used as the yeast seed and the amount of fermentation liquid generated in each stage can be estimated as follows.

[0131] If four earthenware fermentation bottles 10 are used, Figure 2 As shown in (1) to (2), in order to soak 6.5 kg of dry soybeans, 20 L of soft water w was used for each fermentation bottle 10. Therefore, a total of 80 L of soft water w was used for the four fermentation bottles. Next, while adding a small amount of soft water w of about 5 L, the fermented soybeans fs in each fermentation bottle 10 were crushed into a paste to obtain soybean paste gfs. The soybean paste gfs was transferred to Figure 2 (3) The first preliminary fermentation liquid pn1 is mixed with the four other containers 11 shown in FIG. At this time, approximately 10 L of soft water w is added to each container 11. The soybean paste gfs and the first preliminary fermentation liquid pn1, to which 10 L of soft water w has been added, are transferred from each container 11 to the heating kettle 30 and heated. The heated soybean paste gfs and the first preliminary fermentation liquid pn1 (gfs + pn1 + w), equivalent to 35 L per fermentation bottle 10, are transferred to the first culture medium tank 20.

[0132] In the heating kettle 30, 10 L of soft water w was added to each fermentation bottle 10, stirring and heating the 35 L soybean paste gfs and first preliminary fermentation liquid pn1. Next, the soybean paste gfs and first preliminary fermentation liquid pn1 were cooled to the fermentation temperature in the first culture medium tank 20, which contained 100 L of soft water w for cooling. As a result, the amount of soft water w required to produce the first fermentation medium m1 was 180 L (80 L + 20 L + 40 L + 40 L). The amount of soft water w used for cooling in the first culture medium tank 20 was 100 L. Therefore, the resulting first fermentation medium m1 amounted to 240 L (35 L × 4 + 100 L).

[0133] In the two first fermentation tanks 100, soft water w is further added to 240 L of the first fermentation medium m1 as a starter and 270 L to 450 L of the bacterial liquid b.

[0134] If the feed amount of the first fermentation tank 100, i.e., the amount of the second preliminary fermentation liquid pn2, is expected to be 1800 L, then Figure 2 The amount of soft water w in the two first fermentation tanks 100 shown in [c] is 1110 to 1290 L. Therefore, the amount of soft water w required to produce the first fermentation liquid n1 is 1290 L (1110 L + 180 L) to 1470 L (1290 L + 180 L). According to the present invention, in the method for producing a fermentation liquid containing colloidal particles with a particle size of no more than 50 nm and short-chain fatty acids and having an acidity of pH 3 to 4, no other steps using soft water w are performed.

[0135] exist Figure 16 After 50 to 100 days of fermentation in stage 1, Figure 2 [d] or Figure 4 The two first fermentation tanks 100 shown in [a] each contain a sponge layer sp formed by fermentation gas, which acts as an air barrier, and a fibrous first sediment layer dep1 containing the first fermentation medium m1. A translucent first intermediate layer of liquid is formed between these two layers. This translucent first intermediate layer of liquid is the first fermentation liquid n1, which has a pH of 5.3.

[0136] If the feed amount of two first fermentation tanks 100, that is, the amount of the second preliminary fermentation liquid pn2, is expected to be 1800 L, then more than 30% of the second preliminary fermentation liquid is absorbed by the sponge layer sp and the first sediment layer dep1. Therefore, the first fermentation liquid n1 with a pH of 5.3± generated in each first fermentation tank 100 is equivalent to 600 to 650 L.

[0137] Therefore, the first fermentation liquid n1 at pH 5.3±, which is withdrawn from the two first fermentation tanks 100 and transferred to the second fermentation tank 200 by the withdrawal device 110 of the first fermentation tank 100 connected to the second fermentation tank 200, corresponds to 1200 to 1300 L. The first fermentation liquid n1 at pH 5.3± is the initial fermentation liquid for producing a fermentation liquid having an acidity of pH 3 to 4 and containing colloidal particles having a particle size of not more than 50 nm and short-chain fatty acids according to the present invention.

[0138] A fermentation environment without the use of a culture medium is established within the second fermentation tank 200. The fermentation of the first fermentation liquid n1 at pH 5.3± by the fermentation bacteria b1 contained in the first fermentation liquid n1 produces a second fermentation liquid n2 at pH 5.0±. In the second fermentation tank 200, a first upper surface film layer sf1, comprising a supernatant of fermented gas bubbles generated by the fermentation of the first fermentation liquid n1 at pH 5.3±, forms on the upper layer. A second sediment layer dep2 accumulates below, with a translucent second intermediate layer of liquid forming between these two layers.

[0139] The second intermediate layer liquid is the second fermentation liquid n2 with a pH of 5.0. About 5% of the second fermentation liquid n2 is absorbed by the first upper surface film layer sf1 and the second sediment layer dep2 during fermentation, thereby generating about 1140 to 1240 L of the second fermentation liquid n2.

[0140] The first fermentation line f1 ends at this stage. The first fermentation line f1 is equivalent to Figure 16 Stage 1 requires a fermentation time of more than 55 days and no more than about 108 days.

[0141] Figure 1 The second fermentation line f2 shown in [9] to

[12] uses approximately 1140 to 1240 L of the second fermentation liquid n2 at pH 5.0± as a starter. A portion of the second fermentation liquid n2 is added to the second culture medium tank 40. The remaining second fermentation liquid n2 is transferred to the third fermentation tank 300. Based on the inventors' long-term practical experience, it has been found that approximately 8% to 10% of the third fermentation liquid n3 is reduced during fermentation in the third fermentation tank 300. Therefore, the final amount of the third fermentation liquid n3 at pH 4.5± produced is approximately 1050 to 1100 L.

[0142] exist Figure 1 In the 3rd fermentation production line f3 to the 5th fermentation production line f5 shown in

[13] to

[19] , the amount of the 4th fermentation liquid n4 to the 6th fermentation liquid n6 reduced during fermentation due to gasification, etc. is very small, that is, 2% to 3%. The final fermentation liquid of the 6th fermentation liquid n6 with a pH of 3.3± containing colloidal particles with a particle size not exceeding 50nm is about 1000 to 1050L.

[0143] The amount of fermentation liquid produced during each fermentation stage can be estimated as follows. In two first fermentation tanks 100, using 1290-1470 L of soft water w and the first fermentation medium m1 as a starter, a first fermentation liquid n1 with a pH of 5.3±, corresponding to a feed volume of 1800 L, is produced. This first fermentation liquid n1 is used as the next starter to produce approximately 1140-1240 L of a second fermentation liquid n2 with a pH of 5.0±. Next, using the second fermentation liquid n2 and the second fermentation medium m2, approximately 1050-1100 L of a third fermentation liquid n3 with a pH of 4.5± is produced. In the subsequent fermentation, approximately 1050-1100 L of a third fermentation liquid n3 and the third fermentation medium m3 are used to produce a fourth fermentation liquid n4 with a pH of 3.7±. In the fermentation after this stage, a fermentation environment without using a fermentation medium was established, and the fourth fermentation liquid n4 was used to generate the fifth fermentation liquid n5 with a pH of 3.6±. Subsequently, the fifth fermentation liquid n5 was used to finally generate about 1000-1050 L of the sixth fermentation liquid n6 with a pH of 3.3±. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] [ Figure 1 ] is a schematic diagram showing a multi-stage fermentation process including the first to fifth fermentation lines for producing a fermentation liquid with a pH of 3 to 4 and containing colloidal particles with a particle size of not more than 50 nm.

[0145] [ Figure 2 ] is an enlarged schematic diagram showing the first process and the second process including the first preparatory process, the second preparatory process and the third preparatory process constituting the first fermentation production line.

[0146] [ Figure 3 ] is a three-dimensional diagram of a ceramic fermentation bottle with a capacity of not more than 60L and a lid with a valve function provided in the center, and an enlarged schematic diagram showing the structure of the lid.

[0147] [ Figure 4 ] is a schematic diagram showing a situation in which only the first fermentation liquid, which is the first intermediate layer liquid generated in the first fermentation tank, is taken out from the first fermentation tank and moved to the second fermentation tank by opening and closing the opening and closing valve of the removal device installed at the bottom of the first fermentation tank and applying the Pascal principle and the principle of hydrostatic pressure balance.

[0148] [ Figure 5 ] are photos A and B showing a comparative test of the first fermentation liquid A and the first fermentation liquid B, wherein the first fermentation liquid A is produced by fermenting the specimen A of the first fermentation medium prepared by grinding soybeans that have been fermented in advance for 6 days, and the first fermentation liquid B is produced by fermenting the specimen B of the fermentation medium prepared by grinding dry soybeans that have not been fermented in advance and immersed in water in the same manner as the specimen A for 6 days.

[0149] [ Figure 6 ] is a list of seven fermentative bacteria including spore-forming Clostridium species that produce organic acids including short-chain fatty acids. The strains listed in the table were deposited in Japan on May 16, 2019, with the Japan Patent and Microorganisms Depository (NPMD), an independent administrative institution and one of the international depositary agencies (IDAs) under the Budapest Treaty, under the deposit numbers NITE P-02945, NITE P-02946, NITE P-02947, NITE P-02948, NITE P-02949, NITE P-02950, ​​and NITE P-02951. They were transferred to the same institution as international deposits on April 22, 2020, under the international deposit numbers NITE BP-02945, NITE BP-02946, NITE BP-02947, NITE BP-02948, NITE BP-02949, NITE BP-02950, ​​and NITE BP-02951.

[0150] [ Figure 7 ] is a schematic diagram of the second culture medium tank equipped with a drip cap having holes for passage of fermentation gas.

[0151] [ Figure 8 ] is a schematic diagram of a bag-shaped culture medium filter installed in a suspended manner inside a third fermentation tank and used in a second fermentation production line for generating a third fermentation liquid from a second fermentation medium, as well as a stereoscopic view and a top view of the third fermentation tank equipped with the bag-shaped culture medium filter.

[0152] [ Figure 9 ] is a schematic diagram of a third fermentation tank equipped with a circulation pump device used in the second fermentation production line for producing a third fermentation liquid from a second fermentation medium.

[0153] [ Figure 10 ] are photos A and B of a comparative test of the third fermentation liquid A and the third fermentation liquid B. As for the third fermentation liquid A, the second fermentation medium sample A was prepared by pre-fermenting jujubes, wolfberries, and turmeric in a weight ratio of 7 to 4 to 1 using the second fermentation liquid, grinding and crushing them to the extent that only jujube seeds remained, and the sample A was used to ferment for 5 days; as for the third fermentation liquid B, the fermentation medium sample B was prepared by not pre-fermenting jujubes, wolfberries, and turmeric in a weight ratio of 7 to 4 to 1 and grinding them to the extent that only jujube seeds remained in the second fermentation liquid, and the sample B was used to ferment for 5 days in the same manner as the sample A.

[0154] [ Figure 11] is a graph and a comparison table showing the transition of the pH value of the acidity of the third fermentation liquid A and the third fermentation liquid B from the start of fermentation to the fifth day.

[0155] [ Figure 12 ] is a partially enlarged view of a schematic diagram including steps [a] to [g], wherein step [a] represents a second fermentation production line in which the second fermentation liquid is transferred to the third fermentation tank, the second fermentation medium generated in the second culture medium tank is added to a bag-shaped culture medium filter arranged in a suspended state inside the third fermentation tank, a circulation pump device is started to circulate them, and the contents of the third fermentation tank are fermented while circulating to generate the third fermentation liquid; steps [b] and [c] represent steps in which a portion of the third fermentation liquid and an appropriate amount of honey raw material are added to the third culture medium tank, and the fermentation is performed while stirring to generate the third fermentation medium; steps [d] to [g] represent a third fermentation production line in which the third fermentation liquid and the third fermentation medium transferred from the third fermentation tank are added to the fourth fermentation tank, and the fermentation is performed while stirring to generate the fourth fermentation liquid.

[0156] [ Figure 13 ] are photos A and B showing a comparative test of the 4th fermentation liquid A and the 4th fermentation liquid B. The 4th fermentation liquid A is produced by fermenting the 3rd fermentation liquid and the 3rd fermentation medium produced by pre-fermenting the honey raw material for 4 days, and the 4th fermentation liquid B is produced by fermenting the 3rd fermentation liquid and the medium of the honey raw material that has not been pre-fermented for 4 days.

[0157] [ Figure 14 ] are a graph and a comparison table showing the transition of the pH values ​​of the acidity of the fourth fermentation liquid A and the fourth fermentation liquid B from the start of fermentation to the fourth day.

[0158] [ Figure 15 ] is a schematic diagram of an expansion of the 5th fermentation production line in which the 5th fermentation liquid is transferred from the 5th fermentation tank to the preparatory tank, rapidly cooled by a cooling device, and the liquid temperature is managed according to the normal temperature state of the season, and the cooled 5th fermentation liquid is transferred to the 6th fermentation tank to form a fermentation environment without using a fermentation medium, and the 6th fermentation liquid is generated by fermentation of fermentation bacteria based on the 5th fermentation liquid.

[0159] [ Figure 16 ] is a graph and table that divides the fermentation time required for the 1st to 5th fermentation lines and the changes in the pH values ​​of the acidity of the 1st to 6th fermentation broths and the 1st to 3rd fermentation medium into stages 1 to 3, and expresses the changes in the degree of fermentation in terms of pH values ​​of acidity.

[0160] [ Figure 17] are a graph and a table showing the results of cumulative amount analysis of the size of colloidal particles contained in the fourth fermentation broth measured using the supernatant of the heated / sterilized fourth fermentation broth as a specimen.

[0161] [ Figure 18 ] are a graph and a table showing the results of cumulative amount analysis of the size of colloidal particles contained in the sixth fermentation broth, measured using the supernatant of the heated and sterilized sixth fermentation broth as a specimen.

[0162] [ Figure 19 ] is a table showing the analysis results of the sugar content in 100 g [mL] of the fermentation liquid measured by the Somogyi modified method at the Japan Food Analysis Center.

[0163] [ Figure 20 ] is a graph showing the analysis results of the amount of short-chain fatty acids contained in 100 g [mL] of the sixth fermentation liquid measured by high performance liquid chromatography at the Japan Food Analysis Center.

[0164] [ Figure 21 ] Bar graph showing the calcium increase and calcium absorption rate based on the results of the "Calcium absorption investigation test based on the reversed intestinal method" based on the administration of the test substance (the 6th fermentation liquid) using male SD rats as model animals.

[0165] [ Figure 22 ] are graphs and bone density photographs based on the results of an experiment on the effects of the administration of the test substance (the 6th fermentation broth) on the "Gla / Glu-Osteocalcin ratio" and "bone weight, bone strength, and bone density" using osteoporosis model mice that were in an estrogen-deficient state by removing both ovaries.

[0166] [ Figure 23 ] represents the results of an experiment conducted at ITECHLAB Co., Ltd. (experiment leader: Masaki Matsuura) to test the effect on intestinal bacterial flora. The experiment was conducted by collecting colon contents from C57BL / 6 mice (male, 7 weeks old) in the treatment group and the control group, which were orally administered with the test substance (the 6th fermentation broth) for 28 consecutive days, and extracting bacterial DNA from the colon contents.

[0167] [ Figure 24 ] is a graph showing the expression levels of the Krt6b gene in tumor tissues of mice in the test substance (sixth fermentation broth)-administered group and the water for injection-administered group.

[0168] Specific embodiments of the present invention

[0169] Hereinafter, one embodiment of the present invention will be described. One embodiment of the present invention provides a method for producing a fermentation broth having a pH of 3 to 4 and containing colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids through a multi-stage fermentation process. Short-chain fatty acids may include butyric acid, propionic acid, and lactic acid. As for the fermentation broth produced by the method of the present invention, while containing colloidal particles at a content rate of 6.5% to 7.5%, it may contain 0.5 g to 0.6 g of butyric acid per 100 mL of fermentation broth. As for the fermentation broth produced by the method of the present invention, it itself can be used as a functional food or a raw liquid of a functional food.

[0170] [Details of the first fermentation line f1]

[0171] More specifically, the multi-stage fermentation process is a conceptual diagram of the method of the present invention. Figure 1 It starts with the first step p11 of the first fermentation line f1 shown in [1].

[0172] Assuming that the feed amount for producing the first fermentation liquid n1 in the first fermentation line f1, that is, the amount of the second preliminary fermentation liquid pn2, is 1800 L, the feed amount of the starter and the amount produced in each step can be estimated as follows.

[0173] like Figure 2 As shown in (1) and (2), the first step p11 of the first fermentation line f1 includes a first preparatory step pp11 including a first pretreatment step and a second pretreatment step.

[0174] In the first pretreatment step, Figure 3 In each of the four ceramic fermentation bottles 10 shown in the figure, 6.5 kg of dried soybeans ds were immersed in 20 L of soft water w at a pH of 7.3 ± and reduced with water. Each fermentation bottle 10 had a capacity of no more than 60 L and was equipped with a lid 12 having a valve function 11 for allowing fermentation gas to escape from the center. In each fermentation bottle 10, an amount corresponding to the amount of 6.5 kg of dried soybeans ds, more specifically 0.25 kg of sugar chains s and the following were added. Figure 6 A portion of the original bacterial solution b shown in , more specifically 0.14 L of the original bacterial solution b.

[0175] In the second pretreatment step, the content of the fermentation bottle 10 was fermented for 3 days (68 to 74 hours), and 35 L of the first preliminary fermentation liquid pn1 and fermented soybeans fs were produced per fermentation bottle 10 .

[0176] like Figure 2As shown in (3), the first step p11 of the first fermentation line f1 further includes a second preliminary step pp12. In the second preliminary step pp12, the fermented soybeans fs, which constitute the second pretreatment step of the first preliminary step pp11, are removed from each fermentation bottle 10 and ground into a paste by a grinding device (not shown) while adding a small amount of soft water w. The ground fermented soybeans fs are transferred to four containers 11 corresponding to the fermentation bottles 10 and mixed with the first preliminary fermentation liquid pn1 of the second pretreatment step. Each container 11 produces 35 L of the first preliminary fermentation liquid pn1 and soybean paste gfs.

[0177] like Figure 2 As shown in (3), the first step p11 of the first fermentation line f1 further includes a third preliminary step pp13. In the third preliminary step pp13, the first preliminary fermentation liquid pn1 and soybean paste gfs, each equivalent to 35 L produced in each container 11, are transferred from each container 11 to the heating kettle 30, gradually heated to 55-60°C and stirred, and a total of 180 L [(35 L + 10 L) × 4] of the first preliminary fermentation liquid pn1 and soybean paste gfs are produced in the heating kettle 30.

[0178] The second step p12 of the first fermentation line f1 further includes Figure 2 The fourth preliminary step pp14 to the sixth preliminary step pp16 shown in [a] to [d].

[0179] In the fourth preliminary step pp14 of the second step p12, 180 L of the first preliminary fermentation liquid pn1 and soybean paste gfs produced in the heating kettle 30 in the third preliminary step pp13 of the first step p11 are transferred to the first culture medium tank 20 into which 100 L of cooling soft water w have been previously added, without being exposed to the outside air, and stirred to produce 280 L of the first fermentation medium m1 having a pH of 4.5±.

[0180] like Figure 2 As shown in (4), in the fourth preparatory step pp14 of the second step p12, 280 L of the first fermentation medium m1 at pH 4.5 ± in the first culture medium tank 20 is placed without contact with the outside air. Figure 6 270L to 450L of the original bacterial solution b and 1100L to 1300L of soft water w, which are preliminarily stabilized by the fermentation bacteria b1 shown in FIG, are evenly distributed into two first fermentation tanks 100. Figure 2 As shown in [b], the first fermentation medium m1 and the bacterial liquid b are stirred together with soft water w in each first fermentation tank 100 to generate a second preliminary fermentation liquid pn2 having a pH of 6.4± corresponding to a total of 1800 L.

[0181] Figure 2 In the fifth preliminary step pp15 and the sixth preliminary step pp16 of the second step p12 shown in [c] and [d], the second preliminary fermentation liquid pn2 having a pH of 6.4± is sealed in each first fermentation tank 100 so as not to come into contact with the outside air and fermented for 50 to 100 days while maintaining a fermentation state at 37°C to 40°C. Figure 4 The fermentation gas bubbles shown in [a] form a supernatant sponge layer sp. This sponge layer sp prevents oxygen from dissolving into the solution and promotes the secretion and fermentation of acidic substances by the fermentation bacteria b1 within the bacterial solution b. A paste-like first sediment layer dep1, composed of the cellulose of the first culture medium m1 and the fermentation bacteria b1, forms below. Between the sponge layer sp and the first sediment layer dep1, a translucent first intermediate layer forms. This translucent first intermediate layer is the first fermentation solution n1, with a pH of 5.3.

[0182] In each fermentation tank 100, the sponge layer sp, expanded due to long-term fermentation, and the pasty first sediment layer dep1 together occupy a volume equivalent to 300 L. Therefore, the first fermentation liquid n1 with a pH of 5.3±, which is a translucent first intermediate layer, can be equivalent to 600 L. The sponge layer sp and the pasty first sediment layer dep1, which are the upper layers equivalent to 300 L, serve as the base materials for the bacterial liquid b.

[0183] The first fermentation production line f1 may further include a third step p13. In the third step p13, Figure 4 As shown in [b], the first fermentation liquid n1 having a pH of 5.3± corresponding to 600 L of the first intermediate layer liquid produced in each first fermentation tank 100 is transferred to one second fermentation tank 200 .

[0184] In the third step p13, 1200 L of the first fermentation liquid n1 at pH 5.3± transferred to a second fermentation tank 200 is used as the first fermentation seed, and the first fermentation liquid n1 is fermented for 3 to 5 days by the fermentation bacteria b1 of the bacterial liquid b contained therein, thereby generating a second fermentation liquid n2 at pH 5.0±.

[0185] [Technical Issues in Generating the Second Fermentation Liquid n2]

[0186] The fermentation process in step 3 p13 is characterized by fermenting the first fermentation liquid n1 solely by the fermentation bacteria b1 from the original bacterial solution b, without the use of a culture medium or fermentation medium. Here, the technical challenges associated with step 3 p1 of the first fermentation line f1 are examined. The two batches of first fermentation liquid n1, produced in two first fermentation tanks 100, may each have a different fermentation environment due to the symbiotic relationship between the fermentation bacteria b1 from the original bacterial solution b. By transferring the two batches of first fermentation liquid n1 to a single second fermentation tank 200, mixing them, and integrating them, the symbiotic antagonism between the fermentation bacteria b1 from the original bacterial solution b contained in the first fermentation liquid n1 is further stimulated. This can be inferred from the phenomenon in which the pH of the first fermentation liquid n1, which had a pH of 5.3±, is acidified to the pH of the second fermentation liquid n2, which has a pH of 5.0±.

[0187] That is to say, the multi-stage fermentation process of the present invention can also be as follows Figure 1 As shown in the two routes shown in [6] or [8], the fermentation product directly enters the second fermentation line f2 from the second step p12 of the first fermentation line f1 without passing through the third step p13 of the first fermentation line f1.

[0188] If fermentation line f1 is fed directly from step p12 of fermentation line f1 to fermentation line f2, a single first fermentation tank (not shown) of the same size as fermentation tank 200 is used. Based on years of practical experience, the inventors discovered that without passing through step p13 of fermentation line f1, the symbiotic antagonism of fermentation bacteria b1 remains low, meaning that sufficient secretion and fermentation of acidic substances by fermentation bacteria b1 do not progress. Therefore, it is difficult to stably produce 1200 L of first fermentation liquid n1 at a pH of 5.3± in a single first fermentation tank. Therefore, further processing and fermentation days are required to pre-ferment first fermentation liquid n1 at pH 5.3± before converting it to second fermentation liquid n2 at pH 5.0±. Based on this discovery, the inventors devised a solution to this problem by adding step p13 to first fermentation line f1, which resulted in a successful solution.

[0189] In fact, in the third step p13 of the first fermentation line f1, the first fermentation liquid n1, with a pH of 5.3, is fermented solely by the fermentation bacteria b1 for 3 to 5 days while maintaining a fermentation temperature of 37°C to 40°C. This results in the formation of a supernatant first upper surface layer sf1 due to the bubbling of fermentation gases. This first upper surface layer sf1 prevents oxygen from dissolving into the solution, further promoting the secretion and fermentation of the fermentation bacteria b1. A second, pasty sediment layer dep2, containing the residue of the first culture medium m1, forms below. Between the first upper surface layer sf1 and the second sediment layer dep2, a translucent second intermediate layer of liquid, equivalent to 1140 to 1240 L, forms. This translucent second intermediate layer is the second fermentation liquid n2, with a pH of 5.0. Whether or not to include the third step p13 in the first fermentation line f1 is optional.

[0190] [Technical features of the first fermentation line f1]

[0191] The technical features of the first fermentation line f1 differ from conventional soybean fermentation in that dried soybeans ds are fermented in a fermentation flask 10 to produce a first preliminary fermentation liquid pn1 and soybean paste gfs. The first fermentation medium m1 is then generated from the first preliminary fermentation liquid pn1, soybean paste gfs, soft water w, and a portion of the original bacterial liquid b. This pre-fermented first fermentation medium m1, at a pH of 4.5, and soft water w are used to generate a first fermentation liquid n1 at a pH of 5.3.

[0192] Typically, fermentation products using soybeans are produced by immersing the soybeans ds in a liquid such as a fermentation product or water. However, the present inventors focused on a technical solution that produces a first fermentation liquid n1 having a pH of 5.3± by pre-fermenting a first fermentation medium m1 using dried soybeans to produce a fermentation liquid, thereby completing the present invention.

[0193] Figure 5 Photos A and B show a comparative test of the first fermentation broth A and the first fermentation broth B. First fermentation broth A was produced by fermenting Sample A, a first fermentation medium prepared by grinding pre-fermented soybeans, for six days. First fermentation broth B was produced by fermenting Sample B, a fermentation medium prepared by grinding dried soybeans soaked in water without prior fermentation, for six days in the same manner as Sample A.

[0194] Photo A shows the first fermentation broth A produced by fermenting specimen A for six days. Specimen A was prepared by pre-fermenting and grinding 60 g of soybeans to produce a soybean paste, heating the resulting soybean paste and soft water w to 55°C, cooling it to 37°C, adding 0.8 L of the original bacterial solution b, and stirring. As can be seen in Photo A, the resulting first fermentation broth A clearly has three layers: a supernatant sponge layer sp, a sediment layer dep, and a highly transparent intermediate layer n1 (4 L) between these two layers.

[0195] For comparison with Photo A, Photo B shows fermentation broth B produced by fermenting Sample B for six days. Sample B was prepared by directly soaking 60g of soybeans, the same amount as in Sample A, in water for 18 hours, grinding them, and then preparing a soybean paste without prior fermentation. The soybean paste and soft water w were then heated to 55°C, cooled to 37°C, and 0.8L of the original bacterial solution b was added and stirred. Unlike the conditions used in Sample A, fermentation broth B was produced using soybean paste without prior fermentation. As can be seen in Photo B, a low-transparency intermediate layer and a sediment layer formed, but the supernatant sponge layer sp, essential for the fermentation environment of the fermenting bacteria b1, was barely formed.

[0196] Figure 5 The results of the comparative test shown in [ ] demonstrate a significant difference in the fermentation environment between fermentation broth A, obtained by pre-fermenting dried soybeans used as the first culture medium, and fermentation broth B, which was not pre-fermented. More specifically, the comparative test demonstrates the technical necessity of forming a supernatant sponge layer sp and isolating the fermentation environment using the fermentation bacterial colony b1 contained in the original bacterial culture b. In fact, fermentation broth B, which lacks the supernatant sponge layer sp, produces a fishy odor. This is presumably because the fermentation bacteria b1, due to contact with the outside air, decrease in bacterial count, leading to oxidation and decomposition that outpaces fermentation, resulting in the production of the odor.

[0197] from Figure 5 The experimental results of photos A and B clearly show that in the realization of generating the first fermentation liquid n1 with a pH of 5.3± based on the method of the present invention, it is necessary to pre-ferment the first culture medium to generate the first fermentation medium m1 so that the supernatant sponge layer sp is formed.

[0198] Details of the second fermentation line f2

[0199] The second fermentation line f2 consists of the following steps:

[0200] like Figure 1 As shown in [9] and

[10] , a first step p21 of pre-generating a second fermentation medium m2 having a pH of 4.8± in the second culture medium tank 40; and

[0201] In Figure 1

[11] the 3rd fermenter 300 and the 3rd fermentation system s3, the 2nd fermentation medium m2 of pH 4.8± and the 2nd fermentation broth n2 of pH 5.0± generated in the 1st process p21 are used as the 2nd starter, and the 2nd process p22 generates the 3rd fermentation broth n3 of pH 4.5±.

[0202] Here, the starter refers to a raw material.

[0203] The 1st process p21 of the 2nd fermentation line f2 includes, as a preliminary process, a step of preparing the 2nd medium tank 40 equipped with a dropper lid 41 having a hole through which a fermentation gas passes, blocked with a cover lid 42, as shown in an enlarged view of Figure 7 [a].

[0204] The 1st process p21 further includes:

[0205] As shown in Figure 7 [a], either one of the mixed medium pm2 prepared in advance or a part of the 2nd fermentation broth n2 or a part of the 1st fermentation broth n1 in the fermentation process for 1 day to 2 days or so is put into the 2nd medium tank 40 so that the total amount of the mixed medium and the fermentation broth corresponds to 35 L, and they are stirred, and

[0206] By fermenting them for 2 days to 3 days while maintaining the fermentation state at 37°C to 40°C, the preliminary fermentation mixed medium pfm2 and the 3rd preliminary fermentation broth pn3 are generated as shown in Figure 7 [b].

[0207] By a process not shown in the figure, the preliminary fermentation mixed medium pfm2 is ground to the extent that the seeds of the jujube are not ground by a grinding means, and the ground preliminary fermentation mixed medium pfm2 is put back into the 2nd medium tank 40.

[0208] The 3rd preliminary fermentation broth pn3 is mixed into the ground preliminary fermentation mixed medium pfm2, and the 2nd fermentation medium m2 of pH 4.8± is generated.

[0209] The prepared mixed culture medium PM2 consists of sterilized dried plants containing jujube, wolfberry, and turmeric. The weight ratio of 200-210g of jujube, 110-120g of wolfberry, and 25-30g of turmeric is used for the second fermentation liquid N2, equivalent to 35L. Jujube, wolfberry, and turmeric are natural products with medicinal properties and are listed in the Japanese Pharmacopoeia's list of crude drugs. Jujube is the fruit of the jujube tree, wolfberry is the fruit of the wolfberry plant, and turmeric is the rhizome of the ginger family. These plants were selected by the inventors from a wide variety of crude drugs based on years of experience.

[0210] [Technical features of the second fermentation line f2]

[0211] Inventors based on years of trial and error practice, such as Figure 7 As shown in [a] and [b], with respect to the total amount equivalent to 35 L of the second fermentation liquid n2, 200-210 g of jujube, 110-120 g of wolfberry and 25-30 g of turmeric (weight ratio 7:4:1) are mixed to form a mixed culture medium pm2, and the mixed culture medium pm2 is pre-fermented in the second culture medium tank 40 to produce a preliminary fermentation mixed culture medium pfm2 and a third preliminary fermentation liquid pn3. Through a process not shown in the figure, the preliminary fermentation mixed culture medium pfm2 is ground by a pulverizing means to an extent that the jujube seeds are not ground and returned to the second culture medium tank 40. The third preliminary fermentation liquid pn3 is mixed with the ground preliminary fermentation mixed culture medium pfm2 to produce the second fermentation medium m2 with a pH of 4.8±, thereby completing the first process p21 of the second fermentation production line f2.

[0212] When the third step p13 is integrated into the first fermentation line f1, it is preferable to adopt a step that partially overlaps with the fermentation step p13 of the second fermentation liquid n2 as the first step p21 of the second fermentation line f2.

[0213] The first technical reason for arranging the first step p21 and the fermentation step p13 of the second fermentation liquid n2 in parallel, with some overlap, rather than linearly, is to alter the fermentation environment of the fermentation bacteria b1 during the fermentation steps. This altered fermentation environment further promotes the secretion and fermentation of acidic substances by the fermentation bacteria b1, enabling more rapid production of the second fermentation medium m2.

[0214] The second technical reason is to align the start time of fermentation step p22 using the third fermentation liquid n3 in the third fermentation tank 300 with the end time of fermentation step p13 using the second fermentation liquid n2. This allows the start of first step p21 in the second fermentation line f2, which produces the second fermentation medium m2, without waiting for the completion of fermentation step p13 using the second fermentation liquid n2, which is the third step in the first fermentation line f1. As a result, second step p22 in the second fermentation line f2, which produces the third fermentation liquid n3, can be started immediately upon completion of third step p13 for fermenting the second fermentation liquid n2 in the first fermentation line f1.

[0215] The technical significance of the second fermentation line f2 in which the second fermentation medium m2 in which the three selected herbal medicines, the dried plant pm2, are pre-fermented in the first step p21 of the second fermentation line f2 can be seen from the following: Figure 10 The results of the comparative test of specimens A and B of the fermentation broth model of the third fermentation broth n3 shown in FIG, and Figure 11 This is clearly seen in the analysis results showing the difference in the degree of fermentation progress between Sample A and Sample B shown in FIG.

[0216] The second fermentation line f2 includes step p21 for producing the second fermentation medium m2. Step p21 includes a preliminary step pp21. In this preliminary step pp21, a mixed medium pm2 containing 200-210 g of jujube, 110-120 g of wolfberry, and 25-30 g of turmeric (7:4:1 weight ratio) is pre-fermented in the second culture medium tank 40, along with a portion of the second fermentation liquid n2, relative to a total volume of 35 L of the second fermentation liquid n2. This produces a third preliminary fermentation liquid pn3 and a preliminary fermentation mixed medium pfm2. Step p21 further includes a preliminary step pp22. In this preliminary step pp22, the preliminary fermentation mixed medium pfm2 is ground using a pulverizing device until the jujube seeds are not ground, returned to the second culture medium tank 40, and mixed with the third preliminary fermentation liquid pn3, producing the second fermentation medium m2 at a pH of 4.8.

[0217] The second fermentation line f2 further includes a step p22 of producing a third fermentation liquid n3 having a pH of 4.5±. Figure 1Step p22 shown in

[11] and

[12] includes a preliminary step pp21. In the preliminary step pp21, the second fermentation medium m2 produced in step p21 is sealed in a bag-shaped medium filter 320 and suspended in the third fermentation tank 300. The third preliminary fermentation liquid pn3 is transferred from the second medium tank 40 to the third fermentation tank 300, and the second fermentation liquid n2 having a pH of 5.0± is transferred from the second fermentation tank 200 to the third fermentation tank 300. Step p22 further includes a preliminary step pp22. In the preliminary step pp22, the third preliminary fermentation liquid pn3 and the second fermentation liquid n2 are circulated so that the second fermentation medium m2 in the bag-shaped medium filter 320 is not mixed into the fermentation liquid. By fermenting for 8 to 9 days while maintaining a fermentation state at 37°C to 40°C, 1050 L of the third fermentation liquid n3 having a pH of 4.5± is produced.

[0218] Figure 10 Photographs A and B show the results of a comparative test of specimens A and B of the fermentation liquid model of the third fermentation liquid n3.

[0219] Specimen A is fermentation liquid A prepared as follows: 1 L of the second fermentation liquid n2 and a mixed culture medium pm2 compatible with 1 L of the second fermentation liquid n2 and containing 5.71 g of jujube, 3.14 g of wolfberry, and 0.85 g of turmeric (weight ratio of 7:4:1) are pre-fermented, the pre-fermented second fermentation medium m2 is sealed in a herbal medicine filter as a substitute for a bag-shaped culture medium filter, and the filter is suspended in a test container A, the second fermentation liquid n2 corresponding to 1 L of the starter seed and the third preliminary fermentation liquid pn3 are added to the test container A, stirred, and fermented for 5 days while maintaining a fermentation state at 38°C to 40°C, thereby forming a two-layer fermentation liquid A ( Figure 10 Photo A).

[0220] Specimen B is a fermentation liquid B prepared as follows: 1 L of the second fermentation liquid n2 and a mixed culture medium pm2 containing 5.71 g of jujube, 3.14 g of wolfberry, and 0.85 g of turmeric (weight ratio 7:4:1) compatible with 1 L of the second fermentation liquid n2 are sealed in a herbal medicine filter as a substitute for a bag-shaped culture medium filter without prior fermentation, and the mixture is suspended in a test container B. The second fermentation liquid n2 corresponding to 1 L of the starter seed and the third preliminary fermentation liquid pn3 are added to the test container B, stirred, and fermented for 5 days while maintaining a fermentation state at 38°C to 40°C to form a fermentation liquid B containing the third fermentation liquid n3 with almost no supernatant gas layer ( Figure 10 (Photo B) Fermentation broth B differs decisively from fermentation broth A in that a fermentation gas layer having almost no supernatant is formed.

[0221] The results of the comparative test indicate that the difference between fermentation broths A and B may be due to differences in the degree of fermentation caused by the secretory metabolism of acidic substances by fermentation b1. More specifically, fermentation in fermentation broth A was more advanced than in fermentation broth B due to the secretory metabolism of fermentation b1.

[0222] Figure 11 The results show the difference in fermentation levels between fermentation broths A and B over five days, as measured by changes in acidity. The pH of fermentation broth A remained lower than that of fermentation broth B, indicating higher acidity. Until day three, the acidity of fermentation broth A fluctuated between pH 5.11 and 5.09, while that of fermentation broth B fluctuated between pH 5.13 and 5.14. While the acidity of fermentation broth A was higher than that of fermentation broth B, there were no significant changes. However, on days four and five, the acidity of both broths increased significantly, reaching pH 4.76 to 4.68 for fermentation broth A and pH 4.87 to 4.78 for fermentation broth B. Progressive acidification, while acidity in fermentation broth A was higher than that in fermentation broth B, confirmed a certain degree of fermentation due to the secretory metabolism of the anaerobic fermenting bacteria b1.

[0223] also, Figure 11 The figure also shows that using the pre-fermented second fermentation medium m2 in the production of the third fermentation liquid n3 can alter the fermentation environment of the fermentation bacteria b1, increasing the secretion and metabolism of acidic substances by the fermentation bacteria b1, and promoting fermentation. This is the technical significance of using the pre-fermented second fermentation medium m2 in the production of the third fermentation liquid n3.

[0224] In the first preparatory step pp21 of the second step p22 of the second fermentation line f2, as Figure 1 As shown in

[11] ,

[0225] The second fermentation medium m2 with a pH of 4.8± generated in the first step p21 is evenly divided and sealed in a plurality of steam-sterilized bag-shaped medium filters 320 suspended in the third fermentation tank 300.

[0226] The third preliminary fermentation liquid pn3 produced in the first step p21 is transferred from the second culture medium tank 40 to the third fermentation tank 300.

[0227] The second fermentation liquid n2 having a pH of 5.0± was transferred from the second fermentation tank 200 to the third fermentation tank 300 via the extraction device 210 of the second fermentation tank 200 connected to the third fermentation tank 300 .

[0228] In the second preparatory step pp22 of the second step p22 of the second fermentation line f2,

[0229] start up Figure 9 The circulation pump device 330 shown in FIG.

[0230] In the third fermentation tank 300, the second fermentation medium m2 having a pH of 4.8± is sealed in the bag-shaped medium filter 320 suspended therein without being mixed with other materials.

[0231] The third preliminary fermentation liquid pn3 at pH 4.8± and the second fermentation liquid n2 at pH 5.0± are circulated integrally as the second fermentation seed.

[0232] These were fermented for 8 to 9 days while being kept in a fermentation state at 37° C. to 40° C. to generate a third fermentation liquid n3 having a pH of 4.5±, including a turbid liquid having a second upper surface film layer sf2 formed as a supernatant.

[0233] In the second step p22 of the second fermentation line f2, since the second fermentation medium m2 is enclosed in the fine-mesh bag-shaped medium filter 320, the residue of the second fermentation medium m2 does not accumulate at the bottom of the fermentation tank 300. It was confirmed that when the second fermentation liquid n2 is transformed into the third fermentation liquid n3, the acidity of the third fermentation liquid n3 drops to a pH of 5 or less.

[0234] [Details of the third fermentation line f3]

[0235] Figure 1 The third fermentation line f3 shown in

[13] to

[16] includes: a first step p31 for producing a third fermentation medium m3 with a pH of 4.4±, and a second step p32 for producing a fourth fermentation liquid n4 with a pH of 3.7±.

[0236] The overall image of the third fermentation production line f3 is as follows Figure 12 As shown in . Figure 12 The process [a] shows that after the second fermentation liquid n2 produced in the second fermentation line f2 is transferred to the third fermentation tank 300, the second fermentation medium m2 produced in the second culture medium tank 40 is added to the bag culture medium filter 320 provided inside the third fermentation tank 300, and the circulation pump device 330 is started to circulate and ferment for 8 to 9 days, thereby producing the third fermentation liquid n3.

[0237] The third fermentation production line f3 includes a step [c] of adding a portion of the third fermentation liquid n3 of the third culture medium tank 300 and the honey raw material pm3 into the third culture medium tank 50 in appropriate amounts and stirring them, and fermenting them for 2 to 3 days while maintaining a fermentation state of 37°C to 40°C to produce a third fermentation medium m3.

[0238] The third fermentation line f3 further includes steps [d] to [g] of adding the third fermentation liquid n3 removed from the third fermentation tank 300 and the third fermentation medium m3 to the fourth fermentation tank 400 and stirring them to ferment for 30 to 60 days while maintaining a fermentation state of 37° C. to 40° C. to produce a fourth fermentation liquid n4.

[0239] More specifically, the first step p31 of the third fermentation line f3 includes: Figure 12 As shown in [a] to [b], 3 to 5% of the generated 1050 to 1100 L of the third fermentation liquid n3, which is a honey raw material pm3 composed of multi-flower honey and acacia honey in a ratio of 1 to 4, and a portion of the third fermentation liquid n3 equivalent to 4 times the amount of the honey raw material pm3, are transferred to the third culture medium tank 50 in a manner that prevents them from contacting the external air and are stirred. Figure 12 The first preliminary step pp31 of producing the preliminary fermentation medium pfm3 as shown in [c].

[0240] The first step p31 further includes a second preparatory step pp32 of fermenting the preliminary fermentation medium pfm3 produced in the first preparatory step pp31 for 2 to 3 days while maintaining a fermentation state at 37°C to 40°C to produce a third fermentation medium m3 having a pH of 4.4± based on the honey raw material pm3.

[0241] In the second step p32 of the third fermentation production line f3, Figure 12 As shown in [d] to [g],

[0242] The third fermentation medium m3 with a pH of 4.4± is added to the fourth fermentation tank 400.

[0243] The third fermentation liquid n3 with a pH of 4.5± is transferred to the fourth fermentation tank 400 through the extraction device 310 of the third fermentation tank 300 connected to the fourth fermentation tank 400.

[0244] A third fermentation medium m3 at pH 4.4± and a third fermentation liquid n3 at pH 4.5± are mixed and stirred as a fourth fermentation seed, and fermented for 30 to 60 days while maintaining a fermentation state at 37°C to 40°C, forming a third upper surface layer sf3 containing a fermentation gas layer as the supernatant and a third sediment layer dep3 containing a sediment as the lower layer.

[0245] Between the third upper surface layer sf3 and the third sediment layer dep3, a translucent third intermediate layer liquid corresponding to 1000 to 1050 L is generated. The translucent third intermediate layer liquid is the fourth fermentation liquid n4 with a pH of 3.7±.

[0246] [Technical features of the third fermentation line f3]

[0247] Based on years of trial and error, the inventors conceived the third fermentation production line f3 and conducted repeated trial and error. By supplying a certain amount of sugar to the fermentation bacteria b1, the proliferation and secretion metabolism capacity of the fermentation bacteria b1 was improved, and continuous fermentation based on the secretion metabolism of further acidic substances was achieved.

[0248] Through repeated trial and error, the inventors discovered that, based on water activity (if it is too low, bacterial activity in water is inhibited, preventing bacterial growth), the optimal sugar content for maintaining the secretory metabolic capacity of fermentation bacteria B1 is 3-5% of the third fermentation liquid N3. This honey raw material PM3 is mixed with a third fermentation liquid N3 (four times the amount of honey raw material PM3) and stirred to produce a preliminary fermentation medium PFM3. The preliminary fermentation medium PFM3 is fermented for two to three days while maintaining a temperature of 37°C to 40°C to produce the third fermentation medium M3.

[0249] The third fermentation medium m3 was then mixed with a third fermentation liquid n3 (20 times the amount of the third fermentation medium m3) and stirred, and fermented for 30 to 60 days while maintaining a temperature of 37°C to 40°C, thereby promoting the secretion and metabolism of acidic substances by the fermentation bacteria b1. In this way, the inventors successfully generated a fourth fermentation liquid n4 with a pH of 3.7±. This is the third fermentation line f3. The technical features of the third fermentation line f3 are as follows: Figure 13 and Figure 14 As shown in .

[0250] Figure 13 Photographs A and B show the results of a comparative test of specimens A and B of the fermentation liquid model of the fourth fermentation liquid n4.

[0251] Specimen A is fermentation liquid A, which is produced by mixing the third fermentation medium m3 produced by pre-fermenting the honey raw material pm3 with the third fermentation liquid n3 (20 times the amount of the third fermentation medium m3) to make a total of 1 L, stirring, and fermenting for 4 days while maintaining a fermentation state at 38°C to 40°C. Specimen B is fermentation liquid B, which is produced by directly mixing the honey raw material pm3 (third culture medium pm3) with the third fermentation liquid n3 (20 times the amount of the third culture medium pm3) to make a total of 1 L, stirring, and fermenting for 4 days.

[0252] Specimen A is fermentation liquid A prepared by the following steps: A third fermentation medium m3, produced by pre-fermenting honey raw material pm3, is placed in one of the test containers and stirred for four days. This fermentation forms three layers: a third upper surface layer sf3, which is a supernatant fermentation gas layer; a third sediment layer dep3, which contains sediment accumulated below; and a translucent third intermediate layer between the third upper surface layer sf3 and the third sediment layer dep3. The translucent third intermediate layer corresponds to the fourth fermentation liquid n4.

[0253] Specimen B is fermentation liquid B prepared by adding honey raw material PM3 directly to another test container, stirring, and fermenting for 4 days. As can be seen from Photo B, although the third upper surface layer sf3 of the fermentation gas layer is formed in the supernatant, it is much thinner than that of Specimen A. This indicates that the fermentation of Specimen B, caused by the secretion and metabolism of acidic substances by fermentation bacteria B1, is not as active as that of Specimen A. This can be seen in the Figure 14 Confirmed.

[0254] Figure 14 This chart shows the changes in acidity of fermentation broths A and B over four days of fermentation. On day 1, the acidity of both broths A and B was identical, at pH 4.6. On day 2, the pH values ​​rose together (acidity decreased), with broth A reaching pH 4.7 and broth B reaching pH 4.73. From day 3 to day 4, as fermentation progressed, the pH values ​​fell together (acidity increased), with broth A reaching pH 4.6-4.52, while broth B reached pH 4.68-4.6. From day 2 to day 4, the acidity of broth A consistently exceeded that of broth B.

[0255] Figure 14 This graph shows the acidity changes of fermentation broths A and B from the start of fermentation to day 4. The difference between fermentation broths A and B is that the pH of fermentation broth A remains lower than that of fermentation broth B over the four days, indicating that the fermentation process in fermentation broth A is higher than that in fermentation broth B. This demonstrates that the fermentation bacteria B1 in fermentation broth A secretes acidic substances more actively than those in fermentation broth B. Specifically, the inventors modified the fermentation environment of fermentation bacteria B1 by using the third fermentation medium M3, generated by pre-fermenting the honey raw material PM3, rather than directly using the third medium from the honey raw material PM3, when generating the fourth fermentation broth N4. This resulted in the successful activation of fermentation bacteria B1, which enhanced the secretion and metabolism of acidic substances.

[0256] [Details of the 4th fermentation line f4]

[0257] Figure 1In the fourth fermentation line f4 shown in

[17] , a fourth fermentation liquid n4 with a pH of 3.7± is used as the fifth fermentation seed. Fermentation is carried out for 30 to 60 days using only the fermentation bacteria b1 contained in the fourth fermentation liquid n4 while maintaining a fermentation state at 37°C to 40°C, resulting in a fifth fermentation liquid n5 with a pH of 3.6±. This is step p41 of the fourth fermentation line f4. Since there is almost no reduction in volume during the fermentation step, a volume of 1000 to 1050 L of the fifth fermentation liquid n5 is produced.

[0258] Step p41 of the fourth fermentation line f4 is a step of producing a fifth fermentation liquid n5 through the following steps.

[0259] In the first fermentation line f1, a first fermentation liquid n1 is produced using the first fermentation medium m1 in two first fermentation tanks 100 in the first step p11 and the second step p12. This first fermentation liquid n1 is then transferred to a second fermentation tank 200. In the third step p13, a second fermentation liquid n2 is produced in a fermentation environment without using a fermentation medium. In the second fermentation line f2, a third fermentation liquid n3 is produced using the second fermentation liquid n2 produced in the third step p13 of the first fermentation line and the second fermentation medium m2 produced in the second culture medium tank 40. In the third fermentation line f3, a fourth fermentation liquid n4 is produced using the third fermentation liquid n3 produced in the second fermentation line f2 and the third fermentation medium m3 produced in the third culture medium tank 50. In the fourth fermentation line f4, a fifth fermentation liquid n5 is produced in a fermentation environment without using a fermentation medium. In step p41 of the fourth fermentation line f4, the fifth fermentation seed is solely the fourth fermentation liquid n4; no new culture medium or fermentation medium is used.

[0260] [Technical features of the fourth fermentation line f4]

[0261] At first glance, the acidity seems to be unchanged between the pH value of 3.7± in the fourth fermentation liquid n4 and the pH value of 3.6± in the fifth fermentation liquid n5. However, the change in the degree of fermentation in each step based on actual measurement is expressed as the pH value of the acidity. Figure 16 As can be seen from the graph, the acidity of the fifth fermentation liquid n5, which indicates the change in the fermentation degree, gradually increased during the 30th to 60th day of fermentation of the fourth fermentation liquid n4. This demonstrates that the fermentation progressed due to the secretory metabolism of the fermentation bacteria b1 contained in the fourth fermentation liquid n4.

[0262] The technical idea of the 4th fermentation line f4 will be explained. The inventors, in the course of long years of solid trial and error practice, at a certain period, thought that the manufacturing method of the fermentation broth aimed at by the inventors was completed by the 3rd fermentation line f3. At that time, the technical necessity of the 4th fermentation line f4 after the 3rd fermentation line f3 was not foreseen. However, at the end of the 3rd fermentation line f3, the acid substance secreting metabolic activity of the fermentation bacteria b1 was not ended. Therefore, the inventors performed various treatments such as heat sterilization of the fermentation bacteria b1, and tried many times to stably produce the 4th fermentation broth n4 in the 3rd fermentation line f3. As a result, the technical finding obtained by the inventors was that the fermentation bacteria b1 containing spore-forming fermentation bacteria could form spores and survive in a dormant state even under severe conditions such as high temperature, dryness, and deterioration of nutrients, and that such bacteria were difficult to be treated by heat sterilization and the like.

[0263] Based on this technical finding, the inventors focused on the technical problem of whether it was necessary to end the active secretion metabolism of the acid substance of the fermentation bacteria b1 at the end stage of the 3rd fermentation line f3 or to continue it although it would take time. As a result, the inventors found that, based on the technical value of the secretion metabolism of the acid substance of the fermentation bacteria b1, the present invention was successfully completed in the 4th fermentation line f4 and the fermentation process thereafter.

[0264] [Details of the 5th fermentation line f5]

[0265] Figure 1 The 5th fermentation line f5 shown in

[18] to

[19] includes:

[0266] a 1st process p51 of temporarily and rapidly cooling the 5th fermentation broth n5 produced in the 5th fermenter 500 in the 4th fermentation line f4, and returning the rapidly cooled 5th fermentation broth n5 to a normal temperature state; and

[0267] a 2nd process p52 of fermenting the 5th fermentation broth n5 by the fermentation bacteria b1 contained in the 5th fermentation broth n5 in the normal temperature state to produce a 6th fermentation broth n6 corresponding to 1000 to 1050 L of pH 3.3 ±.

[0268] The 6th fermentation broth n6 is produced by fermenting the 5th fermentation broth n5 by only the fermentation bacteria b1 contained in the 5th fermentation broth n5. In this regard, it is the same as in the 4th fermentation line f4 in which the 5th fermentation broth n5 is produced by fermenting the 4th fermentation broth n4 by only the fermentation bacteria b1 contained in the 4th fermentation broth n4. However, the fermentation environment at the time of producing the 6th fermentation broth n6 and the fermentation environment at the time of producing the 5th fermentation broth n5 are decisively different in the following two points. This is the technical feature of the 5th fermentation line f5.

[0269] [Technical features of the 5th fermentation line f5]

[0270] The first point of the first technical feature of the fifth fermentation production line f5 is as follows Figure 15 [a] is a step of transferring the fifth fermentation liquid n5 maintained at a fermentation temperature of 37°C to 40°C in the fifth fermentation tank 500 to the preparatory tank 620 equipped with the cooling device 610 of the sixth fermentation system s6.

[0271] The technical intention of the 5th fermentation production line f5 is as follows Figure 15 As shown in [b], the fifth fermentation liquid n5 is rapidly cooled to a temperature below 4-5°C, which halts the secretion of acidic substances by the fermentation bacteria b1 contained in the fifth fermentation liquid n5. This means that the fermentation environment is switched from one where the fermentation bacteria b1 easily activates its secretory metabolism to one where it is less likely to do so. In this state, the fermentation bacteria b1 enters a dormant state, and its secretory metabolism ceases.

[0272] Will include Figure 15 The 5th fermentation liquid n5 of the fermentation bacteria b1, which has stopped its secretion metabolism due to rapid cooling as shown in [c], is moved from the preparation tank 620 to the 6th fermentation tank 600, and is restored to the normal temperature state according to the season. The fermentation is carried out under the normal temperature environment for 180 to 240 days, and the activity of the fermentation bacteria b1 is gradually promoted, and the secretion metabolism of acidic substances is activated.

[0273] The second technical feature of the fifth fermentation production line f5 is that, instead of the fermentation state of 37°C to 40°C where the fermentation bacteria b1 is easy to move, a normal temperature state is created where the fermentation bacteria b1 is difficult to move. Specifically, a fermentation environment of 5°C to 28°C is created according to the season. Figure 16 The long fermentation period (6-8 months) of 180-240 days, shown in stage 3, promotes the secretion and metabolism of acidic substances by fermentation b1 while simultaneously converging its metabolic activity. Ultimately, fermentation b1 ceases its secretion and metabolism, entering a dormant state, producing a sixth fermentation broth n6 at a pH of 3.3± in the sixth fermentation tank 600.

[0274] Effects of the invention

[0275] In principle, the first fermentation liquid n1 with a pH of 5.3± produced by the present invention is the initial fermentation liquid for producing the sixth fermentation liquid n6 with a pH of 3.3± as the final product. The first fermentation liquid n1 is obtained by using as a fermentation seed a stock solution θ used for producing a fermentation liquid with a pH of 3 to 4 containing colloidal particles with a particle size of no more than 50 nm and short-chain fatty acids. Figure 16As shown in stage 1 of the present invention, fermentation is performed for 50 to 100 days while maintaining a suitable fermentation temperature of 37°C to 40°C. The stock solution θ is a turbid solution (second preliminary fermentation solution pn2) obtained by mixing soft water w, a fermentation medium m1 of soybean paste, and a starting bacterial solution b containing seven types of spore-forming fermentation bacteria b1 (International Deposit Numbers NITE BP-02945 to NITE BP-02951) at suitable volume ratios.

[0276] The turbid liquid (second preliminary fermentation liquid pn2) used as the starter for the first fermentation liquid n1 is referred to as stock solution θ because soft water w and the original bacterial solution b containing spore-forming fermentation bacteria b1 are used only when producing the first fermentation liquid n1. No new soft water w or original bacterial solution b is used when producing the second through sixth fermentation liquids n2 through n6. In each of the steps for producing the second through sixth fermentation liquids n2 through n6, a fermentation environment is established that is suitable for activating the secretion and metabolism of acidic substances by the seven types of fermentation bacteria b1 containing spore-forming fermentation bacteria in the first fermentation liquid n1. This allows individual fermentations to proceed in each step, thereby increasing the acidity of each fermentation liquid.

[0277] According to the present invention, in the method for producing a fermentation liquid having a pH of 3 to 4 and containing colloidal particles having a particle size of not more than 50 nm and short-chain fatty acids, a starting bacterial liquid b containing seven types of fermentation bacteria b1 including spore-forming fermentation bacteria is an essential component. The starting bacterial liquid b is composed of the following seven types of fermentation bacteria b1 including spore-forming fermentation bacteria: Figure 6 The fermentation bacteria shown in the table were deposited on May 16, 2019, with the Japan Patent and Microorganisms Depository (NPMD), an independent administrative institution and one of the international depositary authorities (IDAs) under the Budapest Treaty, under the deposit numbers NITE P-02945, NITE P-02946, NITE P-02947, NITE P-02948, NITE P-02949, NITE P-02950, ​​and NITE P-02951, and were transferred to the same institution as international deposits on April 22, 2020, under the international deposit numbers NITE BP-02945, NITE BP-02946, NITE BP-02947, NITE BP-02948, NITE BP-02949, NITE BP-02950, ​​and NITE BP-02951. If the bacterial liquid b cannot be stably supplied, the present invention cannot be realized.

[0278] The inventors have been repeating trial and error in practice for a long time, and finally discovered and selected, Figure 6Seven types of fermentation bacteria b1 including spore-forming fermentation bacteria that produce organic acids containing short-chain fatty acids including butyric acid, propionic acid and lactic acid shown in were successfully generated, and a starting bacterial liquid b that stably produced the fermentation bacteria b1 was successfully generated.

[0279] The original bacterial liquid b is generated from three layers formed in the two first fermentation tanks 100: a sponge layer sp, a translucent intermediate layer, and a first sediment layer dep. More specifically, the supernatant sponge layer sp and the first sediment layer dep accumulated at the bottom of the first fermentation tanks 100 are removed and mixed to form a paste, which is the original bacterial liquid b. Each of the three layers formed in the two first fermentation tanks 100 contains the seven types of fermentation bacteria b1, including spore-forming fermentation bacteria, that constitute the original bacterial liquid b.

[0280] The sponge layer sp and the first sedimentation layer dep formed in the first fermentation tank 100, excluding the translucent intermediate layer liquid, become the bacterial liquid b. Therefore, if the feed amount of each first fermentation tank 100 is expected to be 900 L, then one-third of the feed amount, that is, 300 L of material, will become the raw material of the bacterial liquid b. If a considerable amount of paste-like bacterial liquid b prepared from this raw material is used as the bacterial liquid b for the next feed, three layers of the same quality as the previous feed will be formed in the first fermentation tank 100, generating a first fermentation liquid n1 with a pH of 5.3±.

[0281] The paste-like starting bacterial liquid b can also be frozen for storage. This is confirmed by the fact that even when a sufficient amount of the frozen starting bacterial liquid b is returned to a fermentation state at 37°C to 40°C, the paste-like starting bacterial liquid b used as the next feed forms three layers of the same quality as the previous feed in the first fermentation tank 100. The acidity of the first fermentation liquid n1, the semi-transparent intermediate layer thus formed, remains stable at pH 5.3±.

[0282] The paste-like bacterial liquid b can be stored for a long time in a large-scale freezing device, but can also be used immediately in the next fermentation line f1 after 50 to 100 days of fermentation while maintaining the fermentation temperature. Figure 16 As can be seen, it takes at least 303 days (10 months) to 477 days (16 months) to produce the sixth fermentation liquid n6, the final product. However, there is no need to wait for production of this final product; continuous production can be carried out at intervals of around 100 days after the completion of production of the first fermentation liquid n1. In other words, the sixth fermentation liquid n6, which has a pH of 3.3, the final product of the present invention, can be produced continuously at intervals of less than 100 days.

[0283] [Fermentation Broth Produced by the Present Invention]

[0284] The fermentation liquid produced by the present invention is produced by fermenting soft water w as a starter; a bacterial liquid b containing seven types of fermentation bacteria b1 including spore-forming fermentation bacteria; dried soybeans; dried plants including jujube, wolfberry, and turmeric as three herbal medicines; and three fermentation media derived from natural raw materials including honey raw materials.

[0285] The following analysis was performed on the sixth fermentation broth n6 with a pH of 3.3±, the final product produced by the present invention, to examine the activity of acidic substances secreted and metabolized by the fermentation bacteria b1 during the fermentation of the first to sixth fermentation broths n1 to n6, and to estimate the elements that constitute the fermentation broth produced by the present invention.

[0286] (1) Analysis of pH Values ​​of the First to Sixth Fermentation Broths

[0287] (2) Particle size analysis of the sixth fermentation broth n6

[0288] (3) Analysis of sugar concentrations in the fourth to sixth fermentation broths n4 to n6

[0289] (4) Analysis of Short-Chain Fatty Acids in Fermentation Broths 2 to 6

[0290] (1) Changes in pH Values ​​of the First to Sixth Fermentation Broths

[0291] The inventors produced the fermentation medium and fermentation liquid according to the present invention every day while monitoring the pH values ​​of the acidity of the fermentation medium and fermentation liquid produced in the above-mentioned steps. Figure 16 This graph shows the average values ​​of acidity between batches, measured and monitored consistently. The ± appended to each pH value indicates pH fluctuations (usually within 0.3) due to variations in the temperature and humidity of the room where the measurement was taken, as well as slight variations in measurement timing. Such fluctuations are common during fermentation.

[0292] like Figure 16 As shown in Figure 1, the evolution of acidity in the first fermentation line f1 is divided into Stage 1. Stage 1 begins with soft water w at pH 7.3 and includes the acidity of the first fermentation medium m1, the first fermentation broth n1, and the second fermentation broth n2. The fermentation time in Stage 1 ranges from a minimum of 55 days to a maximum of approximately 108 days.

[0293] It is noteworthy that the first fermentation medium m1 (pH 4.5±), the starting bacterial liquid b, and soft water w produced in the first culture medium tank 20 were added in large quantities to the two first fermentation tanks 100 and stirred, producing a turbid second preliminary fermentation liquid pn2 (pH 6.4±). This served as the starting point for the fermentation liquid produced by the present invention, also known as the starting fermentation liquid. Subsequently, no further additions of the starting bacterial liquid b and soft water w were made. Therefore, the second preliminary fermentation liquid pn2 (pH 6.4±) corresponded to the stock solution θ of the final product, the sixth fermentation liquid n6 (pH 3.3±), produced by the present invention. The second preliminary fermentation liquid pn2 (pH 6.4±) was then fermented at an appropriate fermentation temperature for 50 to 100 days to produce the first fermentation liquid n1 (pH 5.3±).

[0294] It is noteworthy that in the final part of stage 1, the first fermentation liquid n1 is fermented in the second fermentation tank 200 for 3 to 5 days at an appropriate fermentation temperature using only the fermentation bacteria b1 to produce the second fermentation liquid n2 with a pH of 5.0. This final part overlaps with the process of producing the second fermentation medium m2 with a pH of 4.8 through 2 to 3 days of fermentation in the second fermentation line f2.

[0295] like Figure 16 As shown in Figure 2, the change in acidity in the second to fourth fermentation lines f2 to f4 is divided into Stage 2. Stage 2 begins with the second fermentation broth n2, pH 5.0, in the second fermentation line f2 and ends with the fifth fermentation broth n5, pH 3.6, in the fourth fermentation line f4. Stage 2 also includes the acidity of the third fermentation broth n3, pH 4.5, produced using the second fermentation medium m2 and second fermentation broth n2 from the second fermentation line f2 as starters; the acidity of the fourth fermentation broth n4, pH 3.7, produced using the third fermentation medium m3 and third fermentation broth n3 from the third fermentation line f3 as starters; and the acidity of the fifth fermentation broth n5, pH 3.6, produced using only the fourth fermentation broth n4 from the fourth fermentation line f4 as starters. The fermentation time at the appropriate fermentation temperature for Stage 2 can be as short as 68 days and as long as approximately 129 days.

[0296] The following two changes are noteworthy. The first change is the activation of the secretion and metabolism of acidic substances by the fermentation bacteria b1 in the second fermentation line f2, using the second fermentation medium m2, and the third fermentation line f3, using the third fermentation medium m3. This resulted in an increase in acidity from pH 5.0± in the third fermentation broth n3 to pH 3.7± in the fourth fermentation broth n4. The second change is the production of the fifth fermentation broth n5 in the fourth fermentation line f4, using only the fourth fermentation broth n4 as the starter and fermenting it for 30 to 60 days while maintaining a temperature of 37°C to 40°C. The acidity remained relatively stable, increasing from pH 3.7± in the fourth fermentation broth n4 to pH 3.6± in the fifth fermentation broth n5.

[0297] At the end of the third fermentation line f3, the secretion and metabolism of acidic substances by the fermentation bacteria b1 did not cease. The inventors exploited this fact and, by not halting the active secretion and metabolism of acidic substances by the fermentation bacteria b1 at the end of the third fermentation line f3, discovered the technical value of the fermentation process in the fourth fermentation line f4, which promotes the secretion and metabolism of acidic substances by the fermentation bacteria b1 even during slow fermentation. The fermentation bacteria b1, which include spore-forming, spore-forming bacteria, can form spores and survive in a dormant state even under harsh conditions such as high temperature, dryness, and poor nutrition. Therefore, these bacteria are difficult to treat with heat sterilization or other methods. The inventors recognized the benefit of accelerating the fermentation of the fourth fermentation liquid n4.

[0298] like Figure 16 As shown in Figure 3, the evolution of acidity in the fifth fermentation line f5, using the fifth fermentation liquid n5 with a pH of 3.6± as the starter, is divided into Stage 3. Stage 3 involves rapidly cooling the fifth fermentation liquid n5, maintained at a suitable fermentation temperature of 37°C to 40°C, to a temperature below 4°C, where the secretory metabolism of the fermentation bacteria b1 is difficult to activate. The environment is then switched to a room temperature environment where the secretory metabolism of the fermentation bacteria b1 can be slowly activated. The fifth fermentation liquid n5 is then fermented at room temperature for 180 to 240 days, depending on the season. The fermentation time in Stage 3 will naturally be longer, ranging from a minimum of 180 days (six months) to a maximum of approximately 240 days (eight months).

[0299] The fifth fermentation line f5 appears to be technically identical to the process for maturing the fifth fermentation liquid n5 at pH 3.6. However, in reality, the fifth fermentation line f5 promotes the secretion and metabolism of acidic substances by the fermentation bacteria b1 while simultaneously restraining its metabolic activity. Ultimately, this process halts the secretion and metabolism of the fermentation bacteria b1, causing the spore-forming fermentation bacteria b1 to enter a dormant state, thereby producing the sixth fermentation liquid n6 at pH 3.3.

[0300] The acidity and pH values ​​change during each fermentation period as follows: At the start of the main fermentation in the two first fermentation tanks 100, the acidity is pH 6.4 ± that of the second preliminary fermentation liquid pn2, the stock solution θ. By the end of the first stage, the acidity has increased to pH 5.0 ±. From the end of the first stage, fermentation progresses due to the secretory activity of the fermentation bacteria b1 contained in the second fermentation liquid n2. By the end of the second stage, while maintaining the optimal fermentation temperature of 37°C to 40°C, the acidity has increased to pH 3.6 ±. Even if non-spore-forming fermentation bacteria or aerobic bacteria contained in the primary fermentation liquid b enter the fermentation liquid during these steps or enter during fermentation, the highly acidic environment inhibits the growth of these bacteria, and the dead bacteria accumulate as a precipitate in the lower layer of each fermentation tank.

[0301] During the third stage of prolonged fermentation under a seasonally adjusted, room-temperature fermentation environment, fermentation progresses while promoting the secretory activity of the spore-forming, spore-forming bacteria contained in fermentation bacteria b1. Ultimately, the secretory activity of the spore-forming bacteria contained in fermentation bacteria b1 nearly ceases. Fermentation no longer progresses, and the acidity stabilizes at a pH of ±3.3.

[0302] (2) Particle size analysis of the sixth fermentation broth n6

[0303] Based on years of rigorous practical research, the inventors have noted that the fermented liquid produced by the present invention has an extremely high absorption rate in mammals and other organisms. The inventors commissioned a specialized agency to analyze the composition of the fermented liquid and confirmed that it contains approximately 6.5% to 7.5% colloidal particles. Specifically, the inventors commissioned the Gifu Prefectural Industrial Technology Center to perform particle size distribution analysis using cumulant analysis on the following two fermented liquids produced by the present invention, and the results were obtained.

[0304] The two fermentation broths produced by the present invention were the fourth fermentation broth n4 at pH 3.7 and the sixth fermentation broth n6 at pH 3.3. The particle size distribution analysis based on cumulative amount analysis was prepared as follows. Figure 17 The results of the cumulative amount analysis of the fourth fermentation liquid n4 are shown. Figure 18 The results of the cumulative amount analysis for the sixth fermentation liquid n6 are shown. The cumulative amount analysis is a method for measuring approximately 6.5% to 7.5% of colloidal particles contained in the fermentation liquid.

[0305] Therefore, the samples submitted to the center were the 1st sample (Calbio-5) and the 2nd sample (Calbio-7). The 1st sample (Calbio-5) was prepared by the following procedure: after heat sterilization treatment of the 4th fermentation broth n4 of pH 3.7± generated by fermentation based on fermentation bacteria b1 using fermentation media m1~m3, cooling was performed, it was allowed to precipitate for 1 day, 200 mL of a sample of the supernatant of the precipitated fermentation broth was subjected to a reduction of bacteria, and was divided into 2 bottles of 100 mL bottles. The 2nd sample (Calbio-7) was prepared by the following procedure: after heat sterilization treatment of the 6th fermentation broth n6 of pH 3.3± generated by long-term fermentation of 180 days (6 months)~240 days (8 months) in a fermentation environment at ordinary temperature of the 5th fermentation broth n5 of pH 3.6± fermented by only fermentation bacteria b1 without using a fermentation medium, cooling was performed, it was allowed to precipitate for 1 day, 200 mL of a sample of the supernatant of the precipitated fermentation broth was subjected to a reduction of bacteria, and was divided into 2 bottles of 100 mL bottles.

[0306] Specifically, the 1st sample (Calbio-5) was prepared as follows: the last fermentation medium m3 (pH 4.4±) and the 3rd fermentation broth n3 (pH 4.5±) were used as a starter, fermentation was performed while maintaining a fermentation state of 37°C~40°C for 30 days~60 days, a 3rd upper surface layer sf3 containing a fermentation gas layer, a 3rd precipitate layer dep3 containing a precipitate, and a translucent 3rd intermediate layer liquid between the 3rd upper surface layer sf3 and the 3rd precipitate layer dep3 were formed. The translucent 3rd intermediate layer liquid was the 4th fermentation broth n4 of pH 3.7± which became the 1st sample. The 1st sample corresponds to a fermentation broth generated in the last fermentation process using a fermentation medium.

[0307] Specifically, the 2nd sample (Calbio-7) was prepared as follows: without using a medium or a fermentation medium at all, the 5th fermentation broth n5 of pH 3.6± was used as a starter, the 5th fermentation broth n5 maintained in a fermentation state at a suitable fermentation temperature of 37°C~40°C was temporarily rapidly cooled to 4~5°C or lower which is an environment in which the fermentation bacteria b1 cannot activate a secretion metabolism, and then the environment was switched to an ordinary temperature environment in which the fermentation bacteria b1 can slowly activate a secretion metabolism. Fermentation progressed while promoting the secretion activity of the spore-forming fermentation bacteria contained in the fermentation bacteria b1, and finally the secretion activity of the fermentation bacteria b1 containing the spore-forming fermentation bacteria almost stopped. Fermentation no longer progressed, and the 6th fermentation broth n6 of the final product of pH 3.3± was obtained. The 6th fermentation broth n6 was the 2nd sample.

[0308] Figure 17 The particle size distribution of the 4th fermentation broth n4 is shown. Figure 17These are a graph and table showing the results of a cumulative analysis of the size of approximately 6.5% to 7.5% of colloidal particles contained in the fourth fermentation broth n4, which had been subjected to heat treatment and sterilization. The analysis confirmed that the fourth fermentation broth n4 contained colloidal particles measured in micrometers. Specifically, the graph, with the vertical axis representing frequency y (%) and the horizontal axis representing particle size x (μm) logarithmically, shows the colloidal particle size in micrometers. Specific numerical values ​​can be found in the table showing detailed numerical values. The frequency exceeded 0% between CH numbers 55 and 101. The particle size x of the colloidal particles contained in the first sample can be determined as follows.

[0309] x=0.375μm (y=0.24%)~18.5μm (y=0.17%)

[0310] Figure 18 It shows the particle size distribution of the 6th fermentation liquid n6. Figure 18 These graphs and tables show the results of a cumulative analysis of the size of approximately 6.5% to 7.5% of colloidal particles contained in the sixth fermentation broth n6 after the second sample was heated and sterilized. The analysis confirmed that the sixth fermentation broth n6 contained colloidal particles in the nanometer unit. Specifically, the graph on the upper left, where the vertical axis represents volume distribution y (%) and the horizontal axis logarithmically represents particle size x (nm), shows the colloidal particle size in nanometers. The graph indicates an average particle size of 21.4 nm. Specific numerical values ​​can be found in the table showing detailed numerical values. These values ​​are based on values ​​where the volume distribution frequency exceeds 0%. The particle size x of the colloidal particles contained in the second sample can be inferred as follows.

[0311] x=1.1nm(y=5.7%)~6.6nm(y=0.1%)

[0312] Figure 17 and Figure 18 The difference in the analytical data lies in the difference between the micrometer unit and the nanometer unit which is 1 / 1000 μm. Figure 17 In contrast, the particle size distribution is expressed in microns. Figure 18 The volume distribution frequency is expressed in nanometers.

[0313] At some point, the inventors believed they had achieved their desired method for producing a fermented broth with the third fermentation line f3, and so attempted to treat the fermentation bacteria b1 to halt its metabolic activity, which caused it to secrete acidic substances. The inventors also noted that the spore-forming bacteria contained in the fermentation bacteria b1 could survive in a dormant state by forming spores even under harsh conditions, making it difficult to treat and halt their activity. Through a trial-and-error process, the inventors leveraged the metabolic activity of the active spore-forming bacteria in the fermentation bacteria b1, using the fourth fermentation broth n4 containing micron-sized particles as a starter and fermenting it for 30 to 60 days, resulting in a fifth fermentation broth n5 with a pH of 3.6±.

[0314] The inventors used the fifth fermentation broth, n5, as the final starter, and carried out a long-term fermentation at room temperature for 180 to 240 days (eight months). This allowed the secretory activity of the fermentation bacteria b1 to gradually subside. As a result, the acidity gradually increased through spontaneous fermentation by the fermentation bacteria b1, which included spore-forming bacteria, ultimately stabilizing the acidity at pH 3.3. Furthermore, the inventors achieved the unexpected result of producing the sixth fermentation broth, n6, containing nanometer-sized colloidal particles. When such a fermentation broth is used as a fermented food, it is expected that there will be a significant difference in the in vivo absorption rate between the fermentation broth containing micrometer-sized colloidal particles and the fermentation broth containing nanometer-sized colloidal particles.

[0315] The second performance analysis of the fourth and sixth fermentation broths n4 and n6 involved measuring the sugar content of each broth. The inventors prepared a third fermentation medium m3 from honey raw material pm3. They then mixed 20 times the amount of third fermentation broth n3 into the third fermentation medium m3. The mixture was stirred to establish an optimal fermentation environment with a sugar concentration of 3-5%, ideal for the secretory activity of the fermentation bacteria b1. The fermentation was maintained at 37-40°C for 30-60 days, further promoting the secretion and metabolism of acidic substances by the fermentation bacteria b1. The result was a successful fourth fermentation broth n4 with a pH of 3.7±.

[0316] (3) Analysis of sugar concentrations in the fourth to sixth fermentation broths n4 to n6

[0317] Figure 19 The measurement results are as follows: the sugar mass c1 per 100 mL of the fourth fermentation liquid n4, the sugar mass c2 per 100 mL of the fifth fermentation liquid n5, and the sugar mass c3 per 100 mL of the sixth fermentation liquid n6.

[0318] c1=3.39g / 100mL

[0319] c2=2.88g / 100mL

[0320] c3=1.19g / 100mL

[0321] Based on years of solid trial and error practice, the inventors conceived the third fermentation production line f3. By repeating trial and error, they supplied a certain amount of fermentation medium containing sugars obtained by fermenting honey raw materials to the fermentation bacteria b1, while at the same time improving the proliferation and secretion metabolism capacity of the fermentation bacteria b1, thereby achieving continuous fermentation based on the secretion metabolism of further acidic substances.

[0322] The inventors discovered that, based on water activity, the specific sugar mass required to maintain the secretory metabolism of fermentation bacteria B1 is equivalent to 3-5% of the third fermentation liquid N3 in the honey raw material PM3. This honey raw material PM3 was mixed and stirred with a volume of the third fermentation liquid N3 equal to four times the volume of honey raw material PM3 to produce a preliminary fermentation medium PFM3. The preliminary fermentation medium PFM3 was fermented for two to three days while maintaining a temperature of 37°C to 40°C to produce the third fermentation medium M3. The third fermentation medium M3 was then mixed and stirred with a volume of the third fermentation liquid N3 equal to 20 times the volume of the third fermentation medium M3 and fermented for 30 to 60 days while maintaining a temperature of 37°C to 40°C to promote the secretory metabolism of acidic substances by fermentation bacteria B1. In this way, the inventors successfully produced a fourth fermentation liquid N4 with a pH of 3.7±. This is the third fermentation line F3.

[0323] Figure 13 and Figure 14 This is a technical feature of supplying a certain amount of fermentation medium as fermented sugar to the third fermentation liquid n3 to enhance the growth and secretion metabolic capacity of the fermenting bacteria b1. Figure 13 Photos A and B show the results of a comparative test of specimens A and B, which are fermentation liquid models of the fourth fermentation liquid n4. Specimen A is fermentation liquid A (equivalent to the fourth fermentation liquid n4) prepared by fermenting a third fermentation medium m3, prepared by pre-fermenting honey raw material pm3 at a volume equivalent to 5% of the third fermentation liquid n3, mixed with a third fermentation liquid n3 at a volume 20 times the volume of the third fermentation medium m3 to make a total of 1 L. Specimen B is fermentation liquid B prepared by fermenting honey raw material pm3 directly with a third fermentation liquid n3 at a volume 20 times the volume of the third fermentation medium pm3 to make a total of 1 L.

[0324] In the case of specimen A, three layers were formed in the test container, including a translucent third intermediate layer liquid between the third upper surface layer sf3 and the third sediment layer dep3. This translucent third intermediate layer liquid corresponds to the fourth fermentation liquid n4. In the case of specimen B, as shown in Photo B, although an upper surface layer sf3 was formed in the test container, it was a much thinner layer than that of specimen A. This indicates that the fermentation of specimen B, based on the secretion and metabolism of acidic substances by fermentation bacteria b1, was not as active as that of specimen A. This is due to the fact that Figure 14 It was confirmed in.

[0325] Figure 14 This demonstrates that the fermentation bacteria b1 in fermentation broth A more actively secretes acidic substances. The inventors considered using a third fermentation medium m3, produced by pre-fermenting the honey raw material pm3, rather than directly using the third medium from the honey raw material pm3, when producing the fourth fermentation broth n4. This would alter the fermentation environment of the fermentation bacteria b1 and activate the fermentation bacteria b1 in a manner that enhances the secretion and metabolism of acidic substances.

[0326] The inventors commissioned the Japan Food Analysis Center to analyze the sugar concentrations of samples of the fourth to sixth fermentation broths using the Somogyi method. The fourth fermentation broth, n4, with a pH of 3.7±, was produced as described above. The fifth fermentation broth, n5, with a pH of 3.6±, was produced by fermenting the fourth fermentation broth, n4, with a pH of 3.7±, for 30 to 60 days using only the fermentation bacteria b1, without using a fermentation medium. The sixth fermentation broth, n6, with a pH of 3.3±, was produced by fermenting the fifth fermentation broth, n5, and the fifth fermentation broth, n5, with a pH of 3.6±, for 180 to 240 days (8 months) at room temperature using only the fermentation bacteria b1, without using a fermentation medium. All fermentation broths were heat-sterilized, cooled, and allowed to settle for one day. 200 mL of the supernatant from the settled fermentation broth was sterilized, divided into two 100 mL bottles, and submitted to the Japan Food Analysis Center.

[0327] The analysis results are as follows. Since the fourth fermentation broth, n4, with a pH of 3.7, was produced using 5% honey raw material pm3, 5g of sugar was added per 100mL of the third fermentation broth, n3. After fermentation, the sugar content in the fourth fermentation broth, n4, decreased, reaching a final level of 3.93g per 100mL. This is likely due to the activity of fermentation bacteria b1, which consumed the sugar. Similarly, the sugar content in the fifth fermentation broth, n5, was 2.88g, and in the sixth fermentation broth, n6, was 1.19g. This may also be due to the activity of fermentation bacteria b1, which consumed the sugar.

[0328] The results show that the sugar concentration of the fourth fermentation liquid n4 is close to 4%, the metabolic activity of the fermentation bacteria b1 secreting acidic substances in the third fermentation line f3 has not converged, and the fermentation bacteria b1 is difficult to perform various treatments such as heat sterilization.

[0329] Furthermore, the sugar concentrations of 2.88 g / 100 mL in the fifth fermentation liquid n5 and 1.19 g / 100 mL in the sixth fermentation liquid n6 indicate that prolonged fermentation without forced treatment such as heat sterilization can reduce the sugar concentration in a manner that facilitates the restraint of secretory activity while promoting the active secretory metabolism of the fermentation bacteria b1.

[0330] The process of producing the fifth fermentation liquid n5 through fermentation for 30 to 60 days in the fourth fermentation production line f4, and the process of producing the sixth fermentation liquid n6 through long-term fermentation for 180 days (6 months) to 240 days (8 months) in the fifth fermentation production line f5 are both processes for promoting the secretion activity of spore-forming fermentation bacteria contained in the fermentation bacteria b1 while forming spores, ultimately providing an environment close to a dormant state for the fermentation bacteria b1.

[0331] (4) Analysis of Short-Chain Fatty Acids in Fermentation Broths 2 to 6

[0332] The present invention comprises an acidic substance produced by the secretion activity of seven fermentation bacteria b1 in a bacterial liquid b composed of fermentation bacteria including spore-forming Clostridium (International Deposit Numbers NITE BP-02945 to NITE BP-02951) managed under low temperature. Figure 20 The acidic substances shown in are short-chain fatty acids including butyric acid that form the intestinal ecology of the living body. This can be seen from the comparative analysis of the short-chain fatty acids contained in the second fermentation liquid n2 to the sixth fermentation liquid n6.

[0333] The second fermentation liquid n2 is the final product of the first fermentation line f1. Figure 4 As shown in the conceptual diagram, the first fermentation liquid n1 produced in the second step p12 is transferred from two first fermentation tanks 100 to one second fermentation tank 200. A fermentation environment is established without using a fermentation medium. The first fermentation liquid n1, which has a pH of 5.3, is fermented for three to five days while being maintained at 37°C to 40°C using only the fermentation bacteria b1 from the original bacterial liquid b contained in the first fermentation liquid n1. This produces a second fermentation liquid n2 with a pH of 5.0. The third to sixth fermentation liquids n3 to n6 are the final products of the second to fifth fermentation lines f2 to f5, respectively.

[0334] Regarding the changes in the fermentation broths, the final products of each fermentation production line, the acidic substances produced by the secretion activities of seven fermentation bacteria b1 in the original bacterial liquid b composed of fermentation bacteria including spore-forming Clostridium (International Deposit Numbers NITE BP-02945 to NITE BP-02951) managed under low temperature conditions were observed.

[0335] The samples are as follows:

[0336] (1) The second fermentation liquid n2 with a pH of 5.0± is generated in the first fermentation production line over 55 to 108 days.

[0337] (2) The second fermentation liquid n2 is used as the starter to ferment for 8 to 9 days to produce the third fermentation liquid n3 with a pH of 4.5±,

[0338] (3) The fourth fermentation liquid n4 with a pH of 3.7± is produced by fermenting the third fermentation liquid n3 for 30 to 60 days.

[0339] (4) The 5th fermentation liquid n5 with a pH of 3.6± is produced by fermenting the 4th fermentation liquid n4 for 30 to 60 days.

[0340] (5) The sixth fermentation liquid n6 with a pH of 3.3± was produced by fermenting the fifth fermentation liquid n5 for 180 to 240 days.

[0341] The second fermentation liquid n2(1) to the fifth fermentation liquid n5(4) are all final products of fermentation carried out while maintaining a fermentation state at 37° C. to 40° C. The sixth fermentation liquid n6(5) is the final product of fermentation carried out in a normal temperature fermentation environment different from (1) to (4).

[0342] All fermentation broths (1) to (5) were heat-sterilized and then cooled to allow sedimentation for one day. 200 mL of the supernatant from each fermentation broth that had been precipitated was sterilized and divided into two 100 mL bottles. The samples were then submitted to the Japan Food Analysis Center for short-chain fatty acid analysis using high-performance liquid chromatography (HPLC). Results were obtained for the content of short-chain fatty acids, including lactic acid, propionic acid, and butyric acid, in the second to sixth fermentation broths n2 to n6.

[0343] The results of the test are as follows Figure 20 Specifically, as shown below (unit / 100mL):

[0344]

[0345] In the case of the 2nd fermentation broth n2, lactic acid was not detected, 0.01 g / 100 mL of propionic acid, and 0.18 g / 100 mL of butyric acid were detected. The fermentation broth manufactured by the present application is generated by fermenting natural raw materials. The content of short-chain fatty acids, particularly butyric acid, is 0.30 g / 100 mL in the 3rd fermentation broth n3, 0.54 g / 100 mL in the 4th fermentation broth n4, and 0.60 g / 100 mL in the 5th fermentation broth n5 or the 6th fermentation broth n6 generated by the 4th fermentation broth n4 or the 5th fermentation broth n5 containing only the fermentation bacteria b1 without using a culture medium or a fermentation medium. Evaluation for such a high butyric acid content will be conducted in future basic research, but it can be sufficiently predicted that a butyric acid content of at least 6 g per 1000 mL will have a great impact on the generation of regulatory T cells related to anti-inflammatory effects, bone strength improvement, anti-obesity, anti-cancer effects, and the like.

[0346] Finally, the fermentation metabolite containing short-chain fatty acids accumulated by filtering the 6th fermentation broth n6 with a filter having a mesh size of 0.2 μm is extracted. The extract, as a naturally fermented substance, can be used for various uses including food raw materials or foods themselves. Some of the applicability of the fermentation broth is shown below.

[0347] [Effects of the fermentation broth manufactured in the present application]

[0348] The fermentation broth manufactured by the present application is a fermentation broth of a natural raw material having an acidity of pH 3 to 4, containing colloidal particles having a particle size of not more than 50 nm and butyric acid of not less than 5 g per 1000 mL. Daily intake of the fermentation broth can activate calcium metabolism in a living body, significantly enhance bone strength and bone metabolism. This is confirmed in "Calcium Absorption Exploration Test Based on Inverted Intestinal Tube Test" conducted by Natural Material Exploration Institute, and detailed tests for functional food tests using osteoporosis model mice conducted by the Department of Physiology, Gifu University School of Medicine, and the like.

[0349] Natural Material Exploration Institute (President, Akemi Aoyama) presented a report on "Calcium Absorption Exploration Test Based on Inverted Intestinal Tube Test" in November 2015. The test was conducted on test animals, which were male rats of the system SD, allowed to freely ingest solid feed CRF-1 (Oriental Yeast Co., Ltd.) and distilled water in a water bottle during a preparatory feeding period. The sample was the 6th fermentation broth n6 (Calbio-7).

[0350] The 3 test groups of the test animals were set as follows:

[0351] Control group, containing 5 animals (9 intestinal tubes; 3 intestinal tubes for 30, 60, 90 minute responses, respectively);

[0352] The group supplemented with 0.1% fermentation broth n6 (Calbio-7) consisted of 5 animals (9 intestinal tubes; 3 intestinal tubes for each of the 30-, 60-, and 90-min reactions);

[0353] The group supplemented with 0.5% fermentation broth n6 (Calbio-7) consisted of 5 animals (9 intestines; 3 intestines for each of the 30, 60, and 90 minute reactions).

[0354] The test items and methods are as follows:

[0355] (1) The preparatory rearing period was set to 6 days. Before the start of the main rearing, the animals were preparatory reared and acclimated by free intake of CRF-1 feed.

[0356] (2) Next, an absorption test using the inverted intestinal tube method was performed. This test included: a) preparation of reagents, external and internal solutions, and internal and external solutions; b) preparation of the inverted intestinal tube; c) subjecting the inverted intestinal tube to a reaction for 30, 60, and 90 minutes; d) calcium measurement; and e) statistical analysis. The results were considered significant if the risk ratio was 5% or less.

[0357] The experimental results of the calcium absorption test based on the inverted intestinal tube method are as follows Figure 21 As shown in .

[0358] Figure 21 The graph shows the results of a calcium absorption test using the reversed gut method, using male SD rats as a model animal and administering the test substance Calbio-7. The top graph shows the "calcium absorption amount," and the bottom graph shows the "calcium absorption rate." Comparative studies of the results of calcium absorption using the reversed gut method show that, among various calcium sources, the Calbio-7-supplemented group exhibited significantly higher calcium increases compared to the control group. The bottom graph shows that, among calcium lactate sources, the control group exhibited higher calcium absorption rates. It also shows that, among both calcium citrate and calcium carbonate sources, the group supplemented with 0.5% Calbio-7 exhibited significantly higher calcium absorption rates compared to the control group.

[0359] In summary, the results of this experiment showed that the increase in calcium in the Calbio-7 group at each concentration was significantly higher than that in the control group at all reaction times, with the maximum transfer from the external fluid to the internal fluid at a reaction time of 30 minutes. On the other hand, the calcium absorption rate in the Calbio-7 group was significantly higher than that in the control group at all reaction times, with the highest absorption rate at a reaction time of 60 minutes.

[0360] The experiment using the osteoporosis model mouse was conducted by the Department of Physiology, Faculty of Medicine, Gifu University, and a detailed report of the experiment was made by Abe Tsuyoshi, Department of Physiology, Faculty of Medicine, Gifu University, and was confirmed with a seal by Nagatsuma Shin'ya, Graduate School of Medicine, Gifu University, on February 27, 2017. The experiment was conducted using an osteoporosis model mouse, considering the report of the experiment on November 5, 2015, "Calcium absorption exploration experiment based on the inverted intestinal tube experiment", regarding the effects of administration of the test substance Calbio-7 on bone strength and bone metabolism markers. Hereinafter, a part of the contents will be quoted.

[0361] The experiment was conducted regarding the effects of administration of the test substance on bone density and bone metabolism markers using an osteoporosis model mouse. The outline of the experiment was as follows: In osteoporosis model mice in which estrogen deficiency was induced by bilateral ovariectomy (OVX), the test substance was administered continuously for 8 weeks, and the effects on osteoporosis prevention were explored.

[0362] The experimental method was as follows:

[0363] As experimental animals, 12-week-old rats C57BL / 6 (female, n = 24) were used. Four experimental groups were used, each of which was as follows: 6 rats including normal rats Sham and bilateral ovariectomized rats OVX were included.

[0364] • Sham + water (SWW), n = 6

[0365] • Sham + Calbio-7 (0.5 mL / day) (SCC), n = 6

[0366] • OVX + water (OWW), n = 6

[0367] • OVX + Calbio-7 (0.5 mL / day) (OCC), n = 6

[0368] The administration plan of the test substance was as follows.

[0369] [Table 1]

[0370]

[0371] C7: Calbio-7 of the test substance (6th fermentation broth n6)

[0372] In the case of C7, concentration adjustment was made based on the data of the amount of drinking water so that 0.5 mL was taken on average per day.

[0373] The test project involves bone tissue, including: obtaining data on the ratio of Gla-Osteocalcin, a bone formation marker, and Glu-Osteocalcin, a bone resorption marker, in the blood; and obtaining data on the impact of tests on bone weight, bone strength and bone density.

[0374] Figure 22 The present invention shows the results of a test on the Gla / Glu-Osteocalcin ratio using estrogen-deficient osteoporosis model mice and the results of a test on the effects on bone weight, bone strength, and bone density.

[0375] Figure 22 The effects of test substance administration on the Gla / Glu-Osteocalcin ratio in osteoporosis model mice, which are in an estrogen-deficient state due to bilateral ovariectomy (OVX), are shown for each test group. The bar graph and photographs show the degree of effect on bone weight and strength, and the degree of effect on bone density. Gla-Osteocalcin is a bone formation marker, and Glu-Osteocalcin is a bone resorption marker. Therefore, a high ratio indicates either promoted bone formation or suppressed bone resorption, while a low ratio indicates either suppressed bone formation or promoted bone resorption.

[0376] Indicates the degree of influence on "bone weight and bone strength" Figure 22 The left graph shows the dry bone weight of the femur (weight-adjusted). The right graph shows the bone strength of the femur obtained through a three-point bend test (weight-adjusted). Bilateral ovariectomy (OVX) causes a decrease in dry bone weight. While Calbio-7 ingestion did not restore the decreased bone weight, bone strength tests showed that Calbio-7 ingestion significantly prevented the decrease in bone strength caused by bilateral ovariectomy (OVX).

[0377] Indicates the impact on bone density Figure 22 The left figure shows a typical example of cancellous bone mass within the femur. The right figure shows cancellous bone density (cancellous bone trabeculae weight / cancellous bone cavity volume). A significant decrease in cancellous bone trabeculae density can be observed due to bilateral ovarian removal (OVX). No recovery of this value was observed with Calbio-7 uptake. On the other hand, the SCC group showed insignificantly higher values.

[0378] The above is a partial excerpt from this report. According to the experimenter, the following evaluation results (1) to (3) are recorded in the conclusion section.

[0379] (1) In this experiment, the bone strength of the OCC group was improved by Calbio-7 uptake. However, in terms of dry bone weight and quantitative evaluation of spongy bone trabecula, the OCC group and the OWW group were not different, and thus it can be considered that factors other than bone tissue (trabecular bone density) affect the recovery of bone strength. In addition, regarding the state of bone formation and bone resorption, in the 8th week, the values of the OCC group had a higher tendency compared to the values of the OWW group. It can be considered that calcium metabolism is activated due to certain factors.

[0380] (2) Since the change in the estrogen balance due to bilateral ovariectomy (OVX) is very strong, it can be considered that the amount of calcium consumption cannot be sufficiently supplemented with the amount of calcium contained in the usual feed. Regarding the results of the test on calcium absorption using the inverted intestine method, the amount of calcium increase and the absorption rate were significantly higher than the control group in all calcium sources.

[0381] (3) Considering (1) and (2), and by Calbio-7 uptake, it is possible to significantly prevent the decrease in bone strength due to bilateral ovariectomy (OVX). In order to improve the change in the values such as the increase in bone resorption due to bilateral ovariectomy (OVX), it is particularly preferable to use a feed to which calcium lactate with a significantly high absorption rate and increase rate is added (refer to the "Calcium absorption exploration test based on the inverted intestine test" in November 2015) for additional exploration.

[0382] Regarding the function of the fermentation broth made from natural raw materials containing colloidal particles with a particle size of not more than 50 nm and 5 g or more of caproic acid per 1000 mL, with an acidity of pH 3 to 4, according to the present invention, in May 2014 in Haizen City, Gifu Prefecture, I TECH LAB Co., Ltd. (Test Responsible Person: Masanori Matsura, Report Book Maker: Noboru Nakamura) conducted: (1) a test on the effect of Calbio-7 uptake on intestinal bacterial flora; and (2) a test on the anti-cancer effect, and the test has been reported. The test substance thereof is also Calbio-7.

[0383] In test (1), C57BL / 6 mice (male, 7 weeks old) were divided into a control group and a Calbio-7 administration group (n = 10), and the control group was orally administered with water for injection for 28 consecutive days at 0.5 mL / body, and the Calbio-7 administration group was orally administered with Calbio-7.

[0384] In this experiment (1), colon contents were collected after the end of the dosing period and analyzed or frozen for storage. Bacterial DNA was extracted from the colon contents using a special kit for bacterial nucleic acid extraction. The concentration of the obtained DNA was measured and its purity was confirmed. When extracting DNA, samples from 3 or 4 individuals in the same group were used as a sample pool in equal amounts, and DNA was extracted from the pool. Specific primers for lactic acid bacteria, bifidobacteria and clostridium, and universal primers were used to quantify bacterial DNA. While calculating the relative copy number of each bacterial DNA, the ratio of each relative to the control group was calculated.

[0385] The results of the experiment (1) on the effect of Calbio-7 intake on intestinal bacterial flora are as follows Figure 23 As shown in . Figure 23 The present invention shows the results of a test on the effect of the fermentation liquid (Calbio-7) produced by the present invention on intestinal bacterial flora when administered to mice.

[0386] Figure 23 The graph above shows that among the intestinal flora, the number of beneficial intestinal bacteria, namely "good bacteria", which include at least lactic acid bacteria and bifidobacteria, has doubled. Figure 23 The graph below shows that the number of malignant intestinal bacteria, or "harmful bacteria," including at least Clostridium perfringens, in the intestinal bacterial flora has been reduced by half.

[0387] Figure 24 The results of Experiment (2) are shown. Experiment (2) analyzed gene expression in tumor tissues using real-time PCR. Specifically, after observing gene expression changes using DNA microarray (ITL-13-MO-049), primers specific for the Krt6b (Keratin 6B) gene were designed and quantitatively analyzed using real-time PCR.

[0388] In experiment (2), Lewis lung carcinoma cells, which are epithelial cell cancer cells, were used as tumor tissues in mice administered with Calbio-7. The gene expression of Krt6b, a tumor growth factor, was compared and analyzed in comparison with a control group administered with injection water. As a result, the expression of keratinocyte (keratinocyte) genome, such as keratin, was observed. In short, Krt6b is a marker for distinguishing squamous cell carcinomas in lung cancer, such as Figure 24 As shown in , inhibition of the expression of the Krt6b gene was observed.

[0389] [Summary of test results]

[0390] Regarding the functions of the fermentation liquid produced from natural raw materials according to the present invention, which contains about 6.5% to 7.5% of colloidal particles with a particle size of no more than 50nm and at least 5g of butyric acid per 1000mL and has an acidity of pH 3 to 4, on living organisms, the fermentation liquid can be used as a functional food itself or as a raw liquid for a functional food. The first point of summary is: from Figure 21 The data of "increase amount and absorption rate" as a result of the calcium absorption test based on the reversed intestinal tube method shown in, and Figure 22 The data on the "Gla / Glu-Osteocalcin ratio" and "bone weight, bone strength, and bone density" shown in the results of an experiment using estrogen-deficient osteoporosis model mice indicate that the fermentation liquid acts on the living body, activating calcium metabolism and significantly affecting bone strength and bone metabolism.

[0391] The second point of the summary is: From the increase of "Lactic acid bacteria" and "Bifidobacteria" and the decrease of "Clostridium perfringens", Figure 23 The data show that the fermentation liquid has an effect on the intestinal bacterial flora, causing the benign intestinal bacteria, namely lactic acid bacteria and bifidobacteria, which are called "good bacteria", to double, and the malignant intestinal bacteria, namely Clostridium perfringens, which is called "harmful bacteria", to be halved. Figure 24 As shown in , suppression of Krt6b gene expression was confirmed in tumor tissues (Lewis Lung carcinoma-syngraft model) of Calbio-7-administered mice.

[0392] [Supplementary Materials]

[0393] Seven types of fermentation bacteria deposited and registered with the Japan Patent Microorganisms Collection (NPMD), an independent administrative institution. (Deposited domestically on May 16, 2019, and transferred to an international deposit on April 22, 202)

[0394] Based on the results of partial base sequence analysis (approximately 1500 bp) of the 16S rDNA (16S rRNA gene), which is used by HigherMount Co., Ltd. to isolate, culture, and identify fermentation bacteria when producing fermentation products, the following bacteria have been confirmed to be harmless to humans and animals and can be recovered from dried or freeze-dried forms.

[0395] NITE BP-02945

[0396] Type of microorganism: bacteria

[0397] Cell shape: bacillus

[0398] Characteristics: Negative for spore formation (non-spore forming)

[0399] Taxonomic position Aneurinibacillus sp.

[0400] Culture conditions Standard agar medium

[0401] Culture temperature 30°C

[0402] Culture method Aerobic

[0403] NITE BP-02946

[0404] Type of microorganism Bacterium

[0405] Taxonomic position Brevibacillus sp.

[0406] Culture conditions Standard agar medium Culture temperature 30°C Culture time 48 hours

[0407] Culture method Aerobic

[0408] NITE BP-02947

[0409] Type of microorganism Bacterium

[0410] Cell shape Bacillus

[0411] Characteristics Spore formation negative (asporic)

[0412] Taxonomic position Pseudoclavibacter sp.

[0413] Culture conditions Standard agar medium

[0414] Culture temperature 30°C

[0415] Culture time 72 hours

[0416] Culture method Aerobic

[0417] NITE BP-02948

[0418] Type of microorganism Bacterium

[0419] Cell shape Bacillus

[0420] Characteristics Spore formation positive (sporulated)

[0421] Taxonomic position Paenibacillus sp.

[0422] Culture conditions Standard agar medium

[0423] Cultivation temperature: 30°C

[0424] Cultivation time: 72 hours

[0425] Cultivation method: Aerobic

[0426] NITE BP-02949

[0427] Type of microorganism: bacteria

[0428] Cell shape: bacillus

[0429] Characteristics: Positive for spore formation (spore-forming)

[0430] Taxonomic position Clostridium sp.

[0431] Culture conditions: GAM Broth "Nissui" agar

[0432] Cultivation temperature: 30°C

[0433] Cultivation time: 48 hours

[0434] Culture method: anaerobic

[0435] NITE BP-02950

[0436] Type of microorganism: bacteria

[0437] Cell shape: bacillus

[0438] Characteristics: Positive for spore formation (spore-forming)

[0439] Taxonomic position Clostridium sp.

[0440] Culture conditions: GAM Broth "Nissui" agar

[0441] Cultivation temperature: 30°C

[0442] Cultivation time: 48 hours

[0443] Culture method: anaerobic

[0444] NITE BP-02951

[0445] Type of microorganism: bacteria

[0446] Cell shape: bacillus

[0447] Characteristics: Positive for spore formation (spore-forming)

[0448] Taxonomic position Clostridium sp.

[0449] Culture condition GAM Broth "Nissui" agar

[0450] Culture temperature: 30°C

[0451] Culture time: 48 hours

[0452] Culture method: anaerobism

[0453] Explanation of symbols

[0454] 1 Fermented broth having a pH of 3 to 4 containing colloidal particles having a particle size of not more than 50 nm and short-chain fatty acids

[0455] n1 to n6 1st to 6th fermented broths

[0456] pn1 to pn3 1st to 3rd preliminary fermented broths

[0457] w Soft water

[0458] m Fermentation medium

[0459] m1 to m3 1st to 3rd fermentation media

[0460] ds Dried soybean

[0461] fs Fermented soybean

[0462] gfs Soybean paste

[0463] pm2 Mixed culture medium

[0464] pfm2 Preliminary fermentation mixed medium

[0465] pm3 Honey raw material

[0466] pfm3 Preliminary fermentation medium

[0467] b Primary bacteria solution

[0468] b1 Fermentation bacteria contained in the primary bacteria solution (International Depositary Accession Nos. NITE BP-02945 to NITE BP-02951)

[0469] sp Sponge layer

[0470] sf1 to sf4 1st to 4th upper surface layers

[0471] dep1 to dep4 1st to 4th deposition layers

[0472] f Fermentation stage

[0473] f1~f5 1st fermentation line~5th fermentation line

[0474] p11 to p13 f1 Step 1 to Step 3

[0475] p21 / p22 f2 first step / second step

[0476] p31 / p32 f3 first step / second step

[0477] p41 f4 process

[0478] p51 f5 process

[0479] 10 Fermentation bottles (ceramic)

[0480] 20 f1 first culture medium tank

[0481] 30 F1 heating kettle

[0482] 40 f2 second culture medium tank

[0483] 50 f3 third culture medium tank

[0484] 100 f1 1000 liters first fermentation tank (2)

[0485] 101 The opening and closing valve of the first fermentation tank using Pascal's principle and hydrostatic pressure balance

[0486] 110 The first fermentation tank has an opening and closing valve to remove the device

[0487] 200 f1 second fermentation tank with a capacity of 2000 liters

[0488] 201 The opening and closing valve of the second fermentation tank using Pascal's principle and hydrostatic pressure balance

[0489] 210 The removal device with the opening and closing valve of the second fermentation tank

[0490] 300 f2 2000 liters third fermentation tank

[0491] 301 The opening and closing valve of the third fermentation tank using Pascal's principle and hydrostatic pressure balance

[0492] 310 The third fermentation tank has an opening and closing valve removal device

[0493] 320 f2 bag culture medium filter

[0494] 330 f2 circulation pump device

[0495] 400 f3 4th fermentation tank with a capacity of 2000 liters

[0496] 401 The opening and closing valve of the fourth fermentation tank using Pascal's principle and hydrostatic pressure balance

[0497] 410 The fourth fermentation tank has an opening and closing valve removal device

[0498] 500 f4 is equivalent to the 5th fermentation tank with a capacity of 2000 liters

[0499] 501 The opening and closing valve of the fifth fermentation tank using Pascal's principle and hydrostatic pressure balance

[0500] 510 The removal device with the opening and closing valve of the fifth fermentation tank

[0501] 600 f5 6th fermentation tank with a capacity of 2000 liters

[0502] 610 f5 cooling device

[0503] 620 f5 reserve tank

[0504]

[0505]

[0506]

[0507]

[0508] Fill in the column for the receiving office

[0509]

[0510] International Affairs Bureau Fill-in Column

[0511]

Claims

1. A method for producing a fermentation broth having an acidity of pH 3-4, wherein the fermentation broth contains colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids, wherein the short-chain fatty acids include butyric acid, propionic acid, and lactic acid, the method comprising: Prepare the following materials: Soft water with a pH of 7.0 to 7.6; Three fermentation media, comprising: a first fermentation medium with a pH of 4.2 to 4.8 produced from dried soybeans; a second fermentation medium with a pH of 4.5 to 5.1 produced from dried plants including wolfberries, jujubes, and turmeric; and a third fermentation medium with a pH of 4.1 to 4.7 produced from honey; and A bacterial liquid with a pH of 5.0 to 5.6, comprising fermentation bacteria that produce organic acids including short-chain fatty acids, wherein the fermentation bacteria are composed of seven species with international deposit numbers of NITE BP-02945, NITE BP-02946, NITE BP-02947, NITE BP-02948, NITE BP-02949, NITE BP-02950, ​​and NITE BP-02951. Perform step A comprising the following steps: The soft water, the first fermentation medium, and the original bacterial solution are added to a first fermentation tank at a volume ratio of 1100 L to 1300 L of the soft water, 280 L of the first fermentation medium, and 270 L to 450 L of the original bacterial solution, and mixed to produce a turbid solution with a pH of 6.1 to 6.7; Fermenting the turbid liquid at a fermentation temperature of 37° C. to 40° C. for 50 to 100 days to form three layers including: a sponge layer containing a supernatant of fermented gas, a first sedimentation layer as a lower layer, and an intermediate layer of translucent fermentation liquid with a pH of 5.0 to 5.6 between the sponge layer and the first sedimentation layer; as well as The step of taking out the translucent fermentation liquid from the three layers to generate a first fermentation liquid with a pH of 5.0 to 5.6, Carry out process B comprising the following steps: The step of transferring the first fermentation broth having a pH of 5.0 to 5.6 to a second fermentation tank; Fermenting the first fermentation broth at a fermentation temperature of 37° C. to 40° C. for 3 to 5 days in a fermentation environment without using a fermentation medium, thereby forming three layers including: a first upper surface layer comprising a supernatant of fermented gas bubbles, a second sediment layer as a lower layer on which residues of the first fermentation medium are accumulated, and an intermediate layer of translucent fermentation broth having a pH of 4.7 to 5.3 between the first upper surface layer and the second sediment layer; as well as The step of taking out the translucent fermentation liquid from the three layers to generate a second fermentation liquid with a pH of 4.7 to 5.3, Carry out process C comprising the following steps: The step of transferring the second fermentation broth having a pH of 4.7 to 5.3 to a third fermentation tank; The second fermentation medium is sealed in a bag filter, the bag filter is suspended in the third fermentation tank, and the second fermentation liquid is fermented at a fermentation temperature of 37° C. to 40° C. for 8 to 9 days while circulating the second fermentation liquid to form two layers including a second upper surface layer of a supernatant containing fermentation gas bubbles and a lower layer of a turbid fermentation liquid with a pH of 4.2 to 4.8; and The step of removing the turbid fermentation liquid from the second layer to generate a third fermentation liquid with a pH of 4.2 to 4.8, Performing step D comprising the following steps: The step of transferring the third fermentation broth having a pH of 4.2 to 4.8 to a fourth fermentation tank, and mixing the third fermentation broth and the third fermentation medium at a volume ratio of 20 to 1 to generate a turbid liquid; fermenting the turbid liquid at a fermentation temperature of 37° C. to 40° C. for 30 to 60 days to form three layers including: a supernatant third upper surface layer, a lower third sediment layer, and an intermediate layer of translucent fermentation liquid having a pH of 3.4 to 4.0 between the third upper surface layer and the third sediment layer; and The step of taking out the translucent fermentation liquid from the three layers to generate a fourth fermentation liquid with a pH of 3.4 to 4.0, Performing step E comprising the following steps: The step of transferring the fourth fermentation broth having a pH of 3.4 to 4.0 to a fifth fermentation tank; Fermenting the fourth fermentation broth at a fermentation temperature of 37° C. to 40° C. for 30 to 60 days in a fermentation environment without using a fermentation medium to generate a translucent fermentation broth with a pH of 3.3 to 3.9 and a fourth precipitation layer; and The step of taking out the translucent fermentation liquid to generate a fifth fermentation liquid with a pH of 3.3 to 3.9, Performing step F comprising the following steps: The fifth fermentation liquid with a pH of 3.3 to 3.9 is transferred to a preparatory tank of a cooling device, and the fifth fermentation liquid with a fermentation temperature of 37° C. to 40° C. is rapidly cooled to below 5° C. by the cooling device; The step of returning the rapidly cooled fifth fermentation liquid to room temperature to generate a fermentation liquid at room temperature; fermenting the room temperature fermentation broth at room temperature for 180 to 240 days to produce a transparent fermentation broth with a pH of 3.0 to 3.6; as well as The transparent fermentation liquid is taken out to generate a sixth fermentation liquid with a pH of 3.0 to 3.6, When it is expected that 1200 L of the first fermentation liquid will be produced, the step of producing the first fermentation medium having a pH of 4.2 to 4.8 includes: In four or more fermentation bottles equipped with valve-functioning lids, 6.5 kg of the dried soybeans are immersed in at least 20 L of the soft water, the soybeans are soaked with water, and 250 g of the sugar chain and 140 cc of the original bacterial solution are added to each fermentation bottle; fermenting the contents of the fermentation bottles at a fermentation temperature of 37° C. to 40° C. for 68 to 74 hours to produce 35 L of a first preliminary fermentation liquid and fermented soybeans in each fermentation bottle; The fermented soybeans are taken out from each of the fermentation bottles, ground into a paste by a pulverizing means, and the ground soybeans are mixed with the first preliminary fermentation liquid to produce 35 L of the first preliminary fermentation liquid and soybean paste; The first preliminary fermentation liquid and the soybean paste, each containing 35 L of the fermented liquid, are transferred to a heating kettle, soft water is added, and the mixture is slowly heated to 55-60° C. to produce a total of 180 L of the first preliminary fermentation liquid and the soybean paste; as well as The first fermentation medium is prepared by transferring 180 L of the first preliminary fermentation liquid and the soybean paste from the heating kettle to a first culture medium tank containing 100 L of soft water previously charged for cooling without contact with the outside air, and stirring the mixture to produce 280 L of the first fermentation medium. When it is expected that 1050 to 1100 L of the third fermentation broth will be produced, the method for producing the second fermentation medium having a pH of 4.5 to 5.1 comprises: a step of mixing 200-210 g of jujube, 110-120 g of wolfberry, and 25-30 g of turmeric with the second fermentation broth (35 L in total) to generate a mixed culture medium; The mixed culture medium and 35 L of the second fermentation broth are added to a second culture medium tank, and the mixed culture medium and the second fermentation broth are fermented for 2 to 3 days to generate a preliminary fermentation mixed culture medium with a pH of 4.5 to 5.1 and a third preliminary fermentation broth; as well as The steps of removing the preliminary fermentation mixed medium from the second culture medium tank, crushing the removed preliminary fermentation mixed medium by a crushing means, returning the crushed preliminary fermentation mixed medium to the second culture medium tank, and mixing the preliminary fermentation mixed medium with the third preliminary fermentation liquid to generate 35 L of a second fermentation medium having a pH of 4.5 to 5.1, When it is expected that 1000 to 1050 L of the fourth fermentation broth will be produced, the method for producing the third fermentation medium comprises: The steps of adding the honey raw material equivalent to 3-5% of the third fermentation liquid produced in the third fermentation tank to a third culture medium tank, transferring the third fermentation liquid equivalent to 4 times the honey raw material to the third culture medium tank in a manner not to be exposed to external air, and stirring them to produce a preliminary fermentation medium; as well as The step of fermenting the preliminary fermentation medium at a fermentation temperature of 37° C. to 40° C. for 2 to 3 days to generate a third fermentation medium with a pH of 4.1 to 4.

7.

2. The method according to claim 1, wherein The process A is a process A' comprising the following steps: Steps for preparing two first fermentation tanks; The soft water, the first fermentation medium, and the original bacterial solution are added equally into the two first fermentation tanks at a volume ratio of 1100 L to 1300 L of the soft water, 280 L of the first fermentation medium, and 270 L to 450 L of the original bacterial solution, respectively, and mixed to produce a turbid solution with a pH of 6.1 to 6.7; Fermenting the turbid liquid at a fermentation temperature of 37° C. to 40° C. for 50 to 100 days to form three layers including: a sponge layer comprising a supernatant of fermented gas, a first sedimentation layer as a lower layer, and an intermediate layer of translucent fermentation liquid having a pH of 5.0 to 5.6 between the sponge layer and the first sedimentation layer; as well as The step of taking out the translucent fermentation liquid from the three layers to generate a first fermentation liquid with a pH of 5.0 to 5.6, The process B is a process B' comprising the following steps: The first fermentation broth with a pH of 5.0 to 5.6 is transferred to a second fermentation tank respectively, and mixed to generate a first fermentation broth with uniform acidity; The method further comprises the steps of fermenting the first fermentation broth at a fermentation temperature of 37° C. to 40° C. for 3 to 5 days in a fermentation environment without using a fermentation medium, thereby forming three layers including: a first upper surface layer as a supernatant containing fermentation gas bubbles; a second sediment layer as a lower layer on which residues of the first culture medium are accumulated; and an intermediate layer of translucent fermentation broth having a pH of 4.7 to 5.3 between the first upper surface layer and the second sediment layer; as well as The step of taking out the translucent fermentation liquid from the three layers to generate a second fermentation liquid with a pH of 4.7 to 5.

3.

3. The method according to claim 1 or 2, wherein: The fermentation broth with an acidity of pH 3-4 contains 0.5 g-0.6 g of butyric acid per 100 mL of fermentation broth.

4. The method according to claim 1 or 2, wherein: When it is expected that 1200 L of the first fermentation liquid will be produced, The turbid liquid with a pH of 6.1 to 6.7 produced in the first fermentation tank is 1800 L of a second preliminary fermentation liquid with a pH of 6.1 to 6.7 produced by adding 280 L of the first fermentation medium with a pH of 4.2 to 4.8, 270 L to 450 L of the bacterial liquid with a pH of 5.0 to 5.6, and 1000 L to 1300 L of soft water with a pH of 7.0 to 7.6 into the first fermentation tank, and stirring and mixing them.

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