Lactic acid bacteria preparation and method for producing the lactic acid bacteria preparation

Abstract

To provide a low-cost lactic acid bacteria preparation that includes lactic acid bacteria derived from corn.SOLUTION: A lactic acid bacteria preparation characterized by containing lactic acid bacteria derived from corn.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a lactic acid bacteria preparation characterized by containing lactic acid bacteria derived from corn, and a method for producing the lactic acid bacteria preparation. [Background technology]

[0002] Traditionally, cornstarch has been produced using corn as a raw material. The process of producing cornstarch from corn involves a step called wet milling, in which corn kernels (maize) are soaked in sulfurous acid. Wet milling softens the maize, disrupting the protein membrane surrounding the starch granules in the endosperm, allowing the starch and protein to easily separate, and these soluble components are eluted into the sulfurous acid. Naturally occurring lactic acid bacteria attached to the maize then grow using the eluted components as a nutrient source, allowing lactic acid fermentation to proceed while suppressing spoilage of the soaking liquid. After lactic acid fermentation, the soaking liquid is separated from the maize to obtain corn steep water (CSW). As described above, this CSW contains a large amount of lactic acid bacteria that have grown through fermentation.

[0003] During the cornstarch production process, CSW is concentrated to a concentration of approximately 50 wt% by evaporating the water, producing corn steep liquor (CSL). CSL is mainly mixed with maize husks (fiber) to produce gluten feed. Gluten feed is primarily used as a feed ingredient. In addition to gluten feed, other uses of CSL have been proposed, including as a fertilizer ingredient for plant growth regulators and the like (Patent Document 1) and as a nutrient source for production media for antibiotics and the like (Patent Document 2). However, although CSL and gluten feed contain killed lactic acid bacteria, their content is low, and neither use can be said to fully utilize the functions of lactic acid bacteria. Furthermore, none of these uses fully utilize lactic acid bacteria, as neither of these uses fully utilizes lactic acid bacteria.

[0004] On the other hand, lactic acid bacteria preparations containing beneficial lactic acid bacteria are generally used to improve the natural immune system of living organisms, primarily for the purpose of maintaining human health. Ingestion of lactic acid bacteria maintains and activates the internal environment, particularly the intestinal bacteria, and can enhance the immune system of living organisms.

[0005] However, general lactic acid bacteria preparations have several problems when used as livestock feed. For example, lactic acid bacteria preparations are produced in dedicated facilities through complicated processes such as fermentation (cultivation), purification, concentration, and drying, just to obtain lactic acid bacteria. However, the amount of lactic acid bacteria produced is very small relative to the amount of fermentation liquid, and the production of lactic acid bacteria preparations is very complicated. As a result, lactic acid bacteria preparations are very expensive, making them difficult to use as livestock feed.

[0006] Therefore, it is conceivable that an inexpensive lactic acid bacteria preparation using the lactic acid bacteria contained in CSW, as in the present invention, can be produced that does not require a fermentation process in dedicated equipment just to obtain the lactic acid bacteria, and used as livestock feed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2022-183435 [Patent Document 2] Japanese Patent Application Laid-Open No. 2004-329157 Summary of the Invention [Problem to be solved by the invention]

[0008] As a result of extensive research, the inventors discovered that lactic acid bacteria preparations can be produced efficiently and inexpensively by isolating and concentrating lactic acid bacteria contained in CSW generated during the process of producing corn starch from corn, and thus came up with the present invention.

[0009] An object of the present invention is to provide an inexpensive lactic acid bacteria preparation containing corn-derived lactic acid bacteria. Another object of the present invention is to provide an efficient and inexpensive method for producing a lactic acid bacteria preparation containing corn-derived lactic acid bacteria from CSW. [Means for solving the problem]

[0010] The present invention provides a lactic acid bacteria preparation characterized by containing corn-derived lactic acid bacteria isolated from CSW.

[0011] The lactic acid bacteria are preferably isolated from corn steepwater produced in the wet milling process of maize.

[0012] The lactic acid bacteria preferably include at least Lactobacillus secaliphilus, Lactobacillus amylovorus, and Lactobacillus frumenti.

[0013] The lactic acid bacteria preparation preferably contains 10 billion or more lactic acid bacteria per gram.

[0014] The lactic acid bacteria are preferably killed.

[0015] The lactic acid bacteria preparation preferably contains 10 to 40 parts by mass of salt per 100 parts by mass of concentrated bacteria of the lactic acid bacteria preparation. The concentrated bacterial cells are a liquid containing lactic acid bacteria as the main component, separated from CSW by centrifugal sedimentation or the like.

[0016] The lactic acid bacteria preparation preferably contains 0.5 to 2 parts by mass of a viscosity adjuster relative to 100 parts by mass of concentrated bacteria cells of the lactic acid bacteria preparation.

[0017] The lactic acid bacteria preparation is preferably used as livestock or pet feed containing the lactic acid bacteria preparation.

[0018] The present invention also provides a method for producing a lactic acid bacteria preparation containing corn-derived lactic acid bacteria, which includes a separation step of separating concentrated bacterial cells from corn steep water and a sterilization step of heating the concentrated bacterial cells.

[0019] The method for producing the lactic acid bacteria preparation preferably includes a step of adding salt, and the amount of salt added is preferably 10 to 40 parts by mass per 100 parts by mass of concentrated bacteria in the lactic acid bacteria preparation.

[0020] The method for producing the lactic acid bacteria preparation preferably includes a step of adding a viscosity adjuster, and the amount of the viscosity adjuster added is preferably 0.5 to 2 parts by mass per 100 parts by mass of concentrated bacteria cells of the lactic acid bacteria preparation. [Effects of the Invention]

[0021] The lactic acid bacteria preparation of the present invention can be produced efficiently and inexpensively by using corn steep water generated during the process of producing corn starch from corn. Furthermore, since the lactic acid bacteria preparation of the present invention has excellent immunostimulating effects and intestinal regulating effects, when used as livestock feed containing the lactic acid bacteria preparation, it can enhance the immunity of livestock. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows the immunostimulatory effect of the lactic acid bacteria preparation of the present invention. [Figure 2] FIG. 1 shows the sedimentation rate of the lactic acid bacteria preparation of the present invention when xanthan gum is used as the viscosity adjuster. [Figure 3] FIG. 1 shows the sedimentation rate of the lactic acid bacteria preparation of the present invention when guar gum is used as a viscosity adjuster. DETAILED DESCRIPTION OF THE INVENTION

[0023] The lactic acid bacteria preparation of the present invention is produced using CSW, which is generated during the process of producing corn starch from corn as a raw material, and therefore has the major characteristic of containing lactic acid bacteria derived from corn.

[0024] The form of the lactic acid bacteria preparation of the present invention is not limited, and may be, for example, a powder, a liquid, or a solid tablet.

[0025] The genus of lactic acid bacteria contained in the lactic acid bacteria preparation of the present invention is preferably Lactobacillus. Examples of lactic acid bacteria species within the genus Lactobacillus include Lactobacillus secaliphilus, Lactobacillus amylovorus, and Lactobacillus frumenti.

[0026] The lactic acid bacteria preparation of the present invention preferably contains at least the above-mentioned Lactobacillus secaliphilus, Lactobacillus amylovorus, and Lactobacillus frumenti. By containing these bacterial species, the immunostimulatory effect and the intestinal regulating effect are more effectively exerted.

[0027] The lactic acid bacteria preparation of the present invention preferably contains a large amount of lactic acid bacteria. Specifically, it preferably contains 10 billion or more lactic acid bacteria per gram. When the lactic acid bacteria preparation of the present invention contains 10 billion or more lactic acid bacteria per gram, it effectively exhibits immunostimulatory effects and intestinal regulating effects.

[0028] The lactic acid bacteria contained in the lactic acid bacteria preparation of the present invention are preferably killed. This is because live lactic acid bacteria contained in the lactic acid bacteria preparation may affect the flavor and quality of the lactic acid bacteria preparation. In the present invention, the lactic acid bacteria are killed by heating in the sterilization step described below. Although traces of live bacteria may remain, it is more preferable that all of the lactic acid bacteria contained in the lactic acid bacteria preparation are killed, from the viewpoint of the flavor and quality of the lactic acid bacteria preparation.

[0029] The lactic acid bacteria preparation of the present invention preferably contains salt to improve storage stability at room temperature. Specifically, the lactic acid bacteria preparation preferably contains 10 to 40 parts by mass, preferably 20 to 35 parts by mass, and more preferably 25 to 30 parts by mass of salt per 100 parts by mass of concentrated bacterial cells of the lactic acid bacteria preparation. If the amount of salt contained in the lactic acid bacteria preparation is less than 10 parts by mass per 100 parts by mass of concentrated bacterial cells of the lactic acid bacteria preparation, the storage stability of the lactic acid bacteria preparation is poor. On the other hand, if the amount of salt contained in the lactic acid bacteria preparation is more than 40 parts by mass per 100 parts by mass of concentrated bacterial cells of the lactic acid bacteria preparation, the salt will remain undissolved.

[0030] When the lactic acid bacteria preparation of the present invention is liquid, it is preferable to contain a viscosity adjuster to stabilize the viscosity of the liquid. Specifically, the lactic acid bacteria preparation preferably contains 0.5 to 2 parts by mass, preferably 0.75 to 1.75 parts by mass, and more preferably 1 to 1.5 parts by mass of viscosity adjuster per 100 parts by mass of concentrated lactic acid bacteria. If the amount of viscosity adjuster in the lactic acid bacteria preparation is less than 0.5 parts by mass per 100 parts by mass of concentrated lactic acid bacteria, viscosity stability will deteriorate, and phase separation will occur, especially if the lactic acid bacteria preparation is liquid. On the other hand, if the amount of viscosity adjuster in the lactic acid bacteria preparation is more than 2 parts by mass per 100 parts by mass of concentrated lactic acid bacteria, the viscosity of the lactic acid bacteria preparation will be excessively high.

[0031] Viscosity adjusters used in the present invention include polysaccharides such as pectin, guar gum, xanthan gum, tamarind gum, and carrageenan, as well as propylene glycol, carboxymethyl cellulose, etc. Among these, guar gum and xanthan gum are preferably used from the viewpoint of facilitating the attainment of a desired viscosity.

[0032] When the lactic acid bacteria preparation of the present invention is in the form of a powder or tablet, starch or dextrin may be added as a solidifying agent.

[0033] The lactic acid bacteria preparation of the present invention is not limited to any particular use, but is particularly preferably used in livestock or pet feed. By adding a predetermined amount to livestock or pet feed, it is expected to improve the weight gain and feed conversion ratio of livestock or pets.

[0034] Ingestion of the lactic acid bacteria preparation of the present invention exerts an excellent immunostimulatory effect, which can be confirmed by the production of interleukin-12 (IL-12). The higher the expression level of IL-12, the higher the immunostimulatory activity.

[0035] IL-12 is a heterodimeric cytokine (signaling substance) linked by disulfide bonds. When lactic acid bacteria are ingested, cells present in the body, such as dendritic cells or macrophages, recognize components of the bacteria and produce the signaling substance IL-12. IL-12 induces the activity of NK cells (natural killer cells) and T cells. NK cells and T cells have the ability to attack cancer cells and virus-infected cells, which ultimately leads to the prevention of viral infections and cancer, and provides an immunostimulatory effect.

[0036] IL-12 can be measured by known methods, such as measurement using a commercially available measurement kit.

[0037] Next, the method for producing the lactic acid bacteria preparation of the present invention will be described.

[0038] First, maize, the raw material, is soaked in a sulfurous acid solution and wet-milled to obtain CSW. While known soaking conditions can be used, typically the sulfurous acid concentration of the sulfurous acid solution is 0.1 to 0.3 wt%, the soaking temperature is 45 to 50°C, and the soaking time is approximately 30 to 50 hours. The pH of the resulting CSW is approximately 3.0 to 4.0.

[0039] Next, the bacterial cells contained in the CSW are separated from the obtained CSW to obtain a liquid form of concentrated bacterial cells. Examples of separation methods include known methods such as centrifugal sedimentation (disk type), centrifugal filtration (basket type, decanter), and membrane separation. Among these, centrifugal sedimentation is preferred from the viewpoint of the proportion of solids (amount of bacterial cells) contained in the CSW. Furthermore, when centrifugal sedimentation is used, the rotation speed is preferably 8,000 to 13,000 G, more preferably 9,000 to 12,000 G, and even more preferably 9,000 to 10,000 G. The concentrated bacterial cells obtained after separation usually have a volume of about 1 / 10 to 1 / 30 of the original CSW.

[0040] The concentrated bacterial cells obtained above are different from CSL obtained by concentrating CSW. That is, CSL obtained by concentrating CSW is obtained by simply evaporating the water from the CSW, and most of the components contained in the CSW other than lactic acid bacteria remain in the CSL. In contrast, the concentrated bacterial cells obtained in the process of producing the lactic acid bacteria preparation of the present invention are obtained by selectively separating mainly lactic acid bacteria from CSW, and most of the components contained in the CSW other than lactic acid bacteria remain in the concentrated bacterial cells.

[0041] The concentrated bacterial cells obtained by separation are sterilized by heating. Sterilization kills the lactic acid bacteria in the concentrated bacterial cells, stabilizing the quality of the lactic acid bacteria preparation and also killing other bacteria other than lactic acid bacteria contained in the concentrated bacterial cells.

[0042] The heating temperature in the sterilization treatment is preferably 60 to 95° C., more preferably 60 to 75° C., and even more preferably 60 to 65° C. If the heating temperature is less than 60° C., sterilization will be insufficient and live bacteria will remain in the lactic acid bacteria preparation.

[0043] The heating time in the sterilization treatment is preferably 5 minutes to 1 hour, more preferably 5 minutes to 30 minutes, and even more preferably 5 to 10 minutes.

[0044] The combination of the heating temperature and heating time is preferably 60 to 95°C for 5 minutes to 1 hour, more preferably 60 to 75°C for 5 to 30 minutes, and even more preferably 60 to 65°C for 5 to 10 minutes.

[0045] To improve storage stability at room temperature, salt is preferably added to the concentrated bacterial cells after sterilization. For the same reasons as above, the amount of salt to be added is preferably 10 to 40 parts by mass, preferably 20 to 35 parts by mass, and more preferably 25 to 30 parts by mass, per 100 parts by mass of concentrated bacterial cells after sterilization. Note that the concentrated bacterial cells after sterilization, which are the basis for the amount of salt to be added, contain not only lactic acid bacteria but also components other than lactic acid bacteria, such as dead miscellaneous bacteria.

[0046] It is also preferable to add a viscosity modifier to the concentrated bacterial cells after sterilization to prevent sedimentation. For the same reasons as above, the amount of viscosity modifier to be added is preferably 0.5 to 2 parts by mass, preferably 0.75 to 1.75 parts by mass, and more preferably 1 to 1.5 parts by mass, per 100 parts by mass of concentrated bacterial cells after sterilization. Note that the concentrated bacterial cells after sterilization, which are the basis for the amount of viscosity modifier to be added, contain not only lactic acid bacteria but also components other than lactic acid bacteria, such as dead miscellaneous bacteria.

[0047] The concentrated cells after sterilization are liquid, but if necessary, they can be heated to evaporate the water and turn into powder. When making tablets, the powder obtained by evaporating the water can be solidified by adding a solidifying agent such as starch or dextrin.

[0048] In this manner, the lactic acid bacteria preparation of the present invention can be produced. [Example]

[0049] Example 1: Preparation of lactic acid bacteria preparation The maize raw material was wet milled under the soaking conditions (sulfite concentration, soaking temperature, and soaking time) described above to obtain CSW. The pH of the obtained CSW was 3.8. The obtained CSW was separated into concentrated bacterial cells using a disc centrifuge (Alfa Laval, BTPX-205, 10,000G). The separated concentrated bacterial cells were placed in a container and sterilized by heating in a thermostatic chamber. The heating temperature was 65°C and the heating time was 10 minutes. To the sterilized concentrated bacterial cells, 30 parts of salt and 1.2 parts of xanthan gum were added per 100 parts by mass of the sterilized concentrated bacterial cells, and the mixture was thoroughly stirred to obtain a liquid lactic acid bacteria preparation 1. Furthermore, since the content of lactic acid bacteria, etc., is derived from the raw material maize, three types of maize other than the maize used to obtain lactic acid bacteria preparation 1 were prepared and lactic acid bacteria preparations 2 to 4 were obtained by performing the same procedure as above.

[0050] <Example 2> Measurement of the number of lactic acid bacteria contained in a lactic acid bacteria preparation Lactic acid bacteria preparations 1 to 4 were each diluted 10,000 times with sterilized water, and the number of lactic acid bacteria was measured using a hemocytometer. The results are shown in Table 1.

[0051] [Table 1]

[0052] As shown in Table 1, the number of lactic acid bacteria contained in lactic acid bacteria preparations 1 to 4 was 2.5 × 10 10 , 2.5×10 10 , 1.3 × 10 10 and 1.0 × 10 10 In all lactic acid bacteria preparations, the lactic acid bacteria count was 10 billion or more per gram (1.0 × 10 10 It was found that it contained more than 1000 pieces / g.

[0053] <Example 3> Measurement of immunostimulatory effect The lactic acid bacteria preparation of the present invention was prepared according to the method of Example 1. In addition, as controls, livestock lactic acid bacteria A (Lactohirox (registered trademark), Hirosho Co., Ltd.), livestock lactic acid bacteria B (Bios3Ace, Toa Pharmaceutical Co., Ltd.), and pet food lactic acid bacteria C (EF Power (registered trademark), Combi Corporation) were prepared. A fixed amount of each of these four samples was taken and suspended in RPMI 1640 medium containing 10% FBS to prepare a test solution containing 100 μg / mL of bacterial cells. Separately from the test solution, macrophage-like cell line J774.1 cells (cell number JCRB9108; JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) were prepared and plated at approximately 1.0 × 10 cells per well in a 6-well plate. 6 Four cell line suspensions were prepared by adding 10% FBS-containing RPMI 1640 medium to the cells at a concentration of 1 / mL. The resulting cell line suspensions were placed in an incubator at 5% CO₂ and 37°C and cultured for 48 hours. After culture, the cell line suspensions were removed from the incubator, and the medium (2 mL) was removed while the J774.1 cells were still immobilized on the plate. Approximately 2 mL of each of the four test solutions prepared above was added in place of the removed medium. The cell suspensions with the test solutions added were placed in an incubator at 5% CO₂ and 37°C for 24 hours to induce IL-12. After induction, the suspensions were removed from the incubator, centrifuged (3,000 rpm for 10 minutes), and the culture supernatants were collected and stored frozen at -80°C. After several days, the frozen suspension was thawed, and the IL-12 concentration (IL-12p40 concentration) was measured according to the ELISA method (Biolegend). The results are shown in Figure 1.

[0054] As shown in Figure 1, the IL-12p40 concentration was very low, at 995 pg / mL for livestock lactic acid bacteria A and 5 pg / mL for livestock lactic acid bacteria B. Furthermore, the IL-12p40 concentration was very high at 37,840 pg / mL for lactic acid bacteria C for pet food. On the other hand, the IL-12p40 concentration of the lactic acid bacteria preparation of the present invention was significantly higher at 27,520 pg / mL. This indicates that the lactic acid bacteria preparation of the present invention can be expected to have an immunostimulatory effect similar to that of commercially available lactic acid bacteria C for pet food. Since lactic acid bacteria C for pet food is expensive, the lactic acid bacteria preparation of the present invention can be said to have an excellent balance between cost and immunostimulatory effect.

[0055] Example 4: Study of viscosity adjusters for lactic acid bacteria preparations The concentrated bacterial cells obtained after heat sterilization according to the method of Example 1, to which salt had been added, were divided into 5 g portions in 15 ml test tubes. To the divided concentrated bacterial cells, 0.5 to 3 parts by mass of xanthan gum or guar gum was added per 100 parts by mass of the concentrated bacterial cells after sterilization. After thorough mixing, the sedimentation state of the concentrated bacterial cells in the lactic acid bacteria preparation was observed, and the level of bacterial cell surface height in the test tube was measured to evaluate the degree of phase separation. The day of mixing was counted as day 1, and observations were continued for up to day 4. The sedimentation rate of the concentrated bacterial cells was calculated using the following formula to evaluate the degree of phase separation. A higher sedimentation rate of the concentrated bacterial cells indicates a greater tendency for the lactic acid bacteria preparation to undergo phase separation. The results for the case where xanthan gum was added are shown in Figure 2, and the results for the case where guar gum was added are shown in Figure 3. The results for the control group in Figures 2 and 3 are the same. Sedimentation rate (%) = (bacterial surface height on day 1 - bacterial surface height after N days) / bacterial surface height on day 1

[0056] As a result of the evaluation, in the control group, concentrated bacterial cells settled within a few hours, with the settling rate being 60% on day 1, 70% on day 2, and 80% on days 3 and 4, as shown in Figures 2 and 3. That is, the settling rate gradually increased until the third day, and most of the concentrated bacterial cells had settled.

[0057] In contrast, as shown in Figure 2, in the test group to which 0.5 parts by mass of xanthan gum was added, the sedimentation rate on day 1 was about 20%, which gradually increased thereafter, but was about 30% on days 3 and 4. Compared to the control group, the sedimentation rate of concentrated bacterial cells was lower, and phase separation was suppressed. Furthermore, in the test group to which 1.0 part by mass or more of xanthan gum was added, no sedimentation of concentrated bacterial cells was observed even after 4 days, and phase separation was suppressed.

[0058] Similarly, as shown in Figure 3, in the test section to which 0.5 parts by mass of guar gum was added, the sedimentation rate on day 1 was about 5% and gradually increased thereafter, but on days 3 and 4 the sedimentation rate was about 15%, indicating that the sedimentation rate of concentrated bacterial cells was lower than that of the control section and phase separation was suppressed. Furthermore, in the test section to which 1.0 part by mass or more of guar gum was added, no sedimentation of concentrated bacterial cells was observed even after 4 days, indicating that phase separation was suppressed.

[0059] As described above, by adding xanthan gum or guar gum to concentrated bacterial cells, sedimentation of the concentrated bacterial cells can be suppressed. In particular, by adding 1.0 part by mass or more of xanthan gum or guar gum per 100 parts by mass of concentrated bacterial cells, sedimentation of the concentrated bacterial cells can be almost completely eliminated. Taking into consideration the above results and the ease of handling of the lactic acid bacteria preparation, it is believed that the amount of xanthan gum or guar gum added is particularly preferably 1.0 to 1.5 parts by mass per 100 parts by mass of concentrated bacteria.

Claims

1. A lactic acid bacteria preparation characterized by containing lactic acid bacteria derived from corn.

2. 2. The lactic acid bacteria preparation according to claim 1, wherein the lactic acid bacteria are isolated from corn steep water produced in a maize wet milling process.

3. 2. The lactic acid bacteria preparation according to claim 1, wherein the lactic acid bacteria comprise at least Lactobacillus secaliphilus, Lactobacillus amylovorus, and Lactobacillus frumenti.

4. 3. The lactic acid bacteria preparation according to claim 1, wherein the lactic acid bacteria comprises 10 billion or more cells / g.

5. The lactic acid bacteria preparation according to claim 1 or 2, wherein the lactic acid bacteria are killed.

6. 3. The lactic acid bacteria preparation according to claim 1, wherein said lactic acid bacteria preparation contains 10 to 40 parts by mass of salt per 100 parts by mass of concentrated bacteria.

7. 3. The lactic acid bacteria preparation according to claim 1, further comprising 0.5 to 2 parts by mass of a viscosity adjuster relative to 100 parts by mass of concentrated bacterial cells of the lactic acid bacteria preparation.

8. A livestock or pet feed comprising the lactic acid bacteria preparation according to claim 1 or 2.

9. A method for producing a lactic acid bacteria preparation containing corn-derived lactic acid bacteria, comprising: a separation step of separating concentrated bacterial cells from the corn steep water; and a sterilization step of heating the concentrated bacterial cells.

10. 10. The method for producing a lactic acid bacteria preparation according to claim 9, further comprising the step of adding salt, wherein the amount of salt added is 10 to 40 parts by mass per 100 parts by mass of concentrated bacteria in the lactic acid bacteria preparation.

11. The method for producing a lactic acid bacteria preparation according to claim 9 or 10, further comprising the step of adding a viscosity adjuster, wherein the amount of the viscosity adjuster added is 0.5 to 2 parts by mass per 100 parts by mass of concentrated bacterial cells of the lactic acid bacteria preparation.

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