A method for preparing porcine bile acid by clostridium butyricum fermentation and a granule preparation method

Abstract

The application discloses a method for preparing pig bile acid by Clostridium butyricum fermentation and a granule preparation method. The inventor finds that the Clostridium butyricum (CCTCC M 2018426) has a very high fermentation efficiency for pig bitter gall, close to 20% of bile acid output, by preparing bile acid by fermentation based on an accidental condition, while the bile acid content prepared by other types of Clostridium butyricum is less than 10%, the fermentation efficiency is beyond expectation, the process condition is mild, easy to control, low in energy consumption and does not affect the environment, meets the production concept of green, safe and environmental protection, and is worth popularization and application.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, specifically to a method for preparing porcine bile acids by fermentation of Clostridium butyricum and a method for preparing granules. Background Technology

[0002] Pig bile, a byproduct of pork processing, is a traditional Chinese medicine with significant efficacy and a long history of use. Bile acids, as the main active components of animal bile, have wide applications and high market demand. While bovine bile is the primary source, it is costly and difficult to collect; therefore, pig bile has become a major alternative source of bile acids.

[0003] In actual production of bile acids, saponification is usually used to hydrolyze the bound form into the free form for extraction. Although this method is simple, the process requires a long reaction time with strong alkali under high temperature and high pressure conditions, which is not environmentally friendly and cannot meet the development requirements of safety, energy saving and green environmental protection. Therefore, it needs to be improved. Summary of the Invention

[0004] To address at least one of the aforementioned technical deficiencies, the present invention provides the following technical solution:

[0005] This application discloses a method for preparing porcine bile acids by Clostridium butyricum fermentation, comprising the following steps:

[0006] Clostridium butyricum (CCTCC M 2018426) was inoculated into a culture medium and fermented under alkaline conditions at 30-40℃ for 100-180 h. The culture medium contained porcine bile, and the proportion of porcine bile in the culture medium was 60-85% by mass.

[0007] Furthermore, the culture medium formula is as follows: 15-25g corn steep liquor powder, 8-13g soybean meal, 3-7g glucose, 3-7g yeast extract, 3-8g sodium chloride, 40-180g pig bile, 2-4g KH2PO4, and 4-6g taurine.

[0008] Furthermore, by mass, the culture medium initially contains 25-40% pig bile, and the culture medium is in a liquid state. The remaining pig bile is added in batches during the fermentation process.

[0009] Furthermore, the culture medium initially contained 50g of pig gallbladder, and the remaining pig gallbladder was added in batches and evenly during the fermentation process.

[0010] Furthermore, the samples were incubated statically at pH 8.5 and 36°C.

[0011] Furthermore, spore induction is performed during the culture process.

[0012] Furthermore, the number of viable bacteria to be fermented reaches 10. 8Fermentation ends when CFU / ml and spore rate is above 90%.

[0013] The second aspect of this application discloses a method for preparing Clostridium butyricum bile acid granules, which includes the fermentation broth prepared above, mixing the fermentation broth with montmorillonite and corn cob powder evenly, drying and then pulverizing into granules.

[0014] Furthermore, in terms of quality,

[0015] 45%-60% of the fermentation broth prepared according to any one of claims 1-7

[0016] Montmorillonite (feed) 15%–25%

[0017] Corn cob powder 25%–35%.

[0018] Further drying is required until the moisture content is less than 10%.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. The inventors, by chance, used Clostridium butyricum (CCTCC M 2018426) for the fermentation preparation of bile acids and found that this type of Clostridium butyricum has extremely high fermentation efficiency for pig bile, with a bile acid yield of nearly 20%, while the bile acid content prepared by other types of Clostridium butyricum tested was less than 10%. The fermentation efficiency exceeded expectations and has excellent commercial value.

[0021] 2. This invention prepares bile acids by fermenting pig bile with Clostridium butyricum (CCTCC M 2018426). The process is mild, easy to control, low in energy consumption, and does not affect the environment. It is more in line with the green, safe, and environmentally friendly production concept and is worth promoting and applying. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 These are images of the samples prepared in the examples and control examples;

[0024] Figure 2 This is a microscopic image of Clostridium butyricum;

[0025] Figure 3 It's a picture of frozen pig gallbladders; Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0027] I. Preparation of Fermentation Broth

[0028] Example 1

[0029] Preparation of fermentation broth

[0030] 1) Filtering

[0031] Clostridium butyricum (CCTCC M 2018426) was isolated as single colonies using streaking on Petri dishes. The culture medium composition (g / L) was: 20g cornmeal, 10g soybean meal, 5g glucose, 5g yeast extract, 5g sodium chloride, 3g K₂HPO₄, 3g KH₂PO₄, 0.3g MnSO₄, and agarose (adjusted to pH 7.0). The medium was sterilized at 121℃, and after cooling to 55℃, the plates were inverted. Fermentation was carried out at 36℃ in an anaerobic digester with an oxygen-removing agent added. The mixture was then statically cultured, and single colonies were subcultured for later use.

[0032] 2) Bile acid fermentation

[0033] The selected Clostridium butyricum was inoculated into the culture medium. The initial culture medium composition was as follows (g / L): corn steep liquor 20g, soybean meal 10g, glucose 5g, yeast extract 5g, sodium chloride 5g, frozen pig bile 50g, KH₂PO₄ 3g, taurine 5g. The pH was adjusted to 8.5, and the medium was sterilized at 121℃. The fermentation temperature was 36℃. An additional 50g of frozen pig bile was added to the culture medium at 48h and 96h of fermentation, respectively. Figure 3 As shown, the fermentation broth was examined under a microscope after 96 hours of fermentation. Figure 2 As shown, if no spores are produced, 0.3 g / L MnSO4 solution is added for induction, and the culture is allowed to stand for 144 hours until the viable cell count in the fermentation broth reaches 10. 8 Fermentation ends when CFU / ml is above 90% and spore count is above 90%.

[0034] Several control examples were added. The control examples differed from Example 1 in that the types of Clostridium butyricum were different. Several strains preserved in the laboratory were used to replace Clostridium butyricum (CCTCC M 2018426) in the preparation of fermentation broth.

[0035] II. Detection of bile acid content

[0036] The fermentation broths prepared in Example 1 and several control examples were tested for bile acid content. Samples are shown in the attached figures. Figure 1 As shown, the detection process is as follows:

[0037] 2.1 Experimental Instruments: Agilent 1260II ELSD high-performance liquid chromatograph, ELSD detector; Agilent ZORBAX SB-Aq (5μm 4.6mm×150mm) column; bile acid standards: including five standards: cholic acid, deoxycholic acid, porcine deoxycholic acid, ursodeoxycholic acid, and chenodeoxycholic acid (Note: cholic acid, deoxycholic acid, porcine deoxycholic acid, ursodeoxycholic acid, and chenodeoxycholic acid were purchased from the National Institutes for Food and Drug Control (bile acid standards from October 31 to November 3, 2023); ② Fermentation broth sample ---- dissolve the standards in methanol.

[0038] 2.2 Chromatographic conditions

[0039] ELSD detector nitrogen flow rate: 1.6 L / min; drift tube temperature: 110℃ -- (Changes: evaporator temperature: 60℃; nebulizer temperature: 60℃); Smoothing: 30 (3.0 seconds); PMT Gain: 3.0

[0040] Mobile phase: A is 0.2% formic acid solution, mobile phase C is acetonitrile, gradient elution (0-7 min, 5% C; 7-20 min, 5% → 80% C; 20.1-25 min: 5% C --- return to the initial mobile phase at the later time).

[0041] Flow rate: 1.0 mL / min; Injection volume: 10 μL;

[0042] Column temperature 25℃.

[0043] 2.3 Sample Preparation

[0044] Reference solution: Accurately weigh approximately 0.020 g of bile acid standard, place it in a 10 mL volumetric flask, dissolve it in methanol, and dilute to the mark. Shake well to prepare a 2 mg / mL stock solution. Then, dilute serially. Test solution: Accurately weigh approximately 25 mg (0.025 g) of sample and place it in a stoppered conical flask. Add 50 mL of methanol tightly, weigh, and sonicate (200 W, 40 kHz) for 20 min. Make up the weight loss with methanol, filter, and obtain the final solution.

[0045] 2.4 Negative Solution

[0046] A negative sample (methanol) free of bile acids was prepared according to the prescription ratio and preparation process, and a negative solution was prepared according to the "test solution" preparation method.

[0047] 2.5 Results

[0048] 2.51 Establishment of the bile acid standard curve

[0049] The correspondence between liquid phase number and peak area is shown in Table 1.

[0050] Table 1 Peak area and elution time of bile acids

[0051]

[0052]

[0053] According to the standard curve comparison, the peak elution times of bile acid were 17.428-17.488 min; deoxycholic acid: 19.111-19.178 min; porcine deoxycholic acid: 18.107-18.161 min; chenodeoxycholic acid: 19.026-19.080 min; and ursodeoxycholic acid: 18.206-18.242 min.

[0054] 2.52 Results of high-performance liquid chromatography (HPLC) determination of bile acid content in different fermentation samples prepared in the Examples and Control Examples

[0055]

[0056] The peak times of each substance correspond to those of the standard, and the regression curve equations of peak area and concentration correspond to those of the standard. Among them, fermentation product No. 5, namely the fermentation broth obtained by strain (CCTCC M 2018426) from the decomposition of pig bile, has a bile acid content of 18.94%, which far exceeds the fermentation efficiency of other Clostridium butyricum and has excellent commercial value.

[0057] 3. Preparation of bile acid granules by drying and pulverizing the fermentation broth.

[0058] The fermentation broth prepared by strain 1 (CCTCC M 2018426) in Example 1 was dehydrated to a water content of 15% by centrifugation and other methods. The concentrated fermentation broth with a water content of 15% was then mixed evenly with a carrier (corn cob powder and montmorillonite powder mixed at a ratio of 2:1) using a high-speed mixer. By weight, the composition was 55% Clostridium butyricum fermentation broth, 15% montmorillonite (feed), and 30% corn cob powder.

[0059] The mixed wet bacterial powder is then granulated into uniform particles using a granulator and placed in a fluidized bed dryer for drying. The material temperature is controlled at 33°C during fluidized bed drying, and the processing time for a single batch is about 45 minutes, so that the moisture content of the dried bacterial powder is below 10%. After that, the bile acid content in the bacterial powder product is tested.

[0060] 4. Bile acid content in granular products

[0061] Bile acid content in Clostridium butyricum powder

[0062]

[0063] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing porcine bile acids by fermentation of Clostridium butyricum, characterized in that, Includes the following steps: Preparation of fermentation broth 1) Filtering Clostridium butyricum CCTCC NO: M 2018426 was isolated by streaking in a petri dish. The culture medium was sterilized at 121°C. After cooling to 55°C, the plate was poured out and fermented at 36°C. The plate was placed in an anaerobic tank, an oxygen-removing agent was added, and the culture was allowed to stand. Single colonies were then passaged and prepared for later use. 2) Bile acid fermentation The selected Clostridium butyricum was inoculated into a culture medium. The initial culture medium composition was as follows (g / L): corn steep liquor 20g, soybean meal 10g, glucose 5g, yeast extract 5g, sodium chloride 5g, frozen pig gallbladder 50g, KH2PO4 3g, taurine 5g. The pH was adjusted to 8.

5. The medium was sterilized at 121℃, and fermented at 36℃. An additional 50g of frozen pig gallbladder was added to the culture medium at 48h and 96h of fermentation. After 96h of fermentation, the fermentation broth was examined microscopically. If no spores were produced, 0.3g / L MnSO4 solution was added for induction. The culture was allowed to stand for 144h until the viable cell count reached 10^6. 8 When the concentration of CFU / ml is above 90% and the spore rate is above 90%, fermentation is complete.

2. A method for preparing Clostridium butyricum bile acid granules, characterized in that: The fermentation broth prepared according to claim 1 is mixed evenly with montmorillonite and corn cob powder, dried, and then pulverized into granules.

3. The method for preparing Clostridium butyricum bile acid granules as described in claim 2, characterized in that: In terms of mass, The fermentation broth prepared according to claim 1 contains 45%-60% feed-grade montmorillonite, 15%-25% corn cob powder, and 25%-35% corn cob powder.

4. The method for preparing Clostridium butyricum bile acid granules as described in claim 3, characterized in that: Dry until the moisture content is less than 10%.

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