CN114015740B - Production method of pig bile salt - Google Patents
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- CN114015740B CN114015740B CN202111370590.3A CN202111370590A CN114015740B CN 114015740 B CN114015740 B CN 114015740B CN 202111370590 A CN202111370590 A CN 202111370590A CN 114015740 B CN114015740 B CN 114015740B
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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Abstract
The invention discloses a production method of pig bile salt, which comprises the process steps of special hydrolase preparation, enzyme hydrolysis, deoiling, decoloring, concentrating, drying, crushing and the like, wherein the enzyme hydrolysis comprises the steps of strain selection, fermentation medium preparation, fermentation, hydrolase extraction, hydrolase detection and the like; the process method solves the defects of severe reaction conditions, long time consumption, high energy consumption, difficult removal of grease, complex process and the like in the prior art, and can thoroughly and completely hydrolyze the crude bile acid of the feed after bilirubin is extracted from pig gall and further produce pig gall salt by selecting the used strain to produce hydrolase through self fermentation. The pig bile salt produced by the process has the advantages of simple production process, short time consumption, mild condition, low energy consumption, environmental friendliness and easy realization. The pig bile salt produced by the process has obvious microorganism effect characteristics on BGLB culture medium and intestinal bacteria screening culture medium, and has obvious microorganism effect compared with the pig bile salt production culture medium produced by the prior art.
Description
Technical Field
The invention relates to the technical field of bile salt production, in particular to a production method of pig bile salt.
Background
The pig bile salt is sodium salt formed by bile acid secreted by pig liver cells, and is mainly used as a bacteriostat for gram positive bacteria when being added into a culture medium, and can promote intestinal flora growth.
The pig bile salt is produced by using the crude cholic acid after bilirubin calcium salt is extracted, and the existing common production process is a pure chemical method. The chemical process comprises the following steps: grinding appropriate amount of crude cholic acid extracted from bilirubin calcium salt, adding 9 times of water and 1.5 times of industrial sodium hydroxide, boiling and saponifying for more than 8 hr to hydrolyze bile acid, fat, protein and phospholipid in crude cholic acid completely, cooling, filtering, and discarding small amount of insoluble residue. Decolorizing, acidifying to form sodium salt, concentrating, drying, and grinding to obtain pig bile salt product in light yellow powder. The key point of the pure chemical process is whether the hydrolysis degree is thorough, the hydrolysis process has the characteristics of severe reaction conditions, long time consumption and high energy consumption, and the subsequent production process has the characteristics of difficult removal of grease, poor color and luster, complex process and the like.
Disclosure of Invention
In order to solve the problems mentioned in the background art, the invention provides a production process method of pig bile salt. The pig bile salt is produced by hydrolyzing crude cholic acid with the enzyme by self-made hydrolase, so that the defects of severe reaction conditions, long time consumption, high energy consumption and the like in the prior art can be overcome, and the defects of difficult removal of grease, dark color, poor color, complex process and the like can be overcome by improving the later-stage process.
In order to achieve the above purpose, the present invention provides the following technical solutions:
firstly, preparing special hydrolase, which comprises the following specific steps:
(1) Bacterial strain selection and preparation of bacterial strain liquid: the bacterium ATCC1766 which is discovered by the company during bile discharging bioconversion and has hydrolysis effect on pig bile is selected, and the bacterium is bacillus, gram negative bacterium and is preserved by glycerol pipe. Transferring and passaging to prepare strain liquid;
(2) Preparing a fermentation medium: taking 0.5% of beef extract, 1% of peptone, 0.5% of sodium chloride, 0.5% of lactose, 0.1% of dipotassium hydrogen phosphate and water to prepare a culture medium; ammonia water was adjusted to ph=7.0-7.5, sterilized at 121 ℃,30 minutes, cooled to 60 ℃, and 1.25% sterile kanamycin marker solution was added;
(3) Fermentation: cooling the culture medium in the fermentation tank to 37 ℃, calibrating the DO electrode, adjusting the pH value to 7.0-7.5, aseptically inoculating strain liquid, and stably fermenting until the strain concentration is 9-11%, the metabolism of thalli in the fermentation tank is slow, and ending the fermentation when the enzyme activity is not increased;
(4) Extraction of hydrolytic enzyme: crushing a fermentation liquor cell crusher, centrifuging, collecting supernatant, salting out by ammonium sulfate, stirring, centrifuging again to collect enzyme sludge, and storing below-20 ℃ in a cold storage for later use;
(5) Detection of hydrolytic enzyme: the enzyme amount required for producing 1umolCDCA by hydrolyzing crude cholic acid for 1min with crude cholic acid as substrate at 35-37deg.C and pH5.7-5.9 is defined as 1 enzyme activity unit (U); quantitatively measuring the content by liquid phase differential, and calculating the enzyme activity of the hydrolase;
(II) enzymatic hydrolysis: weighing a certain amount of crude cholic acid, adding 10 times of water of the crude cholic acid, regulating pH to 5.7-5.9 with sodium hydroxide water, controlling the temperature to 35-37 ℃, adding 0.5% of hydrolase of the materials for hydrolysis for 1 hour, and stopping hydrolysis when the liquid phase detection hydrolysis reaches more than 95%;
and (III) regulating the pH value and deoiling: regulating the pH of the hydrolysate in the step (II) to be more than 10, filtering, taking clear liquid, adding petroleum ether with 300% of crude cholic acid, stirring at 45-50 ℃ for extraction and deoiling, standing, and separating liquid to obtain a water phase;
(IV) decoloring: adding 10% active carbon of crude cholic acid into the water phase solution after deoiling in the step (III), and stirring for more than 30 minutes at the temperature of 70-75 ℃ for decolorization;
and (V) concentrating, drying and crushing: filtering the decolorized solution obtained in the step (IV), taking clear solution, concentrating to obtain fluid paste, placing in a 100-130 ℃ oven, drying to less than 5% of dry weight loss, pulverizing into powder, and packaging.
Compared with the prior art, the invention has the following beneficial effects:
the hydrolytic enzyme is produced by selecting the strain which is used for self fermentation, so that the crude cholic acid which is discharged after bilirubin is extracted from pig gall is thoroughly and completely hydrolyzed, and pig gall salt is further produced. The pig bile salt produced by the process has the advantages of simple production process, short time consumption, mild condition, low energy consumption, environmental friendliness and easy realization. The pig bile salt produced by the process has obvious microorganism effect characteristics on BGLB culture medium and intestinal bacteria screening culture medium, and has obvious microorganism effect compared with the pig bile salt production culture medium produced by the prior art.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention.
Example 1:
firstly, preparing special hydrolase, which comprises the following specific steps:
(1) Bacterial strain selection and preparation of bacterial strain liquid: selecting bacterium ATCC1766 which is found by the company when screening bile blanking bioconversion strains and has hydrolysis effect on pig bile, wherein the bacterium is bacillus, gram-negative bacterium and is stored in a glycerol pipe; 200ML of culture medium for preparing strains, which comprises 0.5% of beef extract, 1% of peptone, 0.5% of sodium chloride, 0.5% of lactose and 0.1% of dipotassium hydrogen phosphate, and sterile water; culturing at 35deg.C for 18-22 hr after inoculation, sampling, detecting to be qualified, and placing in a refrigerator at 4deg.C for use;
(2) Preparing a fermentation medium: taking 0.5% of beef extract, 1% of peptone, 0.5% of sodium chloride, 0.5% of lactose, 0.1% of dipotassium hydrogen phosphate and water to prepare 12L of liquid culture medium; adjusting pH to 7.0-7.5 with hydrochloric acid or ammonia water, sterilizing at 121deg.C for 30 min, cooling to 60deg.C, adding 1.25% sterile kanamycin marker solution to reach system concentration of 50ug/ml (0.4 ml1.25% sterile kanamycin is added into 100ml culture medium), and cooling to 40deg.C for use;
(3) Fermentation: the temperature of the culture medium in the fermentation tank is reduced to 37 ℃, the pH is adjusted to 7.0, bacteria are aseptically inoculated, and fermentation parameters are controlled by a fermentation tank control system: air flow rate: 1vvm; stirring: the stirring speed is low, when DO is lower than 30%, stirring is moderately and gradually improved, and dissolved oxygen is ensured to be more than 30%; pressure: 0.01-0.05Mpa; temperature: 35-37 ℃; pH: adjustment is not needed; sampling: sampling after inoculation, and detecting pH and bacterial concentration; recording time, period, temperature, pH, flow, rotating speed, tank pressure, volume, dissolved oxygen and microscopic examination conditions in each hour in the culture process, sampling 4 hours after inoculation, and detecting pH, bacterial concentration and microscopic examination; the fermentation is carried out smoothly until the concentration of the bacteria is 9-11%, the metabolism of the bacteria in the fermentation tank is slow, and the fermentation is ended when the enzyme activity is not increased any more;
(4) Extraction of hydrolytic enzyme: crushing the fermentation material by ultrasonic waves, centrifuging, collecting supernatant, salting out by 30% ammonium sulfate, stirring, centrifuging again to collect enzyme sludge, and storing below minus 20 ℃ in a cold storage for later use;
(5) Detection of hydrolytic enzyme: the enzyme amount required for producing 1umolCDCA by hydrolyzing crude cholic acid for 1min with crude cholic acid as substrate at 35-37deg.C and pH5.7-5.9 is defined as 1 enzyme activity unit (U); quantitatively measuring the content by liquid phase differential, and calculating the enzyme activity of the hydrolase;
(II) enzymatic hydrolysis: weighing a certain amount of crude cholic acid, adding 10 times of water of the crude cholic acid, regulating pH to 5.7-5.9 with sodium hydroxide water, controlling the temperature to 35-37 ℃, adding 0.5% of hydrolase of the materials for hydrolysis for 1 hour, and stopping hydrolysis when the liquid phase detection hydrolysis reaches more than 95%;
and (III) regulating the pH value and deoiling: regulating the pH of the hydrolysate in the step (II) to be more than 10, filtering, taking clear liquid, adding petroleum ether with 300% of crude cholic acid, stirring at 45-50 ℃ for extraction and deoiling, standing, and separating liquid to obtain a water phase;
(IV) decoloring: adding 10% active carbon of crude cholic acid into the water phase solution after deoiling in the step (III), and stirring for more than 30 minutes at the temperature of 70-75 ℃ for decolorization;
and (V) concentrating, drying and crushing: filtering the decolorized solution obtained in the step (IV), collecting clear solution, concentrating to obtain fluid paste, drying in a 100-130deg.C oven until the weight loss is below 5%, pulverizing into powder, and packaging.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or scope of the invention. Accordingly, embodiments of the present invention are exemplary and not limiting. The scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any attachment in a claim should not be construed as limiting the claim concerned.
Claims (1)
1. A production method of pig bile salt is characterized in that self-made hydrolase is used, hydrolysis is completed by a biological enzyme method, and the technological links of acidification and salification in the original technology are removed, and the method comprises the following specific steps:
firstly, preparing special hydrolase, which comprises the following specific steps:
(1) Bacterial strain selection and preparation of bacterial strain liquid: selecting bacterium ATCC1766 which is discovered by the company during bile discharging bioconversion and has hydrolysis effect on pig bile, wherein the bacterium is bacillus, gram-negative bacterium and is stored in glycerol tubes;
(2) Preparing a fermentation medium: taking beef extract 0.5%, peptone 1%, sodium chloride 0.5%, lactose 0.5%, dipotassium hydrogen phosphate 0.1% and water as the other components to prepare a culture medium; ammonia water is used for adjusting PH=7.0-7.5, sterilization is carried out at 121 ℃ for 30 minutes, 1.25% sterile kanamycin marking solution is added after cooling to 60 ℃ to enable the system concentration to reach 50ug/ml, namely 0.4ml of 1.25% sterile kanamycin is added into 100ml of culture medium, and cooling is carried out for later use;
(3) Fermentation: the sterilization of the culture medium in the fermentation tank is finished, the temperature is reduced to 37 ℃, the DO electrode is calibrated, the pH is adjusted to 7.0, bacteria are inoculated aseptically, the fermentation is carried out steadily until the bacterial concentration is 9-11% by a fermentation tank control system, the metabolism of the bacterial in the fermentation tank is slow, and the fermentation is finished when the enzyme activity is not increased any more;
(4) Extraction of hydrolytic enzyme: crushing the fermentation material by ultrasonic waves, centrifuging, collecting supernatant, salting out by ammonium sulfate, stirring, centrifuging again to collect enzyme sludge, and storing below minus 20 ℃ in a cold storage for later use;
(5) Detection of hydrolytic enzyme: the enzyme amount required for producing 1umolCDCA by hydrolyzing crude cholic acid for 1min at 35-37 ℃ and pH5.7-5.9 with crude cholic acid as substrate is defined as 1 enzyme activity unit U; quantitatively measuring the content by liquid phase differential, and calculating the enzyme activity of the hydrolase;
(II) enzymatic hydrolysis: weighing a certain amount of crude cholic acid, adding 10 times of water of the crude cholic acid, regulating pH to 5.7-5.9 with sodium hydroxide water, controlling the temperature to 35-37 ℃, adding 0.5% of hydrolase of the materials for hydrolysis for 1 hour, and stopping hydrolysis when the liquid phase detection hydrolysis reaches more than 95%;
and (III) regulating the pH value and deoiling: regulating the pH of the hydrolysate in the step (II) to be more than 10, filtering, taking clear liquid, adding petroleum ether with 300% of crude cholic acid, stirring at 45-50 ℃ for extraction and deoiling, standing, and separating liquid to obtain a water phase;
(IV) decoloring: adding 10% active carbon of crude cholic acid into the water phase solution deoiled in the step (III), and stirring for more than 30 minutes at the temperature of 70-75 ℃ for decolorization;
and (V) concentrating, drying and crushing: filtering the decolorized solution obtained in the step (IV), collecting clear solution, concentrating to obtain fluid paste, drying in a 100-130deg.C oven until the weight loss is below 5%, pulverizing into powder, and packaging.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111370590.3A CN114015740B (en) | 2021-11-18 | 2021-11-18 | Production method of pig bile salt |
| ZA2022/08774A ZA202208774B (en) | 2021-11-18 | 2022-08-05 | Method for producing pig bile salts |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111370590.3A CN114015740B (en) | 2021-11-18 | 2021-11-18 | Production method of pig bile salt |
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| CN114015740A CN114015740A (en) | 2022-02-08 |
| CN114015740B true CN114015740B (en) | 2023-07-21 |
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| CN114686367A (en) * | 2022-05-06 | 2022-07-01 | 白银赛诺生物科技有限公司 | Electrode protection device for fermentation environment, use method and calibration method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908158A (en) * | 2006-08-30 | 2007-02-07 | 中国农业大学 | Bile salt hydrolase and preparation method and special preparing strain thereof |
| CN103597092A (en) * | 2011-03-25 | 2014-02-19 | 生物梅里埃公司 | Detection of bacteria having a resistance to carbapenems |
| CN103865910A (en) * | 2014-04-09 | 2014-06-18 | 青岛博之源生物技术有限公司 | Method for extracting bile salt hydrolase by fermenting lactobacillus salivarius |
| CN106754844A (en) * | 2016-12-14 | 2017-05-31 | 曹书华 | A kind of method for improving bile salt hydrolase secernment efficiency |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017220486A2 (en) * | 2016-06-20 | 2017-12-28 | Pharmazell Gmbh | Coupled, self-sufficient biotransformation of chenodeoxycholic acid to ursodeoxycholic acid and novel enzyme mutants applicable in said process |
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- 2021-11-18 CN CN202111370590.3A patent/CN114015740B/en active Active
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- 2022-08-05 ZA ZA2022/08774A patent/ZA202208774B/en unknown
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908158A (en) * | 2006-08-30 | 2007-02-07 | 中国农业大学 | Bile salt hydrolase and preparation method and special preparing strain thereof |
| CN103597092A (en) * | 2011-03-25 | 2014-02-19 | 生物梅里埃公司 | Detection of bacteria having a resistance to carbapenems |
| CN103865910A (en) * | 2014-04-09 | 2014-06-18 | 青岛博之源生物技术有限公司 | Method for extracting bile salt hydrolase by fermenting lactobacillus salivarius |
| CN106754844A (en) * | 2016-12-14 | 2017-05-31 | 曹书华 | A kind of method for improving bile salt hydrolase secernment efficiency |
Non-Patent Citations (3)
| Title |
|---|
| 产胆盐水解酶BSH乳酸菌的鉴定及发酵条件优化;惠明;刘勇;张红星;刘慧;;中国食品学报(01);220-227 * |
| 双歧杆菌胆盐水解酶基因的重组表达、纯化与酶学性质;周晓玲;张娟;陈坚;堵国成;;食品与生物技术学报(08);14-22 * |
| 植物乳杆菌Lactobacillu plantarumY1菌株胆盐水解酶基因(bsh)的克隆及重组表达;黄茜;黄璐;潘道东;杨瑶;;南京师大学报(自然科学版)(03);97-102 * |
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| CN114015740A (en) | 2022-02-08 |
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