A preparation method of a whole broth microencapsulation of a recombinant bacillus subtilis fermentation liquor for producing gamma-aminobutyric acid

Abstract

The present application relates to a kind of preparation method of full quality microencapsulation of fermentation liquor of gamma-aminobutyric acid produced by recombinant bacillus subtilis. Recombinant bacillus subtilis (strain preservation in China General Microbiological Culture Collection Center, strain preservation number is CGMCC No.5550) is used as starting strain to carry out fermentation. For recombinant bacillus subtilis fermentation liquor system, ethyl cellulose, glycerol, sucrose, yeast extract, dipotassium hydrogen phosphate and gelatin are selected to carry out microencapsulation preparation, spray drying. The optimal addition concentration (mass / volume) of capsule material in catalytic mixed solution is 2.5% ethyl cellulose, 1.5% glycerol, 2.5% sucrose, 2% yeast extract, 0.15% dipotassium hydrogen phosphate and 0.3% gelatin. The optimal rotation speed when capsule material is mixed with catalytic liquid is 60rpm, and the optimal stirring time is 30min;The optimal speed of high-speed dispersion is 15000rpm, and the optimal dispersion time is 3min. The optimal air inlet temperature of capsule material and catalytic liquid mixed dispersion liquid spray drying is 160℃, the optimal air inlet temperature is 70℃, and the optimal feeding rate is 1.5L / min.

Description

Technical Field

[0001] This invention relates to the field of probiotics biotechnology for food and feed, specifically to a method for preparing microencapsulated probiotics (Bacillus subtilis) and its catalytic product (γ-aminobutyric acid). Background Technology

[0002] Bacillus subtilis is a ubiquitous type of aerobic bacteria. Numerous studies have shown that Bacillus subtilis antagonizes intestinal pathogens and enhances disease resistance. During its growth, Bacillus subtilis produces volatile fatty acids such as acetic acid, propionic acid, and butyric acid. These acids lower the pH value of the animal's intestines, effectively inhibiting the growth of pathogens. Simultaneously, by biologically depleting oxygen in the intestines, it creates an anaerobic environment that promotes the reproduction of dominant intestinal bacteria, maintaining the normal ecological balance of the gut. Furthermore, Bacillus subtilis can metabolize numerous enzyme systems, including proteases, breaking down large molecules into smaller, absorbable molecules in the animal's intestines, significantly improving feed conversion ratio, increasing feed conversion ratio, and reducing farming costs.

[0003] Gamma-aminobutyric acid (GABA) is a non-protein amino acid widely used in the food, pharmaceutical, cosmetic, and livestock industries in countries such as Japan and the United States. Currently, domestic GABA production in China mainly relies on chemical synthesis. Separation and extraction methods and bio-fermentation methods have not yet achieved true industrialization due to technological and raw material limitations. Biomanufacturing, characterized by high efficiency, green practices, low carbon emissions, and sustainability, has become a global strategic emerging industry and is experiencing rapid growth.

[0004] Because GABA has functions such as regulating animal appetite, improving liver and kidney function, and regulating hormone secretion, there are numerous reports on its application in the livestock industry. In piglet breeding, Yang Xiaojun et al. added 400 and 800 mg / kg GABA to the diets of 28-day-old weaned three-way crossbred pigs. After 28 days of feeding, they found that GABA significantly affected daily weight gain and feed intake in the later stages of the experiment, with the 800 mg / kg dose showing better results. In growing-finishing pigs, Wei Xihui et al. added 0, 10, 20, and 40 mg / kg GABA to the diets of approximately 30 kg three-way crossbred pigs for 80 days. The results showed that the daily weight gain of the 10 and 20 mg / kg groups increased by 11.39% and 9.49% respectively, and feed intake increased by 7.82% and 9.47% respectively, while the feed intake and daily weight gain of the 40 mg / kg group decreased. The effects of GABA on the growth performance of poultry have also attracted researchers' attention. Chen Zhong et al. found that adding 0.05% GABA to the drinking water of broiler chickens significantly reduced respiratory rate, increased daily weight gain, and improved feed conversion ratio in heat-stressed chicks. Wu Changxin reported that adding appropriate amounts of GABA to the diets of broiler chicks in the early (50, 100 mg / kg) and later (75, 150 mg / kg) stages both improved daily weight gain and reduced feed conversion ratio, with lower doses showing better results than higher doses. In aquaculture, researchers have conducted studies on the effects of feeding GABA on the growth-promoting and stress-resistant properties of carp, grass carp, crucian carp, goldfish, Japanese flounder, and loach. The results showed that adding a certain amount of GABA can improve the feed conversion ratio and stress resistance of aquatic animals.

[0005] In the early stages, Lu Sha used a safe strain—Bacillus subtilis—as the host cell and constructed a low-energy-consumption, high-GABA-producing genetically engineered bacterium using genetic engineering techniques. This strain was then rationally modified, fermentation conditions optimized, and pilot-scale amplification performed using metabolic engineering. Using glutamic acid as a substrate, biocatalysis for 24 hours resulted in a GABA concentration of 327 g / L. However, because GABA is a small-molecule crystalline amino acid, compared to amino acids in feed protein, it is absorbed too quickly in the intestines of animals and fish / shrimp, rapidly reaching its peak concentration in plasma and undergoing metabolic decomposition, thus failing to sustain its physiological functions. Especially in aquaculture, fish and shrimp feed slowly, and GABA, being readily soluble in water, easily leach from feed pellets into the water, which is detrimental to the effectiveness of crystalline amino acid supplementation. Furthermore, improving the survival rate of Bacillus subtilis during the drying process of microecological preparations, which is prone to heat-induced death, is another key issue.

[0006] Microencapsulation technology utilizes natural or polymeric materials to encapsulate substances into microcapsules with sealed membranes, applicable to pharmaceuticals, food, and animal feed. It is currently a widely used and effective solution to the problems of lactic acid bacteria probiotics' insufficiency to high temperatures and storage. However, differences in encapsulation materials and processes lead to significant variations in the final microencapsulation effect. In summary, this paper addresses the previous issue of how to efficiently and comprehensively utilize GABA catalytic products obtained using Bacillus subtilis as a host and glutamate as a substrate. It proposes a research approach using Bacillus subtilis as a probiotic and GABA as a prebiotic, and rationally designs a sustained-release formulation to develop a novel feed additive. This achieves efficient and comprehensive utilization of GABA catalytic products while obtaining a novel feed additive containing both probiotics and prebiotics. Furthermore, it can significantly reduce wastewater and waste discharge during GABA fermentation and post-processing, contributing to environmental protection. Summary of the Invention

[0007] 1. Purpose of the invention This invention addresses the problem of efficient and complete utilization of the fermentation broth from recombinant Bacillus subtilis in the production of γ-aminobutyric acid (GABA). It rationally designs a microencapsulation preparation method to preserve the activity of Bacillus subtilis while maximizing the utilization of its fermentation catalytic product (γ-aminobutyric acid), reducing costs and minimizing wastewater treatment burden. When taken by humans or animals, it can enhance their tolerance to gastric acid and bile salts, thereby maximizing its reach into the intestines to exert its efficacy. Technical solution

[0008] The main research contents of this project are as follows: Bacillus subtilis was fermented in liquid at 37°C for 48 hours, and the spore rate was >90%.

[0009] Based on the characteristics of the Bacillus subtilis fermentation system, ethyl cellulose, glycerol, sucrose, yeast extract, dipotassium hydrogen phosphate, and gelatin were selected for microencapsulation preparation. The concentrations (mass / volume) of the encapsulating material in the catalytic mixture were 2%~3% ethyl cellulose, 1%~2% glycerol, 2%~3% sucrose, 1.5%~2.5% yeast extract, 0.1%~0.2% dipotassium hydrogen phosphate, and 0.2%~0.4% gelatin.

[0010] (3) When mixing the capsule material with the Bacillus subtilis fermentation liquid in technical solution (2), the rotation speed is 30~90 rpm, the stirring time is 20~40 min, the high-speed dispersion rate is 10000~20000 rpm, and the dispersion time is 2~4 min.

[0011] (4) Spray drying is performed on the well mixed and dispersed materials in technical solution (3). The inlet air temperature of spray drying is 140~180℃ and the outlet air temperature is 60~80℃; the feed rate is 1~2 L / min.

[0012] The Bacillus subtilis strain used in step (1) above is deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, on December 8, 2011, with the strain accession number CGMCC No. 5550 and the classification name Bacillus subtilis.

[0013] In step (1) above, liquid fermentation was performed using Bacillus subtilis as the starting strain. The culture medium used was: glucose 20 g, peptone 10 g, yeast extract 5 g, ammonium sulfate 2.5 g, magnesium sulfate heptahydrate 0.2 g, potassium dihydrogen phosphate 2 g, dipotassium hydrogen phosphate 1 g, pH 7.0. After 48 h of fermentation, the viable count of Bacillus subtilis was ≥3.0 × 10⁻⁶. 9 cfu / mL, spore rate >90%.

[0014] The optimal addition concentration (mass / volume) of the capsule material in the catalytic mixture in step (2) above is 2.5% ethyl cellulose, 1.5% glycerol, 2.5% sucrose, 2% yeast extract, 0.15% dipotassium hydrogen phosphate, and 0.3% gelatin.

[0015] The optimal rotation speed for mixing the capsule material and the catalyst solution in step (3) above is 60 rpm, and the optimal stirring time is 30 min.

[0016] The optimal dispersion rate of the mixture of capsule material and catalyst in step (3) above is 15,000 rpm, and the optimal dispersion time is 3 min.

[0017] The optimal inlet air temperature for spray drying of the mixture of capsule material and catalyst in step (4) above is 160°C, the optimal inlet air temperature is 70°C, and the optimal feed rate is 1.5 L / min.

[0018] Beneficial effects of the present invention This invention primarily targets the fermentation broth system for the production of γ-aminobutyric acid (GABA) from recombinant Bacillus subtilis, and proposes a method for its complete utilization using microencapsulation combined with spray drying. The beneficial results of this invention are manifested in the following aspects: (1) The invention achieves the full utilization of Bacillus subtilis cells and catalytic products (γ-aminobutyric acid). This invention not only has economic value, but also effectively reduces the discharge of waste gas, wastewater, and solid waste during the fermentation process of Bacillus subtilis.

[0019] (2) Research on novel spray-dried microencapsulated formulations: A highly efficient feed additive containing Bacillus subtilis (probiotic) and γ-aminobutyric acid (prebiotic) was developed. The encapsulation rate of Bacillus subtilis was ≥85%, the encapsulation rate of γ-aminobutyric acid was ≥90%, and the moisture content was ≤10%.

[0020] (3) After animals take it, their tolerance to gastric acid can be improved, so that it can reach the intestines to exert its effects to the greatest extent. Detailed Implementation

[0021] Example 1. Liquid fermentation of Bacillus subtilis The fermentation medium for Bacillus subtilis was: glucose 20g, peptone 10g, yeast extract 5g, ammonium sulfate 2.5g, magnesium sulfate heptahydrate 0.2g, potassium dihydrogen phosphate 2g, dipotassium hydrogen phosphate 1g, pH 7.0.

[0022] After 48 hours of fermentation, the viable count of Bacillus coagulans was ≥3.0*10⁻⁶. 9 cfu / mL, spore rate >90%.

[0023] Example 2. Preparation method of microencapsulation of Bacillus subtilis fermentation broth (1) The formulation and addition ratio of the microencapsulation material are as follows (mass / volume): 2.5% ethyl cellulose, 1.5% glycerol, 2.5% sucrose, 2% yeast extract, 0.15% dipotassium hydrogen phosphate, and 0.3% gelatin.

[0024] (2) The speed of mixing the capsule material and the catalyst solution is 60 rpm and the stirring time is 30 min.

[0025] (3) The high-speed dispersion rate of the capsule material and the catalyst solution after mixing is 15,000 rpm and the dispersion time is 3 min.

[0026] (4) The inlet air temperature of the spray drying of the mixture of capsule material and catalyst is 160℃, the inlet air temperature is 70℃, and the feed rate is 1.5 L / min.

[0027] Example 3. Effect test of Bacillus subtilis fermentation broth microcapsules (1) Detection of Bacillus subtilis encapsulation rate 1) Sample processing Accurately weigh 25 g of the sample to be tested into an Erlenmeyer flask containing 225 mL of sterile physiological saline diluent (concentration of 0.9%, W / W), and homogenize it at 10,000 rpm for 1 min using a high-speed homogenizer to open the capsule wall, thus obtaining the bacterial suspension stock solution.

[0028] 2) Dilution Using a sterile pipette tip, draw 1 mL of the diluent into 9 mL of sterile physiological saline, and then dilute sequentially at a volume ratio of 1:10 to the appropriate gradient (10). -8 10 -9 and 10 -10 ).

[0029] Coating method testing Prepare 9 sterile nutrient broth culture plates in advance, and use aseptic techniques in a laminar flow hood to aspirate 1:10... -5 1:10 -6 1:10 -7 Spread 100 μL of each of the three gradient dilutions evenly onto plates, with 3 plates for each gradient, resulting in a 1:10 solution. -5 The corresponding plate is marked as 1×10 -6 1:10 -6 The corresponding plate size is marked as 1:10. -7 1:10 -7 The corresponding plate size is marked as 1:10. -8 .

[0030] Place in a biochemical culture medium and incubate upside down at 37°C for 24 h.

[0031] 4) Colony count After 24 hours of plate incubation, the number of colonies in the plate was recorded. The result with a colony count between 30 and 300 was considered the most reliable result. The average colony count of the corresponding gradient was multiplied by the multiple of the plate label to obtain the viable bacteria content of the sample.

[0032] The test results showed that the encapsulation rate of Bacillus subtilis was >95%.

[0033] γ-aminobutyric acid encapsulation rate detection 1) Sample processing Accurately weigh 25 g of the sample to be tested into an Erlenmeyer flask containing 225 mL of sterile physiological saline diluent (concentration of 0.9%, W / W), and homogenize it at 10,000 rpm for 1 min using a high-speed homogenizer to open the capsule wall, thus obtaining the bacterial suspension stock solution.

[0034] 2) Detection of γ-aminobutyric acid content: carried out in accordance with the national light industry standard for γ-aminobutyric acid (QB / T 4587-2013).

[0035] The test results showed that the γ-aminobutyric acid encapsulation rate was >90%.

[0036] Microcapsule moisture content detection The water content of the microcapsules was determined according to the first method (direct drying method) in the national standard GB5009.3-2016.

[0037] The test results showed that the water content of the microcapsules was <7%.

[0038] Example 4. Gastric acid resistance test of Bacillus subtilis fermentation broth microcapsules Simulated gastric fluid conditions: 0.5 mL (5 mol / L) concentrated hydrochloric acid was pipetted into a sealed container containing 180 mL of distilled water, at which point the pH was approximately 2. 1% pepsin was added and mixed thoroughly. Control group: 1 g of Bacillus subtilis powder and γ-aminobutyric acid (GABA) were added; experimental group: 1 g of Bacillus subtilis microcapsules were added. The mixture was incubated at 37℃ for 2 h. The Bacillus subtilis and GABA detection methods described in Case Study 3 were followed. Results showed that the survival rate of Bacillus subtilis after 2 h of treatment in the control group was 63%, while the survival rate after 2 h of treatment with microencapsulation was 91%, and the GABA retention rate was 99.5%. Microencapsulation significantly improved the gastric acid resistance of Bacillus subtilis.

[0039] Example 5. Test of sustained-release time of Bacillus subtilis fermentation broth microcapsules The treated liquid from Case 4 was introduced into a small intestine simulation vessel, and 1% trypsin and 0.68% dipotassium hydrogen phosphate were added. The pH was adjusted to 6.8 with 1 mol / L sodium hydroxide solution, and the mixture was kept at 37°C. Samples were taken every 0.5 h to detect the content of Bacillus subtilis and γ-aminobutyric acid (GABA). The results showed that the microcapsule release rate was 81% after 0.5 h, 87% after 1 h, and reached 99% after 1.5 h. Therefore, the microencapsulated Bacillus subtilis and GABA can be completely released in the intestine after 1.5 h after passing through the stomach, thus exerting their efficacy.

Claims

1. A method for preparing a fully microencapsulated fermentation broth from recombinant Bacillus subtilis for the production of γ-aminobutyric acid, the process being as follows: (1) Bacillus subtilis was fermented in liquid at 37℃. After 48 h of fermentation, the spore rate was >90%. (2) Based on the characteristics of the Bacillus subtilis fermentation system, ethyl cellulose, glycerol, sucrose, yeast extract, dipotassium hydrogen phosphate, and gelatin were selected for microencapsulation. The concentrations (mass / volume) of the encapsulation materials in the catalytic mixture were 2%~3% ethyl cellulose, 1%~2% glycerol, 2%~3% sucrose, 1.5%~2.5% yeast extract, 0.1%~0.2% dipotassium hydrogen phosphate, and 0.2%~0.4% gelatin. (3) The mixing speed of the capsule material and Bacillus subtilis fermentation broth described in technical solution (2) is 30~90 rpm, the stirring time is 20~40 min, the high-speed dispersion rate is 10000~20000 rpm, and the dispersion time is 2~4 min. (4) Spray drying is performed on the well mixed and dispersed materials in technical solution (3). The inlet air temperature of spray drying is 140~180℃ and the outlet air temperature is 60~80℃; the feed rate is 1~2 L / min.

2. The microbial fermentation according to claim 1, characterized in that... The strain used for Bacillus coagulans fermentation is deposited at the China General Microbiological Culture Collection Center (CGMCC), with the accession number CGMCC No. 5550.

3. The Bacillus subtilis liquid fermentation according to claim 1, characterized in that... The fermentation medium used was: glucose 20g, peptone 10g, yeast extract 5g, ammonium sulfate 2.5g, magnesium sulfate heptahydrate 0.2g, potassium dihydrogen phosphate 2g, dipotassium hydrogen phosphate 1g, pH 7.

0. After 48 h of fermentation, the viable count of Bacillus subtilis was ≥3.0 x 10⁻⁶. 9 cfu / mL, spore rate >90%.

4. The optimal concentration (mass / volume) of the encapsulation material in the catalytic mixture according to claim 1 is 2.5% ethyl cellulose, 1.5% glycerol, 2.5% sucrose, 2% yeast extract, 0.15% dipotassium hydrogen phosphate, and 0.3% gelatin.

5. The optimal mixing speed of the capsule material and the catalyst liquid according to claim 1 is 60 rpm, and the optimal stirring time is 30 min.

6. The optimal dispersion rate of the capsule material and the catalyst liquid according to claim 1 is 15,000 rpm, and the optimal dispersion time is 3 min.

7. The spray drying of the mixture of capsule material and catalyst liquid according to claim 1 has an optimal inlet air temperature of 160°C, an optimal outlet air temperature of 70°C, and an optimal feed rate of 1.5 L / min.