Lactobacillus mucilaginosus FUA033 direct vat set and preparation method thereof

Abstract

The invention belongs to the field of microbial fermentation, and particularly relates to a lactobacillus mucilaginosus FUA033 direct vat set and a preparation method of the lactobacillus mucilaginosus FUA033 direct vat set. The optimal fermentation culture medium for fermenting the lactobacillus mucus FUA033 is an improved MRS anaerobic basic broth culture medium, and the formula comprises 15.83 g / L of peptone, 4.0 g / L of yeast extract powder, 5.0 g / L of beef extract powder, 30.72 g / L of glucose, 1.0 mL / L of Tween 80, 5.16 g / L of dipotassium phosphate, 5.0 g / L of sodium acetate, 0.2 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate and 2.0 g / L of ammonium citrate. The method for preparing the fermented lactobacillus mucus FUA033 microbial inoculum comprises the following steps: culturing FUA033 in an optimized MRS culture medium under the anaerobic condition of 37 DEG C for 48 hours, centrifuging at 12000 * g and 4 DEG C for 10 minutes, removing supernatant, taking out thalli, putting the thalli into a 30mm plate containing a freeze-drying protective agent, covering the thalli with a preservative film with holes, pre-freezing in a refrigerator at the temperature of 80 DEG C for 5 hours, then putting the thalli into a vacuum freeze-drying machine, and freeze-drying for 12 hours, so as to obtain the fermented lactobacillus mucus FUA033 microbial inoculum. The microbial agent is obtained. The content of the microbial inoculum obtained by the method can reach 2.06 * 10 < 10 > CFU / mL, and the survival rate reaches 74.25% compared with that before freeze-drying.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and particularly relates to a fermented mucus lactobacillus FUA033 direct-administered bacterial agent and a preparation method thereof. Background Art

[0002] Direct-throw starter culture refers to a highly concentrated and standardized freeze-dried bacterial strain that can be used directly in production without pre-treatment such as activation and expansion culture. Direct-throw starter culture is available in two forms: frozen and freeze-dried. After the bacterial strain is activated, cultured at high density, centrifuged and mixed with a cryoprotectant medium, it is frozen at low temperature to produce a concentrated frozen starter culture. Frozen starter culture is mainly preserved by deep freezing (-30 to -80°C) and ultra-low temperature freezing (-196°C). Among them, freezing in liquid nitrogen at -196°C is the most ideal method for preserving starter culture. It is reported that the number of viable bacteria in commercial frozen concentrated lactic acid bacteria can reach 10 11 CFU / mL, it can be used directly in production. However, frozen concentrated lactic acid bacteria agents need to be kept frozen during application, and transportation requires special refrigerated trucks and special equipment, which to a certain extent limits its promotion and application in production. The emergence of freeze-dried starter cultures has gradually replaced frozen starter cultures. Freeze-dried starter cultures, also known as solid starter cultures, refer to solid starter cultures that are obtained by high-density culture, centrifugal collection of bacteria, addition of freeze-drying protective agents, and then low-temperature drying, spray drying, or vacuum freeze drying. The number of live bacteria in freeze-dried concentrated lactic acid bacteria starter cultures can even reach 10 12 CFU / mL. The dried fermentation agent is easy to carry and transport, and is easy to realize commercial production and application.

[0003] In recent years, probiotics have been widely used in the pharmaceutical, health food, fermented dairy products and other industries. With the improvement of modern people's health awareness, probiotic products have become more and more popular. Especially in developed Western countries, the proportion of probiotic fermented products in people's dietary structure has become more and more prominent. For example, in Europe, more than 45% of dairy processing companies produce probiotic fermented products. The total sales of dairy products fermented by Lactobacillus acidophilus and Bifidobacterium have increased year by year. In addition to probiotic fermented foods, there are also many probiotic preparations in the form of freeze-dried powder on the market, in which the concentration of probiotics is generally above 100 million units (10 8 CFU / g).

[0004] Ellagic acid in food is difficult to be used by the human body due to its extremely low solubility. It is converted into urolithin A by intestinal microorganisms, which significantly improves its nutritional and health value. Urolithin A is a class of ellagitannin substances with multiple biological activities and high utilization. Urolithin A has a significant effect on inhibiting classic inflammatory factors, such as TNF-α, IL-1β, etc., and plays an important role in the treatment of various inflammations. Urolithin A also has the function of inhibiting the activation of nuclear transcription factors of fibroblasts induced by inflammatory factors, reducing the expression and activity of membrane-bound prostaglandin E2 synthase-1, which will downregulate the level of PGE2; on the other hand, urolithin A can inhibit enteritis by increasing the tight connection between protein and intestine, regulating the energy metabolism of cells, restoring the barrier protection function of the colon, and regulating the intestinal microbiota. Urolithin A can also inhibit D-galactose-related brain aging by activating the SIRT1 / mTOR pathway, and has a certain improvement effect on mouse neurons. At the same time, it also shows the function of improving learning and memory ability and inhibiting the adverse effects caused by oxygen free radicals by regulating the biosynthesis of mitochondria. Urolithin A has significant anti-proliferation and antioxidant effects, can prevent and treat prostate cancer and colorectal cancer, and has preventive and therapeutic effects on bladder cancer, endometrial cancer and pancreatic cancer. Urolithin A can induce mitochondrial autophagy, thereby playing a role in delaying aging and treating cardiovascular diseases. Urolithin A can protect the kidneys and prevent diseases such as colorectal inflammation, osteoarthritis and intervertebral disc degeneration. At the same time, studies have found that urolithin A also has good application value and efficacy in the treatment of neurological diseases. In addition, urolithin A also has a positive effect on the prevention and treatment of many metabolic diseases. Therefore, the probiotics that convert ellagic acid to urolithin A have excellent potential for application, so it is necessary to develop a fermented mucus lactobacillus FUA033 direct-dose bacterial agent that converts ellagic acid to urolithin A to facilitate industrial application. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a fermented Lactobacillus mucilaginosus FUA033 direct-administered bacterial agent and a preparation method thereof.

[0006] The technical scheme of the present invention is as follows: a method for preparing a direct-administered bacterial agent of fermented Lactobacillus mucilaginosus FUA033, comprising the following steps:

[0007] 1) Activation of strains: The fermented Lactobacillus mucilaginosus FUA033 stored in a -80°C refrigerator was activated by streaking on a plate;

[0008] 2) Seed culture: Pick a single activated colony and inoculate it into a seed culture medium to obtain a seed solution;

[0009] 3) Expanded culture: Inoculate the seed liquid of FUA033 into MRS medium and culture under anaerobic conditions; centrifuge and remove the supernatant to obtain the bacterial cells.

[0010] 4) Freeze-drying: Add freeze-drying protective agent to the bacteria, pre-freeze, and then place in a vacuum freeze dryer for freeze drying to obtain the bacterial agent, which is stored at 4°C.

[0011] Preferably, in step 3), the components of the seed culture medium and the fermentation culture medium are: peptone 15.83 g / L, yeast extract powder 4.0 g / L, beef extract powder 5.0 g / L, glucose 30.72 g / L, Tween 80 1.0 mL / L, dipotassium hydrogen phosphate 5.16 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, and ammonium citrate 2.0 g / L.

[0012] Preferably, in step 3), the inoculation amount of the FUA033 seed solution is 2.9%.

[0013] Preferably, in step 3), the culture temperature under anaerobic conditions is 38° C. and the culture time is 20 h.

[0014] Preferably, in the step 3), the initial pH of the culture under anaerobic conditions is 6.8.

[0015] Preferably, in step 3), the centrifugation conditions are 12000×g and 4° C. for 10 min.

[0016] Preferably, in step 4), the lyoprotectant is selected from a combination of skim milk powder, trehalose and maltodextrin in a ratio of 59.9:26.7:13.4, and the ratio of the lyoprotectant to the bacteria is 3:1.

[0017] Preferably, in step 4), the pre-freezing condition is -80°C for 5 hours.

[0018] Preferably, in step 4), the freeze-drying time is 12 hours.

[0019] Beneficial effects of the present invention: The yield of the bacterial agent obtained by the method of the present invention is high, and the bacterial content of the bacterial agent obtained by this method can reach 1.53×10 10 CFU / mL, compared with the survival rate before freeze-drying, it reached 74.25%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Effects of different carbon sources and concentrations on fermentation medium

[0021] Figure 2 Effects of different nitrogen sources and concentrations on fermentation medium

[0022] Figure 3 Effects of different buffer salts and concentrations on fermentation medium

[0023] Figure 4Effects of different nutritional factors on fermentation medium

[0024] Figure 5 The effect of inoculum size on bacterial concentration

[0025] Figure 6 The effect of temperature on bacterial concentration

[0026] Figure 7 Effect of pH on bacterial concentration

[0027] Figure 8 Effects of centrifugation conditions on cell viability

[0028] Fig. 9 Selection of freeze-drying protective agent and concentration

[0029] Fig.10 The survival rate of live bacteria in the inoculant stored at different temperatures DETAILED DESCRIPTION

[0030] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0031] Fermented mucus Lactobacillus FUA033 is disclosed in CN117487707A. The strain has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 13, 2023, with the deposit number CGMCC NO.28447. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the contact number is 010-64807850.

[0032] Optimization of fermentation components and conditions of strain FUA033 of the present invention.

[0033] 1 Effects of different carbon sources on high-density culture of Lactobacillus mucilaginosus

[0034] On the basis of MRS liquid medium, glucose, fructose, lactose, dextrin, maltose, starch and sucrose were selected as carbon sources, with a mass fraction of 5%. The seed liquid was inoculated into the fermentation medium at a 2% inoculation rate, with an initial pH of 6.0 and cultured at 37°C for 18 h. The OD of the fermentation liquid was measured. 600 The optimal carbon source was determined by the value. Five concentrations of 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% were selected respectively. The seed solution was inoculated at 2% in the fermentation medium and cultured at 37°C. The OD 600 The optimal carbon source concentration is determined by the value of glucose. Carbon source is the basis of cell material and the main component of culture medium. 600The value is the highest and most suitable for bacterial growth, which may be because fermentative mucus lactobacillus lacks cells that decompose polysaccharides. After determining that the best carbon source is glucose, the glucose concentration was changed. The results are shown in the attached figure. Figure 1 As shown, when the glucose concentration is 3%, the fermentation effect is the best, so the optimal glucose concentration is 3%.

[0035] 2 Effects of different nitrogen sources on high-density culture of Lactobacillus mucilaginosus

[0036] Based on MRS liquid medium, five nitrogen sources were selected, including peptone, tryptone, casein peptone, soy peptone, and ammonium sulfate, with a mass fraction of 1%. The seed liquid was inoculated into the fermentation medium at a 2% inoculation rate, with an initial pH of 6.0 and cultured at 37°C for 18 h. The OD value of the fermentation liquid was measured. 600 Select five concentrations of 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% respectively, inoculate the seed solution at 2% inoculum in the fermentation medium and culture at 37°C. Determine the OD 600 The nitrogen source is mainly used to form bacterial cell substances and nitrogen-containing metabolites. Commonly used nitrogen sources can be divided into two categories: organic nitrogen sources and inorganic nitrogen sources. MRS medium itself has a relatively rich nitrogen source. This experiment chose to use ammonium sulfate, trypsin, soy peptone, and casein peptone to replace peptone, and kept other components unchanged to optimize the medium. The experimental results are as follows Figure 2 As shown in the figure, the effects of the alternative nitrogen sources are not as good as the initial nitrogen source peptone. After determining that the best nitrogen source is peptone, the peptone concentration is changed. The results are as follows Figure 2 As shown in the figure, when the peptone concentration is 1.5%, the fermentation effect is the best, so the optimal peptone concentration is 1.5%.

[0037] 3 Effects of different buffer salts on high-density culture of Lactobacillus mucilaginosus

[0038] On the basis of MRS liquid medium, Na2HPO4, K2HPO4, NaAC, NaH2PO4, and KH2PO4 were selected as buffer salts, and no buffer salt was added as a blank control. The mass fraction was 0.5%. The seed liquid was inoculated into the fermentation medium at a 2% inoculation rate, with an initial pH of 6.0 and cultured at 37°C for 18 hours. The OD of the fermentation liquid was measured. 600 The optimal buffer salt was determined by the value. Five concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% were selected for inoculation and culture at 37°C. The OD 600 The optimal buffer salt concentration is determined by the value. During the culture process, the growth of fermenting mucus lactobacillus will change the pH of the culture medium, which is not conducive to further culture. Adding buffer salts can greatly delay the change of pH in the culture medium, thereby making the culture performance of the culture medium more superior. Figure 3It can be seen that the five buffer salts have similar effects on the culture effect, among which K2HPO4 has the best effect. Then change its concentration, the experimental results are as follows Figure 3 As shown, when the concentration of K2HPO4 was 0.5%, the growth effect of FUA033 was the best.

[0039] 4 Effects of different growth factors on high-density culture of Lactobacillus mucilaginosus

[0040] Corn syrup, tomato juice, VC, cucumber juice, and carrot juice were selected as growth factors, and no growth factor was added as a blank control. The mass fraction was 0.5% and the seed liquid was inoculated into the fermentation medium at a 2% inoculation rate. The initial pH was 6.0 and the culture was statically cultured at 37°C for 18 h. The OD of the fermentation liquid was measured. 600 value to determine the best growth factor. Some microorganisms must also supplement trace amounts of organic matter in order to grow. These trace organic substances are called growth factors. Their main function is to provide important chemical substances for microbial cells, components of auxiliary factors and participate in metabolism. Growth factors usually include vitamins, amino acids, bases, sterols, amines, etc. Such as yeast extract, peptone, malt extract, corn pulp, animal and plant tissues or cell infusions, etc., are all natural substances that can provide growth factors. On the basis of ordinary MRS culture medium, this experiment added 0.5% tomato juice, cucumber juice, potato juice, carrot juice, corn pulp, and VC to study the effects of different growth factors on the amount of bacteria. The experimental results are as follows Figure 4 The selected growth factors all have certain effects on bacterial growth, among which corn pulp has the best effect, mainly because it contains rich carbohydrates, proteins and cellulose, which to some extent make up for the deficiencies in the original culture medium. After determining that the best growth factor is corn pulp, its addition amount is changed to find the best addition amount. The results are as follows Figure 4 .Depend on Figure 4 It can be seen that with the increase of corn syrup addition, the number of viable bacteria and OD 600 When the addition amount reached 0.5%, the viable count and OD 600 When the addition amount exceeded 0.5%, the viable count and OD 600 The value decreased instead, which shows that the optimal addition amount of corn syrup is 0.5%.

[0041] 5 Optimization of fermentation medium by response surface methodology

[0042] The above-mentioned optimal carbon source, nitrogen source, and buffer salt were selected for response surface test. The mass fractions of carbon source were 1%, 2%, 3%, 4%, and 5%, the mass fractions of nitrogen source were 0.5%, 1%, 1.5%, 2%, and 2.5%, and the mass fractions of buffer salt were 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, respectively. Three factors and three levels were used to optimize the culture medium formula using the response surface method of Design-expert 13 software. The results are shown in the table below.

[0043] Table 1. Response surface experimental factors and levels

[0044]

[0045] Table 2 Response surface experimental design and results

[0046]

[0047]

[0048] The variance analysis was performed on the experimental results, and the pseudo-regression equation was:

[0049] Y=1.60688+0.454375X1+0.680625X2+0.5425X3-0.03675X1X2+0.010125X1X3-0.210375X2X3

[0050] -0.278815X1 2 -0.146815X2 2 -0.12694X3 2

[0051] From the variance analysis table 1.3, we can see that X2, X3, X2X3, X1 2 , X2 2 、X3 2 The effect on the response value was significant (P<0.05), indicating that peptone, glucose, and K2HPO4 are important control factors for the composition of the fermentation medium of Lactobacillus mucilaginosus FUA033. The lack of fit term P=0.070 is not significant. From the model credibility analysis table 4, it can be seen that the regression equation multiple correlation coefficient R 2 =96.44%, indicating that the regression equation has a good fit and a small lack of fit, and can be used for the optimization of the culture medium.

[0052] Table 3 Analysis of variance table of response surface experiment

[0053]

[0054] Table 4 Feasibility analysis of the model

[0055]

[0056]

[0057] In order to further obtain the optimal mass fraction, the regression equation was predicted using software. Finally, the optimal mass fractions predicted were glucose 3.072%, peptone 1.583%, K2HPO4 0.516%, and the predicted OD 600 The value is 1.616.

[0058] Use the best value predicted by the regression model to conduct a verification experiment and obtain OD 600 The viable count was 1.536 and 6.20×10 9 The CFU / mL fit was 95.05%, indicating that there was a good fit between the predicted value and the true value, further verifying the reliability of the model.

[0059] 6. Selection of inoculum size

[0060] The inoculation amount was selected as 1%, 3%, 5%, 7%, and 9%, respectively. The seed liquid was inoculated into the fermentation medium, the initial pH was 6.0, and the culture was kept at 37℃ for 18h. The OD of the fermentation liquid was measured. 600 Value. Figure 5 It can be seen that when the inoculation amount is 2%, the bacterial concentration is the highest and the growth is the best. When the inoculation amount is higher than 3%, the number of viable bacteria is low. If the inoculation amount is too high, the bacterial growth is poor. This may be because the nutrients in the culture medium cannot meet the needs of a large number of bacterial growth in the initial stage of fermentation.

[0061] 7. Temperature selection

[0062] The optimal inoculum size was inoculated according to the above, the initial pH was 6.0, and the fermentation liquid was cultured at 22℃, 27℃, 32℃, 37℃, and 42℃ for 18h, and the OD of the fermentation liquid was measured. 600 Value. Figure 6 It can be seen that the effect of temperature on Lactobacillus plantarum is quite obvious, and the bacterial concentration is the highest at 37°C. Temperature changes will directly affect the activity of proteins and enzymes in the bacteria. Too high temperature will denature intracellular proteins and affect the stability of macromolecules such as ribosomes and RNA; too low temperature will reduce enzyme activity and cause changes in cell membrane fluidity, thus affecting the normal growth of bacteria.

[0063] 8pH Selection

[0064] The pH values ​​were set at 4.0, 5.0, 6.0, 6.5, and 7.0, respectively. The fermentation was carried out in batches in the fermenter. The optimal inoculation amount was used and the optimal temperature was used for culture. The OD of the fermentation liquid was measured. 600 value, select the optimal pH value for fermentation. Figure 7It can be seen that when the pH value is 6.5, the bacteria grow best, while when the pH value is 4.0, the bacteria grow slowly; within the acidic range, the lower the pH value, the greater the impact of acid stress on Lactobacillus plantarum, so the optimal fermentation pH value should be controlled at around 6.5.

[0065] 9 Optimization of culture conditions by response surface methodology

[0066] The above optimal inoculum size, temperature, and pH were selected for response surface test. The inoculum size was selected as 1%, 2%, 3%, 4%, and 5%, the temperature was selected as 0.5%, 1%, 1.5%, 2%, and 2.5%, and the pH was selected as 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%. Three factors and three levels were used to optimize the culture conditions using the response surface method of Design-expert 13 software. The results are shown in the table below.

[0067] Table 5. Response surface experimental factors and levels

[0068]

[0069]

[0070] Table 6 Response surface experimental design scheme and results

[0071]

[0072] The variance analysis was performed on the experimental results, and the pseudo-regression equation was:

[0073] Y=1.5572+0.01775X1-0.003375X2+0.046625X3+0.012X1X2+0.011X1X3+0.01825X2X3-0.0139

[0074] 75X1 2 -0.045225X2 2 -0.056725X3 2

[0075] From the variance analysis table 1.7, we can see that X1, X3, X2X3, X2 2 、X3 2 The effect on the response value was significant (P<0.05), indicating that the inoculation amount, temperature, and pH are important control factors in the fermentation of Lactobacillus mucilaginosus FUA033. The lack of fit term P = 0.8138, which is not significant. From the model credibility analysis table 1.8, it can be seen that the regression equation multiple correlation coefficient R 2 =96.92%, indicating that the regression equation has a good fit and a small lack of fit, and can be used to optimize the culture conditions.

[0076] Table 7 Analysis of variance table of response surface experiment

[0077]

[0078] Table 8 Feasibility analysis of the model

[0079]

[0080] Optimal culture conditions: The optimal conditions obtained by the above response surface method optimization results are as follows: inoculation amount 2.9%, temperature 38℃, pH value 6.8, fermentation time 20h. The fermentation process conditions were optimized by response surface method, and the optimal culture conditions were as follows: inoculation amount 2.9%, temperature 38℃, pH value 6.8, fermentation time 20h, and the predicted OD 600 The value was 1.578. In order to ensure the convenience and feasibility of the experiment, the experimental conditions were modified to 2.9% inoculation, 38°C temperature, 6.8 pH value, and 20h fermentation time for verification test. The results showed that the OD 600 The viable count was 1.569 and 2.06×10 10 CFU / mL, the relative error was <1%, indicating that the equation had a good fit and could be used to predict actual results.

[0081] 2. Preparation of strain FUA033 inoculant

[0082] 1 Effect of centrifugation conditions on cell viability

[0083] The fermentation liquid of Lactobacillus fermentum FUA033 cultured at 37℃ to the stable stage was divided into centrifuge cups, balanced, and placed in a centrifuge. The cell activity was measured at 3000r / min, 3500r / min, 4000r / min, and 4500r / min for 5min, 10min, 15min, and 20min, respectively. After centrifugation, the supernatant was taken for live bacteria count to determine the optimal centrifugation conditions. Figure 8 It can be seen that the longer the centrifugation time and the higher the speed, the less bacterial residues in the supernatant. After centrifugation for 15 or 20 minutes, the amount of bacterial residues in the supernatant at the four speeds was very low, with a concentration order of magnitude of only 10 3 CFU / mL. However, the longer the centrifugation time and the higher the speed, the greater the damage to the bacterial activity. In order to obtain highly active bacterial agents, the centrifugation time should not be too long and the speed should not be too high. Therefore, the optimal centrifugation speed can be selected as 4000r / min and the optimal centrifugation time can be selected as 15min.

[0084] 2. Selection of freeze-drying protective agent concentration

[0085] After selecting several suitable freeze-drying protective agents, the appropriate concentration was determined through single factor experiments. Fig. 9It can be analyzed that 15% skim milk powder has the best protection effect among the single protective agents, and the survival rate of the bacterial agent can reach 60%, while the protection effects of sodium glutamate and D-sorbitol are not good. Therefore, skim milk powder, trehalose and maltodextrin are selected to form a freeze-dried composite protective agent.

[0086] 3. Response surface optimization experiment of lyophilization protectant

[0087] Through the above single factor experiment, several selected freeze-drying protective agents with good protection ability were optimized by response surface method, the number of live bacteria before and after freeze-drying was determined by methylene blue counting method, and the cell survival rate was calculated to determine the best protective agent combination and its concentration. The results are shown in the table below.

[0088] Table 9 Factors and levels of freeze-drying response surface test

[0089]

[0090] Table 10 Response surface experimental design and results

[0091]

[0092]

[0093] Table 11 Analysis of variance table of response surface experiment

[0094]

[0095]

[0096] Table 12 Feasibility analysis of the model

[0097]

[0098] The optimal skim milk powder mass fraction obtained by response surface analysis was 15.714%, the mass concentrations of trehalose and maltodextrin were 7% and 3.5%, and the survival rate predicted by the model was 74.396%. In order to verify the reliability of the prediction results, the optimized composite protective agent was used for the test, and the final viable count was 1.53×10 10 CFU / mL, and the survival rate was 74.25%, which was not significantly different from the predicted value.

[0099] 4. Storage period experiment of bacterial agent

[0100] The viable bacteria count of the prepared bacterium powder was measured after being stored at 4°C and room temperature (about 25°C) for different periods of time, and the survival rate was calculated. Fig.10 As shown. Fig.10It can be seen that during the experimental period, when the inoculum was stored at room temperature, the number of viable bacteria decreased significantly, so this temperature was not suitable for long-term storage of bacterial powder. When stored at 4°C, the number of viable bacteria decreased by less than 8%, and more than 94% of the bacteria in the bacterial powder were still alive after 5 months.

[0101] The composition and post-treatment conditions of the best composite protective agent:

[0102] Peptone 15.83g / L, yeast extract powder 4.0g / L, beef extract powder 5.0g / L, glucose 30.72g / L, Tween 801.0mL / L, dipotassium hydrogen phosphate 5.16g / L, sodium acetate 5.0g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, ammonium citrate 2.0g / L.

[0103] The optimal fermentation conditions were pH 6.8, temperature 38°C, inoculation volume 2.9%, and fermentation time 20 h. The number of viable bacteria in the fermentation broth reached 2.06×10 10 CFU / mL, which was 8.77 times that before optimization.

[0104] The composition of the best composite protective agent is skim milk powder 15.714%, trehalose 7%, maltodextrin 3.5%

[0105] Comparative example: CN117487707A is a patent application for a fermented mucus lactobacillus FUA033 and its application, which mainly uses the strain to ferment and convert ellagic acid to produce urolithin A.

[0106] Patented culture conditions of bacteria:

[0107] Fermentation medium: tryptic peptone 10.0, sodium chloride 5.0, peptone 10.0, L-arginine 1.0, glucose 1.0, yeast extract 5.0, sodium pyruvate 1.0, hemin chloride 0.005, vitamin K10.0005, pH 7.1±0.1 (25°C), the seed liquid was inoculated into the medium containing ellagic acid substrate at a 2% inoculum, and cultured in an anaerobic workstation at 37°C for 72h. At this time, the conversion rate of the strain to convert ellagic acid to urolithin A was the highest, and the bacterial liquid concentration was 1.27×10 9 CFU / mL. This patent is mainly to prepare a direct-injection bacterial agent for fermenting mucus lactobacillus FUA033, optimize the culture medium to make it grow rapidly, and the purpose is different. The culture medium used in this patent has a fermentation time of 20h. The number of viable bacteria in the fermentation liquid reaches 2.06×10 10 CFU / mL.

Claims

1. A method for preparing a direct-administered bacterial agent of fermented Lactobacillus mucilaginosus FUA033, comprising the following steps: 1) Activation of strains: The fermented Lactobacillus mucilaginosus FUA033 stored in a -80°C refrigerator was activated by streaking on a plate; 2) Seed culture: Pick a single activated colony and inoculate it into a seed culture medium to obtain a seed solution; 3) Expansion culture: inoculate the seed solution of FUA033 into MRS medium and culture under anaerobic conditions; Centrifuge and remove the supernatant to obtain the bacterial cells. 4) Freeze-drying: Add freeze-drying protective agent to the bacteria, pre-freeze, and then place in a vacuum freeze dryer for freeze drying to obtain the bacterial agent, which is stored at 4°C.

2. The preparation method according to claim 1, characterized in that: In the step 3), the components of the seed culture medium and the fermentation culture medium are: 15.83 g / L peptone, 4.0 g / L yeast extract powder, 5.0 g / L beef extract powder, 30.72 g / L glucose, 1.0 mL / L Tween 80, 5.16 g / L dipotassium hydrogen phosphate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 2.0 g / L ammonium citrate.

3. The preparation method according to claim 1, characterized in that: In the step 3), the inoculation amount of the FUA033 seed solution is 2.9%.

4. The preparation method according to claim 1, characterized in that: In the step 3), the culture temperature under the anaerobic condition is 38° C. and the culture time is 20 h.

5. The preparation method according to claim 1, characterized in that: In the step 3), the initial pH of the culture under anaerobic conditions is 6.

8.

6. The preparation method according to claim 1, characterized in that: In the step 3), the centrifugation conditions are 12000×g and 4° C. for 10 min.

7. The preparation method according to claim 1, characterized in that: In the step 4), the lyophilization protective agent is selected from a combination of skim milk powder, trehalose and maltodextrin in a ratio of 59.9:26.7:13.4, and the ratio of the lyophilization protective agent to the bacterial cells is 3:

1.

8. The preparation method according to claim 1, characterized in that: In the step 4), the pre-freezing condition is -80°C for 5 hours.

9. The preparation method according to claim 1, characterized in that: In the step 4), the freeze-drying time is 12 hours.

Images