Fermentation process and preparation of lactobacillus reuteri L-01

Abstract

The invention discloses a fermentation process and a preparation of lactobacillus reuteri L-01, and belongs to the field of microorganism preparation.After artificial domestication, bile salt tolerance of the lactobacillus reuteri L-01 is enhanced, the lactobacillus reuteri L-01 can be cultured in gastrointestinal fluid, and the survival rate of 98% or above can still be kept for a long time; the lactobacillus reuteri L-01 disclosed by the invention can play an excellent probiotic function, is beneficial to improving the balance of intestinal flora, has an inhibiting effect on pathogenic bacteria, and reduces the prevalence rate. Besides, after the components of the culture medium are optimized, the domesticated lactobacillus reuteri L-01 also has a certain anti-oxidation effect and is rich in selenium, so that the use value of the lactobacillus reuteri L-01 is increased. The lactobacillus reuteri L-01 fermentation broth is obtained, after a protective agent is added, the more excellent probiotic effect can be guaranteed, the stability of the lactobacillus reuteri L-01 in processing application and transportation storage is enhanced, and the lactobacillus reuteri L-01 fermentation broth has a certain slow-release effect and has a longer acting effect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a fermentation process and preparation of Lactobacillus reuteri L-01. Background Art

[0002] Lactobacillus reuteri is a typical representative species among probiotics. It exists in the intestines of most mammals and vertebrates and is harmless to humans and animals. Lactobacillus reuteri can widely inhibit the growth of yeast, Gram-negative bacteria, Gram-positive bacteria, pathogens and fungi. In 2003, the Ministry of Health of my country approved Lactobacillus reuteri as a species of probiotics for health foods. The fermentation types of lactic acid bacteria can be divided into homolactic fermentation and heterolactic fermentation. The homolactic fermentation strain is mainly based on the glycolysis (EMP) pathway, and 1moL of hexose is converted into 2moL of pyruvate and 2 ATP through the EMP pathway. Pyruvate is further reduced to lactic acid under the action of lactate dehydrogenase. In this process, 90% of the substrate is converted into lactic acid. The heterolactic fermentation strain is mainly based on the phosphoketolase (PK) pathway or the pentose phosphate pathway (HMP), and the EMP pathway is used as a substitute pathway. Heterolactic fermentation strains can metabolize 1 mol of hexose to produce 1 mol of lactic acid, acetic acid, ethanol, CO2 and 1 ATP.

[0003] Lactobacillus reuteri belongs to a heterotypic lactic acid fermentation strain, and its sugar metabolism products include lactic acid, acetic acid, ethanol and CO2. The sugars that Lactobacillus reuteri can ferment include: arabinose, glucose, sucrose, raffinose, fructose, galactose, maltose, and ribose. Among them, glucose and sucrose are the main carbon sources. In addition, when Lactobacillus reuteri uses fructose as a carbon source, it will use fructose as an electron acceptor and eventually produce mannitol. Under anaerobic conditions, Lactobacillus reuteri can also metabolize glycerol to produce Reuterin with broad-spectrum antibacterial activity, whose main components are monomers, hydrates and cyclodimers of 3-hydroxypropionaldehyde (3-HPA).

[0004] In the prior art, the following problems exist in the fermentation application process of Lactobacillus reuteri: a. The strain itself has poor stress resistance. During the application, processing and storage, the stability and survival rate of Lactobacillus reuteri will be affected, which may limit its effectiveness in application; b. The acid and bile salt resistance are poor, and the acid and bile salt resistance determines whether it can reach the intestine and play a key role; c. The strain has poor colonization ability in the intestine and fails to fully exert its probiotic effect. Summary of the invention

[0005] Based on this, in order to solve the technical problems of poor probiotic fermentation effect and stability during processing and application in the prior art, the present invention provides a fermentation process and preparation of Lactobacillus reuteri, and the specific technical scheme is as follows:

[0006] A fermentation process of Lactobacillus reuteri L-01, the fermentation process comprising the following steps:

[0007] Pick a single Lactobacillus reuteri L-01 colony and inoculate it on MRS liquid medium for culture, strictly perform aseptic operation, anaerobically culture it at 30°C to 38°C for 20h to 28h, then inoculate it into a primary culture medium, anaerobically culture it at 25°C to 38°C for 20h to 24h, and obtain a primary seed liquid;

[0008] The first-level seed solution is inoculated into the second-level culture medium, and anaerobically cultured at 30° C. to 38° C. for 20 h to 24 h to obtain the second-level seed solution;

[0009] The secondary seed solution is centrifuged at 2500 r / min to 3000 r / min for 3 min to 5 min, the supernatant is discarded, and then washed with sterile saline for several times, and resuspended with saline to obtain a bacterial suspension;

[0010] Prepare fermentation medium, introduce steam, sterilize the material at 120-125°C; cool to 32-39°C, then inoculate the bacterial suspension into the fermentation medium, at 30-38°C, pH 4.0-6.0, maintain nitrogen pressure at 0.01Mpa-0.03Mpa, rotate at 80r / min-100r / min, perform anaerobic fermentation for 20h-24h to obtain fermentation liquid.

[0011] Furthermore, the Lactobacillus reuteri L-01 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, with a storage address in Guangzhou, China, a storage number of GDMCC NO: 65328, and a classification name of Limosilactobacillus reuteri.

[0012] Furthermore, the MRS liquid culture medium, the primary culture medium and the secondary culture medium are all sterilized under high pressure at 100° C. to 120° C. for 15 min to 20 min.

[0013] Furthermore, the MRS liquid culture medium includes the following components: 15g / L-20g / L of glucose, 8g / L-10g / L of tryptone, 3g / L-5g / L of yeast extract, 1g / L-5g / L of tea polyphenols, 1g / L-2g / L of sodium citrate, 8g / L-10g / L of beef extract, 1g / L-2g / L of Tween-80, 1g / L-2g / L of dipotassium hydrogen phosphate, 0.01g / L-0.03g / L of magnesium sulfate and 0.01g / L-0.05g / L of manganese sulfate.

[0014] Furthermore, the pH of the MRS liquid culture medium is 6.5-6.8.

[0015] Furthermore, the primary culture medium and the secondary culture medium both include the following ingredients: 5g / L-8g / L beef extract, 8g / L-12g / L peptone, 2g / L-5g / L yeast powder, 0.1g / L-0.3g / L L-cysteine ​​hydrochloride, 0.001g / L-0.005g / L sodium selenite, 1g / L-3g / L sodium citrate and 1g / L-3g / L calcium carbonate.

[0016] Furthermore, the pH of the primary culture medium and the secondary culture medium are both 6.5-7.0.

[0017] Furthermore, the fermentation medium includes the following ingredients: 15g / L to 20g / L agar powder, 15g / L to 20g / L soybean meal powder, 1g / L to 5g / L alanine, 5g / L to 8g / L isomaltooligosaccharide, 0.01g / L to 0.03g / L trehalose, 1g / L to 5g / L glycerol, 2g / L to 5g / L hyperbranched glucan and 1g / L to 5g / L sodium trimetaphosphate.

[0018] Furthermore, the effective viable count of the fermentation broth is (1.2-1.8)×10 10 CFU / mL.

[0019] The present application also provides a Lactobacillus reuteri L-01 preparation, and the Lactobacillus reuteri L-01 preparation is obtained by adding the fermentation liquid obtained by the fermentation process into a protective agent, stirring the fermentation liquid, and then atomizing the fermentation liquid to obtain the Lactobacillus reuteri L-01 preparation.

[0020] Compared with the prior art, this application has the following beneficial effects:

[0021] After artificial domestication, the bile salt tolerance of Lactobacillus reuteri L-01 is enhanced, and it can be cultured in gastrointestinal fluid, and the survival rate remains above 98% for a long time, indicating that the Lactobacillus reuteri L-01 of the present application can play an excellent prebiotic function, help improve the balance of intestinal flora, maintain the balance of the internal environment, and have a certain inhibitory effect on pathogens, better provide immune protection, resist pathogen invasion, and reduce the incidence of disease. In addition, after the composition of the culture medium is optimized in the present application, the domesticated Lactobacillus reuteri L-01 also has a certain antioxidant effect and selenium-enriched purpose, thereby increasing the use value of Lactobacillus reuteri L-01. The present application optimizes the fermentation process, obtains the fermentation broth of Lactobacillus reuteri L-01 after culturing and fermenting in different culture media, and after adding a protective agent, can ensure its more excellent probiotic effect, not only enhance its stability in processing application and transportation and storage, but also the activity of the Lactobacillus reuteri L-01 preparation will not be destroyed by the protease, lipase and amylase in the host animal, has stronger stability and tolerance, has a certain tolerance to gastric juice, intestinal juice and bile salts, also has a certain sustained release effect, and has a longer effect. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0024] A fermentation process of Lactobacillus reuteri in one embodiment of the present invention comprises the following steps:

[0025] Pick a single Lactobacillus reuteri L-01 colony and inoculate it on MRS liquid medium for culture, strictly perform aseptic operation, anaerobically culture it at 30°C to 38°C for 20h to 28h, then inoculate it into a primary culture medium, anaerobically culture it at 25°C to 38°C for 20h to 24h, and obtain a primary seed liquid;

[0026] The first-level seed solution is inoculated into the second-level culture medium, and anaerobically cultured at 30° C. to 38° C. for 20 h to 24 h to obtain the second-level seed solution;

[0027] The secondary seed solution is centrifuged at 2500 r / min to 3000 r / min for 3 min to 5 min, the supernatant is discarded, and then washed with sterile saline for several times, and resuspended with saline to obtain a bacterial suspension;

[0028] Prepare fermentation medium, introduce steam, sterilize the material at 120-125°C; cool to 32-39°C, then inoculate the bacterial suspension into the fermentation medium, at 30-38°C, pH 4.0-6.0, maintain nitrogen pressure at 0.01Mpa-0.03Mpa, rotate at 80r / min-100r / min, perform anaerobic fermentation for 20h-24h to obtain fermentation liquid.

[0029] In one embodiment, the Lactobacillus reuteri L-01 was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, with a storage address in Guangzhou, China, a storage number of GDMCC NO: 65328, and a classification name of Limosilactobacillus reuteri.

[0030] In one embodiment, the MRS liquid culture medium, the primary culture medium and the secondary culture medium are all sterilized by high pressure at 100° C. to 120° C. for 15 min to 20 min.

[0031] In one embodiment, the MRS liquid culture medium comprises the following components: 15 g / L to 20 g / L of glucose, 8 g / L to 10 g / L of tryptone, 3 g / L to 5 g / L of yeast extract, 1 g / L to 5 g / L of tea polyphenols, 1 g / L to 2 g / L of sodium citrate, 8 g / L to 10 g / L of beef extract, 1 g / L to 2 g / L of Tween-80, 1 g / L to 2 g / L of dipotassium hydrogen phosphate, 0.01 g / L to 0.03 g / L of magnesium sulfate, and 0.01 g / L to 0.05 g / L of manganese sulfate.

[0032] In one embodiment, the pH of the MRS liquid culture medium is 6.5-6.8.

[0033] In one embodiment, the primary culture medium and the secondary culture medium both include the following ingredients: beef extract 5g / L~8g / L, peptone 8g / L~12g / L, yeast powder 2g / L~5g / L, L-cysteine ​​hydrochloride 0.1g / L~0.3g / L, sodium selenite 0.001g / L~0.005g / L, sodium citrate 1g / L~3g / L and calcium carbonate 1g / L~3g / L.

[0034] In one embodiment, the pH of the primary culture medium and the secondary culture medium are both 6.5-7.0.

[0035] In one embodiment, the inoculation amount is 2% to 5% in the primary culture medium.

[0036] In one embodiment, the inoculation amount is 1% to 5% in the secondary culture medium.

[0037] In one embodiment, the fermentation medium comprises the following ingredients: 15 g / L to 20 g / L agar powder, 15 g / L to 20 g / L soybean meal powder, 1 g / L to 5 g / L alanine, 5 g / L to 8 g / L isomaltooligosaccharide, 0.01 g / L to 0.03 g / L trehalose, 1 g / L to 5 g / L glycerol, 2 g / L to 5 g / L hyperbranched glucan and 1 g / L to 5 g / L sodium trimetaphosphate.

[0038] In one embodiment, the inoculum is inoculated into the fermentation medium at a volume ratio of 2% to 8%.

[0039] In one embodiment, the effective viable count of the fermentation broth is (1.2-1.8)×10 10 CFU / mL.

[0040] The present application also provides a Lactobacillus reuteri L-01 preparation, and the Lactobacillus reuteri L-01 preparation is obtained by adding the fermentation liquid obtained by the fermentation process into a protective agent, stirring the fermentation liquid, and then atomizing the fermentation liquid to obtain the Lactobacillus reuteri L-01 preparation.

[0041] In one embodiment, the protective agent includes 1 to 2 parts of glycerol, 15 to 20 parts of skimmed milk powder, 5 to 7 parts of sodium alginate, 1 to 5 parts of chitosan and 55 to 75 parts of water in parts by weight.

[0042] In one embodiment, the volume ratio of the fermentation broth to the protective agent is (1-3): (7-10).

[0043] In one of the embodiments, the gas velocity of the atomization treatment is 20L / min to 25L / min, the pressure is 120Kpa to 150Kpa, and the liquid flow rate is 5mL / min to 8mL / min.

[0044] The bile salt tolerance of Lactobacillus reuteri L-01 in the above scheme is enhanced, which helps to improve the balance of intestinal flora and enhance the body's immune protection. In addition, the obtained Lactobacillus reuteri L-01 preparation also has excellent processing application and transportation and storage stability.

[0045] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0046] Embodiment 1:

[0047] A screening of Lactobacillus reuteri L-01 comprises the following steps:

[0048] The pig intestine was collected in a sterile manner, homogenized, diluted to 100 μL, spread on MRS solid culture medium (containing 1% CaCO3), and cultured anaerobically at 35°C for 2 days until a single colony grew. The colony was then prepared into a bacterial suspension, and then cultured in nutrient agar medium at 35°C for 24 hours. The antibacterial test of pathogens (Staphylococcus aureus, Escherichia coli) was carried out, and the colony with the largest antibacterial diameter was selected, and the colony had no hemolytic activity.

[0049] The strain was cultured and morphologically observed and identified by simple staining. The results showed that the strains all showed consistent characteristics of smooth, round, and milky white on the agar plate. After staining, the strain was observed under a microscope to be short rod-shaped. It is labeled as Lactobacillus reuteri L-01, and the Lactobacillus reuteri L-01 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, with a preservation address in Guangzhou, China, a preservation number of GDMCC NO: 65328, and a classification name of Limosilactobacillus reuteri.

[0050] Embodiment 2:

[0051] A fermentation process of Lactobacillus reuteri L-01 comprises the following steps:

[0052] A single colony of Lactobacillus reuteri L-01 was selected and inoculated into MRS liquid medium (glucose 20 g / L, tryptone 8 g / L, yeast extract 5 g / L, tea polyphenols 4 g / L, sodium citrate 2 g / L, beef extract 8 g / L, Tween-80 1g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate 0.03g / L, manganese sulfate 0.03g / L, purified water 1000mL), strictly perform aseptic operation, anaerobically culture at pH 6.5, 35°C for 20h, then inoculate in a primary culture medium (beef extract 6g / L, peptone 8g / L, yeast powder 2g / L, L-cysteine ​​hydrochloride 0.1g / L, sodium selenite 0.003g / L, sodium citrate 2g / L, calcium carbonate 2g / L, purified water 1000mL) at an inoculum amount of 2%, at pH 6.5, 32°C, anaerobically culture for 24h to obtain a primary seed solution;

[0053] The primary seed solution was inoculated into a secondary culture medium (beef extract 8 g / L, peptone 10 g / L, yeast powder 5 g / L, L-cysteine ​​hydrochloride 0.3 g / L, sodium selenite 0.004 g / L, sodium citrate 3 g / L, calcium carbonate 3 g / L, purified water 1000 mL) at an inoculum amount of 5%, and the culture was performed at pH 6.5, 35° C., and anaerobically for 24 h to obtain a secondary seed solution;

[0054] The secondary seed solution was centrifuged at 2500 r / min for 3 min, the supernatant was discarded, and then washed several times with sterile saline and resuspended with saline to obtain a bacterial suspension;

[0055] A fermentation medium (20 g / L agar powder, 15 g / L soybean meal powder, 3 g / L alanine, 5 g / L isomaltooligosaccharide, 0.03 g / L trehalose, 5 g / L glycerol, 2 g / L hyperbranched glucan and 5 g / L sodium trimetaphosphate) was prepared, steam was introduced, and the materials were sterilized at 120° C.; the mixture was cooled to 35° C., and the bacterial suspension was inoculated into the fermentation medium at a volume ratio of 4%; the mixture was anaerobic fermented for 24 h at 37° C. and pH 6.0 with nitrogen pressure maintained at 0.01 MPa and a rotation speed of 80 r / min, to obtain an effective viable count of 1.5×10 10 CFU / mL fermentation broth.

[0056] Embodiment 3:

[0057] A fermentation process of Lactobacillus reuteri L-01 comprises the following steps:

[0058] A single colony of Lactobacillus reuteri L-01 was selected and inoculated into MRS liquid medium (glucose 20 g / L, tryptone 10 g / L, yeast extract 5 g / L, tea polyphenols 5 g / L, sodium citrate 2 g / L, beef extract 0 g / L, Tween-80 2g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate 0.03g / L, manganese sulfate 0.03g / L, purified water 1000mL), strictly perform aseptic operation, anaerobically culture at pH 6.5, 35°C for 24h, then inoculate in a primary culture medium (beef extract 8g / L, peptone 10g / L, yeast powder 5g / L, L-cysteine ​​hydrochloride 0.3g / L, sodium selenite 0.004g / L, sodium citrate 3g / L, calcium carbonate 3g / L, purified water 1000mL) at an inoculum size of 4%, anaerobically culture at pH 6.5, 32°C for 24h to obtain a primary seed solution;

[0059] The primary seed solution was inoculated into a secondary culture medium (8 g / L beef extract, 12 g / L peptone, 5 g / L yeast powder, 0.3 g / L L-cysteine ​​hydrochloride, 0.005 g / L sodium selenite, 3 g / L sodium citrate, 3 g / L calcium carbonate, 1000 mL purified water) at an inoculum amount of 5%, and the culture was performed at pH 6.5, 35° C., and anaerobically for 20 h to obtain a secondary seed solution;

[0060] The secondary seed solution was centrifuged at 2500 r / min for 3 min, the supernatant was discarded, and then washed several times with sterile saline and resuspended with saline to obtain a bacterial suspension;

[0061] A fermentation medium (20 g / L agar powder, 18 g / L soybean meal powder, 3 g / L alanine, 8 g / L isomaltooligosaccharide, 0.03 g / L trehalose, 3 g / L glycerol, 3 g / L hyperbranched glucan and 3 g / L sodium trimetaphosphate) was prepared, steam was introduced, and the materials were sterilized at 125° C.; the mixture was cooled to 32° C., and the bacterial suspension was inoculated into the fermentation medium at a volume ratio of 4%; the mixture was fermented anaerobicly for 24 h at 37° C. and pH 4.5 with nitrogen pressure maintained at 0.01 MPa and a rotation speed of 80 r / min, and an effective viable count of 1.6×10 10 CFU / mL of fermentation broth.

[0062] Embodiment 4:

[0063] A method for preparing a Lactobacillus reuteri L-01 preparation comprises the following steps:

[0064] According to the weight ratio, 2 parts of glycerol, 17 parts of skimmed milk powder, 5 parts of sodium alginate, 3 parts of chitosan and 73 parts of water are stirred to obtain a protective agent;

[0065] The fermentation liquid of Example 2 and the protective agent in a volume ratio of 2:8 were stirred and then subjected to a gas velocity of 20 L / min, a pressure of 120 KPa, and a liquid flow rate of 5 mL / min to prepare a Lactobacillus reuteri L-01 preparation.

[0066] Embodiment 5:

[0067] A method for preparing a Lactobacillus reuteri L-01 preparation comprises the following steps:

[0068] According to the weight ratio, 1 part of glycerol, 20 parts of skimmed milk powder, 7 parts of sodium alginate, 5 parts of chitosan and 67 parts of water are stirred to obtain a protective agent;

[0069] The fermentation broth of Example 3 and the protective agent in a volume ratio of 3:7 were stirred and then subjected to a gas velocity of 20 L / min, a pressure of 120 KPa, and a feed liquid flow rate of 5 mL / min to prepare a Lactobacillus reuteri L-01 preparation.

[0070] Comparative Example 1:

[0071] The difference between Comparative Example 1 and Example 5 is that Comparative Example 1 uses commercially available conventional Bacillus reuteri, and the rest is the same as Example 5.

[0072] Comparative Example 2:

[0073] The difference between Comparative Example 2 and Example 5 is that in Comparative Example 2, the fermentation broth of Example 3 with a volume ratio of 3:7 and a chitosan solution with a mass percentage concentration of 25% are mixed uniformly, and the Lactobacillus reuteri L-01 preparation is prepared under the conditions of a gas velocity of 20 L / min, a pressure of 120 Kpa, and a feed liquid flow rate of 5 mL / min.

[0074] The strain of Example 1, the fermentation broth samples of Examples 2-3, the preparation samples of Examples 4-5, and the comparative samples of Comparative Examples 1-2 were subjected to antibacterial tests, and the diameters (mm) of the antibacterial zones at different times were recorded. The results are shown in Table 1 below.

[0075] Table 1: Antibacterial results

[0076]

[0077] From the data analysis of Table 1, it can be seen that the fermentation liquid obtained by the fermentation process of the present application has significant antibacterial activity, and after being prepared into a preparation, it not only maintains the antibacterial activity at a certain temperature, but also has a certain sustained release effect, thereby promoting a certain antibacterial effect.

[0078] The strain of Example 1, the fermentation broth samples of Examples 2-3, the preparation samples of Examples 4-5, and the comparative samples of Comparative Examples 1-2 were subjected to a pig bile salt resistance test. Pig bile salt liquids of different concentrations were prepared and the viable bacterial rates at different concentrations were recorded. The results are shown in Table 2.

[0079] Table 2: Results of pig bile salt tolerance (%)

[0080]

[0081] It can be seen from the viable bacteria rate in Table 2 that the fermentation broth and preparation of the present application have excellent bile salt tolerance.

[0082] The strain of Example 1, the fermentation broth samples of Examples 2-3, the preparation samples of Examples 4-5, and the comparative samples of Comparative Examples 1-2 were subjected to a cell adhesion test, and the results are shown in Table 3 below.

[0083] Table 3: Cell adhesion results

[0084] Group Adherent live bacteria count / 100 cells Example 1 1275±3 Example 2 1608±15 Example 3 1649±10 Example 4 1579±12 Example 5 1597±8 Comparative Example 1 589±5 Comparative Example 2 1255±9

[0085] From the data analysis of Table 3, it can be seen that the live bacteria in the fermentation broth and preparation of the present application can colonize and multiply in large quantities within the cells, thereby ensuring that they play a probiotic role in the body.

[0086] The strain of Example 1, the fermentation broth samples of Examples 2 to 3, the preparation samples of Examples 4 to 5, and the comparative samples of Comparative Examples 1 to 2 were subjected to a feeding test. The fermentation broth samples of Examples 2 to 3, the preparation samples of Examples 4 to 5, and the comparative samples of Comparative Examples 1 to 2 were added to the basal diet, respectively. The results are shown in Table 4 below.

[0087] Table 4: Results of production performance and diarrhea rate of weaned piglets

[0088]

[0089] From the data analysis of Table 4, it can be seen that the Lactobacillus reuteri L01, fermentation broth and preparation of the present application all have the effect of promoting the growth of weaned piglets, and are significantly more effective than the Lactobacillus reuteri on the market, and reduce the diarrhea rate, exerting a more excellent conditioning effect.

[0090] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0091] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A fermentation process of Lactobacillus reuteri L-01, characterized in that: The fermentation process comprises the following steps: Pick a single Lactobacillus reuteri L-01 colony and inoculate it on MRS liquid medium for culture, strictly perform aseptic operation, anaerobically culture it at 30°C to 38°C for 20h to 28h, then inoculate it into a primary culture medium, anaerobically culture it at 25°C to 38°C for 20h to 24h, and obtain a primary seed liquid; The first-level seed solution is inoculated into the second-level culture medium, and anaerobically cultured at 30° C. to 38° C. for 20 h to 24 h to obtain the second-level seed solution; The secondary seed solution is centrifuged at 2500 r / min to 3000 r / min for 3 min to 5 min, the supernatant is discarded, and then washed with sterile saline for several times, and resuspended with saline to obtain a bacterial suspension; Prepare fermentation medium, introduce steam, sterilize the material at 120-125°C; cool to 32-39°C, then inoculate the bacterial suspension into the fermentation medium, at 30-38°C, pH 4.0-6.0, maintain nitrogen pressure at 0.01Mpa-0.03Mpa, rotate at 80r / min-100r / min, perform anaerobic fermentation for 20h-24h to obtain fermentation liquid.

2. The fermentation process according to claim 1, characterized in that: The Lactobacillus reuteri L-01 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, with the collection address in Guangzhou, China, the collection number is GDMCC NO: 65328, and the classification name is Limosilactobacillus reuteri.

3. The fermentation process according to claim 1, characterized in that: The MRS liquid culture medium, the primary culture medium and the secondary culture medium are all sterilized under high pressure at 100° C. to 120° C. for 15 min to 20 min.

4. The fermentation process according to claim 1, characterized in that: The MRS liquid culture medium comprises the following components: 15 g / L to 20 g / L of glucose, 8 g / L to 10 g / L of tryptone, 3 g / L to 5 g / L of yeast extract, 1 g / L to 5 g / L of tea polyphenols, 1 g / L to 2 g / L of sodium citrate, 8 g / L to 10 g / L of beef extract, 1 g / L to 2 g / L of Tween-80, 1 g / L to 2 g / L of dipotassium hydrogen phosphate, 0.01 g / L to 0.03 g / L of magnesium sulfate and 0.01 g / L to 0.05 g / L of manganese sulfate.

5. The fermentation process according to claim 1, characterized in that: The pH of the MRS liquid culture medium is 6.5-6.

8.

6. The fermentation process according to claim 1, characterized in that: The primary culture medium and the secondary culture medium both include the following ingredients: 5g / L-8g / L beef extract, 8g / L-12g / L peptone, 2g / L-5g / L yeast powder, 0.1g / L-0.3g / L L-cysteine ​​hydrochloride, 0.001g / L-0.005g / L sodium selenite, 1g / L-3g / L sodium citrate and 1g / L-3g / L calcium carbonate.

7. The fermentation process according to claim 6, characterized in that The pH of the primary culture medium and the secondary culture medium are both 6.5-7.

0.

8. The fermentation process according to claim 1, characterized in that: The fermentation medium comprises the following components: 15 g / L to 20 g / L agar powder, 15 g / L to 20 g / L soybean meal powder, 1 g / L to 5 g / L alanine, 5 g / L to 8 g / L isomaltooligosaccharide, 0.01 g / L to 0.03 g / L trehalose, 1 g / L to 5 g / L glycerol, 2 g / L to 5 g / L hyperbranched glucan and 1 g / L to 5 g / L sodium trimetaphosphate.

9. The fermentation process according to claim 1, characterized in that: The effective viable bacteria count of the fermentation broth is (1.2-1.8)×10 10 CFU / mL.

10. A Lactobacillus reuteri L-01 preparation, characterized in that: The Lactobacillus reuteri L-01 preparation is obtained by adding the fermentation liquid obtained by the fermentation process according to any one of claims 1 to 9 into a protective agent, stirring the fermentation liquid, and then atomizing the fermentation liquid to obtain the Lactobacillus reuteri L-01 preparation.

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