CN113308389B

CN113308389B - Lactobacillus salivarius and application thereof

Info

Publication number: CN113308389B
Application number: CN202110279176.5A
Authority: CN
Inventors: 贾长生; 王新超; 李珮铷
Original assignee: Zhejiang Ty Health Biotechnology Co ltd
Current assignee: Zhejiang Ty Health Biotechnology Co ltd
Priority date: 2020-10-12
Filing date: 2021-03-16
Publication date: 2022-06-14
Anticipated expiration: 2041-03-16
Also published as: CN113308389A; CN112126605A

Abstract

The invention provides a Lactobacillus salivarius GER106 preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020445, the preservation address is Wuhan university in Wuhan city of Hubei province of the people's republic of China, and the preservation date is 2020, 8 and 24 days. Also provides the application of the strain. The lactobacillus salivarius GER106 provided by the invention is derived from the oral cavity of children, can be used for preparing food, leavening agents, functional food or pharmaceutical compositions, and has the effects of improving immunity and preventing dental caries.

Description

Lactobacillus salivarius and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to lactobacillus salivarius and application of the strain.

Background

The immune system of a person cannot normally play a protective role due to low immunity, and in this case, the person is very easy to infect bacteria, viruses, fungi and the like, so the most direct expression of low immunity is that the person is easy to get ill. The common diseases aggravate the consumption of the organism, and usually cause symptoms such as body weakness cold, qi deficiency cold, lung deficiency cough and asthma, spleen deficiency pool diarrhea, qi deficiency palpitation, body weakness, malnutrition, listlessness, fatigue, weakness, anorexia, sleep disorder, etc. The disease recovery period of the people is long, and the diseases are frequently recurrent, which can lead to poor physical and intelligence development and can also easily induce serious diseases.

Dental caries is a common disease and frequently encountered disease, and has high incidence rate, wide distribution and great harm to human bodies. With the diligent efforts of scientists, bacteria are now generally regarded as the main cause of dental caries, and streptococcus mutans is the main pathogenic bacteria, and the plaque formed by streptococcus mutans is adhered to the tooth surface, grows on the tooth surface, produces acid, corrodes the tooth and finally forms dental caries. The caries preventing products currently used in the market are roughly classified into the following categories: one is a fluorine-containing preparation, and the anticarious principle of the fluorine-containing preparation is to promote calcification of teeth and reduce acid corrosion. This is an indirect effect, with limited efficacy; secondly, the application of antibacterial drugs, such as mouthwash containing chlorhexidine, metronidazole and the like, is limited due to obvious side effects; thirdly, the caries prevention product prepared by the plant extract, such as gargle, chewing gum tablet and the like prepared by the traditional Chinese medicine extract, has certain effect on preventing and treating the caries, but is a medicament, needs to be used under the guidance of doctors and is not suitable for long-term use; fourthly, the product which is widely accepted in the market at present and is produced by sugar substitutes for preventing caries and protecting teeth, such as xylitol and the like, does not fundamentally solve the substantive problem of preventing caries and protecting teeth.

The growing awareness of the relationship between diet and health has led to an increasing demand for products that provide basic nutrition and also enhance health, and studies have shown that the intake of probiotics is beneficial to maintain a delicate microbial balance, but there is currently no mature product for caries-preventing probiotics.

Disclosure of Invention

The purpose of the invention is as follows: in order to ensure the health of people, the invention provides lactobacillus salivarius.

The technical scheme is as follows: the technical scheme adopted by the invention is as follows:

lactobacillus salivarius (Lactobacillus salivarius) GER106 preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020445, the preservation address is Wuhan university in Wuhan city, Hubei province of the people's republic of China, and the preservation date is 09 months and 08 days in 2020.

The gram staining result of the strain shows that: strains in the presence of CaCO₃The diameter of a bacterial colony on the MRS culture medium is 1-2mm, and the bacterial colony is white, convex and provided with a calcium dissolving ring; the strain is gram-positive, and the round end is straight rod-shaped; gram staining characteristics of the cells are shown in FIG. 1. The nucleotide sequence of 16S rDNA of the strain GER106 is shown in SEQ ID NO. 1; through 16S rDNA Gene comparison, the similarity rate of the strain to the Lactobacillus salivarius strain in Gene bank reaches 100 percent; combining with the identification of a microbial system, the GER106 is a Lactobacillus salivarius strain and is named as Lactobacillus salivarius GER 106; the 16S rDNA of the Lactobacillus salivarius GER106 is shown in a sequence table, and the biological evolutionary tree is shown in figure 2.

The invention also provides the lactobacillus salivarius (Lactobacillus salivarius) The large-scale fermentation method of GER106 comprises the following steps of carrying out constant-temperature culture at 37 ℃ in a fermentation culture medium: 10L of pure water, 100g of peptone, 200g of glucose, 50g of yeast extract, 50g of sodium acetate, 2g of dipotassium hydrogen phosphate, 20g of diamine hydrogen citrate, 5.8g of magnesium sulfate, 2.5g of manganese sulfate, 100g of beef extract and 0mL of Tween 8010, sterilizing at 121 ℃ for 30min, and adjusting the pH value to 6.0-6.5.

The invention also provides lactobacillus salivarius (Lactobacillus salivarius) Application of GER106 in preparation of immunity-improving food, medicine or health-care product.

Preferably, Lactobacillus salivarius: (Lactobacillus salivarius) The dosage of GER106 is (0.5-3.0) x10¹⁰CFU/g。

The invention also provides lactobacillus salivarius (Lactobacillus salivarius) The GER106 is applied to the preparation of medicaments or health-care products for preventing decayed teeth.

Preferably, Lactobacillus salivarius: (Lactobacillus salivarius) The dosage of GER106 is (1.0-3.5) x10¹⁰CFU/g。

The invention also provides a pharmaceutical composition, which comprises lactobacillus salivarius (A)Lactobacillus salivarius) GER106, further comprising at least one pharmaceutically acceptable carrierA body, a diluent, an excipient or an adjuvant.

Preferably, the pharmaceutical composition is used for enhancing immunity and/or preventing dental caries; said Lactobacillus salivarius: (Lactobacillus salivarius) The dosage of GER106 is (0.5-3.5) x10¹⁰CFU/g。

Has the advantages that: the Lactobacillus salivarius GER106 provided by the invention is derived from oral cavity of children, can be used for preparing food, leavening agent, functional food or pharmaceutical composition, and has functions of improving immunity and preventing dental caries

Drawings

FIG. 1 is a gram-stained optical micrograph of Lactobacillus salivarius GER 106.

FIG. 2 is a biological evolutionary tree of Lactobacillus salivarius GER 106.

FIG. 3 is a graph showing the change in OD value during fermentation of Lactobacillus salivarius GER 106.

FIG. 4 is a graph showing the results of measurement of cytokines and b-defensin 2 (HBD-2) in the supernatant of Caco-2/TC7 cells after exposure to Lactobacillus salivarius GER 106. Wherein, the compound (A) is IL-6, (B) is IL-8, (C) is IL-10, and (D) is HBD-2. Mean. + -. SE. NS (no significance) P <0.05 and P <0.01 compared to Caco-2/TC7 control.

FIG. 5 shows Lactobacillus salivarius GER106 (10) under different culture conditions⁹cells/mL) relative adhesion of streptococcus mutans to hydroxyapatite in the presence of heat-inactivated cells. (A) Determination protocol: pre-incubating the hydroxyapatite discs with lactobacillus salivarius for 3 hours; after the washing step, the discs were incubated with (C1) Lactobacillus salivarius and Streptococcus mutans, (C2) Streptococcus mutans or (C3) KCl buffer for 2 hours; the final assay dish was washed again (C3) and incubated with streptococcus mutans for an additional 2 hours; finally, a final wash was performed and the Streptococcus mutans was quantified by surface plating. (B) And (5) obtaining an adhesion result. The Streptococcus mutans concentration data were normalized to the average of the bacterial counts in the negative control, and the standard deviation was represented by error bars.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Example 1 isolation, purification and characterization of the strains

The strain is derived from oral cavity of healthy children.

And (3) separating and purifying lactobacillus salivarius strains: taking about 0.2g of sample, adding 1ml of MRS culture medium, vortexing and shaking for 1min, and sequentially diluting to 10 times^-6200 μ L of each diluted concentration was applied to a coating containing 1% CaCO₃Culturing in 37 deg.C anaerobic jar for 48h on MRS culture medium, selecting single colony on the plate, streaking and purifying on MRS solid culture medium, and culturing in 37 deg.C anaerobic jar for 48h to obtain pure colony.

Performing gram stain identification on the purified colonies; strains in the presence of CaCO₃The diameter of a bacterial colony on the MRS culture medium is 1-2mm, and the bacterial colony is white, convex and provided with a calcium dissolving ring; the strain is gram-positive, and the round end is straight rod-shaped; gram staining characteristics of the cells are shown in FIG. 1.

Screening and identifying lactobacillus salivarius strains: identifying the obtained strain GER106 by 16S rDNA sequencing; amplifying and sequencing the 16S rDNA gene sequence of the strain GER106 by using a published 16S universal primer (the primer sequence is 8F: 5'-AGAGTTTGATCCTGGCTCA-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'), sending a PCR amplification product to the company of Biotechnology engineering (Shanghai) GmbH for sequencing, and showing the nucleotide sequence of the 16S rDNA of the strain GER106 as SEQ ID NO. 1; through 16S rDNA Gene comparison, the similarity rate of the strain to the Lactobacillus salivarius strain in Gene bank reaches 100 percent; combining with the identification of a microbial system, the GER106 is a Lactobacillus salivarius strain and is named as Lactobacillus salivarius GER 106; the 16S rDNA of Lactobacillus salivarius GER106 is shown in the sequence table, and the biological evolutionary tree is shown in FIG. 2.

Example 2 Lactobacillus salivarius: (Lactobacillus salivarius) GER106 acid and biliary test

GER106 bacteria were grown overnight in 37 ℃ MRS broth and suspended to approximately 10% in 0.5% w/v bile and in MRS broth adjusted to pH 3⁸CFU/ml cell concentration (SigmaeAldrich, France) for 4 hours. These conditions are selected to represent the time taken to pass through the gastrointestinal system toAnd the pH and bile concentrations found in the stomach and intestine, respectively, of chickens. Bacterial viability was assessed by counting on MRS agar plates, with the results shown in table 1.

Table 1 simulation of acid and bile resistance test results

Test standard	Results
		Acid resistance	Is. Survival after 2h at pH 3 of 95%
Bile resistance	Is. Survival after 4 hours of 0.5% w/v bile

Resistance to acids and bile is generally considered as the basic evaluation criterion for probiotic evaluation, since the strain must survive in the stomach and small intestine. Thus, the survival of Lactobacillus salivarius GER106 was tested for 2h in 0.5% w/v bile at pH 3 for 4h, respectively. The results show that this strain is able to withstand acidic conditions and the presence of 0.5% w/v bile without any significant loss of viability (95% survival).

Example 3 Lactobacillus salivarius: (Lactobacillus salivarius) Large-scale fermentation and freeze-dried powder production of GER106

Preparing a large-scale fermentation culture medium: according to 10L of pure water (reverse osmosis method), 100g of peptone, 200g of glucose, 50g of yeast extract, 50g of sodium acetate, 2g of dipotassium phosphate, 20g of diammonium hydrogen citrate, 5.8g of magnesium sulfate, 2.5g of manganese sulfate, 100g of beef extract, 0mL of Tween 8010, high-temperature high-pressure sterilization at 121 ℃ for 20min, adjusting the pH value to 6.5, and heating and stirring uniformly. Sterilizing at 121 deg.C under high temperature and high pressure for 20 min.

Streaking the strain on a solid MRS culture medium, culturing in an anaerobic tank at 37 ℃ for 24h, selecting individual independent and convex bacterial plaques, inoculating into 500ml of liquid culture medium, and culturing in a thermostat at 37 ℃ for 10-12 h; when the OD value is more than or equal to 1.2, transferring the mixture into a shake flask filled with 5L of liquid culture medium, and culturing the mixture in a 37 ℃ incubator for 8-12 h; when the OD value is more than or equal to 1.2, transferring the mixture into a fermentation tank filled with 50L of liquid culture medium, stabilizing the pH value to 6.0-6.5, and fermenting for 8-12h at 37 ℃; the change trend of OD value in the fermentation process is shown in FIG. 3; when the OD value is more than or equal to 1.2, transferring the mixture into a fermentation tank filled with 500L of liquid culture medium, stabilizing the pH value to 6.0-6.5, and fermenting for 10-18h at 37 ℃; and when the OD value is more than or equal to 1.2, ending the fermentation, quickly reducing the temperature of the fermentation tank to be below 10 ℃, and centrifuging the fermentation liquor to obtain thalli.

Adding freeze-drying protectant (16% skimmed milk, 10% dextrin, 4% polyvinylpyrrolidone, 6% lactose, 0.5% sodium glutamate, 0.5% cysteine, 0.30.8% sodium acetate, and water in balance) into the harvested bacterial paste at a ratio of 1:1, vacuum freeze-drying for 32-50h, and jet-pulverizing the lyophilized bacterial cake to obtain Lactobacillus salivarius GER106 lyophilized powder with viable bacteria content of 2.0 × 10¹¹-8×10¹¹CFU/g, used for experiments, fermentation, food, health products or medicines and the like.

Example 4 Lactobacillus salivarius: (Lactobacillus salivarius) GER106 ability to adhere to intestinal cells

Caco-2/TC7 cells were used between

passages

35 and 50. Cells were routinely grown in DMEM medium supplemented with 15% heat-inactivated fetal bovine serum, 2mM L-glutamine, penicillin and streptomycin at 100U/ml and 1% of each non-essential amino acid. For experimental analysis, cells were plated at approximately 10⁵Individual cell/cm²Is seeded in 24-well tissue culture plates or inserts that allow epithelial differentiation. At 37 deg.C, 5% CO₂Cells were cultured in 95% air atmosphere and the medium was changed periodically. Caco-2/TC7 grown in 24-well tissue culture plates was incubated to early confluence (undifferentiated state), while Caco-2/TC7 cells grown on the inserts were used 21 days after confluence (fully differentiated state).

Harvesting of Lactobacillus salivarius by centrifugationGER106, defined as 10⁸CFU/ml concentrations were resuspended in serum and antibiotic free cell culture media and then applied to confluent Caco-2/TC7 monolayers. At 37 ℃ 5% CO₂After the next 4 hours of incubation, the monolayer was washed with PBS to remove non-adhering bacteria and lysed by incubation with 0.1% Triton X100 for 15 minutes, then the lysate was diluted and plated on MRS agar to determine the number of adhering bacteria.

Statistics were made on the number of adherent bacteria for the three intervention groups, and the results are shown in table 2. It can be seen that Lactobacillus salivarius GER106 has significant effect on intestinal adhesion cells.

Table 2 adhesion results during intervention

Test standard	Results
		Adhesion to intestinal cells	10⁵CFU/ml (1% of the original bacteria)

Adhesion to intestinal epithelial cells is another reliable criterion for selection of probiotics. The results of in vitro tests using Caco-2 cells to assess the ability of intestinal epithelial cells to adhere are reported to correlate well with in vivo results, and this feature is often strain-specific. Lactobacillus salivarius GER106 (10)⁸CFU/ml) was examined by incubating the bacteria with confluent monolayers of Caco-2/TC7 for 4 hours. At the end of the incubation period, non-adhering bacteria were removed by washing and the number of adhering bacteria was determined by plate. Under these conditions, 10 was recovered in Caco-2/TC7 monolayer lysate⁵CFU/ml adhered Lactobacillus salivarius GER106, corresponding to 1% of the starting population.

Examples4 Lactobacillus salivarius (Lactobacillus salivarius) Immune defense capability of GER106

By 10⁸After 24 hours of CFU/ml Lactobacillus salivarius GER106 treatment, the levels of cytokines (IL-6, IL-8, and IL-10) produced by Caco-2/TC7 cells in the culture supernatants were measured using an ELISA Quantikine kit. B-defensin 2 was measured using a b-defensin 2, beta (Human) e ELISA kit.

IL-6, IL-8, IL-10 and B-defensin 2 were measured using an ELISA assay as shown in FIG. 4, with Lactobacillus salivarius GER106 inducing a 1.8 fold increase in IL-8 secretion (FIG. 4B) and a 2.6 fold increase in B-defensin 2 secretion. Secretion of b-defensin 2 compared to untreated Caco-2/TC7 cells (FIG. 4D). In contrast, for IL-6 (FIG. 4A) and IL-10 (FIG. 4C), no change in basal secretion was observed for Caco-2/TC7 cells after exposure to bacteria.

The results show that: lactobacillus salivarius GER106 exhibits in vitro immunomodulatory activity by inducing IL-8. We also observed that Lactobacillus salivarius GER106 induced b-defensin 2 secretion. It is known that inducible bdefensins play an important role in intestinal barrier function, and in vitro studies indicate that clinically effective probiotics can induce the production of antibacterial b-defensin 2. Induction of b-defensin 2 by probiotics, including lactobacillus salivarius GER106, may be an alternative approach to enhance the innate defense mechanism and supplement new strategies.

Example 5 Lactobacillus salivarius: (Lactobacillus salivarius) GER106 inhibition of dental caries-causing Streptococcus mutans

Lactobacillus salivarius samples normalised to 10 in KCl buffer¹⁰cells/mL. Streptococcus mutans cells were concentrated by centrifugation, washed 3 times with sterile KCl buffer, and then resuspended in an appropriate volume of KCl buffer to obtain an OD 600 nm reading equal to 3. Both bacterial suspensions were stored at room temperature for less than 1 hour or at 4 ℃ for up to 6 hours.

Dense hydroxyapatite disks (5 mm x 2 mm) were processed as follows: after three cycles of water washing, the discs were incubated in a lysozyme solution (20 mg/mL) at 37 ℃ for 1 hour and again in a trypsin solution (20 mg/mL) at 37 ℃ for 1 h. The enzyme was removed by thorough washing with water and the discs were treated with 0.2M NaOH for 1 h.

After 3 cycles of washing with water, the discs were UV-sterilized (40 min) and then stored in filter-sterilized KCl at 4 ℃. Adhesion assays were performed in flat-bottomed 96-well microtiter plates (1 disc per well) to which samples of Lactobacillus salivarius (10) were added⁹Individual cells/mL) in 0.2 mL of KCl buffer.

After a pre-incubation period of 3 hours at 37 ℃, the wells were washed with KCl buffer with orbital shaking (100 rpm). Streptococcus mutans (OD 600 nm = 0.3) was then added to the well-filled discs and incubated together at 37 ℃ for 2 hours with orbital shaking (100 rpm). Finally, the discs were transferred to 1.5 mL tubes, where they were washed 5 times with 1mL KCl buffer. After quantification of Streptococcus mutans, the results are expressed as relative (%) adherence. Average streptococcus mutans concentration for negative control (adhesion 100%, lactobacillus salivarius GER 106) data were normalized using the following formula: streptococcus mutans concentration X100/concentration in the negative control of the mean value of Streptococcus mutans.

To eliminate the inhibition of adhesion of Streptococcus mutans, which is a non-specific phenomenon, the assay was repeated without incubation of the microorganism with the hydroxyapatite disk at the same time due to the temporary competition for the hydroxyapatite surface during co-incubation (FIG. 5A). Under these conditions, the ability of Lactobacillus salivarius to inhibit the adhesion of Streptococcus mutans was reduced from 70% to 30% after washing, but the effect could still be detected. Pre-incubation of hydroxyapatite with lactobacillus salivarius cells did also inhibit pathogen adhesion after two additional washing steps (figure 5B).

Example 6 Lactobacillus salivarius: (Lactobacillus salivarius) GER106 application example

1. Leaven for preparing dairy products, bean products and fruit and vegetable products by using lactobacillus salivarius GER106

Fermenting lactobacillus salivarius GER106 in a fermentation tank to mature, centrifuging to obtain bacterial sludge, adding a protective agent for emulsification, standing the emulsified suspension at 37 ℃ for 30min, and freeze-drying by a vacuum freeze-drying method to obtain the leavening agent of dairy products, bean products and fruit and vegetable products.

2. Probiotics-containing food prepared from lactobacillus salivarius GER106

Adding the lactobacillus salivarius GER106 fermentation liquid or lyophilized powder into solid beverage, tea beverage, vegetable juice, tablet candy, etc. at room temperature or low temperature, and making into food containing probiotic.

3. Probiotic beverage for improving human immunity by using lactobacillus salivarius GER106

Mixing the lactobacillus salivarius GER106 freeze-dried powder with prebiotics such as inulin and resistant dextrin, subpackaging into 2g-10g of convenient packages, wherein each package contains 50 hundred million CFU-300 hundred million CFU of lactobacillus salivarius GER106, and making into probiotic beverage food beneficial to improving human immunity, storing at normal temperature or refrigerating, and taking with warm water below 37 deg.C.

4. Probiotic beverage prepared from lactobacillus salivarius GER106 and used for preventing dental caries

Mixing the lactobacillus salivarius GER106 freeze-dried powder with prebiotics such as fructo-oligosaccharide, dietary fiber and the like, subpackaging into 2g-10g of convenient package, wherein each bag contains 100 hundred million CFU-350 hundred million CFU of lactobacillus salivarius GER106, and preparing into probiotic beverage food which is helpful for preventing dental caries, storing at normal temperature or refrigerating, taking with warm water below 37 ℃, pouring into the mouth and chewing optimally.

5. Lactobacillus salivarius GER106 prepared lactobacillus beverage

Mixing the skim milk powder with water according to a ratio of 1:15, fully dissolving, sterilizing at 95 ℃ for 20min, then cooling to 35 ℃, adding lactobacillus salivarius GER106 bacterial liquid according to a ratio of 10:1, fermenting, monitoring the pH value in real time, and when the pH value is reduced at the maximum rate, cooling to 4 ℃ for refrigeration or filling to obtain the lactobacillus beverage containing the live lactobacillus salivarius GER 106.

6. Capsule product prepared from lactobacillus salivarius GER106

The freeze-dried powder of the lactobacillus salivarius GER106 is filled into medicinal microcapsules to obtain a capsule product for intervening intestinal flora, human immunity and the like.

7. Production of fermented milk Using Lactobacillus salivarius GER106

Adding sucrose into fresh milk, dissolving, sterilizing at low temperature (or sterilizing fresh milk sold in market), preheating to 37 deg.C in a constant temperature container, mixing Lactobacillus salivarius GER106, Lactobacillus bulgaricus and Lactobacillus plantarum at equal ratio to obtain starter, adding into preheated fresh milk, adding 1L fresh milk into 1 × 10 of the starter⁸CFU-5×10⁹And (3) uniformly mixing the materials, standing for 4-8h at 35-37 ℃, standing for cold storage for 12h at 4 ℃ after curdling is carried out, thus obtaining the fermented milk, wherein the taste of the fermented milk can be adjusted by adding honey and the like according to personal taste. In addition, after the milk is fermented and curdled, the curdled milk is added into the fresh milk according to the proportion of 1:10 to serve as a fresh milk leavening agent, and the fermentation is carried out in the same way.

Sequence listing

<110> Zhenjiang city Probiotics science and technology Limited

<120> Lactobacillus salivarius and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1050

<212> DNA

<213> Lactobacillus salivarius (Lactobacillus salivarius)

<400> 1

cgaacccata tctgtcacct tagacggctg gctccttgcg gttaccccac cggctttggg 60

tgttacaaac tctcatggtg tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg 120

cgacatgctg attcgcgatt actagcgatt ccgacttcat gtaggcgagt tgcagcctac 180

aatccgaact gagaacggct ttaagagatt agctaaacct cgcggtcttg cgactcgttg 240

taccgtccat tgtagcacgt gtgtagccca ggtcataagg ggcatgatga cttgacgtcg 300

tccccacctt cctccggttt gtcaccggca gtctcgccag agtgcccaac ttaatgctgg 360

caactgacaa caagggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc 420

tgacgacagc catgcaccac ctgtcacttt gtccccgaag ggaaagccta atctcttagg 480

tggtcaaagg atgtcaagac ctggtaaggt tcttcgcgtt gcttcgaatt aaaccacatg 540

ctccaccgct tgtgcgggcc cccgtcaatt cctttgagtt tcaaccttgc ggtcgtactc 600

cccaggcgga atgcttattg cgttagctgc ggcactgaag ggcggaaacc ctccaacacc 660

tagcattcat cgtttacggc gtggactacc agggtatcta atcctgtttg ctacccacgc 720

tttcgaacct cagcgtcagt tacagaccag agagccgctt tcgccactgg tgttcttcca 780

tatatctacg catttcaccg ctacacatgg agttccactc tcctcttctg cactcaagtc 840

ttccagtttc caatgcacta ctccggttaa gccgaaggct ttcacatcag acttaaaaga 900

ccgcctgcgt tccctttacg cccaataaat ccggacaacg cttgccacct acgtattacc 960

gcggctgctg gcacgtagtt agccgtgact tgctgggtta gataccgtca tcgaatgaac 1020

agttactctc actcgtgttc ttctctaaca 1050

Claims

1. A lactobacillus salivarius freeze-dried powder is characterized in that: it contains Lactobacillus salivarius (Lactobacillus salivarius) GER106, deposited in China center for type culture Collection with the deposit number of CCTCC NO: m2020445, the preservation address is Wuhan university in Wuhan city of Hubei province of the people's republic of China, and the preservation date is 09 months and 08 days in 2020;

the preparation method of the freeze-dried powder comprises the following steps:

preparing a large-scale fermentation culture medium: according to 10L of pure water, 100g of peptone, 200g of glucose, 50g of yeast extract, 50g of sodium acetate, 2g of dipotassium hydrogen phosphate, 20g of diamine hydrogen citrate, 5.8g of magnesium sulfate, 2.5g of manganese sulfate, 100g of beef extract and 0mL of Tween 8010, sterilizing at the high temperature and the high pressure of 121 ℃ for 20min, adjusting the pH value to 6.5, heating and stirring uniformly; sterilizing at 121 deg.C under high temperature and high pressure for 20 min;

streaking the strain on a solid MRS culture medium, culturing in an anaerobic tank at 37 ℃ for 24h, selecting individual independent and convex bacterial plaques, inoculating into 500ml of liquid culture medium, and culturing in a thermostat at 37 ℃ for 10-12 h; when the OD value is more than or equal to 1.2, transferring the mixture into a shake flask filled with 5L of liquid culture medium, and culturing the mixture in a 37 ℃ incubator for 8-12 h; when the OD value is more than or equal to 1.2, transferring the mixture into a fermentation tank filled with 50L of liquid culture medium, stabilizing the pH value to 6.0-6.5, and fermenting for 8-12h at 37 ℃; when the OD value is more than or equal to 1.2, transferring the mixture into a fermentation tank filled with 500L of liquid culture medium, stabilizing the pH value to 6.0-6.5, and fermenting for 10-18h at 37 ℃; when the OD value is more than or equal to 1.2, ending the fermentation, quickly reducing the temperature of the fermentation tank to be below 10 ℃, and centrifuging the fermentation liquor to obtain thalli;

adding freeze-drying protective agent into the harvested bacterial sludge, carrying out vacuum freeze-drying for 32-50h, and carrying out air flow crushing on freeze-dried bacterial cake to obtain lactobacillus salivarius GER106 freeze-dried powder, wherein the viable bacteria amount in the freeze-dried powder is 2.0 multiplied by 10¹¹-8×10¹¹CFU/g；

The freeze-drying protective agent comprises 16% of skim milk, 10% of dextrin, 4% of polyvinylpyrrolidone, 6% of lactose, 0.5% of sodium glutamate, 0.5% of cysteine, 0.3-0.8% of sodium acetate and the balance of water.