CN104928219B - A kind of lactobacillus plantarum TH103 and application thereof - Google Patents

Abstract

The invention discloses Lactobacillus plantarum TH103 and its application. Lactobacillus plantarum TH103 has high acid resistance, bile salt resistance and NaCl resistance, and has strong inhibitory effect on the growth of Salmonella typhimurium in vitro. It can inhibit the growth of Salmonella typhimurium, and has a protective effect on the intestinal epithelial cell line HT‑29 cells infected by Salmonella typhimurium. The Lactobacillus plantarum TH103 can be used for fermenting vegetables, preparing food such as silage, fermented sausage and the like.

Description

A kind of Lactobacillus plantarum TH103 and its use

technical field

The invention belongs to the technical field of microorganisms, relates to a plant Lactobacillus (Lactobacillus plantarum) TH103 capable of inhibiting the growth of Salmonella typhimurium, and also relates to the application of the plant Lactobacillus TH103.

Background technique

Salmonella typhimurium (Salmonella typhimurium) is an important intestinal pathogen that harms humans and animals, such as intestinal infection and piglet diarrhea caused by human food-borne contamination. After entering the small intestine through the digestive tract, Salmonella typhimurium first adheres and settles on the epithelial surface of the small intestine, and then invades the cells, mainly causing enterocolitis, which can lead to a series of diseases such as diarrhea and bacteremia in the host. It is the most common salmonella infection in children. By. Salmonella typhimurium has a wide range of natural epidemic sources. Many poultry, livestock, rats, birds and cold-blooded animals are its natural hosts. Flies and fleas can carry the bacteria, and it is often infected orally by contaminated water, milk and food. According to statistics, the number of Salmonella typhimurium infections each year accounts for about 1/5 of the Salmonella infections, and is one of the main pathogenic bacteria threatening human health.

Probiotics refer to the general term of active beneficial microorganisms that can survive in a certain amount and colonize the intestinal tract of the host, and play a beneficial role in the health of the host by regulating the balance of intestinal flora and antagonizing the pathogenic bacteria in the intestinal tract. Many probiotics belong to lactic acid bacteria, which is a general term for a group of Gram-positive bacilli or cocci that can ferment carbohydrates and produce large amounts of lactic acid. Among the more than 400 kinds of microorganisms in the gastrointestinal tract, lactic acid bacteria account for about 10%, and are the main members of the normal intestinal flora. As a normal flora in the intestinal tract of humans and animals, lactic acid bacteria have beneficial functions such as preventing the invasion and colonization of the intestinal tract by pathogenic bacteria, inhibiting pathogenic bacteria and anti-infection, and maintaining the microecological balance of the intestinal tract. Lactic acid bacteria and their metabolites may have a certain preventive or intervention effect on the contamination of Salmonella typhimurium, and have the potential to be used as an adjuvant therapeutic agent for antibiotics. Screening lactic acid bacteria with excellent antagonistic properties is necessary to inhibit the contamination of Salmonella typhimurium.

Contents of the invention

The purpose of this section is to outline some aspects of embodiments of the invention and briefly describe some preferred embodiments. Some simplifications or omissions may be made in this section, as well as in the abstract and titles of this application, to avoid obscuring the purpose of this section, the abstract and titles, and such simplifications or omissions should not be used to limit the scope of the invention.

In view of the above and/or problems existing in the existing Lactobacillus plantarum TH103 and its uses, the present invention is proposed.

Therefore, an object of the present invention is to provide a kind of Lactobacillus plantarum TH103, said bacterial strain has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on April 23, 2015, and its preservation registration number is CGMCC10739, Its classification is named: Lactobacillus plantarum.

Another object of the present invention is to provide an application of Lactobacillus plantarum TH103 in the field of vegetable fermentation.

As a preferred solution for the application of Lactobacillus plantarum TH103 in the field of vegetable fermentation according to the present invention, it includes, after the vegetables are washed and drained, the vegetables are peeled and cut, and 1.0% by weight of the vegetables is added. ~ 1.2% sucrose and 6% ~ 8% salt, stir evenly, pre-cure at room temperature for 36-48 hours, drain the water, and obtain a kind of pre-pickled vegetables; weigh edible spices, and use the spices according to the weight of the spices used 8 to 10 times of water was extracted at a temperature of 75 to 80°C for 4 to 6 hours, and after cooling, 10% white wine by weight of edible spices was added, and mixed to obtain a spice juice; ~3% inoculated in liquid medium, cultured at 37°C for 20-28h, centrifuged at 4500rpm for 10-15min, discarded the supernatant, and resuspended the collected sludge with sterile water to make Lactobacillus plantarum TH103 viable The bacterial concentration reaches above 10 ⁸ cfu/mL, thereby obtaining a bacterial suspension; adding 10-12% of the spice juice and 2-5% by weight of the pre-pickled vegetables to the pre-pickled vegetables The bacteria suspension is mixed evenly and filled into jars, sealed and then fermented at room temperature until the pH value of the fermented liquid reaches 3.0-4.0 to obtain fermented vegetables.

As a preferred scheme for the application of Lactobacillus plantarum TH103 in the field of vegetable fermentation according to the present invention, wherein: the vegetables are cabbage, lettuce, cowpea, carrot, white radish, lettuce, cabbage, sweet pepper, onion or celery one or more of them.

As a preferred scheme for the application of Lactobacillus plantarum TH103 in the field of vegetable fermentation of the present invention, wherein: the spices are one or more of pepper, pepper, ginger, garlic, star anise, fennel, orange peel, and bay leaves. kind.

Another object of the present invention is to provide an application of Lactobacillus plantarum TH103 in the field of working starter, wherein the concentration of Lactobacillus plantarum TH103 in the working starter is above 10 ⁹ cfu/g.

As a preferred scheme for the application of Lactobacillus plantarum TH103 of the present invention in the field of working starters, it includes: inoculating the original strain of Lactobacillus plantarum TH103 in the culture culture medium at 37°C for 20 to 28 hours to activate, and to activate in the same way for 2 to 3 times in a row, and the number of viable Lactobacillus plantarum TH103 in the activated culture was not less than 10 ⁸ cfu/mL; Centrifuge at 2-6°C and 4500rpm for 10-15 minutes, discard the supernatant, and wash the collected sludge with pH 7.2 buffer solution for 3-4 times, then add freeze-dried bacteria to the sludge. For the protective agent suspension, the concentration of the bacterial solution is adjusted to be not less than 10 ⁹ cfu/mL, and after mixing, vacuum freeze-drying is carried out to obtain the Lactobacillus plantarum TH103 starter, wherein the composition of the freeze-dried protective agent suspension is By weight 2% sucrose, 15% skim milk, 5% lactose, 1% glycerin and 75% water.

Another object of the present invention is to provide an application of Lactobacillus plantarum TH103 in the field of fermented silage, which includes kneading and cutting the silage raw material for use; g Lactobacillus plantarum TH103 starter as claimed in claim 7 is mixed evenly with 200～250mL aseptic water, sprayed in the silage raw material, after stirring and mixing evenly, pack into the silage bucket and compact and seal, store under normal temperature condition After 1-2 months, the silage containing Lactobacillus plantarum TH103 was obtained.

Still another object of the present invention is to provide an application of Lactobacillus plantarum TH103 in the field of fermented sausage, which includes mincing meat selected from pork, beef or mutton, and pre-freezing in a refrigerator at 5°C to 8°C After 24-36 hours, fully mix with table salt, white sugar, and sodium nitrite in a weight ratio of 100:4:1.5:0.015; marinate the mixture at a temperature of 0-4°C for 24-48 hours, and add 5-5 10% spices, stir and mix, then add the Lactobacillus plantarum TH103 starter according to claim 7, its addition is 0.5-0.8% by weight of the meat, mix well and pour the meat containing the working starter into the casing The obtained sausage is fermented for 25-30 days at a temperature of 14°C-15°C and a relative humidity of 80%; after the fermentation is completed, it is baked at a temperature of 65-70°C and cooled to obtain the fermented sausage.

The Lactobacillus plantarum TH103 of the present invention has obvious inhibitory effect on Salmonella typhimurium in vitro, has strong tolerance to artificial gastric juice and artificial intestinal juice, and the ability to tolerate NaCl is as high as 10%, and can significantly inhibit typhoid fever in actual food systems Salmonella growth. It has a protective effect on intestinal epithelial cell line HT-29 cells infected by Salmonella typhimurium. The Lactobacillus plantarum TH103 can be used for fermenting vegetables, preparing and producing silage, fermented sausage and other products.

Description of drawings

Fig. 1 is the growth curve of Salmonella typhimurium in milk under the condition of having TH103 and the condition of not adding TH103.

Detailed ways

In order to make the above objects, features and advantages of the present invention more obvious and comprehensible, specific implementations of the present invention will be described in detail below in conjunction with the accompanying drawings.

In the following description, a lot of specific details are set forth in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described here, and those skilled in the art can do it without departing from the meaning of the present invention. By analogy, the present invention is therefore not limited to the specific examples disclosed below.

Second, "one embodiment" or "an embodiment" referred to herein refers to a specific feature, structure or characteristic that may be included in at least one implementation of the present invention. "In one embodiment" appearing in different places in this specification does not all refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

The present invention will be described in more detail below.

The Lactobacillus plantarum TH103 was obtained by the following screening method, and its properties were studied.

1. Screening

The specific implementation steps of the screening method are as follows:

1. Sample collection

The original samples were collected from natural fermented pickles made by local farmers in Heyu Village, Shexian County, Handan. Samples were collected in a sterile Erlenmeyer flask, and the collected samples were stored at a temperature of 0°C. Sample analysis and separation of lactic acid bacteria were carried out within 48 hours of sampling.

2. Isolation of lactic acid bacteria from samples

10 g of sample was placed in 90 mL of normal saline with glass beads, shaken for 30 min, and then serially diluted with normal saline at a volume ratio of 1:10. Using the coating plate method well known to those skilled in the art, 0.1 mL of the bacterial solution of each dilution gradient was coated on the following three medium plates: MRS medium (a medium for cultivating Lactobacillus), cultured at 37°C for 48h. Isolate a single colony from a plate with 30 to 300 colonies, randomly pick 5 to 10 of them with an inoculation loop and place them in 4% NaCl MRS liquid medium, and culture them at the corresponding temperature for 24 hours, then Streak on MRS solid plates with 4% NaCl. The isolates were streaked three times consecutively to obtain pure single colonies, and then subjected to Gram stain and contact enzyme tests well known to those skilled in the art. Inoculate the strains that are Gram-positive and negative in the contact enzyme test into the MRS liquid medium, culture at 37°C for 24 hours, and then inoculate them in the MRS liquid medium according to the inoculation amount of 2-4% based on the weight of the medium , 37 ° C static culture 18 ~ 24h for activation, continuous transfer 3 times, to obtain lactic acid bacteria with good activity. The purified bacterial strain is frozen in 30% sterilized glycerin solution at a temperature of -80°C.

A total of 46 isolates were isolated on the MRS agar medium supplemented with 4% NaCl and bromocresol purple. Three Gram-negatives, one catalase-positive and two yeasts were ruled out by colony morphology, Gram stain, and contact enzyme test. In this way, 32 strains of Lactobacillus were obtained after separation and purification.

3. Screening of lactic acid bacteria that can antagonize Salmonella typhimurium in vitro

The above isolated lactic acid bacteria strains are inoculated in the MRS liquid medium according to the inoculation amount of 2-4% based on the weight of the medium, and fermented and cultured for 18-24 hours under anaerobic and 37° C. culture conditions to obtain a lactic acid bacteria fermentation liquid.

Then, pour the sterilized eosinmethylene blue medium (for cultivating Salmonella typhimurium) into the plate (15 mL per plate, the plate is a 9 cm standard plate), and Salmonella typhimurium (S.typhimurium) ATCC14028 is used as the indicator strain (bacterial strain Purchased from the American Type Culture Collection (ATCC), put 4 Oxford cups (diameter 10mm) into each plate, add 150 μL of the above-mentioned lactic acid bacteria fermentation broth into the Oxford cups, spread at a temperature of 4°C for 6 hours, and then put them into an incubator at an internal temperature After incubation at 37°C for 24 h, the size of the inhibition zone was measured. MRS liquid medium was used as a negative control, and Lactobacillus plantarum ST-III was used as a positive control, and the experiment was repeated 3 times, with 3 parallels each time. Lactic acid bacteria TH103 with a larger inhibition zone than the positive control were screened out. The results of these tests are listed in Table 1.

Table 1 The inhibitory effect of TH103 on Salmonella typhimurium

It can be seen from the results listed in Table 1 that Lactobacillus plantarum (Lactobacillus plantarum) TH103 is a lactic acid bacterium that can significantly inhibit the growth of Salmonella typhimurium.

4. Identification of 16s rDNA sequence of TH103 strain

Lactobacillus plantarum TH103 was inoculated in MRS liquid medium according to the inoculation amount of 2-4% based on the weight of the medium, cultured at 37°C for 12 hours, and 1-1.5mL of fresh bacterial liquid was taken for the extraction of bacterial genomic DNA. Genomic DNA was used as a template, and bacterial identification universal primers 27F and 1492R well known in the art were used as primers, and PCR amplification was carried out in a 50 μL reaction system.

The sequencing of the target gene PCR amplification product was completed by Shanghai Bioengineering Technology Service Co., Ltd. The 16S rDNA sequencing results of Lactobacillus plantarum TH103 were compared with the NCBI nucleic acid database, and the final results showed that the homology between TH103 of the present invention and Lactobacillus plantarum ST-III was as high as 99%. Therefore, the TH103 strain of the present invention was identified It is Lactobacillus plantarum, named Lactobacillus plantarum TH103, which was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on April 23, 2015, and its preservation registration number is CGMCC10739.

In addition, it should be noted that after the first screening and identification of Lactobacillus plantarum TH103, pickle samples were collected again at the sample collection site after several periods of time (45 days, 3 months, 5 months, 7 months). Screening and identification obtained the same strain.

2. Microbiological properties of Lactobacillus plantarum TH103

1. Determination of acid resistance of Lactobacillus plantarum TH103

Preparation of artificial gastric juice: Take 16.4mL of 0.1mol/L hydrochloric acid, add water and shake well, adjust the pH value to 2.0 with concentrated hydrochloric acid or 10% NaOH, then adjust the volume to 1000mL, sterilize at 121°C for 30min, and then add commercial products at a ratio of 1g/100mL Pepsin preparation, mixed to obtain the artificial gastric juice.

Determination of the acid resistance of Lactobacillus plantarum TH103: inoculate the preserved Lactobacillus plantarum TH103 in the MRS medium according to the inoculation amount of 2-4% based on the weight of the MRS medium, cultivate it at a temperature of 37°C for 24 hours, and use the MRS medium in the same way Transplant culture for 2 to 3 times, then take 40 mL of the activated bacterial suspension and add it to a 50 mL centrifuge tube, centrifuge at 4500 rpm for 5 minutes to collect the bacteria, then wash with PBS buffer (pH 7.2, the same below) for 3 times, then add 40 mL of the above prepared Artificial gastric juice was treated in a water bath at 37°C for 3 hours, shaken once every 30 minutes, and the viable bacteria were counted by standard plate colony counting method after treatment for 0 hour, 1.5 hour and 3 hours respectively. The test results of acid resistance of Lactobacillus plantarum TH103 are listed in Table 2.

2. Determination of bile salt tolerance of Lactobacillus plantarum TH103

Preparation of artificial intestinal juice: Take 6.8g of KH ₂ PO ₄ and add 500mL of distilled water to dissolve it, adjust its pH to 6.8 with 0.4% NaOH solution by weight, then add water to make it 1000mL, and add 0.4g of porcine bile salt per 100mL of the solution Pig bile salt was added proportionally, fully dissolved and then sterilized at a temperature of 115° C. for 15 minutes to obtain the artificial intestinal juice.

Bacteria were collected by the same method as described above. The collected bacteria were washed 3 times with PBS buffer (pH 7.2), then added 40 mL of artificial intestinal fluid prepared above, treated in a water bath at 37 °C for 3 h, shaken every 30 min, and treated at 0 h, 1.5 h, 1.5 h, After 3 h, the viable bacteria were counted by the standard plate colony counting method. The test results of Lactobacillus plantarum TH103 tolerance to bile salts are listed in Table 3.

3. Determination of NaCl resistance of Lactobacillus plantarum TH103

Streak the Lactobacillus plantarum TH103 stored in glycerol tubes at -80°C on the MRS solid plate, culture upside down at 37°C until a single colony grows; pick a single colony and inoculate it into the MRS liquid medium, culture it statically at 37°C for 12 hours, and then transfer continuously After being connected 3 times, it is the pre-culture medium.

The above-prepared preculture solution was inoculated with 2% inoculation amount in MRS liquid medium with salt concentrations of 4%, 6%, 8%, 10% NaCl and NaCl-free respectively, cultured statically at 37°C, and tested every 2h. Once the absorbance value is used, the growth curve is drawn according to the absorbance value. Table 4 shows the results of determination of NaCl tolerance of Lactobacillus plantarum TH103.

4. Determination of adhesion of Lactobacillus plantarum TH103 to intestinal epithelial cells HT-29

(1) Recovery, culture and cryopreservation of cells

Quickly put the HT-29 cell cryopreservation tube into a 37°C water bath, centrifuge at 800 rpm for 5 minutes after melting, remove the supernatant and resuspend the cells with fresh culture medium to make them evenly disperse in the culture bottle, in 5% CO ₂ The culture was carried out at 37°C under the gas condition of 95% air, and the culture medium was changed every 48 hours during recovery. When the cells grew well (70% confluence), the HT-29 cells were digested with trypsin-EDTA solution at a temperature of 37°C and cultured in 1:3 flasks. When freezing, add 10% methylene disulfide (DMSO) to the complete culture medium and store in liquid nitrogen. In the adhesion experiment, the cells were digested and the cell concentration was adjusted to 2×10 ⁵ cells/mL, and seeded in a 6-well plate containing coverslips and cultured until the cells grew to a confluence of 80%.

(2) Adhesion test

Centrifuge at 4500rpm for 5min to collect the bacteria grown in the corresponding culture medium; wash the bacteria with saline for 3 times, resuspend the bacteria, and adjust the concentration of the bacteria to 10 ⁸ cfu/mL; add 2 mL of the above bacterial suspension to the Monolayer HT-29 cell coverslips were placed in a 6-well plate, incubated in a 5% CO ₂ incubator at 37°C for 2h; after incubation, washed 3 times with sterile PBS; after 0.4% paraformaldehyde fixation for 0.5h staining.

(3) Adhesion observation and counting

After staining, the bacterial adhesion was observed under an oil microscope and photographed. When observing under the microscope, randomly select about 100 cells in 20 fields of view at each salt concentration, and calculate the number of bacteria adhered to the cells. The average number of bacteria adhered to each cell is the adhesion index. Three replicates, the data analysis was performed after the experiment was repeated 3 times. The test results of Lactobacillus plantarum TH103 adhesion to intestinal epithelial cells HT-29 are listed in Table 4.

According to the same method as the test method of acid resistance, bile salt resistance, NaCl resistance and adhesion intestinal epithelial cell HT-29 performance of Lactobacillus plantarum TH103, a control test was carried out using Lactobacillus plantarum ST-III, and the test results are also listed in Table 2 , Table 3 and Table 4.

Table 2: Determination results of acid resistance characteristics of Lactobacillus plantarum TH103

Table 3: Determination results of bile salt tolerance of Lactobacillus plantarum TH103

Table 4: Determination results of NaCl resistance and adhesion characteristics of Lactobacillus plantarum TH103

The results of Table 2 to Table 4 clearly show that Lactobacillus plantarum TH103 has acid resistance and can grow under pH2.0; it has bile salt tolerance, and the bile salt concentration it can tolerate reaches 0.4%; it has high NaCl tolerance, It can tolerate the concentration of NaCl up to 10%; it has a good ability to adhere to intestinal epithelial cells.

5. Lactobacillus plantarum TH103 inhibits the growth of Salmonella typhimurium in eggs and milk

The cryopreserved Lactobacillus plantarum TH103 was inoculated in the MRS medium according to the inoculation amount of 2-4% based on the weight of the MRS medium, cultured at 37°C for 24 hours, and used the MRS culture medium in the same way to transfer culture for 2-3 times, and then Take 1 mL of the activated bacterial suspension and add it to a 50 mL centrifuge tube, centrifuge at 4500 rpm for 5 min to collect the bacterial cells and resuspend with PBS buffer. Salmonella typhimurium ATCC14028 was treated in the same manner. Then, smear the Salmonella typhimurium ATCC14028 bacterium liquid on the surface of the product egg, and after drying, apply the resuspended Lactobacillus plantarum TH103 bacterium liquid, and use the standard plate colony counting method to count after natural air drying for 5 hours. The control experiment was carried out in the same manner, using sterile MRS liquid medium instead of Lactobacillus plantarum TH103 to smear the egg surface. The results of these tests are listed in Table 5.

Table 5 Inhibitory effect of Lactobacillus lactobacillus TH103 on the surface of eggs on Salmonella typhimurium

The test results show that Lactobacillus plantarum TH103 can inhibit the growth of Salmonella typhimurium on the surface of eggs.

Treat Lactobacillus plantarum TH103 and Salmonella typhimurium ATCC14028 in the same manner as the above method, and then insert them into sterilized milk according to the inoculum amount of 0.5% by milk weight, and use the standard plate colony counting method to count every 2 hours , and the results of these measurements are presented in Figure 1. The test results show that Lactobacillus plantarum TH103 can also inhibit the growth of Salmonella typhimurium in milk.

6. It has a protective effect on the intestinal epithelial cell line HT-29 cells infected by Salmonella typhimurium.

The inhibition of invasiveness is determined by the following test method:

Intestinal epithelial cells HT-29 (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were cultured using RPMI1640 medium (product of Gibco). HT-29 cells were cultured in a 37°C incubator under the gas conditions of 5% CO ₂ and 95% air, and the culture medium was changed every 24 hours, and the culture was continued until the confluence reached 80%. Use 0.2% trypsin solution to digest HT-29 cells at room temperature, then adjust the cell concentration to ² ×105 cells/mL with RPMI1640 culture medium, and distribute them into 6-well cell culture plates, 2 mL per well. Change the cell culture medium every day until a monolayer of cells is formed.

Lactobacillus plantarum TH103 was cultured in MRS liquid medium at 37°C for 12h. The cells were collected by centrifugation at 4500rpm for 5 minutes, washed with PBS buffer three times and resuspended, and the concentration of the cells was adjusted to 10 ⁸ cfu/mL. Salmonella typhimurium ATCC14028 was treated in the same manner as above.

The test was divided into four groups, the first group was the inhibition group, the second group was the inhibition group, the third group was the adhesion control group, and the fourth group was the invasion control group. Aspirate the cell culture medium from the above 6-well cell culture plate, wash the HT-29 cells with PBS buffer three times, add 1 mL of Lactobacillus plantarum TH103 suspension and 1 mL of RPMI1640 culture solution to each well of the first group and the second group, mix Evenly, the multiplicity of infection is about 200:1. Add 2mL RPMI1640 culture solution to each well of the third group and the fourth group, incubate for 1 hour at 37°C, 5% CO ₂ and 95% air, and then wash with PBS buffer three times. All the wells are added with Salmonella typhimurium Suspension 1mL and RPMI1640 culture medium 1mL, mix well. Continue to incubate for 1 h at 37°C under the gas conditions of 5% CO ₂ and 95% air, and wash the obtained monolayer cells with PBS buffer three times, add 0.5mL 0.5% Triton X-100 solution to the first group and the third group, Incubate for 8 min, add 0.5 mL of PBS buffer, and then carry out serial dilution; add 1 mL of RPMI1640 culture solution containing gentamicin (100 μg/mL) after the second group and the fourth group are washed, at 37 ° C, 5% CO ₂ and 95% Continue to incubate for 1 h under the condition of air gas, wash with PBS buffer three times after the incubation, add 0.5 mL 0.5% Triton X-100 solution to incubate for 8 min, add 0.5 mL PBS buffer and then perform gradient dilution. Salmonella typhimurium in all wells was counted by standard plate colony counting method using Salmonella typhimurium culture medium, and three parallel experiments were carried out for each test group, which was repeated three times. Inhibition of adhesion rate=third group of counting results/first group of counting results; inhibition of invasion rate=(fourth group of counting results-third group of counting results)/(second group of counting results-first group of counting results), these The test results are listed in Table 6.

Table 6 Inhibitory effect of Lactobacillus plantarum TH103 on Salmonella typhimurium infecting HT-29 cells

It can be seen from Table 6 that Lactobacillus plantarum TH103 has obvious inhibitory effect on the adhesion and invasion of colon cancer cells by Salmonella typhimurium, therefore, it has a protective effect on the intestinal epithelial cell line HT-29 cells infected by Salmonella typhimurium.

Clearly show by above-mentioned test, described plantarum lactobacillus TH103 has following properties:

(1) It has acid resistance and can still survive and grow at pH 2.0;

(2) have the ability to tolerate bile salts, and the concentration of bile salts that can be tolerated reaches 0.4% (w/v, the same below);

(3) It has the ability to tolerate salt, and the concentration of NaCl is as high as 10%;

(4) It has high adhesion ability to intestinal epithelial cells HT-29;

(5) It has a significant inhibitory effect on the pathogenic bacteria Salmonella typhimurium (Salmonella typhimurium ATCC14028);

(6) Can significantly inhibit the adhesion of Salmonella typhimurium to intestinal epithelial cells HT-29;

(7) It can significantly reduce the invasion of Salmonella typhimurium on intestinal epithelial cells HT-29.

Example 1: Fermented Vegetables

In the present invention, the fermented vegetables are prepared by the following steps:

First, after the vegetables are washed and drained, the vegetables are peeled and cut into pieces, 1.0% to 1.2% of sucrose and 6 to 8% of salt are added according to the weight of the vegetables, and they are evenly stirred and pre-cured at room temperature for 36 to 48 hours , drain and get a pre-marinated vegetable.

Then, take the edible spices, use 8-10 times the water of the used spices by weight, extract at a temperature of 75-80° C. for 4-6 hours, add 10% white wine by the used spices by weight after cooling, and mix well to obtain a Spice sauce.

Next, Lactobacillus plantarum TH103 was inoculated in the MRS liquid medium at 1-3% by volume of the MRS liquid medium, cultured at 37°C for 20-28 hours, centrifuged at 4500rpm for 10-15min, the supernatant was discarded, and the obtained The bacteria sludge was resuspended with sterile water, so that the viable bacteria concentration of Lactobacillus plantarum TH103 reached above 10 ⁸ cfu/mL, thus obtaining a bacterial suspension;

Finally, add 10-12% spice juice and 2-5% bacterial suspension to the obtained pre-pickled vegetables by weight, mix evenly and pack into jars, seal and ferment at room temperature until fermented The pH value of the solution reaches 3.0-4.0, and the fermented vegetables are obtained.

Vegetables described in the present invention are one or more vegetables selected from cabbage, lettuce, cowpea, carrot, white radish, lettuce, cabbage, bell pepper, onion or celery; Spices from chili, pepper, ginger, garlic, star anise, fennel, orange peel, bay leaf.

Embodiment 2: prepare working leavening agent of the present invention

Prepare the described working starter as follows:

Inoculate the original strain of Lactobacillus plantarum TH103 in the MRS liquid medium according to the inoculation amount of 1-2% based on the weight of the MRS liquid medium, culture it at 37°C for 20-28 hours for activation, and continue to activate in the same way 2-3 times until the viable count of Lactobacillus plantarum TH103 in the activated culture is 10 ⁸ cfu/mL or above, then centrifuge at 2-6°C and 4500 rpm for 10-15 minutes, discard the supernatant, and collect After the obtained bacteria slime was washed with PBS buffer solution of pH 7.2 for 3-4 times, the freeze-dried protective agent suspension was added to adjust the concentration of the bacteria solution to 10 ⁹ cfu/mL, and after mixing, vacuum freeze-drying was carried out to obtain the plant Lactobacillus TH103 starter and stored at 4°C. Wherein, the composition of the freeze-drying protective agent suspension is 2% by weight of sucrose, 15% of skim milk, 5% of lactose, 1% of glycerin and 75% of water.

Example 3: Preparation of silage using the working starter of the invention

The silage preparation steps are as follows:

First, knead and cut the silage raw material to 2-5 cm and set it aside.

Then, take by weighing 50～60kg silage raw materials, mix 0.5～0.6g of plantaractobacillus TH103 working starter prepared according to claim 5 with 200～250mL sterile water, spray in the silage raw materials, turn over After mixing evenly, put it into a silage bucket, compact and seal it, and store it for 1-2 months under normal temperature to obtain silage containing Lactobacillus plantarum TH103.

The silage raw material in the present invention is one or more silages selected from rice straw, corn stalk, sorghum stalk, sweet potato seedling, natural pasture grass, cultivated pasture grass, field weeds, aquatic plants or twigs and leaves.

It has been determined that the plant straw feed contains more than 10 ⁶ cfu/g of Lactobacillus plantarum TH103, which can significantly inhibit the growth and survival of Salmonella typhimurium in the feed compared with the same process as in Example 3 without adding TH103.

Example 4: Preparation of fermented sausages using the working starter of the invention

Fermented sausages are prepared as follows:

Mince meat selected from pork, beef or mutton, pre-freeze in a refrigerator at 5°C to 8°C for 24 to 36 hours, and then mix it with salt, white sugar, and sodium nitrite in a weight ratio of 100:4:1.5:0.015. mix. The mixture is marinated at a temperature of 0-4°C for 24-48 hours, adding 5-10% spices according to the weight of the meat, stirring and mixing, and then adding the working starter prepared by the method according to claim 8, the amount of which is based on the meat 0.5-0.8% by weight, after mixing evenly, pour the meat containing the working starter into the casing, and the obtained sausage is fermented for 25-30 days at a temperature of 14°C-15°C and a relative humidity of 80%. Baking is carried out at a temperature of 65-70° C., and the fermented sausage is obtained after cooling. The spices are one or more spices selected from pepper, pepper, ginger, garlic, star anise, fennel, orange peel, bay leaves.

The prepared fermented sausage has a mild sour taste, rich aroma, good color, and good product sliceability. It is produced by the same process as in Example 4, but compared with the fermented sausage without Lactobacillus plantarum TH103, Salmonella typhimurium and The probability of other harmful bacteria is significantly reduced.

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation, although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.

Claims (8)

1. A Lactobacillus plantarum TH103, characterized in that: the bacterial strain has been preserved on April 23, 2015 in the General Microorganism Culture Collection Center of China Microbiology Culture Collection Management Committee, and its preservation registration number is CGMCC10739. 2. An application of Lactobacillus plantarum TH103 according to claim 1 in the field of vegetable fermentation, characterized in that: comprising, After the vegetables are washed and drained, the vegetables are peeled and cut into pieces, 1.0% to 1.2% of sucrose and 6% to 8% of salt are added according to the weight of the vegetables, and the mixture is evenly stirred and pre-cured at room temperature for 36 to 48 hours. Drain the water to obtain a pre-pickled vegetable; weigh the edible spices, use 8-10 times the weight of the spices used, and extract them at a temperature of 75-80°C for 4-6 hours, add edible spices after cooling 10% white wine, mixed to get a kind of spice juice; Plantarum Lactobacillus TH103 is inoculated in the liquid medium at 1-3% by volume of the liquid medium, Cultivate at 37°C for 20-28 hours, centrifuge at 4500rpm for 10-15 minutes, discard the supernatant, and resuspend the collected sludge with sterile water, so that the viable concentration of Lactobacillus plantarum TH103 reaches 10 ⁸ cfu/mL or more. Thereby obtaining a kind of bacterial suspension; Adding 10-12% of the spice juice and 2-5% of the bacterial suspension based on the weight of the pre-pickled vegetables to the pre-pickled vegetables, mixing evenly and packing into cans, sealing and fermenting at room temperature Until the pH value of the fermented liquid reaches 3.0-4.0, fermented vegetables are obtained. 3. The application of Lactobacillus plantarum TH103 in the field of vegetable fermentation according to claim 2, characterized in that: the vegetables are Chinese cabbage, lettuce, cowpea, carrot, white radish, cabbage, sweet pepper, onion or celery one or more of. 4. The application of Lactobacillus plantarum TH103 in the field of vegetable fermentation according to claim 2, characterized in that: the spice is one of pepper, pepper, ginger, garlic, star anise, fennel, orange peel, bay leaves or Several kinds. 5. An application of Lactobacillus plantarum TH103 according to claim 1 in the field of working starter, characterized in that: the concentration of Lactobacillus plantarum TH103 in the working starter is above 10 ⁹ cfu/g. 6. the application of Lactobacillus plantarum TH103 in the field of working starter according to claim 5, is characterized in that: comprising, Inoculate the original strain of Lactobacillus plantarum TH103 in the medium according to the inoculation amount of 1-2% based on the weight of the medium, and activate it by culturing it at 37°C for 20-28 hours, and activate it continuously in the same way for 2-3 times , the number of viable Lactobacillus plantarum TH103 in the activated culture is not less than 10 ⁸ cfu/mL; Centrifuge at a temperature of 2-6°C and a rotational speed of 4500rpm for 10-15 minutes, discard the supernatant, collect the obtained sludge and wash it with pH 7.2 buffer for 3-4 times, and then add Lyoprotectant suspension, adjusting the concentration of the bacterial solution to not less than 10 ⁹ cfu/mL, mixing and vacuum freeze-drying to obtain the Lactobacillus plantarum TH103 starter, wherein the composition of the freeze-drying protective agent suspension is It is 2% by weight of sucrose, 15% of skim milk, 5% of lactose, 1% of glycerin and 75% of water. 7. A use of Lactobacillus plantarum TH103 according to claim 1 in the field of fermented silage, characterized in that: comprising, kneading and cutting the silage raw material into sections for use; weighing 50-60 kg of silage raw material, Mix 0.5～0.6g of the Lactobacillus plantarum TH103 starter as claimed in claim 6 with 200～250mL sterile water, spray it in the silage raw material, mix it evenly, put it into the silage bucket, compact and seal it, The silage containing Lactobacillus plantarum TH103 can be obtained after being stored for 1-2 months under normal temperature conditions. 8. An application of Lactobacillus plantarum TH103 in the field of fermented sausage according to claim 1, characterized in that: comprising, Chopped meat selected from pork, beef or mutton, pre-frozen in a refrigerator at 5°C to 8°C for 24 to 36 hours, then mixed with salt, white sugar, and sodium nitrite in a weight ratio of 100:4:1.5:0.015 mix; Put the mixture at a temperature of 0-4°C and marinate for 24-48 hours, add 5-10% spices according to the weight of the meat, stir and mix, and then add the Lactobacillus plantarum TH103 starter according to claim 7, the amount of which is 0.5-0.8% by weight of the meat, after mixing evenly, pour the meat containing the working starter into the casing, and ferment the sausage obtained at a temperature of 14°C-15°C and a relative humidity of 80% for 25-30 days; After the fermentation is finished, it is baked at a temperature of 65-70° C., and the fermented sausage is obtained after cooling.