CN113604383B

CN113604383B - Lactobacillus casei and application thereof

Info

Publication number: CN113604383B
Application number: CN202110829075.0A
Authority: CN
Inventors: 汪家琦; 周辉; 潘丽娜; 刘成国; 戴智勇; 雷文平; 康文丽; 吴忠坤; 李辉宇
Original assignee: Ausnutria Dairy China Co ltd; Hunan Agricultural University
Current assignee: Ausnutria Dairy China Co ltd; Hunan Agricultural University
Priority date: 2021-07-22
Filing date: 2021-07-22
Publication date: 2023-07-18
Anticipated expiration: 2041-07-22
Also published as: CN113604383A

Abstract

The invention discloses lactobacillus casei and application thereof, the lactobacillus casei taxonomy is named as lactobacillus casei (Lactobacillus casei) AU9077, and the preservation number of the strain is CGMCC No.21663. The strain can regulate immunity, relieve allergy symptoms, has a certain protection effect on the damage of intestinal mucosa structure caused by beta-lactoglobulin induced anaphylactic reaction, and can increase intestinal beneficial flora; in addition, the black tea fermented milk beverage and/or the asparagus fermented milk prepared by using the strain AU9077 has obvious desensitization effect and/or antioxidant activity, which shows that the black tea fermented milk beverage and/or the asparagus fermented milk can be used as probiotics with desensitization effect and/or antioxidant activity to develop various probiotic products.

Description

Lactobacillus casei and application thereof

Technical Field

The invention belongs to the technical field of probiotics, and particularly relates to lactobacillus casei and application thereof.

Background

The concept of Probiotics (Probiotics) was originally derived from greek, meaning "beneficial to life", and is defined by the World Health Organization (WHO) as viable microorganisms, which when ingested in sufficient quantities can bring about a variety of health benefits to the host, including enhancing immunity, regulating intestinal flora, promoting digestive absorption, alleviating irritable bowel syndrome, and lowering cholesterol, etc. The probiotics mainly comprise the following main categories: (1) bifidobacterium (bifidobacterium longum, bifidobacterium infantis, etc.); (2) lactobacillus (lactobacillus casei, lactobacillus acidophilus, lactobacillus rhamnosus, etc.); (3) bacillus (bacillus subtilis, etc.); (4) fungi (yeasts, etc.). Wherein lactobacillus casei (Lactobacillus casei) belongs to the genus lactobacillus, gram-positive bacteria, facultative anaerobism, is one of the important lactic acid bacteria; widely distributed in intestinal tracts and genital tracts of human bodies and animals, fermented fruits and vegetables and dairy products. It has strong acid and bile salt resistance, and some strains have the beneficial functions of regulating immunity, regulating intestinal flora, reducing cholesterol and the like. Lactobacillus casei has a plurality of probiotic properties, can also produce antibacterial active substances, and can inhibit and kill a plurality of putrefying bacteria and pathogenic bacteria in food. The physiological functions of the probiotics are closely related to the life activities of organisms, and the probiotics are widely applied to important fields such as industry, agriculture and animal husbandry, food, medicine and the like.

Currently, allergic diseases have become a ubiquitous health problem with the continual change in the global environment, with more than 30% of people suffering from the effects of allergic diseases. Currently, most of these diseases are treated by drugs, however, the drug treatment has a certain side effect, so that a safer and more effective treatment means are required. Along with the rapid development of microbiology, various researches find that probiotics have effective prevention and treatment effects on human allergic diseases, but the related action mechanism is not clear, and the key technology of screening is still to be researched and broken through.

In addition, most of the probiotics fermented milk is prepared by multi-strain fermentation of different milk sources at present, and the natural extract with health care function (such as dark tea, asparagus and the like) is combined with the probiotics with allergy relieving and/or preventing to prepare the fermented milk, so that the fermented milk with multiple functions is obtained.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides lactobacillus casei and application thereof.

In order to achieve the above purpose, the technical scheme provided by the invention is as follows:

the lactobacillus casei (Lactobacillus casei) AU9077 was deposited in the China general microbiological culture Collection center, address: the preservation number of the strain is CGMCC No.21663, which is the national institute of microbiology, national institute of sciences, no. 3, beijing, chaoyang district, north Chen, west Lu, and the Beijing.

The lactobacillus casei AU9077 can be used for preparing medicines for reducing and/or preventing allergic diseases.

In the present invention, the method for preparing the black tea fermented milk beverage with antioxidant activity comprises the following steps:

(1) Decocting black tea in water, filtering with two layers of gauze, and cooling to obtain black tea soup;

(2) Dissolving skimmed milk powder, high fructose syrup and glucose syrup in black tea soup for primary blending, and fully and uniformly stirring to obtain primary mixed emulsion; the defatted milk powder accounts for 10-12% of the black tea soup in terms of mass volume ratio, the high fructose syrup accounts for 2.5-3% of the black tea soup in terms of mass volume ratio, the glucose syrup accounts for 2.5-3% of the black tea soup in terms of mass volume ratio, and the unit of the mass volume ratio is g/mL;

(3) Preheating the primary mixed emulsion at 65-75 ℃, homogenizing, sterilizing and cooling to obtain sterile mixed emulsion;

(4) Aseptically combining cheeseInoculating lactobacillus into the sterile mixed emulsion according to the inoculum size of 2-4%, fermenting for 44-48 h at 32-35 ℃, and then after-ripening fermenting for 12-24 h at 4 ℃ to obtain the black tea fermented milk base material; the inoculation is to inoculate lactobacillus casei viable bacteria preparation or bacterial suspension (the viable bacteria amount in the bacterial suspension is 1-5 multiplied by 10) ⁸ CFU/mL)；

(5) Adding sterilized secondary blending liquid with the same volume as the black tea fermented milk base material into the black tea fermented milk base material, mixing and stirring, homogenizing, aseptically filling, and cooling to 4 ℃ to obtain black tea fermented milk beverage; the secondary blending liquid is prepared according to the following method: taking water as a solvent, adding white granulated sugar, sodium carboxymethyl cellulose, sodium alginate propylene glycol ester and sodium citrate, uniformly stirring, sterilizing and cooling to obtain the finished product; wherein the mass percentage concentration of the white granulated sugar in the black tea fermented milk beverage is 8-10%, the mass percentage concentration of the sodium carboxymethyl cellulose in the black tea fermented milk beverage is 0.07-0.09%, the mass percentage concentration of the sodium alginate propylene glycol ester in the black tea fermented milk beverage is 0.08-0.1%, and the mass percentage concentration of the sodium citrate in the black tea fermented milk beverage is 0.1-0.15%.

Preferably, the black tea and water mass-volume ratio in the step (1) is 1:100 to 1:150, and the unit of the mass-volume ratio is g/mL.

Preferably, the homogenization conditions in the step (3) and the step (5) are 20-25 mpa and 2-3 times; and (3) sterilizing the secondary blending liquid in the step (5) at 80 ℃ for 15min.

In the invention, the method for preparing the asparagus fermented milk with the antioxidant activity comprises the following steps:

(1) Preparation of asparagus extract: dissolving the freeze-dried asparagus powder in water at 60-65deg.C, preferably 60deg.C

Extracting in water bath for 1.5-2.5 h, preferably for 2h, filtering, collecting filtrate, pre-freezing at-80 ℃ for 5-7 h, preferably for 6h, and quickly freeze-drying in a vacuum freeze dryer for more than 24h to obtain an asparagus extract for later use;

(2) Preparation of asparagus fermented milk: mixing and stirring the skim milk powder, the asparagus extract, the fructose syrup, the glucose syrup and water uniformly at 60-65 ℃ to obtain reconstituted milk; in the reconstituted milk, the mass-volume ratio of the skim milk powder to the reconstituted milk is 10-12%, the mass-volume ratio of the asparagus extract to the reconstituted milk is 0.1-0.5%, the mass-volume ratio of the fructose syrup to the reconstituted milk is 2.5-3%, the mass-volume ratio of the glucose syrup to the defatted reconstituted milk is 2-4%, and the unit of the mass-volume ratio is g/mL; preheating, homogenizing and sterilizing (121 ℃ for 15-20 min) the reconstituted milk at 65-75 ℃, cooling to 37-40 ℃, inoculating lactobacillus casei to the reconstituted milk according to the inoculum size of 2-4%, fermenting for 44-48 h at 32-35 ℃, and fermenting for 12-24 h at 4 ℃ to obtain the asparagus fermented milk; the inoculation is to inoculate lactobacillus casei viable bacteria preparation or bacterial suspension (the viable bacteria amount in the bacterial suspension is 1-5)

×10 ⁸ CFU/mL)。

Preferably, the mass-to-volume ratio of the asparagus powder to the water in the step (1) is 1:10 to 1:40, and the unit of the mass-to-volume ratio is g/mL.

Preferably, the homogenization conditions in step (2) are 20 to 25mpa,2 to 3 times.

Compared with the prior art, the invention has the beneficial effects that:

the lactobacillus casei AU9077 is milky white in colony on MRS solid medium, moist and neat in edge. And gram staining is positive, the rod shape is free of spores, and the 16S rDNA sequence is shown as SEQ ID NO. 1. The lactobacillus casei AU9077 has relatively excellent in vitro probiotics characteristics such as simulated gastrointestinal fluid tolerance, antibacterial activity, cell surface activity, adhesion to Caco-2 cells and the like; and is safe, including not producing common biogenic amines and hemolysin, and is sensitive to most common antibiotics.

In a mouse model sensitized by beta-lactoglobulin, the strain AU9077 can remarkably reduce secretion of histamine, specific IgE, IL-4 and MCP-1 in serum of allergic mice; can also up-regulate the expression of allergy-associated cytokine (IFN-gamma, IL-10, TNF-alpha and TGF-beta) genes in colon tissue of mice, and down-regulate the expression of IL-4 genes; meanwhile, the strain AU9077 can repair the damage of intestinal mucosa structure of the allergic mice; in addition, strain AU9077 can be effective in increasing the intestinal beneficial flora abundance of allergic mice.

In a word, lactobacillus casei (Lactobacillus casei) AU9077 can regulate immunity, relieve allergic symptoms, has a certain protection effect on the damage of intestinal mucosa structure caused by beta-lactoglobulin induced allergic reaction, can effectively increase intestinal beneficial flora of allergic mice, and has effectiveness in preventing and/or relieving allergy.

Drawings

FIG. 1A is a colony morphology diagram of Lactobacillus casei AU 9077;

FIG. 2 is a flow chart of animal experiment design;

FIG. 3 effect of strain AU9077 on mouse immune organ index, igE, histamine and cytokine content (IL-4, MCP-1);

FIG. 4 effect of strain AU9077 on the relative expression of cytokine genes in colon tissue of mice;

FIG. 5 colon histopathological examination of mice (HE X200);

FIG. 6 shows a graph of the intestinal microorganisms Venn of mice;

FIG. 7 is a graph of the structural composition profile of the intestinal microorganisms and the cluster heat of abundance of generic species in mice.

Detailed Description

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1

(1) Isolation and purification of strains

1) And (3) collecting a sample: sampling and numbering in a 50mL sterile centrifuge tube respectively at pastures (different milk stations), a city (dairy company and pasture) and a Ningxiang (dairy company and pasture) in south mountain of Hunan province, and rapidly transferring to a microorganism laboratory by using a sampling box containing an ice bag; together 71 samples were collected from these areas.

2) Separation of lactic acid bacteria: under aseptic condition, 25g of the sample was weighed and mixed with a 500mL conical flask containing 225mL of sterile physiological saline (0.85% NaCl) and diluted to 10 times with a 10-fold gradient ^-2 ～10 ^-6 And in MRS solids (containing 0.5% CaCO) ₃ (w/v)) plates were coated and incubated at 37℃for 48h. Single colonies showing calcium-dissolving rings are picked for gram staining, and positive strains are streaked and purified for three times and are numbered for storage. The obtained strains were all frozen and stored at-80℃in MRS liquid medium containing 40% glycerol.

(2) Evaluation of in vitro probiotic properties and preliminary safety of strains

1) Preparation of bacterial suspension: culturing the strain in MRS liquid culture medium at 37deg.C overnight, activating for 2 times, and centrifuging at 4deg.C for 3min at 8 000r/min; washing with sterile physiological saline for 2 times, and re-suspending to obtain bacterial suspension OD _600nm Approximately 0.8. The control strain lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG, LGG) was cultured and treated in the same manner.

2) Evaluation of acid and bile salts resistance

Inoculating 3% bacterial suspension into MRS liquid culture medium with pH of 3 and cattle bile salt (3.0 g/L), culturing at 37deg.C for 12 hr, and observing whether the culture solution is turbid or not, wherein the turbidity indicates that the strain has tolerance. Lactobacillus casei AU9077 has better tolerance by evaluation of acid resistance and bile salts.

3) Evaluation of simulated gastrointestinal fluid tolerance

Activating the strain AU9077 and preparing into bacterial suspension, then adding 0.5mL of bacterial suspension into 4.5mL of sterile simulated gastric fluid and intestinal fluid respectively, mixing for 15s by vortex, incubating for 3h in the environment of 37 ℃, and measuring the viable count of 0h and 3h respectively by using a plate counting method, wherein the survival rate of the strain in the gastric fluid is calculated as follows:

4) Antibacterial activity

Antibacterial activity assay: the antibacterial activity is measured by an oxford cup method, and the antibacterial capacity of the strain is represented by the diameter of a antibacterial ring, and escherichia coli and listeria are used as indicator strains.

5) Strain surface Activity

Self-aggregation: directly sucking 1mL of bacterial suspension of the screened strain into a 1.5mL centrifuge tube, vortexing for 10s, standing at 37 ℃ for 5h, slowly sucking 200 mu L of upper bacterial liquid, and measuring OD _600nm The values were repeated 3 times and the strain self-aggregation calculation formula was as follows:

wherein: a is that ₁ : OD of the upper layer bacterial liquid after standing for 5h _600nm A value; a is that ₀ : OD of initial bacterial suspension _600nm Values.

Co-aggregation: respectively taking listeria and escherichia coli as indicator bacteria to evaluate the copolymerization collection capacity of the isolated strain; mixing the same volume of the test strain and the bacterial suspension of the indicator bacteria, vortex mixing for 10s, standing at 37 ℃ for 5h, slowly sucking 200 mu L of upper bacterial liquid, and measuring OD _600nm Value (A) _Mixing ) The 3 measurements were repeated and the strain coaggregation calculation formula was as follows:

wherein: a is that ₂ And A ₃ The absorbance values of the test bacteria and the indicator bacteria suspensions, which were treated for 5 hours, respectively, were shown.

Hydrophobicity: 2mL of the bacterial suspension was added to a 10mL centrifuge tube, then an equal volume of organic reagent (each evaluated as hexadecane and ethyl acetate) was added, vortexed and mixed for 2min, and after standing in a fume hood at room temperature for 0.5h, the OD of the aqueous layer solution was determined _600nm The values were repeated 3 times and the calculation formula was as follows:

wherein: a is that _x Indicating the initial OD of the bacterial suspension _600nm Value, A _y Represents OD after 30min of standing _600nm Values.

6) Adhesion to Caco-2 cells

Cell culture: thawing Caco-2 cells preserved in liquid nitrogen in 37deg.C water rapidly, centrifuging at 800r/min for 5min, adding 1mL DMEM high sugar culture medium, mixing, transferring into cell culture bottle, adding DMEM culture medium containing 10% Fetal Bovine Serum (FBS) into 5% CO ₂ Culturing in a 37 ℃ incubator, changing the liquid once in two days, and carrying out passage when the cells grow to more than 90% of the culture flask, wherein the cells are used for test after 3 passages.

Adhesion test: 1mL of Caco-2 cell suspension (1.0X10) ⁵ cells/mL) in 6-well cell culture plates in CO ₂ Culturing in an incubator until the cells grow to a monolayer. The culture broth was aspirated, washed with 2 times with sterile PBS, 1mL of the bacterial suspension (here the bacterial suspension was resuspended in DMEM medium containing 10% FBS) was added, and incubated in an incubator for 2h; then, the bacterial suspension was aspirated, and the cells were washed 3 times with sterile PBS to wash out the non-adherent cells, and finally the cells were collected by cell scraping, and the number of Caco-2 cells in each well and the number of adherent bacteria in each well were measured by a plate count method. The adhesion capacity was calculated as follows:

7) Safety evaluation

Hemolysis: mu.L of the bacterial solution of the test bacteria is inoculated on a blood agar plate, cultured for 72 hours at 37 ℃, and whether the test strain has hemolysis or not is observed, and golden yellow grape balls are used as positive control.

Antibiotic sensitivity and decarboxylase production experiments: the test strain was evaluated for antibiotic susceptibility using a drug susceptibility tablet kit (containing 20 antibiotics).

The test strain was evaluated for biogenic amine activity using a lysine decarboxylase kit.

As shown in table 1, lactobacillus casei AU9077 exhibited probiotic properties that mimic well the gastrointestinal fluid tolerance (survival > 90%), strain surface activity (including self-aggregation, co-aggregation and hydrophobicity), and adhesion to Caco-2 cells, compared to control strain LGG; and it can inhibit common pathogenic bacteria (e.coli and listeria) and can be used as candidate strains of probiotics.

TABLE 1 results of in vitro probiotic properties of Lactobacillus casei AU9077

As shown in table 2, lactobacillus casei AU9077 produced no common biogenic amines and haemolysins and was sensitive to the vast majority of 20 antibiotics, initially judged as a safe strain.

TABLE 2 results of in vitro safety evaluation of Lactobacillus casei AU9077

^a The diameter d of the strain sensitive to antibiotics, "-" d is less than 5mm; "+": d is more than or equal to 5mm and less than 15mm; "++": d is more than or equal to 15mm and less than 25mm; "+++": d is more than or equal to 25mm. ^b "-" is not hemolytic; "alpha" hemolysis; "beta" hemolysis. ^c "-" lysine decarboxylase activity was negative; the "+" lysine decarboxylase activity was positive.

(3) Identification of strains

After lactobacillus casei AU9077 is activated, single colony is obtained by a streaking method, and the characteristics of the strain are observed, wherein the colony is milky white, moist and neat in edge as shown in figure 1; the cell microscopic morphology is rod-shaped, free of spores and positive in gram staining.

Sequencing the PCR amplified product of the 16S rDNA, wherein the sequencing result is shown as SEQ ID NO.1, logging in NCBI website, and performing Blast sequence comparison, and the result shows that the homology of the strain AU9077 and the 16S rDNA of lactobacillus casei (Lactobacillus casei) is more than 99%, and the strain can be determined to be lactobacillus casei (Lactobacillus casei). And the lactobacillus casei is preserved in China general microbiological culture collection center (CGMCC No. 21663) in 2021 month, and the preservation number is the address: the institute of microbiology, national institute of sciences, no. 3, north chen west way 1, the region of korea, beijing, postal code 100101.

Example 2 in vivo evaluation of antiallergic Activity of strains in mice

(1) Activation of strains

After the original strain of lactobacillus casei AU9077 stored in a refrigerator at the temperature of minus 80 ℃ is activated for 2 generations, streaking is carried out on a solid MRS culture medium, single bacterial colony is selected to be inoculated in the MRS liquid culture medium after standing culture for 72 hours at the temperature of 37 ℃, the culture is carried out for 24 hours at the constant temperature of 37 ℃, and the bacterial colony is inoculated in the liquid MRS culture medium according to the inoculum size of 1% (v/v) for 2 generations of continuous culture.

(2) Preparation of bacterial suspension

Inoculating activated Lactobacillus casei AU9077 into MRS liquid culture medium, culturing at 37deg.C for 24 hr, centrifuging at 8 r/min for 4min to collect bacterial precipitate, re-suspending in PBS buffer, and adjusting bacterial concentration to 10 ⁸ CFU/mL、10 ¹⁰ CFU/mL。

(3) Construction of animal models

Experimental animals: BALB/c female mice, about 20g, SPF grade, purchased from Hunan Stokes Lemonda laboratory animal Co., ltd., laboratory animal production license number: SCXK (xiang) 2019-0004; in Hunan drug safety evaluation research center barrier environmental breeding, experimental animals use license number: SYXK (Hunan) 2015-0016.

18-22 g female BALB/c mice are firstly placed in animal houses for proper feeding for one week, the room temperature is maintained at about 25 ℃ and is alternated for 12 hours and day, the ventilation state is maintained, and basic ration and purified water are provided. After one week, mice were randomly grouped, with less than 3g of weight per group, randomly grouped into 6 groups, and the formal trial was started. A blank control group, a model control group, and a lactobacillus casei AU9077 high and low dose group were set. The specific grouping is as in table 3:

table 3 test packets and treatments

Normal mice were routinely kept throughout the experiment, and mice in the blank group were intraperitoneally injected with sterile physiological saline at 7, 14, 21, and 28d, and mice in the experimental group and model group were intraperitoneally injected with 0.2mL of 2mg/mL allergen (1 mL freund's adjuvant+1 mL 2mg/mL beta-lactoglobulin) at 7, 14, 21, and 28d, and the experimental group was perfused with 1mL of bacterial suspension of the corresponding test strain, and the model group was perfused with 1mL PBS buffer. The entire flow is shown in figure 2, four times a week starting on day 7.

Test 34d fasted for 12h, 35d after 1 time of oral excitation with 5mg of beta-lactoglobulin, the condition of the mice was observed, blood was taken from the eyeballs, the mice were sacrificed by neck removal, the spleens and thymus of the mice were taken out for weighing, and the colon and faeces of the mice were collected and stored in liquid nitrogen for the subsequent test.

(4) Determination of allergy-related indicators

1) Determination of the index of the immune organs of mice

Weighing the weight of the mice after oral administration is started for 1 hour, taking blood, and removing the neck and killing the mice; dissecting and taking out spleen and thymus of the mouse, removing peripheral adipose tissues, sucking blood stains on the spleen and thymus by using filter paper, and weighing the blood stains. Spleen and thymus index was calculated as follows: spleen index = mass of spleen (mg)/mouse body weight (g); thymus index = mass of thymus (mg)/mouse body weight (g).

2) Colon pathology observation of mice

Soaking colon tissue of a mouse in 10% formalin solution for fixation, and storing in a refrigerator at 4 ℃; samples were sent to the Hunan province drug safety evaluation research center for histopathological examination by HE staining technique.

3) Histamine, specific IgE and cytokine levels in mouse serum

The ELISA kit is used for detecting the content of histamine, IL-4, MCP-1 and IgE in serum, and the operation is performed according to instructions.

4) PCR detection of colon tissue IL-4, IFN-gamma, IL-10, TNF-alpha, TGF-beta Gene expression

(1) Total RNA extraction (gun head and centrifuge tube were both sterilized by humid heat, without RNase)

Taking a homogenizing tube, adding 1mL of Trizol Reagent, and pre-cooling on ice; taking 100mg of tissue, and adding the tissue into a homogenizing tube; fully grinding by a homogenizer until no visible tissue blocks exist; 12 Centrifuging at 000r/min for 10min to obtain supernatant; adding 250 mu L of chloroform, reversing the centrifuge tube for 15s, fully and uniformly mixing, and standing for 3min; centrifuging at 4deg.C for 10min at 12 000r/min; transferring 400 mu L of supernatant into a new centrifuge tube, adding 0.8 times of isopropanol, and mixing the mixture upside down; placing at-20deg.C for 15min; centrifuging for 10min at 4 ℃ at 12 000r/min, and obtaining white precipitate at the bottom of the tube as RNA. The liquid was removed by suction, and 1.5mL of 75% ethanol was added to wash the precipitate; centrifuging at 4deg.C for 5min at 12 000r/min; the liquid is sucked and removed, and the centrifuge tube is placed on an ultra clean bench for blowing for 3min. Adding 15. Mu.L of RNase-free water to dissolve RNA; incubating at 55 ℃ for 5min; RNA concentration and purity were measured using Nanodrop 2 000: taking 2.5 mu L of RNA solution to be detected on a detection base after the instrument blank is zeroed, putting down a sample arm, and starting detection of a light absorption value by using software on a computer; the RNA with too high concentration is diluted in a proper proportion to make the final concentration 100-500 ng/. Mu.L.

(2) Reverse transcription

Configured according to the system of table 4, gently mixed and centrifuged; reverse transcription program settings refer to table 5; the gun head and the PCR are subjected to wet heat sterilization, and no RNase exists.

TABLE 4 preparation of reverse transcription reaction System (20. Mu.L reaction System)

TABLE 5 reverse transcription procedure settings

(3) Quantitative PCR

A reaction system was prepared by taking 0.2ml PCR tubes and preparing 3 tubes for each reverse transcription product.

(4) PCR amplification

Pre-denaturation at 95℃for 10min

Cycling (40 times) at 95deg.C, 15deg.C to 60deg.C, and 30s

The melting curve is 65 ℃ to 95 ℃, and the primer sequences with the temperature increased by 0.3 ℃ every 15s are shown in Table 6:

TABLE 6 primer sequences

(5) Result processing method

ΔΔct method:

a=ct (target gene, sample to be measured) -CT (internal standard gene, sample to be measured)

B=ct (target gene, control sample) -CT (internal standard gene, control sample)

K＝A-B

Expression fold = 2 ^-K

The effect of low and high doses of lactobacillus casei AU9077 on the immunomodulatory capacity and colon tissue of sensitized mice was evaluated by establishing a beta-lactoglobulin (beta-lg) induced allergic mouse model. As shown in fig. 3-5, the intraperitoneal injection of the mice can significantly reduce the immune organ index and the levels of cytokines MCP-1 and IL-4 in serum, and the content of histamine and ige in the serum of the mice sensitized by the beta-lg is significantly higher than that of the group C (P < 0.05). The beta-lg sensitized mice after the strain AU9077 is subjected to dry prognosis can obviously reduce histamine and sIgE in serum and improve secretion levels of cytokines MCP-1 and IL-4. The relative expression level of the related cytokine genes in colon tissues of mice is analyzed through Q-PCR, and compared with M groups, the expression levels of IL-10 and TGF-beta genes can be obviously increased through low-dose bacterial suspension dry prognosis, and simultaneously the level of IL-4 is obviously reduced and the expression level of IFN-gamma is increased. Meanwhile, the pathological observation of the colon tissue of the mice is carried out by utilizing HE staining, and the intestinal mucosa lamina propria infiltrated by inflammatory cells in the colon tissue of the mice of the test group treated by the strain AU9077 can be seen to have a small amount of mast cells, and the colon tissue has the advantages of complete structure, clear outline, no degranulation phenomenon and similar structure to the group C. These results indicate that the strain has potential functions of alleviating or preventing allergic diseases.

Example 3 effect of beta-lactoglobulin on the intestinal flora of mice

(1) Establishment of mouse model

The mouse models of this example were all constructed as in example 2.

(2) DNA extraction of mouse faeces sample

Fresh feces of each group of mice were collected and frozen at-80℃in time, and genomic DNA was extracted according to instructions using a DNA extraction kit.

(3) PCR amplification

The DNA extracted in the last step is used as a template for amplification, and the amplified region is the V3-V4 region of the bacterial 16S rRNA gene. Primer sequences, amplification systems, amplification conditions are shown in tables 7, 8 and 9.

TABLE 7 primer sequences

TABLE 8 amplification System

TABLE 9 amplification conditions

(4) Detection, combining and purification of PCR products

After amplification, electrophoresis detection is carried out by adopting 1% agarose gel, and electrophoresis conditions are as follows: 1.5% agarose gel, 100V,30min; after concentration comparison of the PCR products using GeneTools Analysis Software (version 4.03.05.0, synGene), the volumes required for each sample were calculated on an equal mass basis and each PCR product was mixed.

(5) Library preparation and on-machine sequencing

Library establishment is followedUltra TM DNA Library Prep Kit for/>And (3) carrying out library building operation by using a standard flow, and carrying out PE250 sequencing on the constructed amplicon library by using an Illumina Nova 6000 platform after completion. The above was delegated to Guangdong Meiger Gene technology Co.

As can be seen from table 10, fig. 6 and fig. 7, the number of species specific to group C is the greatest and group M is the next time in the Venn diagram. The microbial community structure distribution diagram and the clustering heat diagram show that, on the portal level, compared with the C group, the M group of bacteroides and wart micro-bacteria have higher relative abundance, and after the gastric lavage strain AU9077, the bacteroides and the thick-walled bacteria become dominant bacteria in the intestinal tract of the mice; on the department level, the abundance values of different groups are greatly different, and the dominant bacteria of the C group, the M group and the test group are murine, acermaniaceae and lactobacillus respectively; after clustering at the genus level, the same-dose test groups clustered into one group, with the low-dose group being closer to group C. The Alpha diversity analysis result shows that the microbial diversity is increased after the mice are sensitized, and the microbial diversity of the mice is reduced after the mice are filled with the gastric lactobacillus; the test group has a tendency to approach the C group, is far away from the M group, and is found in the gateroad colony aggregation analysis chart that the M group and the C group fall structure are obviously different, but the test group cluster is closer to the M group; the strain AU9077 can effectively improve the change of intestinal flora of mice caused by allergic symptoms and increase the abundance of beneficial bacteria.

TABLE 10 results of analysis of intestinal microbial alpha diversity in mice

Example 4 preparation of dark tea fermented milk beverage

(1) Activation and culture of strains

Inoculating the bacterial liquid of the Lactobacillus casei AU9077 frozen at-80 ℃ into MRS liquid culture medium, culturing at 37 ℃ for 24 hours, and activating for 3 generations. Inoculating the activated strain into liquid MRS culture medium according to a certain inoculation amount (v/v), culturing at constant temperature for 30h, and measuring the viable count and OD of the strain _600nm Values.

(2) Preparation of black tea soup

The black tea small blocks broken by the brick cutter are boiled in water (100 ℃ for 10 min), filtered by two layers of gauze and cooled (65 ℃) to prepare black tea soup for standby. The mass volume ratio of the black tea small blocks to the water is 1:150;

(3) The preparation method of the black tea fermented milk beverage specifically comprises the following steps:

dissolving skimmed milk powder, high fructose syrup and glucose syrup in the prepared black tea soup, performing primary blending, and fully and uniformly stirring to obtain primary mixed emulsion; preheating (65deg.C), homogenizing (20 Mpa,2 times), sterilizing (121deg.C, 15 min), and cooling (40deg.C) to obtain sterile mixed emulsion; inoculating a starter into the mixed emulsion under the aseptic condition, and then fermenting (33 ℃ for 48 hours) and after-ripening (4 ℃ for 24 hours) to obtain a black tea fermented milk base material; adding sterilized secondary blending liquid with the same volume as the black tea fermented milk base material into the black tea fermented milk base material, uniformly stirring, homogenizing (20 mpa,2 times), aseptically filling, and cooling (4 ℃) to obtain the black tea fermented milk beverage.

The skim milk powder accounts for 12% of the black tea soup in terms of mass-to-volume ratio (g/mL), the high fructose syrup accounts for 3% of the black tea soup in terms of mass-to-volume ratio (g/mL), and the glucose syrup accounts for 3% of the black tea soup in terms of mass-to-volume ratio (g/mL);

the secondary blending liquid is prepared according to the following method: taking water as a solvent, adding white granulated sugar, sodium carboxymethyl cellulose, sodium alginate propylene glycol ester and sodium citrate, uniformly stirring, sterilizing and cooling to obtain the finished product; wherein the mass percentage concentration of the white granulated sugar in the black tea fermented milk beverage is 8-10%, the key mass percentage concentration of the sodium carboxymethylcellulose (CMC) in the black tea fermented milk beverage is 0.07-0.09%, the mass percentage concentration of the sodium alginate propylene glycol ester (PGA) in the black tea fermented milk beverage is 0.08-0.1%, and the mass percentage of the sodium citrate in the black tea fermented milk beverage is 0.1-0.15%.

The starter is lactobacillus casei AU9077 bacterial suspension (5×10) ⁸ CFU/mL) and the inoculum size was 3%.

The fructose-glucose syrup, white granulated sugar, CMC, PGA and the sodium citrate are all food grade.

(4) Determination of desensitization and antioxidation capabilities of black tea fermented milk beverage

(1) Preparation of neutral black tea fermented milk beverage (HNM): centrifuging the prepared black tea fermented milk beverage at 4deg.C and 10000r/min under aseptic condition for 10min, collecting supernatant, adjusting pH to neutral with aseptic 1% NaOH solution to obtain aseptic HNM, and temporarily storing at 4deg.C for use.

(2) Evaluation of desensitization

RBL-2H3 cell culture: taking rat basophilic leukemia cells (RBL-2H 3) preserved by liquid nitrogen, thawing rapidly in 37 ℃ water, centrifuging for 5min at 800r/min, removing frozen stock solution, adding 1mL of DMEM (high sugar) culture medium for resuspension, transferring to a cell culture bottle, immediately sucking 5-6 mL of DMEM culture medium (short for complete culture medium) containing 10% FBS, and concentrating at 37 ℃ and 5% CO ₂ Is cultured in a carbon dioxide incubator. 1 time of liquid is changed every 1 day, when the cells grow to more than 90% of the bottle wall, the cells are passaged, and related experiments are carried out after three times of continuous passaging.

Building an RBL-2H3 degranulation model: RBL-2H3 cells after three passages and in vigorous growth phase were digested into cell suspension (1.0X10) ⁵ cells/mL), 1mL of suspension was inoculated into each well in a 6-well plate, 3mL of complete medium was added, and when the cells grew to a monolayer, the cells were tested and divided into test groups (HNM group) and model groups according to the test protocol(group M) and blank (group N); the cell building model is as follows:

sensitization: the cultured RBL-2H3 cells were removed from the culture broth and were grouped as required by the protocol. Then, HNM group and M group were added with 1mL of complete medium containing anti-DNP-IgE (0.5. Mu.L/mL), and N group was replaced with the same amount of complete medium; at 37℃with 5% CO ₂ Culturing in an incubator overnight.

Sample treatment: the overnight sensitized RBL-2H3 cells were removed from the culture. Then, HNM group was added to 1mL HNM, and M group and N group were replaced with equal amount of complete medium in CO ₂ The treatment was carried out in an incubator for 1 hour.

Excitation: the sample-treated RBL-2H3 cells were removed from the culture broth and washed 2 times with PBS. Then, the HNM group and the M group were each added with a complete medium containing DNP-BSA (0.25. Mu.g/mL), and the N group was replaced with an equal amount of complete medium. Collecting supernatant or cells at different excitation time points, after DNP-BSA is excited for 0.5H, determining the inhibition of black tea fermented milk on RBL-2H3 cell beta-hexosaminidase (beta-HEX) release, obtaining culture solution, centrifuging at 1 000r/min and 4 ℃ for 5min, and reserving the supernatant for standby. Adding supernatant (50. Mu.L/well) into 96-well culture plate, adding 50. Mu.L of color development solution to each well, incubating at 37deg.C for 1 hr, adding 200. Mu.L of stop solution (0.1 mol/L Na) ₂ CO ₃ /NaHCO ₂ pH 11.0) to terminate the reaction, and measuring the OD by using an ELISA reader _406nm Values. The calculation formula for the beta-HEX release inhibition rate is as follows:

(3) evaluation of antioxidant Capacity

The total antioxidant capacity (Total antioxidant capacity, T-AOC) and the hydroxyl radical scavenging capacity of the black tea fermented milk were determined with reference to the kit instructions. The DPPH free radical scavenging ability measurement method comprises the following steps: adding 1mL of a sample to be detected (HNM) and 1mL of 0.2mM/L DPPH ethanol solution (prepared by absolute ethanol, stored at 4 ℃ in a dark place and used at present) into a test tube, uniformly mixing, reacting at room temperature in a dark place for 30min, and measuring the absorbance of the solution at 517 nm; 1mL of absolute ethyl alcohol is used to replace 1mL of DPPH ethanol solution to form a blank group; 1mL of PBS buffer solution is used for replacing 1mL of sample to be detected as a control group, and 1mL of PBS buffer solution and absolute ethyl alcohol mixed solution are used for blank zeroing. Each sample was repeated 3 times and averaged. The calculation formula is as follows:

wherein: a is that _s Absorbance of sample group, A _b Absorbance of blank group, A _c Absorbance of control group.

The black tea fermented milk beverage prepared by the method has uniform color and luster and reddish brown color; the sour and sweet taste is palatable, the lubrication is not greasy, the strong tea aroma is achieved, the flavor characteristics of the yoghurt are achieved, and the requirements of most consumers are met; as can be seen from table 11, the black tea fermented milk beverage can significantly inhibit degranulation of RBL-2H3 cells; and the total antioxidant capacity, the hydroxyl radical scavenging capacity and the DPPH radical scavenging capacity are all higher.

TABLE 11 beta-HEX inhibition results and antioxidant Activity of dark tea fermented milk beverages

CK means that no dark tea was added.

EXAMPLE 5 preparation of fermented milk of Germinatus Phragmitis

(1) Activation and culture of strains

(2) Preparation of asparagus extract

Dissolving the freeze-dried asparagus powder in water, extracting for 2 hours in a water bath at 60 ℃, filtering, collecting filtrate, pre-freezing for 6 hours at-80 ℃, rapidly transferring the sample into a vacuum freeze dryer after pre-freezing, and freeze-drying for more than 24 hours to obtain an asparagus extract for later use. The mass volume ratio of the asparagus powder to the water is 1:40.

(3) The preparation method of the asparagus fermented milk comprises the following specific steps:

the method comprises the steps of mixing and stirring skim milk powder, asparagus extract, fructose syrup, glucose syrup and water uniformly at 65 ℃ to obtain reconstituted milk, preheating (65 ℃), homogenizing (20 mpa,2 times), sterilizing, cooling (40 ℃), inoculating a starter, and after-ripening (4 ℃) to obtain the asparagus fermented milk.

The defatted milk powder accounts for 12% of the weight-to-volume ratio (g/mL) of the reconstituted milk, the asparagus extract accounts for 0.5% of the weight-to-volume ratio (g/mL) of the reconstituted milk, the high fructose syrup accounts for 3% of the weight-to-volume ratio (g/mL) of the reconstituted milk, the glucose syrup accounts for 3% of the weight-to-volume ratio (g/mL) of the defatted reconstituted milk, the sterilization temperature is 121 ℃ and the time is 20min, and the starter is a bacterial suspension (5×10) of lactobacillus casei AU9077 ⁸ CFU/mL), the inoculum size was 3%, the fermentation temperature was 33℃and the fermentation time was 48h.

(4) Determination of desensitization and bacteriostasis capacity of asparagus fermented milk

(1) Preparation of neutral asparagus fermented milk (LNM): centrifuging the prepared asparagus fermented milk for 5min at 4 ℃ under the aseptic condition of 8 000r/min, collecting supernatant, regulating pH value to be neutral by using an aseptic 1% NaOH solution, and obtaining an aseptic LNM for temporary storage at 4 ℃ for later use.

(2) Evaluation of desensitization

The measurement was performed as in example 4

(3) Evaluation of bacteriostatic ability

The antibacterial activity is measured by an oxford cup method, and the antibacterial capacity of the strain is represented by the diameter of a antibacterial ring, and escherichia coli and listeria are used as indicator strains.

The asparagus fermented milk prepared by the method is brown, uniform and consistent; has pleasant asparagus flavor and no peculiar smell. And as shown in table 12, as the addition amount of the asparagus extract increases, the viable count and titrating acidity of the asparagus fermented milk also increase, which indicates that the asparagus can promote the growth of the strain. In addition, as shown in table 13, the asparagus fermented milk supernatant was able to significantly inhibit degranulation of RBL-2H3 cells as well; and can inhibit common pathogenic bacteria (e.coli and listeria). Along with the continuous increase of the addition amount of the asparagus extract, the total antioxidant capacity, the hydroxyl radical scavenging capacity and the DPPH radical scavenging capacity of the supernatant of the asparagus fermented milk are gradually improved, which indicates that the asparagus fermented milk prepared by lactobacillus casei AU9077 has stronger antioxidant capacity.

TABLE 12 results of viscosity, titrated acidity, pH and viable count of asparagus fermented milk

TABLE 13 beta-HEX inhibitory effect, antioxidant and bacteriostatic Activity of fermented milk of Germinatus Phragmitis

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Sequence listing

<110> Australian milk industry (China) Limited, hunan agricultural university

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Claims

1. A lactobacillus casei is characterized in that the lactobacillus casei is named as lactobacillus casei in taxonomyLactobacillus casei) AU9077, deposited in China general microbiological culture Collection center with a strain deposit number of CGMCC No.21663.

2. Use of lactobacillus casei as claimed in claim 1 in the manufacture of a medicament for reducing and/or preventing allergic disease.

3. A method for preparing a black tea fermented milk beverage having antioxidant activity using lactobacillus casei as claimed in claim 1, comprising the steps of:

(1) Boiling black tea in water, filtering, and cooling to obtain black tea soup;

(3) Preheating the primary mixed emulsion, homogenizing, sterilizing and cooling to obtain sterile mixed emulsion;

(4) Inoculating lactobacillus casei into the sterile mixed emulsion according to the inoculum size of 2-4% under the aseptic condition, fermenting for 44-48 h at 32-35 ℃, and then after-ripening fermenting for 12-24 h at 4 ℃ to obtain the black tea fermented milk base material;

4. A method according to claim 3, wherein the dark tea and water in step (1) are in a mass to volume ratio of from 1:100 to 1:150, the mass to volume ratio being in g/mL.

5. A method according to claim 3, wherein the homogenisation conditions in step (3) and step (5) are 20 to 25mpa,2 to 3 times; and (3) sterilizing the secondary blending liquid in the step (5) at 80 ℃ for 15min.

6. A method for preparing an asparagus fermented milk having antioxidant activity using lactobacillus casei as claimed in claim 1, comprising the steps of:

(1) Preparation of asparagus extract: dissolving the freeze-dried asparagus powder in water, extracting for 1.5-2.5 hours in a water bath at 60-65 ℃, filtering, collecting filtrate, pre-freezing for 5-7 hours at-80 ℃, and quickly freeze-drying for more than 24 hours in a vacuum freeze dryer after pre-freezing to obtain an asparagus extract for later use;

(2) Preparation of asparagus fermented milk: mixing skimmed milk powder, germinatus Phragmitis extract, fructose syrup, glucose syrup and water, and stirring to obtain reconstituted milk; in the reconstituted milk, the mass-volume ratio of the skim milk powder to the reconstituted milk is 10-12%, the mass-volume ratio of the asparagus extract to the reconstituted milk is 0.1-0.5%, the mass-volume ratio of the fructose syrup to the reconstituted milk is 2.5-3%, the mass-volume ratio of the glucose syrup to the reconstituted milk is 2-4%, and the mass-volume ratio is in g/mL; preheating, homogenizing, sterilizing and cooling the reconstituted milk at 65-75 ℃, inoculating lactobacillus casei to the reconstituted milk according to the inoculum size of 2-4%, fermenting for 44-48 hours at 32-35 ℃, and fermenting for 12-24 hours at 4 ℃ to obtain the asparagus fermented milk.

7. The method of claim 6, wherein the mass to volume ratio of asparagus powder to water in step (1) is from 1:10 to 1:40, said mass to volume ratio being in g/mL.

8. The method according to claim 6, wherein the homogenization conditions in the step (2) are 20 to 25mpa,2 to 3 times.