CN113604383A - Lactobacillus casei and application thereof - Google Patents
Lactobacillus casei and application thereof Download PDFInfo
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- CN113604383A CN113604383A CN202110829075.0A CN202110829075A CN113604383A CN 113604383 A CN113604383 A CN 113604383A CN 202110829075 A CN202110829075 A CN 202110829075A CN 113604383 A CN113604383 A CN 113604383A
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- fermented milk
- lactobacillus casei
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- asparagus
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Abstract
The invention discloses Lactobacillus casei and application thereof, wherein the taxonomic name of the Lactobacillus casei is Lactobacillus casei (Lactobacillus casei) AU9077, and the preservation number of the strain is CGMCC No. 21663. The strain can regulate immunity, relieve allergy symptoms, protect intestinal mucosa structure damage caused by beta-lactoglobulin induced anaphylaxis, and increase intestinal beneficial flora; in addition, the dark green tea fermented milk beverage and/or asparagus fermented milk prepared by the strain AU9077 has obvious allergy reduction effect and/or antioxidant activity, and shows that the dark green tea fermented milk beverage and/or asparagus fermented milk can be used as probiotics with allergy reduction effect and/or antioxidant activity to develop various probiotic products.
Description
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, meant as "beneficial to life" and defined by the World Health Organization (WHO) as a viable microorganism that, when ingested in sufficient quantities, can confer a variety of health benefits to the host, including enhancing immunity, regulating gut flora, promoting digestive absorption, alleviating irritable bowel syndrome, and lowering cholesterol. Probiotics mainly include the following major groups: bifidobacterium (bifidobacterium longum, bifidobacterium infantis, etc.); ② lactobacillus (lactobacillus casei, lactobacillus acidophilus, lactobacillus rhamnosus, etc.); (iii) Bacillus (Bacillus subtilis, etc.); (iv) fungi (yeasts and the like). Wherein, the Lactobacillus casei (Lactobacillus casei) belongs to the Lactobacillus, gram-positive bacteria and facultative anaerobe, and is one of important Lactobacillus species; widely distributed in intestinal tract and reproductive tract of human body and animals, fermented fruits and vegetables and dairy products. It has strong acid and bile salt resistance, and some strains also have the beneficial functions of immunoregulation, intestinal flora regulation, cholesterol reduction and the like. The lactobacillus casei has a plurality of probiotic properties, can also produce bacteriostatic active substances, and can inhibit and kill a plurality of spoilage bacteria and pathogenic bacteria in food. The physiological functions of these probiotics are closely related to the vital activities of the organism, and are widely applied to important fields such as industry, agriculture and animal husbandry, food, medicine and the like.
At present, with the continuous change of global environment, allergic diseases have become a ubiquitous health problem, and more than 30% of people are suffering from the effects of allergic diseases. Currently, most of the diseases are treated by drugs, however, the drug treatment has certain side effects, so that a safer and more effective treatment means is urgently required to be searched. With the rapid development of microbiology, various researches find that probiotics have effective prevention and treatment effects on human allergic diseases, but the relevant action mechanism is still unclear, and the key technology of screening is yet to be researched and broken through.
In addition, most of probiotic fermented milks are prepared by fermenting different milk sources with multiple strains at present, and natural extracts with health-care functions (such as dark tea, asparagus and the like) are combined with probiotics with the functions of relieving and/or preventing allergy to prepare the fermented milks, so that the fermented milks with multiple functions are rarely reported.
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 purpose, the technical scheme provided by the invention is as follows:
the Lactobacillus casei (Lactobacillus casei) AU9077 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) at 18 th of 2021-01-month, and the address is as follows: the microbial research institute of China academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, and Hostwood, the accession number of the strain is CGMCC No. 21663.
The lactobacillus casei AU9077 can be used for preparing medicines for reducing and/or preventing allergic diseases.
In addition, the lactobacillus casei AU9077 can be used for preparing food with desensitization effect and/or antioxidant activity, wherein the food comprises health-care food.
Preferably, the food is black tea fermented milk beverage and/or asparagus fermented milk.
In the invention, the method for preparing the dark green tea fermented milk beverage with desensitization effect and/or antioxidant activity comprises the following steps:
(1) boiling black tea in water, filtering with two layers of gauze, and cooling to obtain black tea soup for use;
(2) dissolving the skim milk powder, the high fructose corn syrup and the glucose syrup in the dark tea soup for primary blending, and fully and uniformly stirring to obtain a primary mixed emulsion; the mass volume ratio of the defatted milk powder to the black tea soup is 10-12%, the mass volume ratio of the high fructose corn syrup to the black tea soup is 2.5-3%, the mass volume ratio of the glucose syrup to the black tea soup is 2.5-3%, and the unit of the mass volume ratio is g/mL;
(3) preheating the primary mixed emulsion at 65-75 ℃, and then homogenizing, sterilizing and cooling to obtain sterile mixed emulsion;
(4) under the aseptic condition, inoculating lactobacillus casei into the aseptic mixed emulsion according to the inoculation amount of 2-4%, fermenting for 44-48 h at 32-35 ℃, and performing after-ripening fermentation for 12-24 h at 4 ℃ to obtain the black tea fermented milk base material; when inoculating, inoculating lactobacillus casei viable bacteria preparation or bacterial suspension (viable bacteria amount in bacterial suspension is 1-5 × 10)8CFU/mL)；
(5) Adding the sterilized secondary blending liquid which is equal to the volume of 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 the black tea fermented milk beverage; the secondary preparation liquid is prepared by the following method: adding white granulated sugar, sodium carboxymethylcellulose, sodium alginate propylene glycol ester and sodium citrate into water as a solvent, uniformly stirring, sterilizing and cooling to obtain the sodium alginate-propylene glycol ester-sodium alginate-sodium citrate-sodium alginate-sodium citrate; the mass percentage concentration of the white granulated sugar in the dark green tea fermented milk beverage is 8-10%, the mass percentage concentration of the sodium carboxymethylcellulose in the dark green tea fermented milk beverage is 0.07-0.09%, the mass percentage concentration of the sodium alginate propylene glycol ester in the dark green tea fermented milk beverage is 0.08-0.1%, and the mass percentage concentration of the sodium citrate in the dark green tea fermented milk beverage is 0.1-0.15%.
Preferably, the mass-to-volume ratio of the water to the dark tea in the step (1) is 1:100 to 1:150, and the unit of the mass-to-volume ratio is g/mL.
Preferably, the homogenizing condition in the step (3) and the step (5) is 20-25 Mpa for 2-3 times; and (5) sterilizing the secondary preparation solution at 80 ℃ for 15 min.
In the invention, the method for preparing the asparagus fermented milk with desensitization effect and/or antioxidant activity comprises the following steps:
(1) preparation of asparagus extract: dissolving freeze-dried asparagus powder in water, extracting in water bath at 60-65 ℃ and preferably 60 ℃ for 1.5-2.5 h and preferably 2h, carrying out suction filtration, collecting filtrate, pre-freezing at-80 ℃ for 5-7 h and preferably 6h, and quickly freeze-drying in a vacuum freeze-drying machine for more than 24h after pre-freezing 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 high fructose corn syrup, the glucose syrup and water uniformly at the temperature of 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 skim reconstituted milk is 2-4%, and the unit of the mass-volume ratio is g/mL; preheating the reconstituted milk at 65-75 ℃, homogenizing, sterilizing (121 ℃, 15-20 min), cooling to 37-40 ℃, inoculating lactobacillus casei to the reconstituted milk according to the inoculation amount of 2-4% for fermentation for 44-48 h at 32-35 ℃, and performing after-ripening fermentation for 12-24 h at 4 ℃ to obtain the asparagus fermented milk; when inoculating, live bacteria preparation or bacterial suspension of lactobacillus casei is inoculated (live bacteria amount in bacterial suspension is 1-5 × 10)8CFU/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 homogenizing condition in the step (2) is 20-25 Mpa for 2-3 times.
Compared with the prior art, the invention has the beneficial effects that:
the bacterial colony of the lactobacillus casei AU9077 on the MRS solid culture medium is milky white, moist and neat in edge. And gram-positive, rod-shaped, spore-free, 16S rDNA sequence is shown in SEQ ID NO. 1. The lactobacillus casei AU9077 has excellent in-vitro probiotic characteristics such as simulated gastrointestinal fluid tolerance, bacteriostatic activity, cell surface activity, adhesion to Caco-2 cells and the like; and is characterized by safety, including the absence of the production of the common biogenic amines and hemolysins, and sensitivity to the vast majority of the commonly used antibiotics.
In a mouse model sensitized by beta-lactoglobulin, the strain AU9077 can obviously reduce the secretion of histamine, specific IgE, IL-4 and MCP-1 in the serum of an allergic mouse; can also up-regulate the expression of allergy-related cytokines (IFN-gamma, IL-10, TNF-alpha and TGF-beta) genes in colon tissues of mice, and down-regulate the expression of IL-4 genes; meanwhile, the strain AU9077 can repair the damage of the intestinal mucosa structure of the allergic mouse; in addition, the strain AU9077 can effectively increase the abundance of beneficial intestinal flora in allergic mice.
In a word, the Lactobacillus casei (Lactobacillus casei) AU9077 can regulate immunity, relieve allergic symptoms, has a certain protection effect on damage of an intestinal mucosa structure caused by beta-lactoglobulin-induced anaphylactic reaction, can also effectively increase the beneficial intestinal flora of allergic mice, and has effectiveness in preventing and/or relieving allergy; in addition, the dark green tea fermented milk beverage and/or asparagus fermented milk prepared by the strain AU9077 has obvious desensitization effect and/or antioxidant activity. The probiotic bacteria can be used as probiotics with allergy reduction effect to develop various probiotic products.
Drawings
FIG. 1 morphology of Lactobacillus casei AU9077 colony;
FIG. 2 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 relative expression of mouse colon tissue cytokine genes;
FIG. 5 mouse colon histopathological examination (HE X200);
FIG. 6 is a Venn diagram of mouse intestinal microorganisms;
figure 7 composition profile of mouse gut microbiome structure and generic species abundance clustering heatmap.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather construed as limited to the embodiments set forth herein.
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 in the description of the invention herein 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 the strains
1) Collecting a sample: sampling and numbering by using 50mL sterile centrifuge tubes in south China pastures (different milk stations), a quaysian (dairy company and pasture) and Ningxiang (dairy company and pasture) in Hunan province, and rapidly transferring to a microorganism laboratory by using a sampling box containing an ice bag; a total of 71 samples were collected from these regions.
2) Separation of lactic acid bacteria: 25g of the sample was aseptically weighed into a 500mL Erlenmeyer flask containing 225mL of sterile physiological saline (0.85% NaCl) and mixed, and diluted to 10-fold with a gradient of 10-2～10-6And in MRS solids (containing 0.5% CaCO)3(w/v)) plates were plated and incubated at 37 ℃ for 48 h. And (4) selecting a single colony with a calcium-dissolving ring for gram staining, streaking and purifying the positive strain for three times, and numbering and storing the positive strain. The obtained strains were all stored in MRS liquid medium containing 40% glycerol at-80 deg.C by freezing.
(2) In vitro probiotic characteristics and preliminary safety evaluation of strains
1) Preparation of bacterial suspension: culturing the strain in MRS liquid culture medium at 37 deg.C overnight, activating for 2 times, and centrifuging at 4 deg.C and 8000 r/min for 3 min; by using sterilityWashing with physiological saline for 2 times, and resuspending to obtain bacterial suspension OD600nmApproximately 0.8. The control strain Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG, LGG) was the same culture and treatment.
2) Acid and bile salt resistance evaluation
3% of bacterial suspension is respectively inoculated into MRS liquid culture medium with the pH value of 3 and containing the bovine bile salt (3.0g/L), the mixture is cultured for 12 hours at 37 ℃, and whether the culture solution is turbid or not is observed, and the turbidity indicates that the strain has tolerance. The lactobacillus casei AU9077 has better tolerance through evaluation of acid resistance and bile salt resistance.
3) Evaluation of simulated gastrointestinal fluid tolerance
Activating and preparing a strain AU9077 into bacterial suspension, respectively adding 0.5mL of bacterial suspension into 4.5mL of sterile simulated gastric fluid and intestinal fluid, mixing in a vortex manner for 15s, incubating for 3h in an environment at 37 ℃, respectively determining the viable count of 0h and 3h by using a plate counting method, and calculating the survival rate of the strain in the gastrointestinal fluid according to the following mode:
4) bacteriostatic activity
And (3) determining the antibacterial activity: the bacteriostatic activity is measured by an Oxford cup method, the bacteriostatic ability of the strain is expressed by the diameter of a bacteriostatic circle, and escherichia coli and listeria are used as indicator strains.
5) Surface activity of the strain
Self-aggregation: directly sucking 1mL of bacterial suspension of the screened strain into a 1.5mL centrifuge tube, swirling for 10s, standing for 5h at 37 ℃, slowly sucking 200 mu L of upper layer bacterial liquid, and determining OD600nmValue, 3 replicates, and the strain self-aggregation calculation formula is as follows:
in the formula: a. the1: OD of upper layer bacterial liquid after standing for 5h600nmA value; a. the0: OD of the initial bacterial suspension600nmThe value is obtained.
Co-aggregation: respectively taking listeria monocytogenes and escherichia coli as indicator bacteria to evaluate the copolymerization integration capability of the separated strains; mixing the bacterial suspension of the test strain and the indicator strain with the same volume, vortex mixing for 10s, standing at 37 ℃ for 5h, slowly sucking 200 mu L of upper layer bacterial liquid, and measuring OD600nmValue (A)Mixing of) The determination was repeated 3 times, and the strain co-aggregation calculation formula was as follows:
in the formula: a. the2And A3Respectively representing the absorbance value of the test bacteria and the indicator bacteria which are respectively treated for 5 hours.
Hydrophobicity: adding 2mL of the bacterial suspension into a 10mL centrifuge tube, adding an equal volume of organic reagent (evaluated by hexadecane and ethyl acetate respectively), vortex mixing for 2min, standing in a fume hood at normal temperature for 0.5h, and determining OD of the aqueous solution600nmValue, 3 replicates, calculated according to the formula:
in the formula: a. thexIndicating the initial OD of the bacterial suspension600nmValue, AyDenotes OD after 30min of standing600nmThe value is obtained.
6) Adhesion ability to Caco-2 cells
Cell culture: rapidly thawing Caco-2 cells preserved with liquid nitrogen in 37 deg.C water, centrifuging at 800r/min for 5min, adding 1mL DMEM high sugar medium, mixing, transferring into cell culture bottle, and culturing with DMEM medium containing 10% Fetal Bovine Serum (FBS) in 5% CO2And culturing in an incubator at 37 ℃, changing the culture solution once every two days, carrying out passage when the cells grow to more than 90% of the culture flask, and carrying out experiment after passage for 3 times.
Adhesion test: 1mL of Caco-2 cell suspension (1.0X 10)5cells/mL) in 6-well cell culture plates in CO2The cells were cultured in an incubator until the cells grew to a monolayer. The culture was aspirated and washed 2 times with sterile PBS, and 1mL of bacterial suspension was added (this wasSuspending the strain bacterial suspension by using DMEM (DMEM) containing 10% FBS), and incubating for 2h in an incubator; then sucking off the bacterial suspension, washing with sterile PBS for 3 times to wash off the bacterial bodies which can not adhere, finally collecting cells by cell scraping, and respectively measuring the Caco-2 cell number of each hole by using a blood counting plate and measuring the adhering bacterial number of each hole by using a plate counting method. The adhesion capability calculation formula is as follows:
7) evaluation of safety
Hemolysis: and (3) inoculating 2 mu L of bacterial liquid of the tested bacteria on a blood agar plate, culturing for 72h at 37 ℃, observing whether the tested bacterial strain has hemolysis or not, and taking a golden yellow staphylococcus as a positive control.
Antibiotic susceptibility and decarboxylase production experiments: the sensitivity of the test strains to antibiotics was evaluated using a drug sensitive tablet kit (containing 20 antibiotics).
The biogenic amine activity of the test strain was evaluated using a lysine decarboxylase kit.
As shown in table 1, lactobacillus casei AU9077 exhibited probiotic properties that well mimicked gastrointestinal fluid tolerance (> 90% survival), strain surface activity (including self-aggregation, co-aggregation and hydrophobicity) and adhesion to Caco-2 cells, compared to the control strain LGG; and it can inhibit common pathogenic bacteria (Escherichia coli and Listeria), and can be used as candidate strain of probiotic bacteria.
TABLE 1 Lactobacillus casei AU9077 in vitro probiotic characteristics results
As shown in Table 2, Lactobacillus casei AU9077 does not produce common biogenic amine and hemolysin, is sensitive to most of 20 antibiotics, and is initially judged as a safe strain.
TABLE 2 Lactobacillus casei AU9077 in vitro safety evaluation results
aThe diameter d of the strain sensitive to antibiotics, "-" d < 5 mm; "+": d is more than or equal to 5mm and less than 15 mm; "++": d is more than or equal to 15mm and less than 25 mm; "+++": d is more than or equal to 25 mm.b"-" non-hemolytic; hemolysis of "α"; "beta" hemolysis.c"-" lysine decarboxylase activity was negative; "+" lysine decarboxylase activity was positive.
(3) Identification of strains
Activating lactobacillus casei AU9077, obtaining a single bacterial colony by a scribing method, and observing the characteristics of the bacterial colony, wherein as can be seen from figure 1, the bacterial colony is milky white, moist and neat in edge; the microscopic morphology of the cells is rod-shaped, free of spores and positive in gram stain.
Sequencing is carried out through a PCR amplification product of 16S rDNA, the sequencing result is shown in SEQ ID NO.1, the NCBI website is logged in, and Blast sequence comparison is carried out, the result shows that the 16S rDNA homology of the strain AU9077 and the Lactobacillus casei (Lactobacillus casei) reaches more than 99 percent, and the strain can be determined to be the Lactobacillus casei (Lactobacillus casei). The lactobacillus casei is preserved in the China general microbiological culture collection center in 2021 months with the preservation number of CGMCC No.21663 and the address: the microbial research institute of the national academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
EXAMPLE 2 in vivo evaluation of anti-allergic Activity of Strain in mice
(1) Activation of the Strain
Activating original strain of Lactobacillus casei AU9077 stored in a refrigerator at-80 deg.C for 2 generations, streaking on a solid MRS culture medium, performing static culture at 37 deg.C for 72h, selecting a single colony, inoculating into an MRS liquid culture medium, performing constant temperature culture at 37 deg.C for 24h, inoculating into the liquid MRS culture medium according to 1% (v/v), and continuously culturing for 2 generations.
(2) Preparation of the bacterial suspension
Inoculating activated Lactobacillus casei AU9077 into MRS liquid culture medium, culturing at 37 deg.C for 24 hr, centrifuging at 8000 r/min for 4min, collecting thallus precipitate, suspending in PBS buffer solution, and adjusting thallus concentration to 108CFU/mL、1010 CFU/mL。
(3) Construction of animal models
Experimental animals: BALB/c female mice, about 20g, SPF grade, purchased from Spanish Sischerda laboratory animals Limited, Hunan, laboratory animals production license number: SCXK (Xiang) 2019-; the method is characterized in that the method is carried out in the barrier environment of the drug safety evaluation research center in Hunan province, and experimental animals use license numbers: SYXK (xiang) 2015-.
18-22 g of female BALB/c mice are firstly placed in an animal room for proper feeding for a week, the room temperature is maintained at about 25 ℃, the daily and nights are alternated for 12 hours, the ventilation state is kept, and basic daily ration and purified water are provided. One week later, mice were randomly divided into groups with a difference of less than 3g between groups in body weight, and randomly divided into 6 groups, and the official experiment was started. Setting blank control group, model control group, and Lactobacillus casei AU9077 high and low dose groups. The specific grouping is as in table 3:
TABLE 3 test grouping and treatment
The mice of the normal group are daily fed in the whole experimental process, the mice of the blank group are injected with sterile normal saline in the intraperitoneal cavity at the 7 th, 14 th, 21 th and 28 th days, the mice of the experimental group and the mice of the model group are injected with 0.2mL of allergen of 2mg/mL (1mL of Freund's adjuvant +1mL of beta-lactoglobulin of 2 mg/mL) in the intraperitoneal cavity at the 7 th, 14 th, 21 th and 28 th days, the experimental group is intragastrically filled with 1mL of bacterial suspension of the corresponding test strain, and the model group is intragastrically filled with 1mL of PBS buffer solution. The whole process is shown in FIG. 2, starting on day 7 and four times per week.
After the 34 th day of the experiment is fasted for 12h, and the 35 th day of the experiment is orally stimulated with 5mg of beta-lactoglobulin for 1 time, the conditions of the mice are observed, blood is taken out through eyeballs, the mice are killed by removing necks, spleens and thymus of the mice are taken out and weighed, and colons and excrement of the mice are collected and stored in liquid nitrogen for subsequent experiments.
(4) Determination of allergy-related indicators
1) Determination of mouse immune organ index
After oral excitation for 1h, weighing the weight of the mouse, taking blood, removing the neck and killing the mouse; dissecting and taking out spleen and thymus of mouse, cleaning peripheral adipose tissue, sucking blood stain on spleen and thymus with filter paper, and weighing. Spleen and thymus indices were calculated as follows: spleen index ═ mass of spleen (mg) per mouse body weight (g); thymus index is mass of thymus (mg)/body weight of mouse (g).
2) Pathological observation of colon in mouse
Taking a colon tissue of a mouse, soaking the colon tissue of the mouse in 10% formalin solution for fixation, and storing the colon tissue in a refrigerator at 4 ℃; the samples were sent to the pharmaceutical safety evaluation research center of Hunan province for histopathological examination by HE staining technique.
3) Histamine, specific IgE and cytokine levels in mouse serum
Detecting by using an ELISA kit, detecting the contents of histamine, IL-4, MCP-1 and IgE in serum, and operating according to the instruction.
4) PCR detection of colon tissue IL-4, IFN-gamma, IL-10, TNF-alpha and TGF-beta gene expression
Extracting total RNA (gun head and centrifugal tube are sterilized by moist heat without RNA enzyme)
Taking a homogenate tube, adding 1mL of Trizol Reagent, and placing on ice for precooling; adding 100mg of tissue into a homogenizing tube; fully grinding by a homogenizer until no tissue block is visible; centrifuging at 12000 r/min for 10min to obtain supernatant; adding 250 μ L chloroform, reversing the centrifuge tube for 15s, mixing, and standing for 3 min; centrifuging at 12000 r/min at 4 deg.C for 10 min; transferring 400 mu L of supernatant into a new centrifuge tube, adding isopropanol with the volume of 0.8 time, and reversing and uniformly mixing; standing at-20 deg.C for 15 min; centrifuging at 12000 r/min at 4 deg.C for 10min to obtain white precipitate as RNA. Absorbing the liquid, adding 1.5mL of 75% ethanol, washing the precipitate; centrifuging at 12000 r/min at 4 deg.C for 5 min; the liquid was aspirated off and the centrifuge tube was placed on a clean bench and blown for 3 min. Adding 15 μ L of RNase-free water to dissolve the RNA; incubating at 55 deg.C for 5 min; RNA concentration and purity were determined using Nanodrop 2000: after blank zero setting of the instrument, taking 2.5 mu L of RNA solution to be detected on a detection base, putting down a sample arm, and starting light absorption value detection by using software on a computer; the RNA with the excessive concentration is diluted with a proper proportion, so that the final concentration is 100-500 ng/. mu.L.
② reverse transcription
Preparing according to a system in a table 4, slightly mixing uniformly and centrifuging; reverse transcription program settings refer to table 5; the gun head and the PCR are sterilized by moist heat without RNA enzyme.
TABLE 4 reverse transcription reaction System preparation (20. mu.L reaction System)
TABLE 5 reverse transcription program settings
③ quantitative PCR
A0.2 ml PCR tube was used to prepare the following reaction system, and 3 tubes were prepared for each reverse transcription product.
(iv) PCR amplification
Pre-denaturation at 95 deg.C for 10min
The cycle (40 times) was 95 ℃, 15s → 60 ℃, 30s
Melting curve 65 ℃ → 95 ℃, and primer sequence at 0.3 ℃ per 15s heating is shown in table 6:
TABLE 6 primer sequences
Fruit treatment method
Δ Δ CT method:
CT (target gene, sample to be tested) -CT (internal standard gene, sample to be tested)
CT (target gene, control sample) -CT (internal standard gene, control sample)
K＝A-B
The influence of low and high dose of Lactobacillus casei AU9077 on immunoregulatory capacity and colon tissues of sensitized mice is evaluated by establishing a beta-lactoglobulin (beta-lg) induced allergic mouse model. As shown in FIGS. 3-5, the injection of beta-lg into the abdominal cavity of mice can significantly reduce the immune organ index and the levels of cytokines MCP-1 and IL-4 in serum, and the contents of histamine and sIgE in the serum of beta-lg sensitized mice are significantly higher than those of group C (P < 0.05). And the beta-lg sensitized mice subjected to strain AU9077 dry prediction can obviously reduce histamine and sIgE in serum and improve the secretion levels of cell factors MCP-1 and IL-4. The relative expression of related cytokine genes in colon tissues of mice is analyzed by Q-PCR, and compared with an M group, the expression of IL-10 and TGF-beta genes can be obviously increased after the dry prognosis of a low-dose bacterial suspension, the level of IL-4 is obviously reduced, and the expression level of IFN-gamma is increased. Meanwhile, the pathological observation of the colon tissues of the mice by HE staining shows that the intestinal mucosa lamina propria infiltrated by inflammatory cells in the colon tissues of the mice in the test group treated by the strain AU9077 can see a small amount of mast cells, has complete structure and clear outline, does not have the degranulation phenomenon, and has the structure similar to that of the group C. These results indicate that the strain has potential functions of relieving or preventing allergic diseases.
Example 3 Effect of beta-lactoglobulin-sensitized mouse intestinal flora
(1) Establishment of mouse model
The mouse models of this example were all established as in example 2.
(2) Mouse fecal sample DNA extraction
Fresh excrement of each group of mice is collected and is frozen at-80 ℃ in time, and the DNA extraction kit is used for carrying out genome DNA extraction according to the instruction.
(3) PCR amplification
And (3) amplifying by using the DNA extracted in the last step as a template, wherein the amplified region is a V3-V4 region of the 16S rRNA gene of the bacteria. The primer sequences, amplification system, amplification conditions are shown in tables 7, 8 and 9.
TABLE 7 primer sequences
TABLE 8 amplification System
TABLE 9 amplification conditions
(4) Detection, pooling and purification of PCR products
After amplification, carrying out electrophoresis detection by using 1% agarose gel, wherein the electrophoresis conditions are as follows: 1.5% agarose gel, 100V, 30 min; after the concentration of the PCR products was compared by using GeneTools Analysis Software (version4.03.05.0, SynGene), the volume required for each sample was calculated according to the equal mass principle, and the PCR products were mixed.
(5) Library preparation and on-machine sequencing
Build a warehouse according to
Ultra TM DNA Library Prep Kit for
Performing library construction operation in standard process, and using Illumina Nova 6000 platformPE250 sequencing of the constructed amplicon library. The above contents were made by Guangdong Meige Gene science and technology Co.
As can be seen from Table 10, FIG. 6 and FIG. 7, in the Venn diagram, the number of species unique to group C is the largest, and M is the lowest. The microbial community structure distribution map and the clustering heatmap show that in the phylum level, compared with the group C, the relative abundance of bacteroidetes and wart microsomycota of the group M is higher, and the bacteroides and the firmicutes become dominant bacteroides in intestinal tracts of mice after the gavage strain AU 9077; on the family level, the difference of different family abundance values of each group is large, and the dominant bacteriaceae of the group C, the group M and the test group are respectively the family of the Bacillaceae, the family of the Ekemanaceae and the family of the Lactobacillaceae; after clustering on the genus level, the groups are clustered into a group with the dose test group, and the low dose group is closer to the group C. The Alpha diversity analysis result shows that the microbial diversity of the sensitized mice is increased, and the microbial diversity of the mice is reduced after the lactobacillus gastrectasis is performed; the test group has the tendency of approaching the group C, is far away from the group M, and finds that the group M and the group C have obvious difference in colony structure composition in a phylum community aggregation analysis chart, but the cluster of the test group is closer to the group M; 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 analysis results of the alpha-diversity of intestinal microorganisms in mice
Example 4 preparation of fermented milk beverage with dark tea
(1) Activation and culture of bacterial species
Inoculating the liquid of Lactobacillus casei AU9077 frozen at-80 deg.C into MRS liquid culture medium, culturing at 37 deg.C for 24 hr, 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 determining viable count and OD of the strain600nmThe value is obtained.
(2) Preparation of dark tea soup
Boiling small pieces of black tea broken with a brick knife in water (100 deg.C, 10min), filtering with two layers of gauze, and cooling (65 deg.C) to obtain black tea soup. The mass volume ratio of the small black tea blocks to water is 1: 150;
(3) the preparation method of the dark green tea fermented milk beverage comprises the following specific steps:
dissolving the skim milk powder, the high fructose corn syrup and the glucose syrup in the prepared dark tea soup for primary blending, and fully and uniformly stirring to obtain a primary mixed emulsion; preheating the obtained primary mixed emulsion (65 deg.C), homogenizing (20Mpa for 2 times), sterilizing (121 deg.C, 15min), and cooling (40 deg.C) to obtain sterile mixed emulsion; inoculating a starter into the obtained mixed emulsion under aseptic conditions, and fermenting (33 deg.C, 48 hr) and after-ripening (4 deg.C, 24 hr) to obtain black tea fermented emulsion base material; adding the sterilized secondary mixed solution with the same volume as the black tea fermented milk base material into the black tea fermented milk base material, uniformly stirring, homogenizing (20Mpa for 2 times), aseptically filling, and cooling (4 ℃) to obtain the black tea fermented milk beverage.
The mass volume ratio (g/mL) of the defatted milk powder to the black tea soup is 12%, the mass volume ratio (g/mL) of the fructose syrup to the black tea soup is 3%, and the mass volume ratio (g/mL) of the glucose syrup to the black tea soup is 3%;
the secondary preparation liquid is prepared by the following method: adding white granulated sugar, sodium carboxymethylcellulose, sodium alginate propylene glycol ester and sodium citrate into water as a solvent, uniformly stirring, sterilizing and cooling to obtain the sodium alginate-propylene glycol ester-sodium alginate-sodium citrate-sodium alginate-sodium citrate; the mass percentage concentration of the white granulated sugar in the dark green tea fermented milk beverage is 8-10%, the mass percentage concentration of sodium carboxymethylcellulose (CMC) in the dark green tea fermented milk beverage is 0.07-0.09%, the mass percentage concentration of sodium alginate propylene glycol ester (PGA) in the dark green tea fermented milk beverage is 0.08-0.1%, and the mass percentage concentration of sodium citrate in the dark green tea fermented milk beverage is 0.1-0.15%.
The starter is a bacterial suspension (5 multiplied by 10) of lactobacillus casei AU90778CFU/mL), the inoculum size was 3%.
The high fructose corn syrup, the glucose syrup, the white granulated sugar, the CMC, the PGA and the sodium citrate are all food grade.
(4) Determination of desensitization and antioxidant capacity of dark green tea fermented milk beverage
Preparation of neutral dark tea fermented milk beverage (HNM): centrifuging the prepared black tea fermented milk beverage for 10min under the aseptic condition of 10000 r/min at 4 ℃, collecting supernatant, adjusting the pH value neutrality by using an aseptic 1% NaOH solution to obtain aseptic HNM, and temporarily storing the aseptic HNM at 4 ℃ for later use.
Evaluation of desensitization
RBL-2H3 cell culture: rapidly thawing rat basophilic leukemia cells (RBL-2H3) preserved by liquid nitrogen in water at 37 ℃, centrifuging at 800r/min for 5min, removing frozen stock solution, adding 1mL of DMEM (high-sugar) culture medium for basic suspension, transferring into a cell culture bottle, immediately sucking 5-6 mL of DMEM culture medium (complete culture medium for short) containing 10% FBS, and culturing at 37 ℃ and 5% CO2The carbon dioxide incubator of (1). The liquid is changed every 1 day, and when the cells grow to more than 90% of the bottle wall, the cells are passaged, and the related experiments are carried out after three successive passages.
Establishing an RBL-2H3 degranulation model: RBL-2H3 cells after three passages and in vigorous growth stage were digested into cell suspension (1.0X 10)5cells/mL), inoculating 1mL of suspension into each well in a 6-well plate, adding 3mL of complete culture medium, and when the cells grow to a monolayer, performing the test, and dividing the test into a test group (HNM group), a model group (M group) and a blank group (N group) according to the test scheme; the cell was modeled as follows:
sensitization: the cultured RBL-2H3 cells were removed from the culture medium and grouped according to protocol requirements. Then 1mL of complete medium containing anti-DNP-IgE (0.5. mu.L/mL) was added to both HNM group and M group, and the N group was replaced with the same amount of complete medium; at 37 ℃ with 5% CO2The culture was carried out overnight in an incubator.
Sample treatment: overnight sensitized RBL-2H3 cells were removed from the culture medium. Then, 1mL HNM was added to the HNM group, and the M and N groups were replaced with equal amounts of complete medium in CO2The culture box is used for treating for 1 h.
Excitation: the sample-treated RBL-2H3 cells were removed from the culture medium and washed 2 times with PBS. Then, complete medium containing DNP-BSA (0.25. mu.g/mL) was added to both HNM and M groups, and the N group was replaced with an equal amount of complete medium. Collecting the supernatant or cells at different excitation time points,after DNP-BSA is excited for 0.5H, the inhibition of the fermented dark tea milk on the release of RBL-2H3 cell beta-aminocaprosaminidase (beta-HEX) is measured, a culture solution is obtained and centrifuged at 1000 r/min and 4 ℃ for 5min, and a supernatant is reserved for later use. Adding the supernatant (50. mu.L/well) into a 96-well culture plate, adding 50. mu.L of developing solution into each well, incubating at 37 deg.C for 1h, and adding 200. mu.L of stop buffer (0.1mol/L Na)2CO3/NaHCO2pH of 11.0), and determining OD by an enzyme-linked immunosorbent assay406nmThe value is obtained. The calculation formula for the beta-HEX release inhibition rate is as follows:
③ evaluation of antioxidation ability
And (3) determining the Total antioxidant capacity (T-AOC) and the hydroxyl free radical scavenging capacity of the black tea fermented milk by referring to the kit specification. The method for measuring the DPPH free radical scavenging capacity comprises the following steps: adding 1mL of a sample to be detected (HNM) and 1mL of a 0.2mM/L DPPH ethanol solution (prepared by absolute ethyl alcohol, stored in a dark place at 4 ℃ and used as the preparation at present), uniformly mixing, reacting for 30min in a dark place at room temperature, and measuring the absorbance of the solution at 517 nm; replacing 1mL of DPPH ethanol solution with 1mL of absolute ethanol to obtain a blank group; 1mL of PBS buffer solution is used as a control group instead of 1mL of the sample to be detected, and the blank is zeroed by using a mixed solution of 1mL of PBS buffer solution and absolute ethyl alcohol. Each sample was repeated 3 times and averaged. The calculation formula is as follows:
in the formula: a. thesAbsorbance of the sample set, AbAbsorbance of blank set, Ac-control absorbance.
The dark green fermented milk beverage prepared by the method is uniform in color and is reddish brown; the sour and sweet taste is palatable, is smooth and not greasy, has the characteristics of rich tea aroma and yoghourt flavor, and is suitable for the requirements of most consumers; as can be seen from Table 11, the fermented milk beverage containing dark green tea significantly inhibited degranulation of RBL-2H3 cells; and the total antioxidant capacity, the hydroxyl free radical scavenging capacity and the DPPH free radical scavenging capacity are high. Therefore, the black tea fermented milk prepared from the lactobacillus casei AU9077 has certain desensitization and oxidation resistance.
TABLE 11 beta-HEX inhibition results and antioxidant Activity of dark tea fermented milk beverages
CK, which indicates that no dark tea is added.
Example 5 preparation of fermented milk with Asparagus
(1) Activation and culture of bacterial species
Inoculating the liquid of Lactobacillus casei AU9077 frozen at-80 deg.C into MRS liquid culture medium, culturing at 37 deg.C for 24 hr, 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 determining viable count and OD of the strain600nmThe value is obtained.
(2) Preparation of asparagus extract
Dissolving freeze-dried asparagus powder in water, extracting in 60 deg.C water bath for 2h, vacuum filtering, collecting filtrate, pre-freezing at-80 deg.C for 6h, quickly transferring the sample into vacuum freeze-drying machine, and freeze-drying for more than 24h to obtain asparagus extract. The mass volume ratio of the asparagus powder to the water is 1: 40.
(3) The asparagus fermented milk is prepared by the following specific steps:
mixing and stirring the skim milk powder, the asparagus extract, the high fructose corn syrup, the glucose syrup and water uniformly at 65 ℃ to obtain reconstituted milk, preheating (65 ℃), homogenizing (20Mpa for 2 times), sterilizing, cooling (40 ℃), inoculating a fermentation agent, and after-ripening (4 ℃, 24 hours) to obtain the asparagus fermented milk.
The mass-volume ratio (g/mL) of the defatted milk powder to the reconstituted milk is 12%, the mass-volume ratio (g/mL) of the asparagus extract to the reconstituted milk is 0.5%, the mass-volume ratio (g/mL) of the high fructose corn syrup to the reconstituted milk is 3%, and the mass-volume ratio (g/mL) of the glucose syrup to the defatted reconstituted milk(g/mL) is 3%, the sterilization temperature is 121 ℃, the sterilization time is 20min, and the leavening agent is a bacterial suspension (5 multiplied by 10) of lactobacillus casei AU90778CFU/mL), the inoculation amount is 3%, the fermentation temperature is 33 ℃, and the fermentation time is 48 h.
(4) Determination of desensitization and bacteriostatic ability of asparagus fermented milk
Preparation of neutral asparagus fermented milk (LNM): centrifuging the prepared fermented asparagus milk for 5min at 4 ℃ and 8000 r/min under aseptic conditions, collecting supernatant, adjusting pH to neutral with sterile 1% NaOH solution to obtain sterile LNM, and temporarily storing at 4 ℃ for later use.
Evaluation of desensitization
The measurement was carried out in accordance with the method in example 4
③ evaluation of bacteriostatic ability
The bacteriostatic activity is measured by an Oxford cup method, the bacteriostatic ability of the strain is expressed by the diameter of a bacteriostatic circle, and escherichia coli and listeria monocytogenes 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. As shown in table 12, the number of viable bacteria and the titer acidity of asparagus fermented milk increased with the increase of the amount of the asparagus extract, indicating that asparagus can promote the growth of the bacterial strain. In addition, as shown in table 13, asparagus fermented milk supernatant was able to also significantly inhibit degranulation of RBL-2H3 cells; and can inhibit common pathogenic bacteria (Escherichia coli and Listeria). Therefore, the asparagus fermented milk prepared by the lactobacillus casei AU9077 has a certain desensitization effect. With the increasing of the adding amount of the asparagus extract, the total antioxidant capacity, the hydroxyl radical scavenging capacity and the DPPH radical scavenging capacity of the asparagus fermented milk supernatant are gradually improved, which indicates that the asparagus fermented milk prepared by the 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 asparagus fermented milk
Sequence listing
<110> Australian milk industry (China) Co Ltd, Hunan university of agriculture
<120> lactobacillus casei and application thereof
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Claims (10)
1. The Lactobacillus casei is characterized in that the taxonomic name of the Lactobacillus casei is Lactobacillus casei (AU 9077) which is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the culture preservation number of CGMCC No. 21663.
2. Use of lactobacillus casei as claimed in claim 1 in the manufacture of a medicament for the reduction and/or prevention of allergic disease.
3. Use of lactobacillus casei as claimed in claim 1 for the preparation of a food product having desensitizing effects.
4. The use according to claim 3, wherein the food is a dark tea fermented milk beverage and/or an asparagus fermented milk.
5. Method for preparing a dark tea fermented milk beverage with desensitizing effect and/or antioxidant activity using lactobacillus casei according to claim 1, characterized in that it comprises the following steps:
(1) boiling the dark tea in water, filtering and cooling to prepare dark tea soup for later use;
(2) dissolving the skim milk powder, the high fructose corn syrup and the glucose syrup in the dark tea soup for primary blending, and fully and uniformly stirring to obtain a primary mixed emulsion; the mass volume ratio of the defatted milk powder to the black tea soup is 10-12%, the mass volume ratio of the high fructose corn syrup to the black tea soup is 2.5-3%, the mass volume ratio of the glucose syrup to the black tea soup is 2.5-3%, and the unit of the mass volume ratio is g/mL;
(3) preheating the primary mixed emulsion, homogenizing, sterilizing and cooling to obtain sterile mixed emulsion;
(4) under the aseptic condition, inoculating lactobacillus casei into the aseptic mixed emulsion according to the inoculation amount of 2-4%, fermenting for 44-48 h at 32-35 ℃, and performing after-ripening fermentation for 12-24 h at 4 ℃ to obtain the black tea fermented milk base material;
(5) adding the sterilized secondary blending liquid which is equal to the volume of 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 the black tea fermented milk beverage; the secondary preparation liquid is prepared by the following method: adding white granulated sugar, sodium carboxymethylcellulose, sodium alginate propylene glycol ester and sodium citrate into water serving as a solvent, uniformly stirring, sterilizing and cooling to obtain the sodium alginate-containing calcium carbonate; the mass percentage concentration of the white granulated sugar in the dark green tea fermented milk beverage is 8-10%, the mass percentage concentration of the sodium carboxymethylcellulose in the dark green tea fermented milk beverage is 0.07-0.09%, the mass percentage concentration of the sodium alginate propylene glycol ester in the dark green tea fermented milk beverage is 0.08-0.1%, and the mass percentage concentration of the sodium citrate in the dark green tea fermented milk beverage is 0.1-0.15%.
6. The method according to claim 5, wherein the mass-to-volume ratio of the water to the dark tea in the step (1) is 1:100 to 1:150, and the unit of the mass-to-volume ratio is g/mL.
7. The method according to claim 5, wherein the homogenization conditions in the steps (3) and (5) are 20 to 25MPa for 2 to 3 times; and (5) sterilizing the secondary preparation solution at 80 ℃ for 15 min.
8. Method for preparing asparagus fermented milk with desensitizing effect and/or antioxidant activity using lactobacillus casei according to claim 1, characterized in that it comprises the following steps:
(1) preparation of asparagus extract: dissolving freeze-dried asparagus powder in water, extracting in a water bath at 60-65 ℃ for 1.5-2.5 h, performing suction filtration, collecting filtrate, pre-freezing at-80 ℃ for 5-7 h, and quickly freeze-drying in a vacuum freeze-drying machine for more than 24h after pre-freezing 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 high fructose corn syrup, the glucose syrup and water uniformly 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 high fructose corn syrup to the reconstituted milk is 2.5-3%, the mass-volume ratio of the glucose syrup to the skim reconstituted milk is 2-4%, and the unit of the mass-volume ratio is g/mL; preheating the reconstituted milk at 65-75 ℃, homogenizing, sterilizing and cooling, inoculating lactobacillus casei to the reconstituted milk according to the inoculation amount of 2-4%, fermenting for 44-48 h at 32-35 ℃, and performing after-ripening fermentation for 12-24 h at 4 ℃ to obtain the asparagus fermented milk.
9. The method of claim 8, wherein the mass-to-volume ratio of asparagus powder to water in step (1) is 1:10 to 1:40 in g/mL.
10. The method according to claim 8, wherein the homogenization conditions in step (2) are 20 to 25MPa for 2 to 3 times.
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| CN101144065A (en) * | 2007-09-03 | 2008-03-19 | 江南大学 | Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof |
| CN106399162A (en) * | 2016-09-08 | 2017-02-15 | 济南康多宝生物技术有限公司 | Novel lactobacillus casei and application thereof |
| US20210128648A1 (en) * | 2017-03-23 | 2021-05-06 | Ku Leuven Research & Development | A novel probiotic lactobacillus casei strain and its uses |
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| CN101144065A (en) * | 2007-09-03 | 2008-03-19 | 江南大学 | Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof |
| CN106399162A (en) * | 2016-09-08 | 2017-02-15 | 济南康多宝生物技术有限公司 | Novel lactobacillus casei and application thereof |
| US20210128648A1 (en) * | 2017-03-23 | 2021-05-06 | Ku Leuven Research & Development | A novel probiotic lactobacillus casei strain and its uses |
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