CN111281895A

CN111281895A - Lactic acid bacteria for treating colitis and application thereof

Info

Publication number: CN111281895A
Application number: CN201911341928.5A
Authority: CN
Inventors: 顾青; 顾容铖; 郦萍; 刘娜娜
Original assignee: 顾青
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2020-06-16
Also published as: CN114657083A

Abstract

The invention discloses a lactic acid bacterium for treating colitis and application thereof, wherein the lactic acid bacterium for treating colitis comprises lactobacillus plantarum ZJ316, and the lactobacillus plantarum ZJ316 can treat colitis safely, effectively and with small side effect.

Description

Lactic acid bacteria for treating colitis and application thereof

Technical Field

The invention relates to the field of treatment of colitis, and further relates to a lactic acid bacterium for treating colitis and application thereof.

Background

Ulcerative Colitis (UC) is an inflammatory disease of the intestinal tract with a complex etiology and a risk of concurrent colon cancer caused by allergy and physicochemical factors, and is classified into acute Ulcerative Colitis and chronic Ulcerative Colitis according to the course of the disease. The lack of effective treatment measures for a long time results in not only serious harm to human health, but also double stress and burden on life and economy of patients.

The amino salicylic acid mainly comprises mesalamine (5-aminosalicylic acid) and sulfasalazine, which are the first choice drugs for treating mild and moderate colitis, wherein the mesalamine and sulfasalazine can inhibit the expression of proinflammatory cytokines such as IL-1 β and TNF- α, inhibit lipoxygenase pathways, remove free radicals and oxides and inhibit the activation of NF-kB, but the drugs can cause complications such as headache, nausea and the like of patients commonly used, antibiotics can promote the increase of beneficial bacteria quantity, reduce the proportion of pathogenic bacteria, although the pathogenic antibiotics are effective in regulating microecological dysregulation of colitis, the disadvantage that the glucocorticoid can inhibit inflammation and immune response, the important effect of the glucocorticoid can inhibit inflammation and immune response, and the important effect of the glucocorticoid can be achieved by using a large amount of antibiotics to treat severe acute colitis, acute pyogenic infection, acute hemorrhage, and severe hemorrhage, and the side-toxic edema of patients can be caused by using a large amount of muco-inflammatory, the muco-inflammatory diseases caused by using a large amount of muco-inflammatory, the antibiotic can be used for treating patients with severe acute pyogenic infection, acute colitis, acute pyogenic infection, acute edema, acute pyogenic infection, acute edema caused by using a large amount of mucosis, acute pyogenic infection, acute edema caused by using a severe tuberculosis, and the acute pyogenic infection caused by using a large amount of mucosis, and the acute pyogenic infection caused by using the muco, the acute pyogenic infection, and the acute pyogenic infection.

The research of Shen Z.H and other people finds that the lactic acid bacteria can reduce the transcription of the level of proinflammatory cytokines such as tumor necrosis factor (TNF- α) and interleukin 1(IL-1 β) through a monokine NF-kB signal pathway and up-regulate the expression of the anti-inflammatory factor IL-10 so as to improve the symptoms of IBD.

Riley S.A and others find that some lactobacillus in lactobacillus can generate small molecular substances involved in immune regulation, and the small molecular substances can induce the expression of anti-inflammatory cytokines such as IL-10 and the like, so that the small molecular substances have an anti-inflammatory effect on UC patients. In addition, when lactic acid bacteria enter the intestinal tract, they interact with other microbial communities and bind to each other, thereby affecting metabolism and exerting a probiotic effect. For example, it has been found that bifidobacteria can metabolize carbohydrates through specific fermentation pathways to produce other metabolites such as acetic acid in varying proportions, thereby slowing down colitis. It has been reported that the anti-inflammatory cytokine IL-10 is increased, while the proinflammatory cytokines IL-2 and IL-12 are significantly decreased in mice perfused with Lactobacillus delbrueckii, subspecies bulgaricus and Lactobacillus casei. In addition, experiments have proved that lactic acid bacteria can change the flora structure of Irritable Bowel Syndrome (IBS) patients, reduce the abundance of harmful bacteria and increase the diversity of probiotics. Although the efficacy of probiotics in inflammatory diseases such as IBD has been demonstrated, the mechanism is not completely determined and a great deal of research is still needed to elucidate the mechanism.

In conclusion, the existing treatment methods are not satisfactory and suitable for long-term treatment. Therefore, further research into colitis pathogenesis and providing safe and effective treatment to prevent and alleviate colitis is imminent.

Disclosure of Invention

An object of the present invention is to provide a lactic acid bacterium for treating colitis and use thereof, wherein the lactic acid bacterium for treating colitis is lactobacillus plantarum ZJ316, which can treat colitis safely, effectively, with little side effect.

The invention aims to provide a lactobacillus for treating colitis and application thereof, wherein the lactobacillus plantarum ZJ316 can improve the DSS-induced colitis in mice.

One aspect of the present invention provides a lactic acid bacterium for treating colitis, comprising: lactobacillus plantarum ZJ 316.

The lactic acid bacterium for use in the treatment of colitis according to an embodiment, wherein said Lactobacillus plantarum ZJ316 is selected from infant faeces.

The lactic acid bacterium for use in the treatment of colitis according to one embodiment, wherein said Lactobacillus plantarum is used in the treatment of colitis in acute and chronic colitis mice induced by DSS.

Another aspect of the invention provides the use of lactobacillus plantarum ZJ316, or a pharmaceutical composition comprising fermented milk thereof, for the manufacture of a medicament for the treatment or prevention of colitis.

The use according to one embodiment, wherein the lactobacillus plantarum ZJ316 is used for down-regulating the expression level of proinflammatory cytokines in the colon of a DSS colitis mouse and up-regulating the expression level of inflammatory cytokines.

According to one embodiment, when preparing the fermented milk, a certain amount of fresh cow milk is measured, 6% of lactobacillus plantarum ZJ316 bacterial suspension prepared in advance is added, 7% of white granulated sugar is added, and the mixture is placed in a yogurt machine to be fermented for 8-12 hours after being uniformly stirred.

The use according to one embodiment, wherein in the culturing of lactobacillus plantarum ZJ316, lactobacillus plantarum ZJ316 frozen in glycerol tubes at-80 ℃ is streaked on MRS solid plate, is recovered by anaerobic culture at 37 ℃ for 24h, and is activated by two passagesTransferring to MRS liquid, culturing bacteria liquid overnight for 16-18h, centrifuging at 6000r/min for 10min, collecting fermentation supernatant, washing bacterial sludge with 0.85% sterilized normal saline twice, and resuspending with normal saline to obtain bacterial suspension with viable count of about 2.5 × 10⁹CFU/mL。

Another aspect of the present invention provides a lactic acid bacteria fermented milk for treating colitis, comprising: lactobacillus plantarum ZJ316 fermented milk is obtained by fermenting Lactobacillus plantarum ZJ 316.

The lactobacillus fermented milk for treating colitis according to an embodiment, wherein the lactobacillus plantarum ZJ316 fermented milk is used to promote an increase in the content of acetic acid, propionic acid, isobutyric acid, butyric acid, and valeric acid in the colon of DSS colitis mice.

According to one embodiment, the lactobacillus fermented milk for treating colitis is prepared by measuring a certain amount of fresh milk, adding 6% of lactobacillus plantarum ZJ316 bacterial suspension prepared in advance, adding 7% of white granulated sugar, stirring uniformly, and fermenting in a yogurt machine for 8-12 hours.

Drawings

FIG. 1 shows the survival rate of Lactobacillus plantarum ZJ316 treated with NaCl at various concentrations.

FIG. 2 shows the survival rate of Lactobacillus plantarum ZJ316 treated with ethanol at various concentrations.

FIG. 3 shows the survival rate of Lactobacillus plantarum ZJ316 at different bile salt concentrations.

FIG. 4 shows the survival rate of Lactobacillus plantarum ZJ316 in artificial gastric juice at various pH.

Figure 5 shows the body weight dynamics of mice in different groups of the acute group.

Figure 6 shows the body weight dynamics of different groups of mice in the chronic group.

Figure 7 illustrates the effect of treatment groups on colon length in mice for the acute group.

FIG. 8 shows the effect of treatment groups on colon length in mice for the chronic group.

Figure 9 illustrates the effect of treatment groups on colon weight in mice in the acute group.

FIG. 10 shows the effect of treatment groups on colon weight in mice in the chronic group.

FIGS. 11A-11B show the H & E staining results of the colon of mice in each treatment group of the acute group.

Figure 12 illustrates the acute group tissue damage score for each treatment group.

FIGS. 13A-13C show H & E staining results for colon of mice in each treatment group of the chronic group.

Fig. 14 shows the tissue damage scores for each treatment group for the chronic group.

FIGS. 15A-15D are graphs showing colonic inflammatory cytokine transcription levels in mice from each treatment group of the acute group.

FIGS. 16A-16E are graphs showing the effect of treatment groups on mouse colon cytokine transcription levels in the chronic group.

FIGS. 17A-17E are graphs showing short chain fatty acid content in the colon of mice in each treatment group of the acute group.

FIGS. 18A-18E are graphs showing short chain fatty acid content in the colon of mice from each treatment group of the chronic group.

FIG. 19 illustrates a sample sequencing dilution curve.

FIG. 20 is a graph showing the distribution of the mouse colonic microflora at the "portal" level.

FIG. 21 is a graph showing the distribution of the mouse colonic microflora at the "genus" level.

FIG. 22 illustrates α diversity index.

FIGS. 23A-23B illustrate PCoA analysis.

FIGS. 24A-24B illustrate acute group LEfse analysis.

FIGS. 25A-25B are schematic illustrations of chronic group LEfse analysis.

Detailed Description

The following description is presented to disclose the invention so as to enable any person skilled in the art to practice the invention. The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art. The basic principles of the invention, as defined in the following description, may be applied to other embodiments, variations, modifications, equivalents, and other technical solutions without departing from the spirit and scope of the invention.

The invention provides a lactobacillus for treating colitis and application thereof, wherein the lactobacillus for treating colitis is lactobacillus plantarum screened from infant feces and named lactobacillus plantarum ZJ316(Lactobacillus plantarum ZJ316), and the lactobacillus plantarum ZJ316 is a deposited strain.

1. Environmental stress resistance and intestinal tolerance of lactobacillus plantarum ZJ316

The physiological functions of lactic acid bacteria are closely related to the vital activities of the body, and thus are widely used in the food processing industry to produce a great variety of delicious and healthy foods, but an ideal lactic acid bacteria strain with industrial application potential must be safe and reliable and be able to resist harsh conditions such as high concentration ethanol and high osmotic pressure during fermentation. The gastrointestinal tract of animals is one of the main sites where microorganisms exist, and lactic acid bacteria must be able to overcome the strong acid and bile salt environment of the digestive system of the gastrointestinal tract of animals to exert their probiotic effects. The pH of the gastric acid of the animal can reach 1.5 at the lowest, but after eating, the pH can be rapidly increased to even exceed 6.0, but the pH is usually between 2.5 and 3.5, and the digestion time of the food is 1.5 to 4.0 hours. The mass fraction of bile salts in different parts of the digestive tract of the animal body is different, but is usually 0.03-0.30%. The lactobacillus can play its physiological function only after passing through various physiological barriers in human and animal bodies and maintaining a higher survival rate of the lactobacillus in the gastrointestinal tract environment, thereby effectively improving the intestinal health of human and animal and balancing the intestinal flora. The acid resistance and the bile salt resistance of the lactic acid bacteria are two important indexes of resisting the intestinal environment, and the survival number of the strains in the gastrointestinal digestive tract is directly influenced by the tolerance capacity of the lactic acid bacteria.

1.1 test strains

The strain used in the experiment is lactobacillus plantarum ZJ 316. This strain was the deposited strain.

1.2 Main reagents and consumables

Peptone, yeast extract, anhydrous glucose, diammonium hydrogen citrate, beef extract, dipotassium hydrogen phosphate, sodium acetate, sodium chloride, anhydrous ethanol, magnesium sulfate heptahydrate, manganese sulfate, tween 80 and bile salt are all purchased from Hangzhou evergreen chemical Co.

Pepsin was purchased from kaihe lake biotechnology limited.

The 0.22 mu m water system/organic microporous filtering membrane is purchased from Hangzhou evergreen chemical industry Co.

1.3 laboratory instruments and apparatus

TABLE 1-1 Main instruments and Equipment

1.4 preparation of culture Medium and related solutions

1.4.1 preparation of the culture Medium

Preparation of MRS liquid culture medium: accurately weighing 10g of peptone, 5g of yeast extract, 20g of anhydrous glucose, 10g of beef extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate and 1mL of Tween-80, dissolving with ultrapure water, fixing the volume to 1L, and sterilizing at 121 ℃ for 15 min.

Preparation of MRS solid culture medium: adding 1-1.5% agar based on MRS liquid culture medium formula, dissolving with ultrapure water, metering to 1L, and sterilizing at 121 deg.C for 15 min.

MRS culture medium containing 3%, 6% and 8% NaCl is prepared: respectively adding 30g, 60g and 80g NaCl on the basis of a formula of a common MRS liquid culture medium, dissolving with ultrapure water, metering to 1L, and sterilizing at 121 ℃ for 15 min.

MRS liquid medium formulation with 2.5%, 5%, 7.5%, 10% and 12.5% ethanol (98%): after sterilization, 25mL, 50mL, 75mL, 100mL and 125mL of ethanol (98%) are added into the ordinary MRS culture medium respectively to obtain ethanol MRS culture medium with corresponding concentration.

0.1%, 0.3% and 0.5% bile salt MRS culture medium preparation: on the basis of a formula of a common MRS liquid culture medium, 1g, 3g and 5g of NaCl are respectively added, dissolved by ultrapure water and subjected to constant volume to 1L, and the solution is sterilized for 15min at 121 ℃.

1.4.2 preparation of other solutions

0.1mol/L PBS solution: 80g of NaCl, 2.2g of KCl and 9.9g of Na are accurately weighed₂HPO₄，2.0g KH₂PO₄800mL of H was added₂Dissolving O completely, adjusting pH to 7.4 with HCl, diluting to 1L, and sterilizing at 121 deg.C for 15 min.

Preparing artificial gastric juice: accurately weighing 1g of pepsin, adding the pepsin into 100mL of ultrapure water, stirring uniformly, fully dissolving, subpackaging into four blue-mouth bottles, respectively adjusting the pH to 2.0, 2.5, 3.0 and 3.5, and filtering and sterilizing by using a filter membrane.

1.5 Experimental methods and content

1.5.1 Strain activation and culture

Streaking and inoculating lactobacillus plantarum ZJ316 frozen at minus 80 ℃ to an MRS solid plate, performing anaerobic culture at 37 ℃ for 24h for recovery, transferring MRS liquid after two passage activation, culturing bacterial liquid overnight for 16-18h, centrifuging at 6000r/min for 10min, discarding fermentation supernatant, washing bacterial sludge twice with sterile physiological saline (0.85%), and then re-suspending with the same volume of sterile physiological saline buffer.

1.5.2 determination of osmotic pressure resistance of Strain

After the strain is cultured for 16-18h in liquid, the strain is centrifuged at 6000r/min for 10min at 4 ℃, and bacterial sludge is resuspended by equal volume of sterilized normal saline after being washed twice by the sterilized normal saline (0.85%). Taking 100 mu L of the resuspended thallus suspension, inoculating the thallus suspension into MRS culture media containing NaCl with different concentrations, culturing for 24h at 37 ℃, taking 50 mu L of culture solution under the aseptic condition, diluting with sterile water in a gradient manner, coating on an MRS solid culture medium plate, culturing at 37 ℃ until bacterial colonies obviously appear, accurately counting, making three parallel in each group, and using a strain cultured by the MRS culture media without NaCl as a control.

1.5.3 determination of ethanol resistance of Strain

After the strain is cultured for 16-18h in liquid, the strain is centrifuged at 6000r/min for 10min at 4 ℃, and bacterial sludge is washed twice by sterile normal saline (0.85 percent) and then resuspended by the same volume of the sterile normal saline (0.85 percent). Taking 100 mu L of the resuspended thallus suspension, inoculating the suspension into MRS liquid culture media with ethanol concentrations of 2.5%, 5%, 7.5% and 10%, culturing for 24h in an incubator at 37 ℃, taking 50 mu L of the culture solution under aseptic condition, diluting with sterile water in a gradient manner, coating on an MRS solid culture medium plate, culturing at 37 ℃ until bacterial colonies obviously appear, accurately counting, and making three parallel in each group, and using the strain cultured by the MRS culture media without ethanol as a control.

1.5.4 determination of artificial gastric pancreatic juice resistance of strain

After the strain is cultured for 16-18h in liquid, the strain is centrifuged at 6000r/min for 10min at 4 ℃, and bacterial sludge is washed twice by sterile normal saline (0.85 percent) and then resuspended by the same volume of the sterile normal saline to prepare a bacterial suspension. Respectively inoculating 1mL of thallus suspension into artificial gastric juice with pH of 2.0, 2.5, 3.0 and 3.5, mixing well, and culturing in biochemical incubator at 37 deg.C. Sampling 50 μ L at 0h, 2h and 4h respectively, diluting with sterile water, coating on MRS solid culture medium plate, culturing at 37 deg.C until bacterial colony appears obviously, counting accurately, making each group in three parallel, and using strain without artificial gastric juice treatment as control.

1.5.5 determination of the bile salt resistance of the Strain

Inoculating lactobacillus plantarum ZJ316 frozen at minus 80 ℃ on an MRS solid plate in a streak manner, performing anaerobic culture at 37 ℃ for 24h for resuscitation, transferring MRS liquid after two passage activation, culturing bacterial liquid overnight for 16-18h, respectively inoculating the bacterial liquid into MRS liquid culture media containing bile salts with different concentrations according to the inoculation amount of 3% (v/v), and culturing in a biochemical incubator at 37 ℃. Sampling 50 mu L in 0h, 2h and 4h respectively, diluting with sterile water in a gradient manner, coating on an MRS solid culture medium plate, culturing at 37 ℃ until bacterial colonies obviously appear, accurately counting, making three parallels in each group, and using a culture medium without bile salt as a control.

1.6 results and analysis

1.6.1 osmotic pressure resistance of Lactobacillus plantarum ZJ316

High osmotic pressure resistance is one of the prerequisites for the commercial use of lactic acid bacteria strains. During the fermentation process, the strain produces lactic acid, increases the osmotic pressure of cells, converts free acid into salt and can prevent the pH value in the system from being excessively reduced. As can be seen from FIG. 1, the survival rates of Lactobacillus plantarum ZJ316 at NaCl concentrations of 3%, 6% and 8% were 89.03%, 69.53% and 18.44%, respectively, indicating that Lactobacillus plantarum ZJ316 can survive in high osmotic pressure environments and has a high survival rate, which is similar to the osmotic pressure resistance of most LAB strains reported by Masuda et al.

1.6.2 ethanol tolerance of Lactobacillus plantarum ZJ316

In industrial fermentation and food processing, bacteria must overcome various physical and chemical barriers to exert their effects, and like high osmotic pressure resistance, the tolerance of lactic acid bacteria to ethanol is also often used as one of the indicators for evaluating their industrial applications. As shown in fig. 2, the survival rate of lactobacillus plantarum ZJ316 gradually decreases with increasing ethanol concentration, and when the ethanol concentration is 5%, the survival rate of lactobacillus plantarum ZJ316 is as high as 71%, and when the ethanol concentration is 10%, the survival rate of lactobacillus plantarum ZJ316 is still maintained at about 40%, thereby indicating that the ethanol tolerance of lactobacillus plantarum ZJ316 is good. The study by Masuda et al found that most LAB strains were very poorly tolerated and have a very low survival rate at ethanol concentrations above 5%.

1.6.3 bile salt tolerance of Lactobacillus plantarum ZJ316

The precondition for lactic acid bacteria to exert their probiotic effect in the digestive tract of animals is their ability to tolerate gastric acid and bile salts and colonize the gastrointestinal tract. The mass fraction of bile salts varies from one part of the digestive tract to another in the animal, but is usually in the range of 0.03% to 0.30%, and the colonization and metabolic activity of intestinal bacteria of the host is usually 1 to 4 hours, and bile salts affect the intestinal flora by their antibacterial activity. As shown in FIG. 3, after the treatment of 0.3% bile salt for 2h and 4h, the survival rate of Lactobacillus plantarum ZJ316 was 81% and 73%, respectively, and after the treatment of 0.5% bile salt for 2h and 4h, the survival rate of Lactobacillus plantarum ZJ316 was still high, 62% and 48%, respectively. Therefore, when the mass fraction of the bile salts is within the range of 0.03-0.30%, the lactobacillus plantarum ZJ316 keeps high survival rate, which indicates that the lactobacillus plantarum ZJ316 has strong bile salt resistance.

1.6.4 gastric acid resistance of Lactobacillus plantarum ZJ316

Gastric juice is considered to be the primary physiological challenge for lactic acid bacteria due to the low pH profile and antibacterial action of pepsin, and furthermore, acid resistance is an important condition for the selection of probiotics. The pH of the gastric acid of the animal can reach 1.5 at the lowest, and when food is ingested, the pH can be rapidly increased to even exceed 6.0, but the pH is between 2.5 and 3.5 under the normal condition. As can be seen from FIG. 4, when the pH was in the range of 2.0 to 3.5, the survival rate of the strain increased as the pH increased. The survival rate of the strain was still relatively high at 53% and 47% when treated in a pH 2 environment for 2h and 4h, respectively. The lactobacillus plantarum ZJ316 keeps high survival rate in the environment with pH of 3.5, the survival rate is 96% after 2h incubation, and the survival rate is 92% after 4h incubation. Therefore, lactobacillus plantarum ZJ316 can tolerate gastric acid and can enter the intestinal tract through the stomach. This is consistent with the results mentioned in the previous report that most lactobacilli show good survival at pH3.5, but less viable at pH 2.

Lactic acid bacteria can only ensure their value in industry if they overcome harsh external environments. Through in vitro simulation environmental stress resistance experiments (salt tolerance experiments and ethanol tolerance experiments), the lactobacillus plantarum ZJ316 still maintains a high survival rate in the environment of high salt (8%) and high concentration ethanol (12.5%), namely the lactobacillus plantarum ZJ316 has strong environmental stress resistance, and a certain foundation is laid for industrial utilization. The survival rates of the animal are respectively 62% and 48% after the animal digestive tract simulation experiment shows that after the animal digestive tract; after the lactobacillus plantarum ZJ316 is incubated for 2 hours in artificial gastric juice (pH3.5), the survival rate is 96%, and after the lactobacillus plantarum ZJ is incubated for 4 hours, the survival rate is 92%, so that the lactobacillus plantarum ZJ316 has good acid resistance and bile salt resistance.

In conclusion, lactobacillus plantarum ZJ316 has good environmental stress resistance and can be selected as an industrially used strain. Meanwhile, the lactobacillus plantarum ZJ316 can overcome the harsh environment of the animal digestive tract, and the possibility of generating a probiotic effect in the animal intestinal tract is preliminarily explained.

2. Effect of Lactobacillus plantarum ZJ316 and fermented milk thereof on DSS-induced colitis murine

Researches find that the probiotics can obviously improve the flora disturbance phenomenon in human intestinal tracts, improve the human immunity and protect intestinal mucosa, thereby having very positive significance for relieving and improving the inflammatory bowel diseases.

According to the invention, the intestinal environment tolerance of lactobacillus plantarum ZJ316 is researched, and the intestinal tolerance is found to be good. Based on the result, 180 BALB/c male mice with the age of 8 weeks are selected, DSS acute and chronic colitis models are established to explore the improvement effect of lactobacillus plantarum ZJ316 and fermented milk thereof on ulcerative colitis, and a technical basis is provided for probiotics to control and improve enteritis and related diseases.

The invention mainly evaluates the treatment effect of the lactobacillus plantarum ZJ316 and the fermented milk thereof on the acute and chronic colitis of the mice induced by DSS from four aspects of the influence of the lactobacillus plantarum ZJ316 and the fermented milk thereof on the inflammatory manifestations of the colitis of the mice induced by DSS, the influence of the lactobacillus plantarum ZJ316 and the fermented milk thereof on the transcription levels of inflammatory cytokines (IL-1 β, IL-6, IL-8, IL-10, TNF- α and TGF- β) of the colon of the mice induced by DSS, the influence of the lactobacillus plantarum ZJ316 and the fermented milk thereof on the short-chain fatty acid content (acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid) of the colon of the mice induced by DSS, and the influence of the lactobacillus plantarum ZJ316 and the fermented milk thereof on the intestinal flora of the colon of the mice induced by DSS.

3 Experimental materials and methods

3.2.1 test strains

Lactobacillus plantarum ZJ316(Lactobacillus plantarum ZJ316), Lactobacillus casei Shirata. Both strains were screened from infant faeces and euthanasia.

3.2.2 Experimental animals

The mice used for the experiments were male SPF (Specific pathogen free) grade BALB/c mice 8 weeks old, purchased from Shanghai Sphere-Bikay laboratory animals Co. The mice are raised in an environment with temperature of 25 +/-2 ℃ and day and night alternation of 12h, and are subjected to free diet, and the experiment is carried out after the ordinary diet adapts to one week.

3.2.3 Experimental reagents and consumables

TABLE 3-1 Experimental reagents and consumables

3.2.4 preparation of related solutions

3.2.4.1 preparation of the Medium

3.2.4.2 preparation of other solutions

(1) 2.5% (m/v) Dextran Sodium Sulfate (DSS) solution formulation: 2.5g dextran sodium sulfate was accurately weighed and dissolved in 100mL ultrapure water and shaken with a vortex shaker to dissolve it thoroughly.

(2) Preparing an internal standard solution: taking a proper amount of 2-ethyl butyric acid, and preparing an internal standard solution with the concentration of 10 mu g/mL by using a 6% phosphoric acid solution.

(3) Preparing an acetic acid control solution: 20mg of acetic acid is precisely weighed and diluted by 6 percent phosphoric acid solution to prepare 2mg/mL reference substance stock solution I. And (3) diluting the reference substance stock solution I step by step to prepare a series of standard yeast solutions, precisely taking 1mL of solution to a headspace bottle, adding 1mL of internal standard solution, and sealing.

(4) Propionic acid, butyric acid, isobutyric acid, valeric acid control solutions: accurately measuring a proper amount of propionic acid, isobutyric acid, butyric acid and valeric acid, diluting with 6% phosphoric acid solution to prepare a reference substance stock solution II of 2mg/mL, diluting the reference substance stock solution II step by step to prepare a series of standard solutions, accurately measuring 1mL of solution to a headspace bottle, adding 1mL of internal standard solution, and sealing.

3.2.5 Experimental apparatus and equipment

TABLE 3-2 Experimental instruments and Equipment

3.2.6 Experimental methods and contents

3.2.6.1 cultivation of Lactobacillus plantarum ZJ316, preparation of bacterial suspension and fermentation supernatant.

Inoculating Lactobacillus plantarum ZJ316 frozen in a glycerol tube at the temperature of minus 80 ℃ on an MRS solid plate in a streak manner, performing anaerobic culture at the temperature of 37 ℃ for 24h for resuscitation, transferring to MRS liquid after two passage activation, culturing bacterial liquid overnight for 16-18h, centrifuging at 6000r/min for 10min, collecting fermentation supernatant, washing bacterial sludge twice with sterilized normal saline (0.85 percent), and then re-suspending with normal saline to enable the viable count of bacterial suspension to reach about 2.5 multiplied by 10⁹CFU/mL。

3.2.6.2 cultivation of Lactobacillus casei strain Shirota and preparation of bacterial suspension.

Inoculating Lactobacillus casei strain Shirota frozen in glycerol tube at-80 deg.C to MRS solid plate, performing anaerobic culture at 37 deg.C for 48 hr for resuscitation, activating by passage twice, transferring MRS liquid, culturing bacterial liquid overnight for 18-20 hr, centrifuging at 8000r/min for 15min, removing supernatant, washing bacterial mud with sterilized normal saline (0.85%) twice, and resuspending with normal saline to obtain bacterial suspension with viable count of about 2.5 × 10⁹CFU/mL。

3.2.6.3 preparation of Lactobacillus plantarum ZJ316 fermented milk

Weighing a certain amount of fresh cow milk, adding 6% of lactobacillus plantarum ZJ316 bacterial suspension prepared in advance, adding 7% of white granulated sugar, stirring uniformly, and fermenting in a yogurt machine for 8-12 hours. After fermentation, storing in a refrigerator at 4 ℃ for later use.

3.2.6.4 establishment, grouping and administration of enteritis model.

One week after normal feeding acclimatization, mice were randomly divided into 3 groups:

(1) blank control (Ctrl) (n ═ 10): normal feeding was performed until the end of the experiment.

(2) DSS acute enteritis group (n 95): under normal diet conditions, pure water drunk by the mice is changed into 2.5 percent DSS solution for 7 consecutive days, and the DSS solution is changed every two days in the period, so that the acute colitis model of the mice is established.

(3) DSS chronic enteritis group (n 95): under normal diet condition, the purified water drunk by the mice is changed into 2.5 percent DSS solution for continuous 7 days, and then the purified water is changed back for drinking for 7 days, so that the purified water is taken as a period, three periods are circulated, and a chronic colitis model of the mice is established^[120]。

After the DSS colitis model was successfully established, the acute enteritis group and the chronic enteritis group were divided into 9 groups, and different intervention drugs were administered to the groups for intragastric administration.

1) Acute modeling (JZ) (n is 5): after successful molding, the mice were sacrificed. The colon tissue is taken and stored for standby.

2) Acute normal diet group (JY) (n ═ 10): after the model was successfully created, the mice were fed normally for 5 weeks until the end of the experiment, sacrificed and colon tissue was collected and stored for further use.

3) Acute MRS group (JN) (n ═ 10): after the model is successfully made, the contrast is kept for 5 weeks in 200 muL/10 g of MRS culture medium by gavage, until the experiment is finished, the mice are sacrificed, and colon tissues are taken for storage and use.

4) Acute lactobacillus plantarum ZJ316 fermentation supernatant group (JM) (n ═ 10): after successful modeling, the fermented supernatant of lactobacillus plantarum ZJ316 was inoculated at 200 μ L/10g for 5 weeks as a control, and the mice were sacrificed until the end of the experiment, and the colon tissue was collected and stored for future use.

5) Acute lactobacillus plantarum ZJ316 bacterial suspension group (JX) (n ═ 10): after the molding is successful, the suspension of the lactobacillus plantarum ZJ316 of the gavage is 200 mu L/10g (about 2.5 multiplied by 10)⁹CFU/mL) for 5 weeks until the end of the experiment, mice were sacrificed and colon tissue was removed and stored for future use.

6) Acute lactobacillus plantarum ZJ316 fermented milk group (JH) (n ═ 10): after the molding is successful, lactobacillus plantarum ZJ316 fermented milk 200 mu L/10g is perfused for 5 weeks, until the experiment is finished, the mice are sacrificed, and colon tissues are taken for storage and standby.

7) Acute bovine milk group (JF) (n ═ 10): after successful molding, the cow's milk was gavaged at 200. mu.L/10 g for 5 weeks as a control until the end of the experiment, the mice were sacrificed and the colon tissue was collected and stored for future use.

8) Acute mesalazine group (JG) (n ═ 10): after the model building is successful, gavage (30.25mg/mL) of 200 muL/10 g is used as a positive control and lasts for 5 weeks until the experiment is finished, the mouse is killed, and colon tissues are taken out and stored for later use.

9) Acute Lactobacillus casei strain Shirota group (JS) (n ═ 10): after successful molding, 2535200 μ L/10g (about 2.5X 10) of Lactobacillus casei was injected into the stomach⁹CFU/mL) as a positive control for 5 weeks until the end of the experiment, mice were sacrificed and colon tissue was removed and stored for use.

10) Chronic modeling block (MZ) (n 5): after successful molding, the mice were sacrificed. The colon tissue is taken and stored for standby.

11) Chronic normal diet group (MH) (n ═ 10): after the model was successfully created, the mice were fed normally for 5 weeks until the end of the experiment, sacrificed and colon tissue was collected and stored for further use.

12) Chronic MRS group (MS) (n ═ 10): after the model is successfully made, the contrast is kept for 5 weeks in 200 muL/10 g of MRS culture medium by gavage, until the experiment is finished, the mice are sacrificed, and colon tissues are taken for storage and use.

13) Chronic lactobacillus plantarum ZJ316 fermentation supernatant group (MY) (n ═ 10): after successful modeling, the fermented supernatant of lactobacillus plantarum ZJ316 was inoculated at 200 μ L/10g for 5 weeks as a control, and the mice were sacrificed until the end of the experiment, and the colon tissue was collected and stored for future use.

14) Chronic lactobacillus plantarum ZJ316 bacterial suspension group (MQ) (n ═ 10): after the molding is successful, the suspension of the lactobacillus plantarum ZJ316 of the gavage is 200 mu L/10g (about 2.5 multiplied by 10)⁹CFU/mL) for 5 weeks until the end of the experiment, mice were sacrificed and colon tissue was removed and stored for future use.

15) Lactobacillus plantarum ZJ316 fermented milk group (MM) (n ═ 10): after the molding is successful, lactobacillus plantarum ZJ316 fermented milk 200 mu L/10g is perfused for 5 weeks, until the experiment is finished, the mice are sacrificed, and colon tissues are taken for storage and standby.

16) Chronic cow's Milk Group (MG) (n ═ 10): after successful molding, the cow's milk was gavaged at 200. mu.L/10 g for 5 weeks as a control until the end of the experiment, the mice were sacrificed and the colon tissue was collected and stored for future use.

17) Chronic mesalazine group (MF) (n ═ 10): after the model building is successful, gavage (30.25mg/mL) of 200 muL/10 g is used as a positive control and lasts for 5 weeks until the experiment is finished, the mouse is killed, and colon tissues are taken out and stored for later use.

18) Chronic Lactobacillus casei strain Shirota group (MN) (n ═ 10): after successful molding, 2535200 μ L/10g (about 2.5X 10) of Lactobacillus casei was injected into the stomach⁹CFU/mL) as a positive control for 5 weeks until the end of the experiment, mice were sacrificed and colon tissue was removed and stored for use.

3.2.6.5 index determination

(1) Weight change and growth activity of mice during the experiment.

For the duration of the animal experiment, mice were weighed once a week, and the weight of each mouse was averaged three times as the weight of the mouse on that day, and the mice were observed for growth activity.

(2) Mouse colon length measurement.

After the experiment, 5 mice were selected per group according to body weight, killed by cervical dislocation, and after immersion and sterilization with benzalkonium bromide, the whole colon (from the tail end of the cecum to the anus) was dissected in an ultraclean work under sterile conditions, and the length was measured.

(3) Determination of colon weight in mice.

After the experiment, 5 mice were selected per group according to body weight, killed by cervical dislocation, soaked in benzalkonium bromide for sterilization, the mice were dissected aseptically in ultraclean work to obtain the whole colon (from the tail end of the cecum to the anus), the feces of the colon were removed, and then weighed.

(4) Paraffin sections of colon and H & E staining and tissue damage scoring.

1) Preparation of Colon Paraffin sections

The preparation process of the colon paraffin section mainly comprises the steps of sampling, fixing, removing fixing liquid, dehydrating, transparentizing, waxing, finishing and slicing.

The method comprises the following steps: sampling and fixing: 1cm of the distal colon is fixed in 10% formalin for 16-18 h.

Step two: removing the stationary liquid: the fixed tissue was then rinsed with running water for 24h to remove the fixative.

Step three: and (3) dehydrating: ethanol with different gradients is selected as a dehydrating agent to dehydrate the colon tissue from which the fixing solution is removed. 70%, 80%, 90% (30 min each gradient), 95% and 100% (20 min each gradient, performed twice).

Step four: and (3) transparency: placing the dehydrated colon tissue in mixed solution of alcohol and xylene at equal ratio for 15min, and soaking in xylene I and xylene II for 15min to complete transparency;

step five: waxing, trimming and slicing: the colon tissue after the transparency was paraffin-embedded by EG1160 paraffin embedding machine (come card). The embedded colon tissue is solidified by paraffin, and the paraffin block is trimmed according to the position of the small tissue block; the trimmed paraffin blocks were sliced (4 μm) using an RM2235 rotary slicer (come card).

2) Colon sections H & E staining

The colon section H & E dyeing step mainly comprises the steps of spreading and fishing slices, baking slices, hydrating, dehydrating, hematoxylin dyeing, differentiating, rinsing, eosin counterdyeing, dehydrating, transparentizing and sealing slices, and the specific operation is as follows:

the method comprises the following steps: and (3) displaying and fishing pieces: placing the cut colon slices in a constant-temperature water bath at 45 ℃ for spreading, and carefully fishing out the spread slices by using a glass slide;

step two: baking slices: placing the slices in an oven at 37 ℃ for baking overnight;

step three: hydration: xylene I15min, xylene II 5min, xylene: anhydrous ethanol (1:1) for 2min

Step four: and (3) dehydrating: 100% ethanol I for 5min, 100% ethanol II for 5min, 80% ethanol for 5min, and distilled water for 5min

Step five: staining sappan wood semen: washing with hematoxylin staining solution for 5min and 30min

Step six: differentiation and rinsing: 1% hydrochloric acid ethanol 30s, water washing 30s, and distilled water washing 5s

Step seven: eosin counterstaining: dyeing with 0.5% eosin solution for 1-3min

Step eight: and (3) dehydrating: washing with distilled water for 30s, 80% ethanol for 30s, 95% ethanol I for 1min, 95% ethanol II for 1min, anhydrous ethanol I for 3min, and anhydrous ethanol II for 3min

Step nine: and (3) transparency: xylene I3 min, xylene II 3min

Step ten: sealing: encapsulating with neutral gum.

After the end of mounting, the plate was observed by an optical microscope with NIKON Eclipse Ci and photographed. The imaging system is as follows: NIKONdigital sight DS-FI2, MADE IN JAPAN, photographic magnification: 25X, 100X, 200X. 3 intervention groups are respectively selected to carry out tissue damage scoring on colon tissue slices, the tissue damage scoring comprises four aspects of inflammation degree, lesion depth, crypt destruction and lesion range, and specific standards are shown in tables 3-3.

TABLE 3-3 tissue injury score criteria

3.2.6.6 Total RNA extraction, reverse transcription and real-time fluorescent quantitative PCR of colon tissue

(1) Extraction of total RNA from colon tissue

1) 1mL trizol was added to the prepared enzyme-free tube according to the sample amount. Samples were removed in a-80 ℃ freezer and inserted on ice. Approximately 100mg (mung bean size) of animal tissue was quickly placed in Trizol solution and marked. The remaining tissue was returned to the-80 ℃ freezer as soon as possible and the samples were broken in a tissue disruptor at 70Hz for 90 s. The mixture was left at room temperature for 10 minutes.

2) Adding 0.2mL of chloroform (1 mL of Trizol), turning upside down, mixing, standing at room temperature for 5min, and separating the solution into upper and lower layers;

3) centrifuging at 12000rpm for 15min at 4 deg.C;

4) carefully aspirate the supernatant (ca. 0.5mL) into a new centrifuge tube;

5) adding isopropanol with the same volume, gently reversing, mixing uniformly, standing at room temperature for 10min, and observing white precipitate;

6) centrifuging at 4 deg.C and 12000rpm for 10min, removing supernatant, and collecting RNA precipitate;

7) adding 1mL of 75% ethanol (diluted without enzyme water) to wash the RNA (the white precipitate is floated by turning upside down), and centrifuging at 7500rpm for 5min4 ℃;

8) pouring out 75% ethanol, performing instantaneous centrifugation, and centrifuging all liquid on the tube wall to the tube bottom. The liquid was gently aspirated away with a white tip (without aspirating off the white precipitate). Drying at 37 deg.C, adding 20-100 μ L (according to precipitation amount) RNase-free or high temperature sterilized DEPC water to dissolve; the RNA was dissolved thoroughly by standing at 55 ℃ for 5-10 min.

9) After the samples are uniformly mixed, 1 mu L of the mixture is taken to measure the concentration and the purity of the samples, wherein 260/280 is between 1.8 and 2.1, and 260/230 is preferably between 1.8 and 2.1; taking about 1 mu g of RNA for agarose gel electrophoresis, and detecting whether the RNA is degraded or not and whether the RNA is polluted or not;

10) the RNA samples were stored at-80 ℃ for future use or subjected to further reverse transcription.

(2) Preparation of cDNA

PrimeScript using a real-time fluorescent quantitative PCR instrument^TM1st Strand cDNA Synthesis Kit mouse tissue total RNA was reverse transcribed into cDNA using the reverse transcription system as follows: (10. mu.L)

And (3) incubating the 10 mu L reverse transcription system at 37 ℃ for 15min, and storing the cDNA in a refrigerator at-80 ℃ for later use.

(3) Real-time fluorescent quantitative PCR

Preparing a Real-time fluorescent quantitative PCR (RT-qPCR) System according to the requirements, carrying out RT-qPCR by using a Bio-Rad Real-time System instrument, and evaluating the data quality of the Real-time fluorescent quantitative PCR by a dissolution curve and an amplification curve. Taking GAPDH as an internal reference gene, and standardizing the level of each gene; the gene levels of the acute and chronic blank groups were set to 1, and the gene levels of the remaining groups were expressed relative to the blank group. By using 2^-ΔΔCtThe method analyzes the data. The RT-qPCR temperature program was: 95 ℃ for 3min, 95 ℃ for 10sec, 55 ℃ for 10sec, and 72 ℃ for 30sec40 cycles were performed and the qPCR primer sequences used are shown in tables 3-4.

RT-qPCR system was as follows (20. mu.L):

TABLE 3-4 RT-qPCR primer sequences

3.2.6.7 measurement of short-chain fatty acids (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid) in Colon of mice

Changes in short chain fatty acids in the colon were determined by GC-MS in each intervention group of mice.

(1) Sample processing

Precisely weighing 50mg of tissue sample, adding 1mL of 6% phosphoric acid solution for homogenizing, transferring all the tissue samples to a 20mL headspace sample bottle, adding 1mL of internal standard solution, and sealing.

(2) Gas chromatography mass spectrometry conditions

Adopting a temperature programming mode: the initial temperature is 80 ℃, the temperature is increased to 220 ℃ at the speed of 8 ℃/min, the temperature is maintained for 0.5 min, the carrier gas is high-purity helium, the flow rate is 1mL/min, the split ratio is 10:1, the temperature of a sample inlet is 200 ℃, and the temperature of an FID detector is 250 ℃. Static headspace sampling is adopted, the incubation temperature is 85 ℃, the incubation time is 30min, the sampling needle temperature is 95 ℃, and the sampling volume is 1 mL. Mass spectrum conditions: ion source temperature 250 ℃, and adopting a selective ion monitoring mode: acetic acid m/z 60, propionic acid m/z 74, isobutyric acid m/z 743, butyric acid m/z 60 valeric acid m/z 60, 2-ethylbutyric acid m/z 55.

(3) Drawing of standard curve

And (3) respectively injecting sample from low concentration to high concentration, collecting and extracting a GC-MS ion flow diagram, recording each peak area, and drawing a standard curve.

(4) Sample assay

Analyzing the sample according to the gas chromatography conditions, collecting and extracting a GC-MS ion flow graph, recording each peak area, and substituting the peak area ratio of each peak into the standard curve to calculate the content of each component in the sample.

3.2.6.8 extraction of flora DNA in colon and 16S rRNA gene amplicon sequencing in each intervening group of mice

Colonic flora DNA was extracted using a (Zymo Research Corp) kit. After the experiment was completed, mice were sacrificed and dissected, the colon was removed, fresh colon tissue was treated with liquid nitrogen, ground in a mortar, and then sample DNA extraction was performed according to the kit instructions. The extracted total DNA of the tissue is purified and recovered by a purification column of the kit, the purified DNA is verified by agarose gel electrophoresis, and then the DNA is sent to Beijing Nuo-He biogenic bioinformation technology company Limited for sequencing.

3.3 data processing and analysis method

Data analysis and mapping were performed using SPSS 23.0 statistical software and OriginPro 9.0 mapping software. The analysis and comparison of the significance difference among the groups adopts a one-way analysis of variance (AVOVA), the result is expressed by means of means +/-SEM, the difference of upper-level letters in the graph represents the significance difference, and the significance level is P < 0.05.

3.4 results and analysis

3.4.1 dynamic changes in body weight and growth activity of mice during the experiment

The body weight and growth activity of the mice are important signs of the success of molding and whether the intervention drug has an improvement effect on the DSS-induced enteritis. Seven days after the adaptive growth of the mice, the mice are weighed and marked, and the molding is started, wherein the weight of the mice reaches 20 +/-1.8 g. During the molding period, the weight of the mouse is weighed, and the condition of the excrement of the mouse is observed, and the occult blood and the condition of the bloody stool of the excrement are detected. The next day of molding, the mice began to loose stool, hair curly, lazy movement, and weight loss. As the molding time goes on, the weight of the mouse continuously decreases, excrement is not formed and adheres to the anus, excrement of the mouse is detected by excrement occult blood test paper, and the result shows that most of the excrement occult blood test paper of the mouse is weak and positive, namely, the colon begins to bleed, but no obvious excrement blood phenomenon is observed by naked eyes, and the mouse continuously drinks the DSS solution. And (3) detecting the mouse feces on the seventh day of molding, wherein all the mouse feces occult blood test paper shows a positive phenomenon, and a part of the test paper shows a macroscopic hematochezia phenomenon, and the weight is seriously reduced, so that the acute molding is successful, the gastric lavage intervention drug is started, and the dynamic change of the weight of the mouse during the experiment is shown in figure 5. And after the mice in the chronic group drink the DSS solution for seven days, the DSS solution is changed into free drinking water, the model is continuously made in one period, after three periods of circulation, the model is successfully made, and the growth activity of the mice in the model making period is the same as that in the acute group. After the model building was successful, the gavage intervention drug was started, and the dynamic body weight changes of the mice during the experiment are shown in fig. 6. After the intervention drug started gavage, the acute and chronic group of lactobacillus ZJ316 and its fermented milk group mice were more active than the other groups.

As shown in FIG. 5, after the modeling was successful, the weight of the mice in the acute group was significantly reduced from 20 + -1.8 g to 15 + -2.5 g in the first week, but the weight of the mice in each treatment group was increased after the gastric lavage with the intervention drugs. At the end of the experiment at week eight, the body weight of the mice in the blank control group increased from 20 + -1.8 g to 24 + -0.9 g in week one; the weight of the lactobacillus plantarum ZJ316 and the fermented milk group thereof is increased rapidly compared with other groups, and the weight of the mice at the end of the experiment is 23.34 +/-1.32 g and 24 +/-0.72 g respectively. The rising trend of the mesalazine group and the lactobacillus paracasei 2535 group of the two positive control groups is slightly weaker than that of the lactobacillus plantarum ZJ316 and the lactobacillus paracasei fermented milk group, but is faster than that of the other intervention groups, and the body weight of the mice increases to 23.10 +/-0.26 g and 23.01+0.10g respectively from 15 +/-2.5 g after the molding success by the end of the eighth week experiment. The body weight of the mice in the normal diet group, the cow milk group, the lactobacillus plantarum ZJ316 fermentation supernatant and the MRS group is increased to 21.35 +/-0.27 g, 21.01 +/-1.30 g, 21.21 +/-1.35 g and 21.64 +/-1.32 g respectively. Overall, mouse body weights were not statistically significant (p) between treatment groups>0.05), but at the end of the experiment, the mice gavaged with lactobacillus plantarum ZJ316 and its fermented milk weighed slightly more than the other intervention groups. Thus, lactobacillus plantarum ZJ316 and fermented milk thereof have efficacy of promoting weight recovery of mice induced by DSS. And M.V.

The same effect was found in Lactobacillus casei.

As can be seen from FIG. 6, the weight of the mice after the chronic modeling was successful was 19.9. + -. 0.80 g. After the gastric lavage intervention drug, the weight of the mice of each treatment group is increased. Similar to the acute group, the body weight of the mice in the blank control group increased from 20. + -. 1.8g to 26.12. + -. 0.34g at the end of the experiment. The weight gains of the mice of the lactobacillus plantarum ZJ316 and the fermented milk group are obviously larger than those of other intervention groups, and the weights of the mice of the two groups are larger than those of a blank control group from the tenth week, and the weights are 27.01 +/-0.81 g and 27.11 +/-0.61 g respectively at the end of the experiment; in the positive control group, the amplification of the mesalazine group and the lactobacillus paracasei 2535 group is slightly weaker than that of the lactobacillus plantarum ZJ316 and the lactobacillus fermentum group, but the amplification is quicker than that of other intervention groups, the body weights of two groups of mice are respectively 26.31 +/-0.32 g and 27.13 +/-0.24 g at the end of the experiment, wherein the lactobacillus paracasei 2535 group is larger than that of the blank control group; at the end of the experiment, the weights of the mice in the normal diet group, the cow milk group, the lactobacillus plantarum ZJ316 fermentation supernatant and the MRS group are 25.35 +/-0.50 g, 25.01 +/-0.58 g, 25.21 +/-0.35 g and 25.21 +/-0.33 g respectively. The body weights of the mice in the groups did not differ significantly during the experiment (p >0.05), but from the end results it was seen that lactobacillus plantarum ZJ316, lactobacillus paracasei 2535 and fermented milk thereof may contribute to the diet and thus to the growth of the mice. In the research process of the lactobacillus plantarum ZS2058 for improving DSS-induced colitis in mice by producing conjugated linoleic acid, Wangjuntong et al also found that the lactobacillus plantarum ZS2058 can also recover the weight of mice of the enteritis model, which is consistent with the result that the lactobacillus plantarum ZJ316 can promote the weight recovery of mice with DSS-induced colitis.

In conclusion, lactobacillus plantarum ZJ316 and its fermented milk were able to promote weight recovery in mice with acute and chronic colitis induced by DSS.

3.4.2 Colon Length changes in mice

DSS-induced colitis shortened the colon of mice, and thus colon length was one of the indicators that characterize the degree of inflammation of DSS-induced colitis. After the experiment, the mice were sacrificed and dissected, and the length of the colon was measured as shown in fig. 7 (length of colon in acute group of mice) and fig. 8 (length of colon in chronic group of mice).

As shown in FIG. 7, the colon length (4.41. + -. 0.56cm) of the mice in the model group was significantly decreased compared to the control group (9.62. + -. 0.41cm), and the tendency of the colon to be shortened was prevented to a different extent in each of the intervention groups. As seen from the figure, the lengths of the colon were significantly maintained in the Lactobacillus plantarum ZJ316 group and the Lactobacillus plantarum ZJ316 fermented milk group, the colon was prevented from being shortened, and there was no significant difference from the blank control group, and the lengths of the colon of the mice were 9.12. + -. 0.38cm and 9.32. + -. 0.58cm, respectively. Compared with the blank control group, the two groups of positive control groups, namely the mesalamine group and the lactobacillus paracasei 2535 group, have no significant difference, namely the mesalamine group and the lactobacillus paracasei 2535 group also significantly prevent the colon from shortening, but the lactobacillus paracasei 2535 group has no effect as compared with the lactobacillus plantarum ZJ316 group and the fermented milk thereof (P <0.05), and the colon lengths are 8.70 +/-1.20 cm and 8.01 +/-0.30 cm respectively; compared with the modeling group, the colon length of the mice in the normal diet group, the cow milk group and the lactobacillus plantarum ZJ316 fermentation supernatant group is remarkably increased (P is less than 0.05), the colon length is respectively 7.78 +/-0.72 cm, 7.71 +/-1.03 cm and 7.08 +/-1.01 cm, but the colon length of the mice is obviously different (P is less than 0.05) compared with the lactobacillus plantarum ZJ316 and the lactobacillus plantarum fermented milk group. Therefore, the lactobacillus plantarum ZJ316 group and the lactobacillus plantarum ZJ316 fermented milk group can effectively promote the colon length recovery of the mice with the DSS acute colitis.

As shown in FIG. 8, the colon length (6.41. + -. 0.26cm) of the mice in the model group was significantly decreased (p <0.05) compared to the normal control group (9.62. + -. 0.41cm), whereas the tendency of the colon to be shortened was prevented to a different extent by each of the intervention drugs except the Lactobacillus plantarum ZJ316 fermentation supernatant group. It can be seen that, in the lactobacillus plantarum ZJ316 and lactobacillus plantarum ZJ316 fermented milk groups, the length of the colon was significantly maintained, the colon was prevented from being shortened, and there was no significant difference from the blank control group, and the lengths of the colon of the mouse were 8.12 ± 0.38cm and 9.01 ± 0.58cm, respectively. The two groups of positive control group mesalamine group and lactobacillus paracasei 2535 group have no significant difference compared with the blank control group, namely mesalamine and lactobacillus paracasei 2535 also significantly prevent the colon from shortening, but the effect of lactobacillus paracasei 2535 is significantly inferior to that of lactobacillus plantarum ZJ316 and fermented milk thereof (p <0.05), and the colon lengths are 8.11 +/-0.20 cm and 7.01 +/-0.30 cm respectively; compared with the model-making group, the colon lengths of the mice in the normal diet group, the cow milk group and the MRS group are respectively 7.58 +/-0.12 cm, 7.51 +/-0.03 cm and 7.46 +/-0.31 cm, but the colon length is far less than that of the lactobacillus plantarum ZJ316 and the fermented milk thereof (p is less than 0.05); the colon length of the lactobacillus plantarum ZJ316 fermentation supernatant group is similar to that of the modeling group, and has no significant difference, and the colon length is 6.41 +/-0.01 cm. Therefore, similar to the acute group, the lactobacillus plantarum ZJ316 group and the lactobacillus plantarum ZJ316 fermented milk group can effectively promote the colon length recovery of the mice with the DSS chronic colitis.

The results show that the lactobacillus plantarum ZJ316 group and the lactobacillus plantarum ZJ316 fermented milk group can effectively prevent the shortening of the mouse colon caused by DSS, and compared with a blank control group, the two groups have no significant difference. This is similar to the finding by Yoda K et al that the lactic acid bacterium Lactobacillus rhamnosus GG and its fermented milk can prevent UC from occurring. It can also be seen that the normal diet group did not have any treatment, but the colon length was also restored compared to the model, probably due to the self-recovery ability of the mice in the DSS established colitis model. Due to the different modeling methods, the colon length of the chronic modeling group is longer than that of the acute modeling group.

3.4.3 changes in Colon weight in mice

As with length, colon weight also provides an important index for evaluating DSS-induced colitis inflammatory status, and Lactobacillus bulgaricus OLL1181 found by Takamura T et al can promote the recovery of colon weight of DSS colitis mice. The lactobacillus fermented milk also has the effect of improving the DSS mouse colitis. Inspired by this, we also investigated the effect of lactobacillus plantarum ZJ316 and its fermented milk on the colon of DSS colitis mice. After the intervention experiment was completed, the mice were sacrificed and dissected, the entire colon was removed, and the weight was measured after removing the feces. The results are shown in FIG. 9 (colon weight in acute group mice) and FIG. 10 (colon weight in chronic group mice), respectively.

As shown in FIG. 9, the colon weight (0.22. + -. 0.04g) of the mice in the model group was significantly reduced compared to the normal control group (0.54. + -. 0.12g), and the colon weight was recovered to various degrees in each intervention group after a period of intervention. As can be seen from the figure, in which the colon weights of the lactobacillus plantarum ZJ316 group, the lactobacillus plantarum ZJ316 fermented milk group and the two positive control groups (mesalamine group and lactobacillus paracasei 2535 group) were similar to those of the blank control group, there was no significant difference, and the colon weights of the mice were 0.48 ± 0.12g and 0.52 ± 0.05g, 0.51 ± 0.12g and 0.47 ± 0.11g, respectively; compared with the modeling group, the normal diet group, the cow milk group and the MRS group have significant differences in colon weight, namely the normal diet, cow milk, MRS and the fermentation supernatant of the lactobacillus plantarum ZJ316 can also recover the colon weight of the DSS acute colitis mice, but the effects are obviously inferior to that of the lactobacillus plantarum ZJ316 and the fermented milk thereof (P <0.05), and the colon weight is respectively recovered to 0.36 +/-0.09 g, 0.43 +/-0.03 g, 0.38 +/-0.06 g and 0.41 +/-0.02 g at the end of the experiment; the results of colon weight recovery and colon length maintenance were consistent for each intervention group.

As shown in FIG. 10, the colon weight (0.34. + -. 0.04g) of the mice in the model group was significantly reduced by 79% as compared with that of the normal control group (0.43. + -. 0.12 g). After the intervention of the medicine for a period of time after the gavage, the colon weight of each intervention group except the normal diet group, the MRS group and the lactobacillus plantarum ZJ316 fermentation supernatant group is recovered to different degrees. As seen from the figure, the colon weights of the mice in the Lactobacillus plantarum ZJ316 group and the Lactobacillus plantarum ZJ316 fermented milk group are similar to those of the blank control group, and have no significant difference, and the colon weights of the mice are 0.40 +/-0.12 g and 0.41 +/-0.05 g respectively; the colon weights of the mice of the two positive control groups (mesalamine group and lactobacillus paracasei 2535 bacterial suspension group) are also recovered, but the colon weights are also significantly different (P <0.05) compared with those of the blank control group, and the colon weights are respectively recovered to 0.37 +/-0.02 g and 0.35 +/-0.11 g at the end of the experiment; the colon weight (0.37 +/-0.12 g) of the mice in the cow milk group has certain recovery compared with the modeling group, but has a remarkable difference (P <0.05) compared with the lactobacillus plantarum ZJ316 fermented milk group; compared with the modeling group, the normal diet group, the MRS and lactobacillus plantarum ZJ316 fermentation supernatant group have no significant difference (p is more than 0.05) in colon weight, namely the normal diet, the MRS and lactobacillus plantarum ZJ316 fermentation supernatant have no effect on the recovery of the colon weight of the DSS chronic colitis mice, and at the end of the experiment, the colon weight is respectively 0.34 +/-0.02 g, 0.33 +/-0.05 g and 0.34 +/-0.06 g; therefore, the lactobacillus plantarum ZJ316 and the fermented milk thereof can recover the colon weight of the mice with the DSS chronic colitis.

In conclusion, the lactobacillus plantarum ZJ316 and the fermented milk thereof can effectively promote the colon weight recovery of mice with acute and chronic colitis induced by DSS.

3.4.4 Colon H & E pathological staining observation and tissue injury scoring of mice

Histopathological observation and tissue damage scoring are effective means for diagnosing the severity of colitis, and are therefore widely used in the medical field. The subject is to observe the influence of each treatment group on the pathological change of mouse colon tissues by H & E staining of the mouse colon. After the mice were sacrificed, 1cm of distal colon was taken, H & E staining was performed and observed by photographing, and the results were shown in FIGS. 11A-11B (acute group mouse H & E staining result) and FIGS. 13A-13C (chronic group mouse H & E staining result). The results of scoring the colon tissue section by tissue injury of 3 mice selected from each intervention group according to the histopathology scoring standard are shown in fig. 12 (the results of scoring the tissue injury of the mice in each intervention group in the acute group) and fig. 14 (the results of scoring the tissue injury of the mice in each intervention group in the acute group).

3.4.4.1 Colon H & E pathological staining observation and tissue damage scoring of acute group mice

As shown in fig. 11A-11B, the colon epithelium structure of the blank control group (I blank control group) was intact, the villus structure was closely and regularly arranged, and contained abundant goblet cells, the crypts were regularly arranged, the gland structure was intact, and no inflammatory cell infiltration was observed; from the staining results of model building groups H & E, DSS successfully induces the occurrence of mouse ulcerative colitis (II model building group), the lesion range reaches about 70% (shown by B1 red arrow), intestinal tissues in visual field are visible to be ulcerated, local intestinal glands and epithelial structures of mucosa are completely lost, villus and crypt structures disappear, and a large amount of connective tissues of mucosa are proliferated (shown by B2 brown arrow); with massive inflammatory cell infiltration, (B3 indicated by yellow arrows); inflammation invasion and submucosa, (B2 blue arrow); mild dilation of part of the intestinal gland lumen, (shown in green head of B2); exfoliated epithelial cells are visible in the intestinal lumen, (indicated by black arrows in B3), and the colon tissue damage score of the molding group is 12.50 ± 0.57, which is significantly elevated relative to the normal control group (tissue damage score of 0); compared with the model building group, each of the other intervention groups reduces the symptoms of colitis inflammation to different degrees, thereby reducing the pathological changes of colon tissues caused by DSS, but compared with the blank control group, each intervention group has any inflammation. The lactobacillus plantarum ZJ316 and lactobacillus plantarum ZJ316 fermented milk remarkably relieves the colon tissue lesion caused by DSS, the lesion range is greatly reduced, most intestinal epithelial structures are intact, intact crypt structures exist, a large number of goblet cells are reserved, but a small amount of inflammatory cell infiltration (shown by a red arrow D3 and a yellow arrow G3) is still remained, the inflammatory cell infiltration does not reach the submucosa, and the tissue injury scores of the lactobacillus plantarum ZJ316 and the fermented milk group are respectively 2.25 +/-0.75 and 2 +/-0.65; the lesion range of the two positive control groups (mesalamine group and lactobacillus paracasei 2535 bacterial suspension group) is reduced, but the lesion range is partially infiltrated by inflammatory cells (shown by blue arrows I2, I3 and J3), and the tissue damage infiltrated into the submucosa (shown by yellow arrows I3 and J3) is respectively 2.3 +/-0.75 and 2.6 +/-0.65; the normal diet group, the MRS group, the Lactobacillus plantarum ZJ316 fermentation supernatant group and the cow milk group have wide lesion range, weak effect of improving the colon tissue damage caused by DSS, and more inflammatory cells infiltrate (shown by yellow arrows C3, E3, F1 and H1), the crypt structure of the mucosa disappears, the epithelial structure is destroyed (shown by red arrows C2, E2, F2 and H2), and a large amount of inflammatory cells infiltrate into the submucosa (shown by green arrows C3, E3, F3 and H3), and the tissue damage scores are respectively 5.20 +/-0.31, 5.31 +/-0.22, 7.31 +/-0.16 and 5.11 +/-0.23.

In conclusion, the lactobacillus plantarum ZJ316 and the lactobacillus plantarum ZJ316 fermented milk group can effectively improve the damage condition of the colon mucosa of the mice with DSS acute colitis.

3.4.4.2 Colon H & E pathological staining observation and tissue damage scoring of chronic group mice

As shown in fig. 13A-13C, similar to the acute group, the colon epithelium structure of the chronic blank control group (I blank control group) was intact, the villi structure was closely and regularly arranged, the crypts were regularly arranged, the gland structure was intact, but a part of the intestinal gland cavities were slightly dilated, (K3 green head), and the tissue damage score was (1.6 ± 0.4); from the staining results of model building groups H & E, DSS successfully induces the occurrence of ulcerative chronic colitis in mice (II model building group), the lesion range reaches about 40% (shown by red arrow L1), ulcer can be seen in intestinal tissues in visual field, local intestinal glands and epithelial structures of mucosa are completely lost, villus and crypt structures disappear, and a large amount of connective tissues of mucosa are proliferated (shown by brown arrow L3); with massive inflammatory cell infiltration, (shown by the yellow arrow at L3); inflammation invasion and submucosa, (shown by the blue arrow at L3); mild dilatation of part of the intestinal gland lumen, (shown in green head of L3); exfoliated epithelial cells are visible in the intestinal lumen, (indicated by black arrows at L3), and the colon tissue damage score of the model group is 11.50 ± 0.17, which is significantly elevated (P <0.05) relative to the normal control group (tissue damage score of 1.5 ± 0.01); after a period of time of gastric lavage by the therapeutic drug, the symptoms of chronic colitis inflammation are slowed down to different degrees in each intervention group, thereby alleviating colon tissue lesion caused by DSS, but compared with the control group, the inflammation of different degrees exists in each intervention group. The lactobacillus plantarum ZJ316 remarkably relieves the colon tissue lesion caused by DSS, the lesion range is greatly reduced, most intestinal epithelial structures are complete, complete crypt structures exist, a large number of goblet cells are regularly arranged, but partial inflammatory cell infiltration (shown by an N3 yellow arrow) still exists, the inflammatory cell infiltration does not reach submucosa, desquamated epithelial cells can be seen in an intestinal cavity (shown by an N2 blue arrow), and the lactobacillus plantarum ZJ316 tissue lesion score is 4.17 +/-0.15; the lactobacillus plantarum ZJ316 fermented milk group also obviously relieves the colon tissue lesion caused by DSS, the lesion range can be greatly reduced, a large number of ulcers disappear, most intestinal epithelium structures in the visual field are complete, complete crypt structures exist, a large number of goblet cells are regularly arranged, but a small part of inflammatory cell infiltration (shown by a black arrow Q2) in the inherent layer of the mucosa layer is still visible, the inflammatory cell infiltration reaches the submucosa (shown by a yellow arrow Q2), the injury does not invade the muscularis, and the tissue injury score is 5.56 +/-0.65; in addition, compared with the two positive control groups, the lactobacillus paracasei 2535 bacterial suspension has better improvement effect on mouse ulcerative colitis induced by DSS, and tissue damage is respectively 6.83 +/-1.02 and 5.33 +/-0.03; the lesion range of the normal diet group, the MRS group, the Lactobacillus plantarum ZJ316 fermentation supernatant group and the cow milk group is still wide (shown by red arrows M1, Q1, P1 and R1), the improvement effect on the colon tissue injury caused by DSS is weak, more inflammatory cells infiltrate (shown by yellow arrows M3, Q3, P3 and R3), the crypt structure of the mucosa disappears, the epithelial structure is destroyed (shown by black arrows M2, Q2, P2 and R2), and a large amount of inflammatory cells infiltrate into the muscle layer (shown by green arrows M3, Q2, P3 and R3) and the tissue injury is respectively evaluated as 8.16 +/-0.31, 6.31 +/-1.02, 8.71 +/-0.16 and 8.16 +/-0.23;

in conclusion, the Lactobacillus plantarum ZJ316 and the fermented milk thereof can effectively improve the damage condition of the colon mucosa of the DSS colitis mice, which is consistent with the result that Matsumoto S et al finds that Lactobacillus and Lactobacillus-fermentgmik can promote the repair of the intestinal mucosa of the DSS mice. In addition, comparing the results of H & E pathological staining and tissue injury scoring of the colon of the acute and chronic groups of mice, it can be seen that the effect of the lactobacillus plantarum ZJ316 and lactobacillus plantarum ZJ316 fermented milk on the improvement of DSS-induced acute ulcerative colitis is better.

3.4.5 transcriptional levels of inflammatory cytokines in Colon of mice of each treatment group

The research shows that the pathogenesis of Inflammatory Bowel Disease (IBD) is greatly related to hyperactive and unbalanced intestinal mucosal immune reaction, the transcription level of each inflammatory factor can indirectly reflect the severity of inflammation, proinflammatory cytokine interleukin 1 β (IL-1 β) and interleukin 8(IL-8) play an important role in the pathogenesis of inflammatory bowel disease, wherein IL-1 β is an inflammation-causing cytokine and is widely involved in various pathological injury processes such as animal tissue damage, edema formation and the like, IL-8 can stimulate the chemotaxis of neutrophils, T lymphocytes and eosinophils, thereby damaging endothelial cells, leading the tissues to necrosis and causing the organ function damage^[125]. White and fineInterleukin 6 (IL-6) synergizes with TNF- α and IL-1 to produce the inflammatory response characteristic of IBD^[131]Tumor necrosis factor (TNF- α) is a key proinflammatory cytokine in the pathogenesis of IBD, and TNF- α is one of the key targets in the treatment of IBD, it induces cytokine synthesis and produces a cascade that further promotes the production of other proinflammatory cytokines, such as IL-1 β, IL-6, and IL-8, etc., ultimately leading to tissue inflammation^[132]Interleukin 10(IL-10) is mainly produced by Th2 cells and mononuclear macrophages, can inhibit the production of proinflammatory cytokines, can stimulate the differentiation and proliferation of B cells, and promotes the production of antibodies.A transforming growth factor (TGF- β) has important effects mainly in inflammation, tissue repair, embryonic development and other aspects, and can inhibit the production of cytokines IFN-gamma, TNF- α and the like^[133]。

In the invention, changes of mRAN levels of proinflammatory cytokines (TNF- α, IL-1 β, IL-6 and IL-8) of colon tissues of mice in each treatment group of an acute group and proinflammatory cytokines (TNF- α, IL-1 β and IL-6) and inflammatory inhibitory cytokines (IL-10 and TGF- β) in each treatment group of a chronic group are respectively detected to explore the influence of the lactobacillus plantarum ZJ316 and fermented milk thereof on DSS-induced colitis mice colon immune response, and the results are respectively shown in FIGS. 15A-15D (transcription level of inflammatory cytokines in each treatment group of the acute group) and 16A-16E (transcription level of inflammatory cytokines in different treatment groups of the chronic group).

Note that FIGS. 15A-D are the relative transcript levels of IL-1 β, IL-6, TNF- α, and IL-8, respectively;

the upper letters in the figure indicate significant differences, P < 0.05.

As shown in the graphs of 15A-15D, the expression levels of the pro-inflammatory cytokines IL-1 α, IL-6, TNF- α 1 and IL-8 in colon of the acute model mouse are not significantly increased by the expression levels of the pro-inflammatory cytokines IL-1 α, IL-6, TNF- α 1 and IL-8 in colon of the lactobacillus plantarum blank group (MRS-1 + -0), the expression levels of the pro-inflammatory cytokines IL-1 + -0 + -0.05 + -0, the expression levels of the pro-inflammatory cytokines IL-1 + -0 + -7, the expression levels of the pro-inflammatory cytokines IL-8 in colon of the lactobacillus blank group (MRS-7), the expression levels of the lactobacillus blank group + -0 + -0.05 + -0, the milk-12, the milk-7, the milk-12, the milk cow-7 + -0 + -0.05 + -0, the milk-7, the milk-6, the milk-7 + -0.13, the milk-8, the milk-2 + -0 + -0.05 + -0, the milk-7, the milk-8, the milk-6, the milk-7, the milk-6, the milk-8, the milk-7, the milk-0, the milk-7, the milk-2, the milk-7, the milk-4, the milk-2, the milk-4, the milk-0, the milk-7, the milk-2, the milk-7, the milk-4, the milk-7, the milk-0, the milk-7, the milk-0, the milk-7, the milk-4, the milk-0, the milk-7, the milk-2, the milk-0, the milk-7, the milk-0, the milk-7, the milk-0, the milk-2, the milk-7, the milk-0, the milk-2, the milk-7, the milk-2, the milk-7, the milk-0, the milk-2, the milk-0, the milk-7, the milk-0, the milk-7, the milk-0, the milk-7, the milk-2, the milk-0, the milk-7, the milk-0, the milk-2, the milk-0, the milk-7, the milk-0, the milk-2, the milk-0, the milk-7, the milk-2, the milk-0, the milk-7, the milk-0, the milk-2, the milk-0, the milk-7, the milk-2, the milk-0, the milk-7, the milk-0, the milk-7.

It is known from the results of the experiment that DSS causes the expression levels of proinflammatory cytokines IL-1 β, TNF- α, IL-6 and IL-8 in the colon of mice to be remarkably up-regulated, possibly causing immune disorder of the colon of the mice, aggravating inflammation and destroying colon tissues, while Lactobacillus plantarum ZJ316 and Lactobacillus plantarum ZJ316 can remarkably down-regulate the expression of proinflammatory cytokines by fermented milk, thereby demonstrating that the fermented milk of the Lactobacillus plantarum ZJ316 and the Lactobacillus plantarum ZJ316 also has a regulating effect on the immune response of a host.

Note that FIGS. E-I are the relative transcript levels of IL-1 β, IL-6, TNF- α, IL-10 and TGF- β, respectively;

the upper-marked letters in the figure represent significant differences, and P <0.05

Note：Figure a-d shows the relative transcriptional levels of IL-1β、IL-6、TNF-α、IL-10and TGF-β,respectively Superscript with different letters onthe bars are significantly different by Duncan's mμLtiple range test(P<0.05).

As shown in the figure 16A-16E, the transcriptional levels of the proinflammatory cytokines IL-1 α, IL-6 and TNF- α 0 in colon of the mice of the chronic model group (1.77 + -0.10, 0.39 + -0.02 and 0.63 + -0.10) are respectively significantly increased compared with those of the mice of the placebo group (IL-1 β, IL-6 and TNF- α) and the transcriptional levels of the proinflammatory cytokines IL-1 α, IL-6 and TNF- α 0 (1.77 + -0.04, 1.60 + -0.11 and 1.61 + -0.12) of the mice of the fermented milk model group (1 + -0) are respectively significantly increased after the gastrointestinal injection of the fermented milk protein of the lactobacillus coli (1 + -0.04 + -0.9), the gastrointestinal protein of the mice of the fermented milk protein.

In conclusion, the Lactobacillus plantarum ZJ316 and fermented milk thereof can remarkably reduce mRAN transcription levels of proinflammatory cytokines IL-1 β, IL-6 and TNF- α in mouse chronic colon induced by DSS and remarkably promote expression of the anti-inflammatory cytokines IL-10 and TGF- β.

3.4.6 Effect of Lactobacillus plantarum ZJ316 and its fermented milk on the content of short-chain fatty acids in Colon of DSS-induced colitis mice

Short chain fatty acids (SFCAs) in the gut are produced primarily by microbial degradation of undigested carbohydrates and small amounts of protein and are critical to gut health. The invention utilizes a GC-MS method to respectively detect the contents of acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid in colon tissues of mice of acute and chronic treatment groups, so as to explore the influence of lactobacillus plantarum ZJ316 and lactobacillus plantarum ZJ316 fermented milk on the content of short-chain fatty acids in the colon of the mice of each treatment group. The results are shown in FIGS. 17A to 17E (short-chain fatty acid content in colon of mice in each treatment group in acute group) and FIGS. 18A to 18E (short-chain fatty acid content in colon of mice in each treatment group in chronic group).

Note: FIGS. 17A-E show the amounts of acetic acid, propionic acid, isobutyric acid, butyric acid, and valeric acid, respectively, in colon tissue of mice in each treatment group

As shown in FIGS. 17A-17E, the levels of acetic acid (1.85 + -0.89 mg/g), propionic acid (387.46 + -11.72 μ g/g), isobutyric acid (14.93 + -4.86 μ g/g), butyric acid (238.67 + -22.54 μ g/g) and valeric acid (6.38 + -1.74 μ g/g) in colon tissue of mice of the model group were all significantly reduced compared to the blank control group (acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid levels: 1.85 + -0.37 mg/g, 387.46 + -36.12 μ g/g, 14.93 + -32.12 and 6.38 + -2.73 μ g/g, respectively), indicating that the levels of short chain fatty acids in colon of mice were significantly reduced after DSS induction. After 5 weeks of each intervention drug intervention, the contents of acetic acid (3.14 +/-0.97 mg/g), propionic acid (336.95 +/-17.23 mu g/g), isobutyric acid (19.93 +/-5.89 mg/g), butyric acid (374.24 +/-16.72 mu g/g) and valeric acid (21.62 +/-7.86 mu g/mg) in colon of the lactobacillus plantarum ZJ316 mice are all obviously increased, and the contents of the other four short-chain fatty acids are all obviously higher than that of a blank control group (p is more than 0.05) except that the propionic acid is slightly lower than that of the blank control group. Lactobacillus plantarum ZJ316 fermented milk also increased the content of acetic acid (1.17. + -. 0.19mg/g), propionic acid (143.41. + -. 14.72. mu.g/g), isobutyric acid (8.26. + -. 2.86. mu.g/mg), butyric acid (130.66. + -. 12.54. mu.g/g) and valeric acid (8.70. + -. 1.79. mu.g/g), but was less effective than Lactobacillus plantarum ZJ 316. In the two positive control groups, the contents of acetic acid (0.75 +/-0.17 mg/g), propionic acid (147.25 +/-19.23 mu g/g), isobutyric acid (7.01 +/-2.89 mg/g), butyric acid (50.60 +/-16.72 mu g/g) and valeric acid (1.96 +/-0.86 mu g/mg) in the colon of the mice in the mesalazine group are similar to those in the modeling group, and have no significant difference (p is more than 0.05), namely, the mesalazine does not influence the change of the content of the short-chain fatty acid in the colon of the mice with acute ulcerative colitis induced by DSS. And the lactobacillus paracasei 2535 can increase the contents of acetic acid (3.96 +/-1.17 mg/g), propionic acid (315.04 +/-27.23 mu g/g), isobutyric acid (10.64 +/-2.85 mg/g), butyric acid (482.61 +/-55.72 mu g/g) and valeric acid (15.87 +/-2.86 mu g/mg) in colon of a colitis mouse, and the contents of the other three short-chain fatty acids except the propionic acid and the isobutyric acid are obviously higher than that of a blank control group (p < 0.05). In the colon of the mouse after the fermentation supernatant of the lactobacillus plantarum ZJ316 is perfused, the contents of acetic acid (1.22 +/-0.17 mg/g), propionic acid (135.71 +/-37.23 mu g/g), isobutyric acid (9.74 +/-2.85 mg/g), butyric acid (284.18 +/-35.72 mu g/g) and valeric acid (12.81 +/-2.46 mu g/mg) are all increased, and the contents of other three short-chain fatty acids except the acetic acid and the propionic acid are all obviously higher than that of a model group (p <0.05), because the lactobacillus plantarum ZJ316 can produce short-chain fatty acids after being fermented, and the short-chain fatty acids exist in the fermentation supernatant, but the influence on the short-chain fatty acids in the colon of the mouse is not as much as that of the lactobacillus plantarum ZJ 316. In the colon of mice in the normal diet group and MRS group, propionic acid (143.98 + -23.43 mu g/mg and 164.72 + -25.17 mu g/mg), isobutyric acid (10.78 + -3.45 and 12.45 + -3.14 mu g/mg), butyric acid (74.18 + -19.27 mu g/mg and 200.21 + -77.23 mu g/mg) and valeric acid (9.58 + -2.63 mu g/mg and 12.22 + -4.23 mu g/mg) were all increased, but the contents of acetic acid (0.77 + -0.12 mu g/mg and 0.76 + -0.12 mu g/mg) were similar to those in the model group and were hardly increased. In the colon of the cow's milk mice, the contents of acetic acid (0.62 + -0.17 mg/g), propionic acid (117.32 + -21.23 μ g/g), isobutyric acid (5.63 + -0.85 mg/g), butyric acid (40.59 + -1.72 μ g/g) and valeric acid (1.09 + -0.46 μ g/mg) were similar to those of the model group, and were not significantly different (p > 0.05).

In conclusion, the lactobacillus plantarum ZJ316 and the fermented milk thereof can promote the generation of short-chain fatty acids in the colon of the mice with acute ulcerative colitis induced by DSS.

As shown in fig. 18A-18E, fig. 18A-18E show the content of acetic acid, propionic acid, isobutyric acid, butyric acid, and valeric acid, respectively, in the colon tissue of each treatment group of mice, taken as a whole, compared with a blank control group (the contents of acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid are respectively 3.32 +/-0.97 mg/g, 530.35 +/-56.12 mu g/g, 17.40 +/-4.11 mu g/g, 569.74 +/-42.13 mu g/g and 34.16 +/-6.73 mu g/g), the contents of acetic acid (0.88 +/-0.19 mg/g), propionic acid (128.79 +/-44.72 mu g/g), isobutyric acid (5.96 +/-1.86 mu g/mg), butyric acid (79.37 +/-12.54 mu g/g) and valeric acid (4.06 +/-1.34 mu g/g) in colon tissues of mice in a chronic modeling group are all obviously reduced (p is less than 0.01), which indicates that the induction can cause the short-chain fatty acid metabolism obstruction in the colon of the mice. Lactobacillus plantarum ZJ316 was found to increase the content of acetic acid (1.77. + -. 0.87mg/g), propionic acid (292.65. + -. 37.23. mu.g/g), isobutyric acid (19.67. + -. 5.79mg/g), butyric acid (556.09. + -. 36.72. mu.g/g), valeric acid (19.56. + -. 8.86. mu.g/mg) in the colon of mice after 5 weeks of each intervention drug intervention, wherein the content of isobutyric acid, butyric acid and valeric acid was significantly increased (p < 0.05). Of the two positive control intervention drugs, mesalamine increased the contents of propionic acid (200.80 + -33.23 μ g/g), isobutyric acid (22.03 + -4.89 mg/g), butyric acid (147.77 + -16.92 μ g/g), valeric acid (17.57 + -3.86 μ g/g) and acetic acid (0.63 + -0.13 mg/g) in the colon of the mice in the group, but not the acetic acid content. And the content of acetic acid (1.26 +/-0.17 mg/g), propionic acid (211.16 +/-29.23 mu g/g), isobutyric acid (13.86 +/-3.87 mg/g), butyric acid (314.48 +/-35.72 mu g/g) and valeric acid (14.91 +/-2.56 mu g/g) in the colon of the mouse can be increased by the lactobacillus paracasei 2535. Similar to the acute group, after the fermentation supernatant of the lactobacillus plantarum ZJ316 is perfused, the contents of acetic acid (1.41 +/-0.72 mg/g), propionic acid (221.96 +/-23.42 mu g/g), isobutyric acid (15.20 +/-3.85 mg/g), butyric acid (378.01 +/-34.12 mu g/g) and valeric acid (20.08 +/-5.46 mu g/mg) in the colon of the mouse can be increased (p is less than 0.05), wherein the contents of the isobutyric acid, the butyric acid and the valeric acid are obviously increased, but the influence on the content of short-chain fatty acids in the colon of the mouse is lower than that of the lactobacillus plantarum ZJ 316. Compared with the modeling group, the contents of isobutyric acid (14.96 +/-2.45 mu g/mg), butyric acid (364.94 +/-29.27 mu g/mg) and valeric acid (15.18 +/-3.63 mu g/mg) in the colon of the mice in the normal diet group are all obviously increased (p <0.05), and the contents of acetic acid (1.48 +/-0.24) and propionic acid (227.45 +/-23.50 mu g/mg) are increased to some extent but have no obvious difference (p > 0.05). Therefore, the DSS chronic colitis mouse may recover the short chain fatty acid metabolic system in the self-recovery process. The cow milk can also promote the content of acetic acid (1.33 +/-0.07 mg/g), propionic acid (217.64 +/-21.23 mu g/g), isobutyric acid (11.28 +/-0.35 mg/g), butyric acid (196.11 +/-11.72 mu g/g) and valeric acid (19.15 +/-3.46 mu g/mg) in the colon of the mouse, but the effect is far less than that of lactobacillus plantarum ZI316 fermented milk (p < 0.05). The contents of acetic acid (0.76 +/-0.12 mg/g) and propionic acid (164.72 +/-22.31 mu g/g) in the colon of the mice in the MRS group are similar to those of the modeling group and are hardly increased, and the contents of isobutyric acid (12.45 +/-4.73 mu g/g), butyric acid (200.21 +/-23.15 mu g/g) and valeric acid (12.22 +/-2.14 mu g/g) are remarkably increased (p is less than 0.05) compared with the modeling group, but are far lower than that of the Lactobacillus plantarum ZJ316 group (p is less than 0.01).

Zhao L et al, when studying the pathogenesis of ulcerative colitis, found that when the metabolism of fatty acids in the colon mucosa of mice is hindered, the related intestinal diseases are caused. Research reports that the content of fatty acid in intestinal tracts of patients with UC is low, particularly the content of acetic acid with anti-inflammatory effect is very low, because the permeability of the intestinal tracts is increased in the morbidity process of the UC, pathogenic bacteria invade the intestinal tracts and probiotics lose advantages, the number of strains producing short-chain fatty acid is reduced, the metabolism of the fatty acid is hindered, and the intestinal diseases such as colitis are finally caused. The experimental results show that the lactobacillus plantarum ZJ316 and the fermented milk thereof can effectively promote the generation of short-chain fatty acids in the colon of mice with acute and chronic colitis induced by DSS, which indicates that the lactobacillus plantarum ZJ316 can generate the short-chain fatty acids through metabolism, so that the metabolic balance of fatty acids in the intestinal tract of the mice is adjusted, and the symptoms of the DSS colitis are relieved.

3.4.7 Effect of Lactobacillus plantarum ZJ316 and its fermented milk on intestinal flora of DSS-induced colitis mice

3.4.7.116S rRNA gene high-throughput sequencing result analysis

16S rDNA (V3-V4 high-mutation region) is carried out on microbial communities in 105 colon samples of 21 groups (blank control group, acute group and chronic group) of mice by adopting an Illumina sequencing platform for high-throughput sequencing, obtained data are screened, optimized and spliced, the spliced sequence is further subjected to quality control such as chimera removal, a high-quality sequence is obtained, the high-quality sequence is clustered under the similarity of 0.97, the clustered sequence is subjected to chimeric filtering, OTU (optical Transform Unit) for species classification is obtained, finally, abundance information in each sample OTU is counted, and the next-step analysis is carried out.

Whether the sequencing depth meets the requirement and whether the sequencing data volume is reasonable is determined by a sequencing dilution curve. The dilution curve is mainly used for reflecting whether the sequencing data volume meets the requirement or not, can indirectly reflect the abundance degree of species in the sample, and indicates that the sequencing depth meets the requirement when the curve tends to be flat, and the sequencing data volume is reasonable; if the difference is contrary, the species diversity in the sample is higher, the sequencing depth is not enough, and more species which are not detected exist. A dilution curve of 21 individual mouse stomach samples for high throughput sequencing is shown in FIG. 19. From the figure, it can be seen that with the increase of sequence sampling, the OTUS curve tends to be smooth, indicating that the sequencing depth has substantially covered all the species in the sample, i.e. the sequencing depth reaches the experimental requirements.

3.4.7.2 influence of Lactobacillus plantarum ZJ316 and its fermented milk on the constitution of intestinal flora of mice with DSS-induced colitis.

(1) The effect of lactobacillus plantarum ZJ316 and its fermented milk on the structural composition of the colonic flora of mice at the "phylum" level.

Results annotated by the species of OTU, we plotted a histogram at the bacterial "phylum" level against the relative abundance of top10 in colon samples from 21 groups of mice and compared for analysis, by the proportion of the flora constituents of the different components. As shown in fig. 20, the dominant bacterial groups in the colon of each treatment group of mice were Firmicutes, Bacteroidetes, and Proteobacteria, wherein Firmicutes and Bacteroidetes are the most predominant bacterial groups, accounting for an average of about 50% of the total OUT of each sample. It can be seen from the figure that in the acute group, the proportion of Firmicutes is decreased and the proportion of bacteroides and Proteobacteria is increased in the model group (JZ) as compared with the blank control group (Ctrl). In the colon of the lactobacillus plantarum ZJ316(JX) and the fermented milk group (JH) thereof, the proportion of Firmicutes is more, and the proportion of bacteroides and Proteobacteria is less; compared with the modeling group, in the two positive control groups, the proportion of Firmicutes in the colon of the mesalamine (JG) mice is increased, and the proportion of Bacteroides and Proteobacteria is reduced, but the proportion of Bacteroides is higher than that of the Lactobacillus plantarum ZJ316 and the fermented milk group thereof, while the proportion of Firmicutes in the Lactobacillus paracasei 2535(JS) group is reduced, and the proportion of Bacteroides and Proteobacteria is increased. In the chronic group, the proportions of the three main flora Firmicutes, bacteroides and Proteobacteria in the model building group (MZ), the blank control group (Ctrl), lactobacillus plantarum ZJ316(MQ) and its fermented milk group (MM), Mesalazine (MF) and lactobacillus paracasei 2535 group (MN), respectively, were similar to the acute group. In summary, after induction of DSS, the firmicutes proportion in the mouse colon decreases, the bacteroides and proteobacteria proportion increases, and after gavage with lactobacillus plantarum ZJ316 and fermented milk thereof, the firmicutes proportion in the mouse colon increases, and the bacteroides and proteobacteria decrease, indicating that lactobacillus plantarum ZJ316 and fermentation thereof can inhibit the propagation of pathogenic bacteria.

(2) The effect of lactobacillus plantarum ZJ316 and its fermented milk on the structural composition of the colonic flora of mice at the "genus" level.

We plotted a histogram at the bacterial "genus" level against the population of relative abundance top10 in 21 groups of mouse colon samples, the results are shown in fig. 21. In the 21 group samples, the flora in the acute modeling group (JZ) samples mainly contained multiple genera including Bacteroides, Helicobacter, unidentified lachnospiraceae, and Alistipes, while Lactobacillus and Faecalibacterium were not detected, in terms of the flora composition at the flora level. In samples of blank control group (Ctrl), Lactobacillus plantarum ZJ316(JX) and its fermented milk group (JH), the flora included genera Bacteroides, Helicobacter, unidentified _ lactinospiraceae, alisistipes, Faecalibacterium and Lactobacillus, and in addition, the three genera of Bacteroides, Helicobacter and alisistipes all decreased in proportion as compared to the manufactured group. The bacterial groups in the colon of mice of two positive control groups, mesalamine (JG) and Lactobacillus paracasei 2535(JS), mainly comprise genera of Bacteroides, Helicobacter, unidentified _ lactinospiraceae, Alistipes and Lactobacillus, and the proportion of the three genera of Bacteroides, Helicobacter and Alistipes is reduced compared with that of the artificial model. In samples from each treatment group of the chronic group, the structural composition of the mouse intestinal flora at the genus level was similar to that of the acute group. The genus Bacteroides is a main pathogenic bacterium in the intestinal tract, the genus Alisipes can cause intestinal inflammation, and the genus Helicobacter can cause wound infection, even peritonitis and the like. Some strains in the genus Faecalibacterium have an anti-inflammatory function, and the genus Lactobacillus can maintain the intestinal health of animals. In conclusion, the lactobacillus plantarum ZJ316 and the fermented milk thereof are beneficial to increasing the number of beneficial flora in the colon of mice with DSS-induced colitis; meanwhile, the compound preparation also has a certain inhibiting effect on the growth of pathogenic bacteria, thereby improving disordered intestinal flora.

Bacteroidetes in the intestine is mainly of the bacteroidetes class and is the source of many conditional pathogens in the intestine. Firmicutes, which include the class bacilli (bacillales and lactobacillales), can help polysaccharide fermentation in the gut, where the main role of bacillus is to maintain the health of the animal's gut. Proteobacteria are the largest of bacteria, including pathogenic bacteria such as Escherichia coli, Salmonella, Vibrio cholerae, and helicobacter pylori, which are susceptible to cause diarrhea in animals. Medical researchers have found that the phylum posterior parietal bacteria in the intestine of patients with UC or other types of inflammatory bowel disease are significantly lower than those of healthy people, while the phylum proteobacteria and bacteroidetes are correspondingly increased. After the mice with DSS colitis were gavaged with Lactobacillus plantarum, the number of Bacteromural phyla in the colon of the mice increased and the colitis of the mice improved.

3.4.7.3 analysis of diversity of bacteria in colon α in mice from each treatment group

α diversity (α diversity) is mainly used to evaluate the diversity of microbial flora in a single sample, which is a comprehensive index reflecting the richness and uniformity. α diversity is mainly related to two factors, i.e. the number of species, i.e. the richness, and the second, i.e. the uniformity of distribution of individuals in the community.the Community richness index mainly includes the Chao1 index, the observed species index and the ACE index, the larger these three indexes indicate the richness of the Community, the index of Community diversity (com diversity) includes the Shannon index and the Simpson index, wherein the higher the Shannon index value indicates the higher the diversity of the Community, and the larger the Simpson index value indicates the lower the Community diversity index, in addition, the PD-hue-tree index reflects the species-to-historical preservation differences in the sample, the PD-hue-tree index indicates the species have more significant evolutionary differences in the historical preservation, the species, the prognosis of the sample is made by making a diversity map of the sample, wherein the ZJ 82 is shown by the Zhan J82, and the fermented milk group is made by making the experimental study of the sample, wherein the sample and the sampleIncrease the abundance and diversity of intestinal flora in DSS colitis mice, which is in contrast to Qiu L^]It was found by others that lactobacillus plantarum could improve colitis with consistent results by increasing the abundance and diversity of flora in the intestinal tract of mice.

Note: FIGS. 22(A-F) are ACE index, Chao1 index, observed speces index, PD-while-tree index Shannon index and Simpson index, respectively.

3.4.7.4 diversity analysis of β of the flora in the colon of mice from each treatment group.

β diversity is used for comparison of diversity between different ecosystems, i.e. overall difference between samples, β diversity uses evolutionary relationship and abundance information between sequences of each sample to calculate the distance between samples, which reflects whether there is difference in microbial colony structure between different samples (groups). β diversity calculation is based mainly on colony comparison method of OTU, wherein one of the most commonly used algorithms is Unifrac distance method, which considers evolutionary distance between sequences, the larger Unifrac index indicates the greater difference between samples, wherein Unifrac results are divided into weighted uniweighted Unifrac (unweighed Unifrac) and unweighted Unifrac (unweighted unic), which considers the abundance of sequences, while unweighted Unifrac considers only evolutionary distance between sequences, the no consideration of sequence abundance, the Unifrac results analysis has various methods, wherein main coordinate analysis (princcorandinatys) is performed for the difference between series, i.e. the difference between right and empty colon, (right) is considered for the difference between the dominant colon bacillus coli groups, i.e. the difference between the left and the left colon of lactobacillus coli group, i.e.e. the left lactobacillus coli and the left lactobacillus sane. the left lactobacillus sane.e. the left lactobacillus sane.a, the left lactobacillus sane.e.e.a is considered to be observed by the difference between the left lactobacillus sane left lactobacillus sane.g. the left lactobacillus sane left lactobacillus sane.g. the left lactobacillus sane.a of fermented milk lactobacillus sane.g. the fermented milk-fermented milk.

Fig. 23B shows the result of principal coordinate analysis based on weighting of 105 colon samples of 21 groups of mice, and it can be seen that when the species abundance is considered, the individual difference of the bacterial flora in the colon tissue of the same group of mice is large, but it can be generally seen that there is still a certain difference in the colon microbial flora of different groups of mice.

Note: FIG. 23A is a PcoA analysis based on unweighted distance, and FIG. 23B is a PcoA analysis based on weighted distance; the percentage represents the degree of contribution of the principal component to the difference of the sample; each point in the graph represents a sample and the samples of the same group are represented using the same color.

3.4.7.5 analysis of species significant differences in the colon of mice in each treatment group.

To specifically analyze species with significant differences in abundance between treatment groups (Biomarke), we chose the LEfSe assay. The LEfse analysis, namely LDA Effect Size analysis, is a linear discriminant analysis method used to estimate the influence of the abundance of each species on the difference Effect, so as to find out the communities or species that have significant difference influence on the sample division.

(1) Analysis of species significant differences in Colon of mice in treatment groups of acute groups

As shown in 24A-24B, the groups with significant differences in colonic flora of mice in the acute treatment groups were identified by LEfSe analysis, including the model group (JZ), the blank control group (Ctrl), the Lactobacillus plantarum ZJ316 group (JX), the Lactobacillus plantarum ZJ316 fermented milk group (JH), and the Lactobacillus paracasei 2535 group (JS). Wherein the flora with significantly increased appearance in colon microbial flora of model building block (JZ) mice is Bacteroides-sartorii on 'species' level; the groups of significantly different flora in the blank control group were lactobacillus at the "seed" level; the Lactobacillus plantarum ZJ316 group had a significant increase in both the flora of oribacter and unidentified lachnospiraceae at the "genus" level. (ii) a The flora significantly increased at the "family" level was Marinilabiliale, and the colonies significantly increased at the "species" level were closteriale-bacterium-CIEAF-020 and lachnospiraceae-bacterium-COE 1; the significantly increased flora of lactobacillus plantarum ZJ316 fermented milk group was Prevotellaceae at the "family" level and alloprevolella at the "genus" level; the groups of lactobacillus paracasei 2535 mice with significant differences in colon were tanderellaceae at the "family" level and Parabacteroides at the "genus" level.

Bacteroides-sartorii belongs to the main pathogenic bacteria of the intestinal tract of animals; the bacterium lachnospiraceae belongs to the phylum firmicutes, is a resident flora in intestinal tracts and can maintain intestinal tract health; the Odoribacter is a common bacterium in intestinal tracts of healthy animals, belongs to the phylum firmicutes, is a resident bacterium group in the intestinal tracts and can maintain the health of the intestinal tracts; unidentified-lachnospiraceae belongs to probiotics and can maintain intestinal health; clotridial-bacterium-CIEAF-020 belonging to the genus clostridium is associated with the development of enteritis; allopretella is a genus of Prevotella and participates in the terminal metabolism of carbohydrates; the Parabacteroidides is a common bacterium in intestinal tracts of healthy animals.

In conclusion, the lactobacillus plantarum ZJ316 and the fermented milk thereof can promote the beneficial bacteria population to be remarkably increased in the colon of the mice with the DSS acute colitis.

Note: fig. 24A is a clustering tree diagram, in which different colors represent different groups, nodes of different colors represent microorganisms playing important roles in the groups represented by the colors, yellow represents microorganisms having no significant difference, and inner-to-outer circles represent species classification levels of phyla, classes, orders, families, genera, and species, respectively. FIG. 24B is a chart of LAD scores obtained by LAD analysis of statistically significant microorganism groups in different groups, with different colors representing different groups.

FIGS. 25A-25B show the LEfse analysis of the bacterial flora in the colon of mice in each treatment group of the chronic group, and it can be seen from the graphs that the groups with significant differences in the bacterial flora in the colon of mice are the chronic modeling group (MZ), the blank control group (Ctrl), the Lactobacillus plantarum ZJ316 group (MQ), the Lactobacillus plantarum ZJ316 fermented milk group (MM), the Lactobacillus paracasei 2535 group (MN), the normal diet group (MH), the cow Milk Group (MG) and the Lactobacillus plantarum ZJ316 fermented supernatant group (MX). Wherein, the flora with obvious difference in the colon of the model-making mouse is Alloprovella on the 'genus' level, Bacteroides-sterorosoisis and Bacteroides-acidiface on the 'species' level; the flora with significant difference in the blank control group was lachnospiraceae-bacterium-DW17 on the "genus" level; the lactobacillus plantarum ZJ316 group had a significantly increased flora at the "family" level of muribacterium µae, and had significantly different flora at the "genus" level of feacalibacter and agathobacterium; the flora of lactobacillus plantarum ZJ316 significantly different in the colon of the fermented dairy mice was lachnospiraceae-bacterium-a4 at the "genus" level; the groups 2535 of lactobacillus paracasei significantly increased in flora at the "family" level were Microbacteriaceae and unidentified0-oceanospirillales, the groups significantly increased in flora at the "genus" level were Microbacterium, Marinilabiliale, pantoea and Thiopseudomonas, and the groups significantly different at the "species" level were laevenifiormans and Acinetobacter; a significantly increased flora appeared in the colon of mice in the normal diet group, Bacteroidia at the "class" level, bacteroides at the "mesh" level, Bacteroidetes at the "phylum" level; the flora with significant differences in bovine milk groups was Helicobacter-sp MIT-01-6451 at the "seed" level; the significantly increased flora in the colon of mice in the lactobacillus plantarum ZJ316 fermentation supernatant group were unidentified-lactonespiraceae at the "genus" level and helicibactee-rodentium at the "species" level, respectively.

Alloprovella is a Bacteroides, can participate in the terminal metabolism of carbohydrates in intestinal tracts, belongs to Bacteroides-acidificians, and is a source of main pathogenic bacteria in intestinal tracts; the lactobacillus-bacillus-DW 17 is a resident flora in healthy intestinal tract and can maintain intestinal tract health; feacallibacter has anti-inflammatory properties in the intestine, and Agathobacterium is a common bacterium in healthy intestine; microbacteriaceae belongs to probiotics in the family of micro-rod bacteria; the Unidentified0-oceanospirillales belong to enterobacteria and are related to intestinal metabolism; microbacterium is a common bacterium in cow milk and has a probiotic effect; pantoea is a opportunistic pathogen of the animal gut; thiopseudomonas belongs to the genus thiomonomonas and is an intestinal pathogenic bacterium; laeveniformans belongs to Vibrio laviensis and is an intestinal pathogenic bacterium; acinetobacter belongs to intestinal pathogenic bacteria; helicobacter-sp MIT-01-6451 belongs to Helicobacter, and is intestinal pathogenic bacteria; helicobacter-rodentium can cause hepatitis, cecal colitis and the like in mice.

Note that fig. 25A is a plot of the LAD scores obtained by LAD analysis of the significant microorganism populations in different groups, with different colors representing different groups. Fig. 25B is a clustering tree diagram, in which different colors represent different groups, nodes of different colors represent microorganisms playing important roles in the groups represented by the colors, yellow represents microorganisms having no significant difference, and inner-to-outer circles represent species classification levels of phyla, classes, orders, families, genera, and species, respectively.

In a word, after acute or chronic colitis is induced by DSS, harmful flora in the colon of a mouse is remarkably increased, and after lactobacillus plantarum ZJ316 and fermented milk thereof are dried, the harmful flora in the colon of the mouse is remarkably reduced, while beneficial flora is remarkably increased, so that lactobacillus plantarum ZJ316 and fermented milk thereof can regulate the flora disorder phenomenon in the colon of the mouse, and further improve colitis symptoms. This is consistent with previous studies that lactobacillus can alleviate Ulcerative Colitis (UC) by modulating the intestinal micro-ecology of mice.

The invention mainly develops researches around the influence of lactobacillus plantarum ZJ316 and fermented milk thereof on the acute and colitis of BALB/c mouse DSS, evaluates the improvement effect of lactobacillus plantarum ZJ316 and fermented milk thereof on the colitis symptoms of mice by detecting each index of colitis inflammation, and obtains the following main conclusions:

(1) the lactobacillus plantarum ZJ316 and the fermented milk thereof can effectively promote the weight recovery of mice with acute and chronic colitis induced by DSS, can obviously prevent the colon from shortening and lightening, and can alleviate tissue injury, thereby obviously improving the symptoms of colitis.

(2) Lactobacillus plantarum ZJ316 and fermented milk thereof remarkably down-regulate the expression level of proinflammatory cytokines (IL-1 β, TNF- α, IL-6 and IL-8) in colon of DSS colitis mice, and up-regulate the expression level of inflammatory cytokines (IL-10 and TGF- β).

(3) The lactobacillus plantarum ZJ316 and fermented milk thereof can promote the increase of the contents of acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid in the colon of the DSS colitis mouse.

(4) The lactobacillus ZJ316 and the fermented milk thereof can increase the diversity and abundance of beneficial flora in the colon of a DSS colitis mouse, inhibit the development of pathogenic flora, further regulate the intestinal flora disorder caused by DSS, and enable the colon microecology of the mouse to be in a more healthy and stable state.

It will be appreciated by persons skilled in the art that the embodiments of the invention described above and shown in the drawings are given by way of example only and are not limiting of the invention. The objects of the invention have been fully and effectively accomplished. The functional and structural principles of the present invention have been shown and described in the examples, and any variations or modifications of the embodiments of the present invention may be made without departing from the principles.

Claims

1. A lactic acid bacterium for use in the treatment of colitis, comprising: lactobacillus plantarum ZJ 316.

2. Lactic acid bacterium for use in the treatment of colitis according to claim 1, wherein said Lactobacillus plantarum ZJ316 is selected from infant faeces.

3. Lactic acid bacterium for use in the treatment of colitis according to claim 1, wherein said Lactobacillus plantarum is used in the treatment of colitis in acute and chronic colitis mice induced by DSS.

4. Lactic acid bacterium for use in the treatment of colitis according to claim 1, wherein said Lactobacillus plantarum ZJ316 is able to tolerate gastric acid, being able to pass from the stomach into the intestinal tract.

5. The lactic acid bacteria for treating colitis according to claim 1, wherein in the cultivation of Lactobacillus plantarum ZJ316, Lactobacillus plantarum ZJ316 frozen in a glycerol tube at-80 ℃ is streaked on an MRS solid plate, and is recovered by anaerobic cultivation at 37 ℃ for 24 hours, and is transferred to MRS liquid after two passage activation, the strain liquid is centrifuged for 16 to 18 hours at 6000r/min overnight, the fermentation supernatant is collected, and the bacterial sludge is washed twice with sterilized normal saline with a concentration of 0.85%, and then is resuspended with normal saline, so that the viable count of the bacterial suspension reaches about 2.5 x 10⁹CFU/mL。

6. The lactic acid bacterium for use in the treatment of colitis according to claim 1, wherein said Lactobacillus plantarum ZJ316 is used to down-regulate the amount of pro-inflammatory cytokine expression and up-regulate the amount of inflammatory cytokine expression in the colon of DSS colitis mouse.

7. Use of lactobacillus plantarum ZJ316 or a pharmaceutical composition comprising fermented milk thereof for the manufacture of a medicament for the treatment or prevention of colitis.

8. The use of claim 7, wherein the Lactobacillus plantarum ZJ316 is used for down-regulating the expression level of proinflammatory cytokines in the colon of a DSS colitis mouse and up-regulating the expression level of inflammatory cytokines.

9. The use according to claim 7, wherein when preparing the fermented milk, a certain amount of fresh cow milk is measured, 6% of lactobacillus plantarum ZJ316 bacterial suspension prepared in advance is added, 7% of white granulated sugar is added, and after uniform stirring, the mixture is placed in a yogurt machine for fermentation for 8-12 hours.

10. The use of claim 7, wherein in the culturing of the lactobacillus plantarum ZJ316, the lactobacillus plantarum ZJ316 frozen in a glycerol tube at-80 ℃ is streaked and inoculated in an MRS solid plate, the anaerobic culture is carried out at 37 ℃ for 24h for recovery, the lactobacillus plantarum ZJ316 is transferred to MRS liquid after two passage activation, the cultured bacterium liquid is cultured overnight for 16-18h and is centrifuged at 6000r/min for 10min, the fermented supernatant is collected, the bacterium mud is washed twice with sterilized normal saline with the concentration of 0.85 percent and then is re-suspended with the normal saline, so that the viable count of the bacterium suspension reaches about 2.5 x 10⁹CFU/mL。

11. The use according to claim 10, wherein the formulation of MRS liquid medium: accurately weighing 10g of peptone, 5g of yeast extract, 20g of anhydrous glucose, 10g of beef extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate and 1mL of Tween-80, dissolving with ultrapure water, fixing the volume to 1L, and sterilizing at 121 ℃ for 15 min.

12. The use according to claim 10, wherein the formulation of MRS solid medium: adding 1-1.5% agar based on MRS liquid culture medium formula, dissolving with ultrapure water, metering to 1L, and sterilizing at 121 deg.C for 15 min.

13. The use according to claim 7, wherein the Lactobacillus plantarum ZJ316 is selected from infant faeces.

14. Lactic acid bacteria fermented milk for the treatment of colitis, comprising: lactobacillus plantarum ZJ316 fermented milk is obtained by fermenting Lactobacillus plantarum ZJ 316.

15. The lactic acid bacteria fermented milk for use in the treatment of colitis according to claim 14, wherein the lactobacillus plantarum ZJ316 fermented milk is used to promote an increased content of acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid in the colon of DSS colitis mice.

16. The lactic acid bacteria fermented milk for treating colitis according to claim 14, wherein in preparation, a certain amount of fresh milk is measured, 6% of lactobacillus plantarum ZJ316 bacterial suspension prepared in advance is added, 7% of white granulated sugar is added, and after being uniformly stirred, the mixture is placed in a yogurt machine for fermentation for 8-12 hours.

17. The lactic acid bacteria fermented milk for use in the treatment of colitis according to claim 14, wherein the lactobacillus plantarum fermented milk is used in the treatment of colitis in acute and chronic colitis mice induced by DSS.

18. The lactic acid bacteria fermented milk for use in the treatment of colitis according to claim 14, wherein said lactobacillus plantarum ZJ316 is used to down-regulate the expression level of pro-inflammatory cytokines and up-regulate the expression level of inflammatory cytokines in the colon of DSS colitis mice.

19. The lactic acid bacteria fermented milk for treating colitis according to claim 14, wherein in the culturing of lactobacillus plantarum ZJ316, lactobacillus plantarum ZJ316 frozen in a glycerol tube at-80 ℃ is streaked on an MRS solid plate, and is recovered by anaerobic culture at 37 ℃ for 24 hours, and is transferred to MRS liquid after being activated by two passages, the bacteria liquid is cultured overnight for 16 to 18 hours, and is centrifuged at 6000r/min for 10 minutes, and the fermented supernatant is collected, and the bacterial sludge is washed twice with sterilized normal saline with a concentration of 0.85%, and then is resuspended in normal saline, so that the viable count of the bacterial suspension reaches about 2.5 x 10⁹CFU/mL。

20. Lactic acid bacteria fermented milk for use in the treatment of colitis according to claim 19, wherein the formulation of MRS liquid medium: accurately weighing 10g of peptone, 5g of yeast extract, 20g of anhydrous glucose, 10g of beef extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate and 1mL of Tween-80, dissolving with ultrapure water, fixing the volume to 1L, and sterilizing at 121 ℃ for 15 min.

21. Lactic acid bacteria fermented milk for use in the treatment of colitis according to claim 19, wherein MRS solid medium formulation: adding 1-1.5% agar based on MRS liquid culture medium formula, dissolving with ultrapure water, metering to 1L, and sterilizing at 121 deg.C for 15 min.