WO2018112741A1 - Lactobacillus acidophilus, culture method therefor and application thereof - Google Patents

Abstract

Provided are a Lactobacillus acidophilus, a culture method therefor and an application thereof. The Lactobacillus acidophilus AM13-1 is preserved in Guangdong Microbiological Culture Collection Center, and the preservation number is GDMCC No: 60091. The Lactobacillus acidophilus AM13-1 can effectively reduce cholesterol and significantly alleviate ulcerative enteritis.

Description

Lactobacillus acidophilus and its culture method and application Technical field

The invention relates to the technical field of microorganisms, in particular to a Lactobacillus acidophilus and a culture method and application thereof.

Background technique

Crohn's disease (UC) and Ulcerative colitis (CD) are two types of Inflammatory bowel disease (IBD), a chronic intestinal inflammatory disease with unknown pathogenesis. disease. The site of inflammation of ulcerative enteritis is mainly in the colon and rectum, and the main lesions are in the colonic mucosa and submucosa. At present, the pathological study of ulcerative enteritis mainly believes that its pathogenesis is related to susceptibility genes, mucosal immunity and intestinal microbes. Its clinical pathological manifestations include persistent abdominal pain, diarrhea and mucous bloody stools, and the condition is repeated. With the improvement of living standards and changes in dietary structure, the incidence of UC is on the rise.

Because the pathological mechanism is not clear, the clinical treatment is also lack of specificity and specificity. Clinically, there are mainly nutritional treatment, surgical treatment and drug treatment. Among them, drug treatment is the most important treatment. The clinical application of UC is mainly water. Salicylic acid, glucocorticoids, immunological preparations. Salicylic acid drugs can better inhibit the synthesis of prostaglandins and scavenge oxygen free radicals to achieve the purpose of relieving inflammation. The main western salicylate drugs for the treatment of UC are sulfasalazine (SASP), mainly for light Patients with moderate, moderate, and chronic UC; glucocorticoids are the first choice for patients with severe or explosive UC, such as betamethasone; immunosuppressive agents such as cyclosporine can inhibit immune response by inhibiting IL-2 production by T cells. Progress, thereby inhibiting UC.

All of the above three drugs can alleviate UC to a certain extent, but there are certain side effects. The side effects of salicylic acid are gastrointestinal reactions, headache, reticulocyte increase, sperm reduction and rash caused by allergic reactions. Hepatotoxicity, leukopenia, anemia, etc. Glucocorticoids cause the machine Side effects such as body metabolism disorder and water retention can only be used as an emergency medication and cannot be taken for a long time. Immunosuppressive therapy is highly dependent on drugs, has a long treatment period, and is prone to cause nephrotoxicity and secondary infection. It can only be used as a means of adjuvant therapy.

Summary of the invention

The present invention provides a novel species of Lactobacillus acidophilus which has the effect of preventing and/or treating ulcerative enteritis. The present invention further provides a method for cultivating the new gut bacterial species, a product thereof, and an application thereof.

The invention includes the following technical solutions:

According to a first aspect of the present invention, there is provided a Lactobacillus acidophilus AM13-1 deposited at the Guangdong Provincial Collection of Microorganisms and Cultures under the accession number GDMCC 60091.

According to a second aspect of the present invention, the present invention provides a method for culturing the Lactobacillus acidophilus AM13-1 of the first aspect, wherein the Lactobacillus acidophilus AM13-1 is inoculated into a PYG medium for anaerobic culture.

According to a third aspect of the invention, there is provided a microecological preparation comprising Lactobacillus acidophilus AM13-1 and/or a metabolite thereof of the first aspect.

As is generally understood in the art, all formulations which promote the growth and reproduction of normal microbial populations and inhibit the growth and reproduction of pathogenic bacteria are referred to as "microecological preparations". In the present invention, the term "microecological preparation" refers to a preparation prepared by using Lactobacillus acidophilus AM13-1 and/or its metabolite, which has an effect of regulating the intestinal tract and rapidly constructs an intestinal microecological balance. A typical microecological preparation may be a probiotic preparation for the prevention/treatment of ulcerative enteritis. As a probiotic of the present invention, Lactobacillus acidophilus AM13-1 has the effect of treating ulcerative enteritis, and can be further modified by different types of probiotic preparations, using different packaging and processing methods, such as embedding technology to maintain the activity of the strain to reach the corresponding The therapeutic effect, or the addition of prebiotics (bacteria powder, oligosaccharide, etc.) combined with Lactobacillus acidophilus AM13-1 to treat ulcerative enteritis can achieve the same therapeutic effect. In addition, the probiotic Lactobacillus acidophilus AM13-1 of the present invention can alleviate ulcerative enteritis, and may also exert its treatment in other inflammation-related diseases (common enteritis, gastritis, etc.). Therapeutic effect.

According to a fourth aspect of the present invention, the present invention provides a food composition, a health supplement or an auxiliary additive comprising the Lactobacillus acidophilus AM13-1 of the first aspect and/or a metabolite thereof.

The food composition of the present invention may contain, in addition to Lactobacillus acidophilus AM13-1 and/or its metabolites, various food materials, food additives, and the like, such as milk, white sugar, and vitamins. Excipient additives in the present invention, such as various edible additives.

According to a fifth aspect of the present invention, the present invention provides a pharmaceutical composition comprising the Lactobacillus acidophilus AM13-1 of the first aspect and/or a metabolite thereof.

The pharmaceutical composition of the present invention may contain, in addition to Lactobacillus acidophilus AM13-1 and/or its metabolites, various pharmaceutically acceptable carriers and/or adjuvants including, but not limited to, lactose and yeast powder. , peptone, purified water, starch, vitamins, etc., may also contain various excipients, and can be formulated into tablets or capsules. Further, the pharmaceutical composition of the present invention may further contain a substance which contributes to the maintenance of the activity of Lactobacillus acidophilus AM13-1, such as a protective agent, and a typical, but non-limiting protective agent is vitamin C.

In the pharmaceutical composition of the present invention, the content of Lactobacillus acidophilus AM13-1 may be, for example, but not limited to, typically 1 × 10 ^-1 to 1 × 10 ²⁰ cfu, based on the total volume or total weight of the pharmaceutical composition. /mL or cfu/g of Lactobacillus acidophilus AM13-1, preferably containing 1 x 10 ⁴ to 1 x 10 ¹⁵ cfu/mL or cfu/g of Lactobacillus acidophilus AM13-1.

According to a sixth aspect of the present invention, the present invention provides the use of the Lactobacillus acidophilus AM13-1 of the first aspect for the preparation of a medicament for preventing and/or treating ulcerative colitis.

According to a seventh aspect of the present invention, the present invention provides the use of the Lactobacillus acidophilus AM13-1 of the first aspect in the preparation of a cholesterol-lowering drug.

According to an eighth aspect of the present invention, the present invention provides the use of the Lactobacillus acidophilus AM13-1 of the first aspect in the preparation of a microecological preparation.

According to a ninth aspect of the present invention, the present invention provides the use of the Lactobacillus acidophilus AM13-1 of the first aspect in the preparation of a food composition, a health care product or an auxiliary additive.

According to a tenth aspect of the present invention, the present invention provides the use of the Lactobacillus acidophilus AM13-1 of the first aspect for producing a short-chain fatty acid or an organic acid.

The organic acid in the present invention, such as formic acid, acetic acid, butyric acid, etc., the organic acid may further include 3-methylbutyric acid, valeric acid, quinic acid, lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, One or more of succinic acid, fufuic acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid, and L-ascorbic acid.

According to an eleventh aspect of the present invention, the present invention provides a method for preventing and/or treating ulcerative colitis comprising administering a subject of the first aspect of the Lactobacillus acidophilus AM13-1 or the pharmaceutical composition of the fifth aspect .

According to a twelfth aspect of the invention, the invention provides a method of lowering blood lipids, controlling a decrease in body weight of a mammal, and/or reducing a disease activity index (DAI) of a mammal, comprising administering to the subject a first aspect Lactobacillus acidophilus AM13-1 or a pharmaceutical composition of the fifth aspect.

In the present invention, the subject may be a human or other mammal.

The Lactobacillus acidophilus AM13-1 of the present invention belongs to a new strain discovered by the inventors. It has been found that Lactobacillus acidophilus AM13-1 can effectively lower cholesterol and has obvious relief effect on ulcerative enteritis, which can be significantly improved. The apparent state of mice with ulcerative colitis reduces the index of disease activity in mice and improves the changes in the colon of mice.

Deposit information

Name of the strain: Lactobacillus acidophilus AM13-1

Date of deposit: October 13, 2016

Depository: Guangdong Provincial Microbial Culture Collection (GDMCC)

Deposit address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong, China

Deposit number: GDMCC 60091

DRAWINGS

Figure 1 shows a picture of a colony of Lactobacillus acidophilus AM13-1 cultured for 48 hours. The colonies are white, convex, viscous, opaque, round, with neat edges and a diameter of about 2-3 mm.

Figure 2 shows the Gram stain image (1000 times) of Lactobacillus acidophilus AM13-1 under the microscope. The microscopically amplified 1000-fold AM13-1 cells are rod-shaped, Gram-positive, non-spore and flagella. .

Figure 3 shows the cholesterol standard curve, using phthalaldehyde colorimetric method (OPA method) for cholesterol detection by using different concentrations (20ug/mL, 40ug/mL, 60ug/mL, 80ug/mL) of cholesterol and OPA The reaction was developed to obtain a standard curve. The equation of linear regression was: y=0.0085x+0.0072; the correlation coefficient R ² was 0.9992.

Figure 4 shows changes in body weight of control, model group, VSL ^# 3 and AM13-1 treated mice.

Figure 5 shows changes in the DAI index of the control, model group, VSL ^# 3 and AM13-1 treated mice.

detailed description

The present invention will be further described below in conjunction with the embodiments.

Example 1: Isolation and Identification of Lactobacillus Acidophilus AM13-1

1, separation and culture

The separated sample was from a stool sample of a healthy male in Shenzhen. The separation process of Lactobacillus acidophilus AM13-1 was as follows:

(1) Transfer the sample to an anaerobic chamber, and take about 0.2 g of the sample in 1 ml of sterile PBS (phosphate buffer), mix well, and then perform gradient dilution;

(2) Take 100ul of the diluted solution on the PYG plate (see Table 1 for the composition of PYG medium, purchased from Huanqi Microbiology Technology Co., Ltd.), then apply it, apply it evenly, and place it in an anaerobic environment at 37 °C for cultivation. The composition of the anaerobic gas is: nitrogen: hydrogen: carbon dioxide = 90: 5: 5;

(3) After culturing for 4 days, after the colonies grow on the plate, single colonies are selected for scribing and pure, and anaerobic culture at 37 ° C;

(4) Glycer storage and vacuum freeze-drying preservation of the pure single bacteria.

Table 1-1

Component	Content (1L)
Peptone	5g
Trypsin	5g
yeast	10g
Beef cream	5g
glucose	5g
K 2 HPO 4	2g
Tween 80	1ml
Cysteine-HCl·H 2 O	0.5g
Sodium sulfide	0.25g
Heme	5mg
Inorganic salt solution	40ml
Resazurin	1mg
Distilled water	950ml

Table 1-2 Formulation of inorganic salt solution

Inorganic salt component	Content (1L)
CaCl 2 ·2H 2 O	0.25g
MgSO 4 ·7H 2 O	0.5g
K 2 HPO 4	1g
KH 2 PO 4	1g
NaHCO 3	10g

NaCl

2g

2. Microbial characteristics and physiological and biochemical characteristics of AM13-1

Colony morphological characteristics statistics, Gram staining, spore and flagella staining, and physiological and biochemical identification of the strain AM13-1 of the present invention showed that AM13-1 has the following microbiological characteristics:

(1) Colony morphology

The colonies of AM13-1 cultured on a PYG plate at 37 ° C for 2 days were white, convex, viscous, opaque, round, with neat edges and a diameter of about 2-3 mm (Fig. 1).

(2) Microscopic morphology of the cells

The microbial cells magnified 1000 times AM13-1 under the microscope were rod-shaped, Gram-positive, and did not produce spores and flagella (Fig. 2).

(3) Physiological and biochemical characteristics

AM13-1 is negative for catalase, negative for oxidase, and facultative anaerobic. Substrate utilization of AM13-1 API 20A (purchased from Mérieux, France) The results of the experiment are shown in Table 2 (+, indicating a positive reaction; -, indicating a negative reaction; w indicating a weak positive reaction).

Table 2

Numbering	reaction	result	Numbering	reaction	result
1	吲哚produce	-	11	Gelatin hydrolysis	-
2	Urea (urease)	-	12	Qiyeling	+
3	glucose	+	13	glycerin	w
4	Mannitol	w	14	Cellobiose	+
5	lactose	+	15	Mannose	+
6	sucrose	+	16	Pine syrup	w
7	maltose	+	17	Cottonseed	w
8	Salicyl alcohol	+	18	Sorbitol	w

9	Xylose	w	19	D	w
10	Arabic candy	w	20	Trehalose	+

3. 16S rDNA identification of AM13-1

Genomic DNA was extracted and 16S rDNA amplification was performed using DNA as a template, and 16S rDNA universal primers (8F-AGAGTTTGATCATGGCTCAG (SEQ ID NO: 1) and 1492R-TAGGGTTACCTTGTTACGACTT (SEQ ID NO: 2)) were used, and the amplification conditions were 95 °C. Pre-denaturation for 4 min, then denaturation at 95 ° C for 30 s, annealing at 57 ° C for 40 s, extension at 72 ° C for 1 min 30 s, 30 cycles. The amplified PCR product was purified and sequenced at 3730 to obtain the full length 16S rDNA sequence of AM13-1 (SEQ ID NO: 3). By aligning the 16S rDNA sequence of AF13-1 in the NCBI database, it can be concluded that the species with the highest 16S rDNA homology with AM13-1 is Lactobacillus acidophilus with a similarity of 100%. It can be confirmed that AM13-1 is acidophilic. Lactobacillus.

Example 2: Bioactive substance of Lactobacillus acidophilus AM13-1

The bioactive substances of AM13-1 mainly investigate the production of short-chain fatty acids (SCFA) and organic acids.

1, sample pretreatment

The AM13-1 was cultured for 48 hours, and 1 ml of the bacterial solution was centrifuged at 10,000 r/min for 5 minutes, and the supernatant was taken to prepare for detection of short-chain fatty acids (SCFA) and organic acids.

2. Determination of SCFA

The short-chain fatty acid was determined by an external standard method, and acetic acid, propionic acid, butyric acid, and valeric acid were used to prepare a standard curve. Using Agilent Meteorological Chromatograph (GC-7890B, Agilent), HP-INNOWax (Cross-Linked PEG), 30m × 0.25mm × 0.25um capillary column was used for analysis. The detector was a hydrogen flame ion detector, and the GC parameter was set to Column temperature: 180～200°C; gasification chamber temperature: 240°C; detection temperature: 210°C; injection volume: 2μL; carrier gas flow rate: N ₂ , 50mL/min; hydrogen flow rate: 50mL/min; air flow rate: 600 ~700ml/min.

3. Determination of organic acids

The selection criteria for organic acids are: 3-methylbutyric acid, valeric acid, quinic acid, lactic acid, oxalic acid, and propylene Acid, benzoic acid, maleic acid, succinic acid, fufuic acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid and L-ascorbic acid. Still using Agilent Meteorological Chromatograph (GC-7890B, Agilent), the column is selected from 122-5532G DB-5ms (40m × 0.25mm × 0.25um), column temperature: 270 ~ 290 ° C; inlet temperature: 250 ° C; gas Flow rate: 0.86 ml/min.

4. The experimental results are detailed in Table 3 below:

table 3

Example 3: Antibiotic sensitivity of Lactobacillus acidophilus AM13-1

To investigate the sensitivity of AM13-1 to common 20 antibiotics, the experiment was carried out using the drug-sensitive paper method. 100 μl of the bacterial solution of the AM13-1 cultured in the log phase was plated, and the antibiotic susceptibility sheet was attached to the surface of the plate, and cultured at 37 ° C for 48 hours, and the size of the inhibition zone was measured. The results are shown in Table 4.

Table 4

Drug susceptibility tests showed that AM13-1 was sensitive to antibiotics other than oxacillin and cefazolin.

Example 4: Tolerance of Lactobacillus acidophilus AM13-1

1. Acid tolerance of AM13-1

PYG medium with pH 2.5 was prepared, and AM13-1 cultured to log phase was inoculated into PYG medium of pH 2.5 according to 10% inoculation amount. After incubation for 2 hours at 37 °C, AM13 cultured in the same amount of normal PYG medium was taken. -1 Bacterial solution and AM13-1 culture solution cultured in PYG medium of pH 2.5 were plated and counted, and the survival rate of AM13-1 bacteria treated under the condition of pH 2.5 was calculated according to the following formula:

pH2.5 treatment survival rate = (number of bacterial liquid plate coated colonies in PYG medium cultured at pH 2.5 / number of bacterial liquid plate coated colonies in normal PYG medium culture) × 100%

The results showed that the survival rate of AM13-1 treated at pH 2.5 for 2 h was 92%.

2. Bile salt tolerance of AM13-1

0.3% bile salt medium was prepared. By adding 0.3% bile salt to PYG, AM13-1 cultured to log phase was inoculated to 0.3% PYG bile salt medium at 10% inoculation, and cultured at 37 °C. After 2 h, the AM13-1 broth cultured in an equal amount of normal PYG medium and the AM13-1 broth cultured in 0.3% PYG bile salt medium were plated and counted, and the condition of 0.3% bile salt was calculated according to the following formula. Survival rate of the treated AM13-1 strain:

0.3% bile salt treatment survival rate = (0.3% of PYG bile salt medium cultured bacterial liquid plate coated colony number / normal PYG medium cultured bacterial liquid plate coated colony number) × 100%

The results showed that the survival rate of AM13-1 treated with 0.3% bile salt for 2 h was 85%.

Through the above tolerance experiments, AM13-1 maintained a high survival rate (92% and 85%) under the conditions of pH 2.5 and 0.3% bile salt, respectively, indicating that AM13-1 has a strong acid and Bile salt tolerance, the vast majority of live bacteria can reach their function through the stomach juice and small intestine of the human body.

Example 5: Cholesterol-lowering properties of Lactobacillus acidophilus AM13-1

1, bile salt hydrolase activity of AM13-1

The bile salt hydrolase was detected by TDCA method, and the TDAC plate was prepared. 4% of TDAC (sodium taurodeoxycholate) and 0.37 g/L of CaCl ₂ were added to the PYG solid medium, and AM13-1 was cultured to a concentration of about 10 ⁸ cfu/ml, take 10ul of bacteria droplets on a 0.6mm diameter filter paper, place the filter paper on the surface of the TDAC plate, and incubate at 37 °C for 2 days to observe the white precipitate produced around the filter paper. The diameter of the white precipitate represents the gallbladder. Salt hydrolase activity.

By measurement, the white precipitate of AM13-1 had a diameter of 12 mm, indicating that AM13-1 has a bile salt hydrolase activity.

2, AM13-1 cholesterol reduction in vitro

The method for determining the content of cholesterol is determined by the o-phthalaldehyde colorimetric method (OPA method), and the degradation ability of cholesterol is examined by the change of the cholesterol content of the strain in a certain concentration of cholesterol medium for a period of time. The specific method is as follows:

(1) Preparation of cholesterol medium and cultivation of experimental strains

A certain amount of cholesterol was weighed and dissolved in ethanol at a concentration of 10 mg/mL, and sterilized by filtration. Add the prepared PYG medium to 10 mg/mL bile salt (autoclave), 10% mass concentration of sodium thioglycolate (filter sterilization) and cholesterol, mix well, and then wait for 3% of the inoculum. The test strain was inoculated into the medium. In addition to AM13-1, another strain of commercial cholesterol-lowering probiotic Lactobacillus plantarum Lp299v (purchased from Probi, Sweden) was used for comparison. Both bacteria were anaerobic at 37 °C. Incubate for 72 h under the conditions.

(2) Production of standard curve

Accurately measure 0.5mg/mL cholesterol standard solution 40uL, 80uL, 120uL, 160uL, 200uL in a clean tube, add absolute ethanol to 1mL, add OPA 4mL (0.5mg phthalaldehyde to each tube) 1mL glacial acetic acid), shake and mix, let stand at room temperature for 10min, then add 2mL of concentrated sulfuric acid and mix, let stand for 10min, measure the absorbance at 550nm. Taking the concentration as the abscissa and the absorbance as the ordinate to draw a standard curve (Fig. 3), by calculation, the equation of linear regression is: y = 0.0085 + 0.0072; the correlation coefficient R ² is 0.9992.

(3) Determination of cholesterol in the medium

The culture solution containing the cholesterol-containing PYG medium was centrifuged at 10,000 r/min, and the supernatant was collected for cholesterol detection, and the uninoculated cholesterol PYG medium was used as a blank control group. Take 1ml of the sample to be tested in a clean test tube, add 6ml of 95% ethanol and 4ml of 50% KOH, shake and mix, then saponify in a 60 °C water bath for 10min, rapidly cool, add 10ml of n-hexane for extraction, fully Mix well, let stand for 20 min at room temperature, measure 8 ml organic phase (n-hexane layer) into another clean test tube, then dry it in nitrogen in a 60 ° C water bath, add 4 ml 0.5 g / L phthalaldehyde acetic acid solution, room temperature After standing for 10 min, 2 ml of concentrated H ₂ SO _{4 was added} for 10 min, and finally, the absorbance at 550 nm was measured.

(4) Calculation of cholesterol degradation rate

The cholesterol content in the medium before and after culture was calculated according to the standard curve. The degradation rate of cholesterol was calculated according to the following formula:

L=(A-B)/A×100%

L: cholesterol degradation rate; A: cholesterol content in the cholesterol medium not inoculated; B: cholesterol content in the culture medium for 48 hours after the test strain is cultured.

(5) Cholesterol degradation results

By calculation, the cholesterol degradation rate of AM13-1 was 78%, and the degradation rate of Lp299v was 70%, which indicated that AM13-1 had stronger cholesterol degradation ability than Lp299v.

Example 6: Effect of Lactobacillus acidophilus AM13-1 on ulcerative enteritis in mice

1. Experimental mice and grouping

The experimental mice were C57bl/6 mice (purchased from Hubei Medical Experimental Animal Center), 8 weeks old, weighing 20g±2g, and the mouse breeding environment was SPF grade, and the feeding was performed for 1 week. The modeling method was performed using a dextran sulfate sodium (DSS) method, and the mice were self-drinking 0.2% DSS for 7 days. The experiment was divided into 4 groups of 12 mice each, as follows:

(1) Control group (blank control group): Normally reared mice were not subjected to DSS induction;

(2) Model group: DSS-induced UC model, each mouse was intragastrically administered with 200 ul of PBS per day;

(3) VSL#3 treatment group: DSS-induced UC model, each mouse was intragastrically administered with 200 ul of VSL#3 bacterial agent (purchased from Sigma Tau, USA);

(4) AM13-1 treatment group: DSS-induced UC model, each mouse was intragastrically administered with 200 ul of AM13-1 bacteria per day.

2. Intervention strain preparation

(1) VSL#3: Take a certain amount of VSL#3 powder dissolved in PBS, mix well, adjust the bacteria to 10 ⁹ cfu/ml;

(2) AM13-1: AM13-1 was cultured to logarithmic growth phase, and the bacterial solution was centrifuged at 8000 r/min, and the cells were suspended in PBS to adjust the concentration to 10 ⁹ cfu/ml.

3. Experimental process

DSS induction and intragastric intervention were performed according to the grouping of mice. The intervention method was performed by side-molding. The body weight, diet and drinking water were recorded every day. The fecal traits and fecal occult blood were observed on the first day. The disease activity index (DAI) of the mice was calculated on the 3rd, 5th, and 7th days. The DAI scores are shown in Table 5. The experiment lasted for 7 days, and the daily gavage amount of probiotics and PBS was 200 ul/mouse. At the end of the experiment, the mice were sacrificed. All mice were bled, necked, colonic, photographed, weighed, and the length of the colon was measured. Colon tissue was stored in a -80 ° C refrigerator and paraformaldehyde.

Table 5 DAI Index Score Sheet

Stool characteristics in the table: normal stool - forming stool; loose stool - paste-like, semi-formed stool that does not adhere to the anus; loose stool - a thin watery stool that can adhere to the anus.

Blood in the stool: normal mice have positive blood in the stool; the blood of the naked eye is red or brown; the occult blood is positive for the naked blood, which is detected by tetramethylbenzidine.

The DAI index is equal to the sum of the three points of weight, stool traits, and fecal occult blood/weak blood.

4. Experimental results

(1) Effect of AM13-1 on DSS-induced changes in body weight of UC mice, as shown in Table 6 and Figure 4.

Table 6

DSS-induced ulcerative enteritis model mice caused weight loss. On day 3, the mice in the model group showed a significant decrease in body weight (*P<0.05). On the fifth day, the body weight of the model group became extremely different from that of the control group. Significant (**P<0.01). The intervention of probiotic AM13-1 could effectively control the decrease of body weight in mice. On the 7th day, the weight loss of AM13-1 and VSL ^# 3 mice was effectively controlled compared with the model group ( ^▲ P<0.05). This indicates that the two groups of probiotics can control the weight loss caused by UC. By comparing the body weight values of the groups on the 7th day, it was found that the body weight of the AM13-1 group was higher than that of VSL ^# 3, indicating that the ability of AM13-1 to control body weight loss in UC mice was better than that of VSL ^# 3.

(2) AM13-1 improves the DAI index of DSS-induced UC mice

The DAI index is an important indicator for judging UC mice. It characterizes the severity of the disease in this mouse. DSS-induced UC model mice cause weight loss in mice, inflammation and ulceration in the colon, bleeding, and affecting stool traits. , causing the DAI index to rise. The DAI values of the mice in each group during the experiment are shown in Table 7 and Figure 5.

Table 7

It can be found from Table 7 and Figure 5 that the DAI index of the mice gradually increased with the induction of DSS, and the DAI of the DSS-induced mice (model group) began to change significantly on the third day relative to the control group (*P< 0.01). Under the intervention of probiotics, the DAI rise of the mice was alleviated. On the 7th day, the DAI index of AM13-1 and VSL ^# 3 mice was significantly lower than that of the model group ( ^▲ P<0.05), from the 5th and 7th days. The DAI values showed that the DAI value of AM13-1 intervention mice was slightly lower than that of VSL#3, indicating that AM13-1 is superior to VSL ^# 3 in controlling disease in mice.

(3) AM13-1 control of colon length shortening induced by DSS in UC mice

The colon of UC mice induced by DSS caused the colon to shorten due to inflammation and ulceration. Therefore, the shortening of the length of the colon can be used as an important indicator of the severity of UC. The end of the experiment, the colon length of each group of mice is shown in Table 8. .

Table 8

The results showed that the colon length of the model group was shortened more severely (P<0.01 compared with the control group), and the colon shortening was controlled to some extent by the intervention of probiotics, compared with the model group, VSL ^# 3 and AM13-1. Significant control of colon length shortening ( ^▲ P < 0.05). The colon length of the AM13-1 group was longer than that of the VSL ^# 3 group, indicating that AM13-1 is superior to the VSL ^# 3 group in controlling colonic shortening in mice, and therefore the ability of AM13-1 to control UC inflammation and ulcers. Better.

Example 7: Food composition containing Lactobacillus acidophilus AM13-1

The raw materials are shown in Table 9.

Table 9

raw material	Percentage of mass (%)
Lactobacillus acidophilus AM13-1	0.5
milk	90.0
White sugar	9.0
Vitamin C	0.5

Mix milk and sugar according to the above formula ratio, stir until completely mixed, preheat, homogenize at 20Mpa, sterilize for 5-10 minutes at 90 °C, cool to 40-43 °C, mix with protective agent vitamin C, inoculate 1-100×10 ⁶ cfu/g of Lactobacillus acidophilus AM13-1 bacteria, that is, a food composition containing Lactobacillus acidophilus AM13-1 bacteria.

Example 8: Pharmaceutical composition containing Lactobacillus acidophilus AM13-1

The ratio of raw materials is shown in Table 10.

Table 10

raw material	Percentage of mass (%)
Lactobacillus acidophilus AM13-1	1.0
lactose	2.0
yeast	2.0
Peptone	1.0
pure water	93.5
Vitamin C	0.5

According to the ratio, lactose, yeast powder and peptone are mixed uniformly with purified water, preheated to 60-65 ° C, homogenized at 20 Mpa, sterilized at 90 ° C for 20-30 minutes, cooled to 36-38 ° C, mixed with protective agent vitamin C, Connected to Lactobacillus acidophilus AM13-1 live bacteria (1-500×10 ⁶ cfu/mL), fermented to pH 6.0 at 36-38 ° C, centrifuged, freeze-dried to a moisture content of less than 3%, ie prepare acidophilus Lactobacillus AM13-1 bacteria freeze-dried. 0.5 g of Lactobacillus acidophilus AM13-1 lyophilized product was weighed in an equal amount with maltodextrin, and then filled into a capsule to prepare a pharmaceutical composition containing Lactobacillus acidophilus AM13-1.

Example 9: Preparation method of medicine for treating ulcerative enteritis (UC)

1. Preparation of bacterial solution: Lactobacillus acidophilus AM13-1 (1×10 ⁹ cfu/ml) was subjected to anaerobic culture, and anaerobic medium was cultured in PYG medium, and subjected to anaerobic fermentation at 37 ° C for 2-3 days.

2. Preparation of growth factors: Mixing skim milk and casein, centrifugation and ultrafiltration to obtain crude extracts of milk growth factors (nutrient substances containing vitamins, terpenoids and pyrimidines).

3. Preparation of pharmaceutical dosage form: Add 5 volumes of growth factor and 1 volume of protective agent microorganism C to 100 volumes of AM13-1 fermented bacterial solution, mix well and mix, then add starch adjuvant (such as maltodextrin) to prepare. Pharmaceutical dosage form.

The above is a further detailed description of the present invention in connection with the specific embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (15)

1. A Lactobacillus acidophilus AM13-1 deposited in the Guangdong Provincial Collection of Microorganisms and Cultures under the accession number GDMCC 60091.
2. A method for culturing Lactobacillus acidophilus AM13-1 according to claim 1, wherein the Lactobacillus acidophilus AM13-1 is inoculated into a PYG medium for anaerobic culture.
3. A microecological preparation comprising the Lactobacillus acidophilus AM13-1 and/or a metabolite thereof according to claim 1.
4. A food composition, a health supplement or an auxiliary additive comprising the Lactobacillus acidophilus AM13-1 and/or a metabolite thereof according to claim 1.
5. A pharmaceutical composition comprising the Lactobacillus acidophilus AM13-1 and/or a metabolite thereof according to claim 1.
6. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition contains 1 × 10 ^-1 to 1 × 10 ²⁰ cfu/mL or cfu, based on the total volume or total weight of the pharmaceutical composition. / g of Lactobacillus acidophilus AM13-1, preferably containing 1 x 10 ⁴ to 1 x 10 ¹⁵ cfu/mL or cfu/g of Lactobacillus acidophilus AM13-1.
7. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant.
8. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition further contains a substance which contributes to the maintenance of the activity of Lactobacillus acidophilus AM13-1.
9. Use of the Lactobacillus acidophilus AM13-1 according to claim 1 for the preparation of a medicament for preventing and/or treating ulcerative colitis.
10. Use of Lactobacillus acidophilus AM13-1 according to claim 1 for the preparation of a cholesterol-lowering drug.
11. Use of Lactobacillus acidophilus AM13-1 according to claim 1 for the preparation of a microecological preparation.
12. The Lactobacillus acidophilus AM13-1 according to claim 1 for preparing a food composition, a health supplement or a supplement Application in feed additives.
13. Use of the Lactobacillus acidophilus AM13-1 of claim 1 for the production of short chain fatty acids or organic acids.
14. A method for preventing and/or treating ulcerative colitis, which comprises administering the pharmaceutical composition according to claim 1 or Lactobacillus acidophilus AM13-1 or the pharmaceutical composition according to claim 5.
15. A method of lowering blood lipids, controlling a decrease in body weight of a mammal, and/or reducing a disease activity index of a mammal, characterized in that the subject is administered the Lactobacillus acidophilus AM13-1 or claim according to claim 1. 5 The pharmaceutical composition described.

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