CN111004734A - Lactobacillus rhamnosus capable of regulating and controlling relative abundance of acinetobacter in intestinal tract - Google Patents
Lactobacillus rhamnosus capable of regulating and controlling relative abundance of acinetobacter in intestinal tract Download PDFInfo
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- CN111004734A CN111004734A CN201910217270.0A CN201910217270A CN111004734A CN 111004734 A CN111004734 A CN 111004734A CN 201910217270 A CN201910217270 A CN 201910217270A CN 111004734 A CN111004734 A CN 111004734A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
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- A23V2400/175—Rhamnosus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention discloses lactobacillus rhamnosus capable of regulating and controlling relative abundance of acinetobacter in intestinal tracts, and belongs to the technical field of microorganisms and medicines. The Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 obtained by screening can improve the intestinal health, and is specifically embodied in that: (1) can utilize fructo-oligosaccharide, xylo-oligosaccharide and galacto-oligosaccharide, and has good oligosaccharide utilization capability; (2) in vitro metabolism can generate a plurality of short chain fatty acids such as acetic acid, propionic acid, butyric acid and the like; (3) can reduce the relative abundance of Acinetobacter in intestinal tracts, so that the Lactobacillus rhamnosus CCFM1039 has great application prospect in preparing products (such as food, medicines or health care products and the like) for improving the intestinal tract health.
Description
Technical Field
The invention relates to lactobacillus rhamnosus capable of regulating and controlling relative abundance of acinetobacter in intestinal tracts, and belongs to the technical field of microorganisms and medicines.
Background
The intestinal tract is the digestion place of the human body and the largest immune organ of the human body, is rich in huge and complex microbial colonies, has over 800 strains of 7000 bacteria, and shows great diversity on the subspecies or strain level. The intestinal microorganisms have important influence on human health, can help host cell metabolism, provide nutrition for intestinal epithelial cells, enhance immune function and resist invasion of foreign microorganisms. Normally, the intestinal microorganisms are in symbiotic state with human body, and the microorganisms draw nutrients required for growth from the host, and the host can degrade some substances which cannot be decomposed and utilized by the intestinal microorganisms.
However, in the modern society, the problem of unbalanced intestinal flora of people is increasingly highlighted due to the wide application of antibacterial drugs. The main reason is that the antibacterial drugs can inhibit intestinal sensitive bacteria, while the growth advantage of the original bacteria or the pass-through bacteria which are easy to generate resistance to the antibacterial drugs (the original bacteria or the pass-through bacteria which are easy to generate resistance to the antibacterial drugs are many pathogenic bacteria, mainly including escherichia coli, typhoid bacillus, dysentery bacillus and vibrio cholerae) can be generated, and the two bacteria cause the structural disorder of intestinal flora together, so that the intestinal microorganisms can influence the health of hosts from various ways such as energy absorption, endotoxemia, chain-broken fatty acid, choline and bile acid metabolism.
Acinetobacter belongs to conditional pathogenic bacteria which exist in human intestinal tracts and are easy to generate resistance to antibiotics, and if the intestinal flora of an organism is unbalanced, the Acinetobacter can multiply in the organism in large quantity to further cause diseases such as respiratory tract infection, septicemia, meningitis, endocarditis, wound and skin infection, urogenital tract infection and the like of the organism; in addition, because antibiotics cannot selectively inhibit Acinetobacter, the Acinetobacter is easy to acquire drug resistance in the antibiotic application process, and the human body is difficult to treat once infected with the Acinetobacter.
Epidemiological data show that 30-35% of people at home and abroad suffer from septicemia, and more than 70% of people suffer from urinary infection and intestinal infection. Therefore, there is an urgent need to find a method for inhibiting Acinetobacter in intestinal tract.
Currently, some studies have been conducted to obtain some probiotics with improved intestinal flora imbalance by screening, for example, patent application publication No. CN107312726A discloses a lactobacillus plantarum which can inhibit the growth of harmful bacteria such as escherichia coli, salmonella, streptococcus suis and staphylococcus aureus in intestinal tract; patent application publication No. CN104232515A discloses Bifidobacterium animalis which is capable of increasing the number of bifidobacteria and lactobacilli in the intestinal tract and, at the same time, of decreasing the number of bacteroides and coliform bacteria in the intestinal tract.
However, the probiotics lack pertinence to Acinetobacter, and at present, no probiotics capable of obviously inhibiting the Acinetobacter in human intestinal tracts exists, so that the treatment of Acinetobacter infection is undoubtedly prevented.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problem of providing a Lactobacillus rhamnosus strain which can obviously inhibit the relative abundance of Acinetobacter (Acinetobacter) in human intestinal tracts; the relative abundance is the percentage of the number of a particular species of bacteria in a sample relative to the total number of bacteria in the sample.
[ solution ]
In order to solve the problems, the invention provides a Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039, wherein the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 is preserved in Guangdong province microbial strain preservation center in 2018, 11 and 20 days, the preservation number is GDMCC No.60490, and the preservation address is No. 59 building 5 of Miyaolu 100 of Guangzhou city.
The Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1039 is obtained by separating a neonatal excrement sample from obstetrics and gynecology department of Huaxing hospital of Tianjin hong Kong, the 16S rRNA sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis, and the sequence obtained through sequencing is compared with the nucleic acid sequence in NCBI, so that the result shows that the strain is Lactobacillus rhamnosus and is named as Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM 1039.
The Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 has the following characteristics:
(1) the colony after 48 hours of culture on the MRS culture medium is round, white, opaque and smooth;
(2) resistance to simulated gastrointestinal fluids: after 3 hours of culture in 3g/L of pepsin-containing physiological saline with pH of 3, the survival rate is as high as 103.19 +/-2.01 percent; after 4h of incubation in physiological saline at pH 8 containing 1g/L trypsin and 0.3% (m/v) (i.e. 0.3g/100mL) bile salts, the survival rate was 12.79. + -. 0.14%; after the culture is carried out for 3 hours in physiological saline with pH 3 and 3g/L pepsin, the culture is continued for 4 hours in physiological saline with pH 8 and containing 1g/L trypsin and 0.3% (m/v) bile salt, and the survival rate is still 13.19 +/-0.40 percent;
(3) can produce acid by utilizing fructo-oligosaccharide, xylo-oligosaccharide and galacto-oligosaccharide;
(4) in vitro metabolism can generate a plurality of short chain fatty acids such as acetic acid, propionic acid, butyric acid and the like;
(5) can reduce the relative abundance of Acinetobacter in the intestinal tract.
The invention provides application of the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 strain in preparation of a product for improving intestinal health.
In one embodiment of the invention, the product is lactobacillus rhamnosus (Lactob)Acillusrhamnosus) CCFM1039 has viable cell count of not less than 1 × 106CFU/mL or 1X 106CFU/g。
In one embodiment of the invention, the viable count of lactobacillus rhamnosus (lactobacillus rhamnosus) CCFM1039 in the product is not less than 1 × 108CFU/mL or 1X 108CFU/g。
In one embodiment of the invention, the product comprises a food, pharmaceutical or nutraceutical product.
In one embodiment of the invention, the medicament comprises lactobacillus rhamnosus (lactobacillus rhamnosus) CCFM1039, a pharmaceutical carrier and/or a pharmaceutical excipient.
The invention provides a product for improving intestinal health, which contains the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM 1039.
In one embodiment of the invention, the viable count of lactobacillus rhamnosus (lactobacillus rhamnosus) CCFM1039 in the product is not less than 1 × 106CFU/mL or 1X 106CFU/g。
In one embodiment of the invention, the viable count of lactobacillus rhamnosus (lactobacillus rhamnosus) CCFM1039 in the product is not less than 1 × 108CFU/mL or 1X 108CFU/g。
In one embodiment of the invention, the product comprises a food, pharmaceutical or nutraceutical product.
In one embodiment of the invention, the medicament comprises lactobacillus rhamnosus (lactobacillus rhamnosus) CCFM1039, a pharmaceutical carrier and/or a pharmaceutical excipient.
Has the advantages that:
the invention screens out a strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039, the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 can improve the intestinal health, and the specific expression is as follows:
(1) functional oligosaccharide can not be digested and absorbed by stomach and small intestine, but can be utilized by probiotics such as bifidobacterium, Lactobacillus and the like in large intestine to proliferate beneficial bacteria, inhibit pathogenic bacteria and adjust intestinal flora balance, and Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 can utilize fructo-oligosaccharide, xylo-oligosaccharide and galacto-oligosaccharide, so that the oligosaccharide utilization capability is good;
(2) the short-chain fatty acid can reduce the pH value in the intestinal tract, improve the acidic environment of the intestinal tract, proliferate beneficial bacteria, inhibit harmful bacteria to regulate intestinal flora and improve the intestinal function, the in vitro metabolism of the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 can generate a plurality of short-chain fatty acids such as acetic acid, propionic acid and butyric acid, and the content of the short-chain fatty acid in the mouse excrement is obviously increased after the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM103915 days;
(3) acinetobacter belongs to conditional pathogenic bacteria which exist in human intestinal tracts and are easy to generate resistance to antibiotics, and can cause respiratory tract infection, septicemia, meningitis, endocarditis, wound and skin infection, urogenital tract infection and other diseases of organisms, and the relative abundance of Acinetobacter in intestinal tracts can be reduced by the Lactobacillus rhamnosus CCFM1039 of the invention, after the Lactobacillus rhamnosus CCFM103915 of the invention is perfused, the relative abundance of Acinetobacter in mouse excrement is obviously reduced,
therefore, the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 has a huge application prospect in preparing products (such as food, medicines or health products and the like) for improving intestinal health.
Biological material preservation
A strain of Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1039 is classified and named as Lactobacillus rhamnous, is preserved in Guangdong province microorganism strain preservation center in 2018, 11 and 20 days, and has the preservation number of GDMCC No.60490 and the preservation address of No. 59 floor 5 of Michelia furiosa No. 100 of Guangzhou city.
Drawings
FIG. 1: culturing different strains of lactobacillus rhamnosus for 3h under simulated gastric juice, and then continuously culturing for 4h under simulated intestinal juice; wherein the abscissa is the number of Lactobacillus rhamnosus LR1, LR2, LR3, LR4, LR5, and CCFM 1039.
FIG. 2: the yield of total short-chain fatty acid of different strains of lactobacillus rhamnosus in the in vitro culture process; wherein the abscissa is the number of Lactobacillus rhamnosus LR1, LR2, LR3, LR4, LR5, and CCFM 1039.
FIG. 3: the yield of acetic acid of different strains of lactobacillus rhamnosus in the in vitro culture process; wherein the abscissa is the number of Lactobacillus rhamnosus LR1, LR2, LR3, LR4, LR5, and CCFM 1039.
FIG. 4: the yield of propionic acid of different strains of lactobacillus rhamnosus in the in vitro culture process; wherein the abscissa is the number of Lactobacillus rhamnosus LR1, LR2, LR3, LR4, LR5, and CCFM 1039.
FIG. 5: the yield of butyric acid of different strains of lactobacillus rhamnosus in the in vitro culture process; wherein the abscissa is the number of Lactobacillus rhamnosus LR1, LR2, LR3, LR4, LR5, and CCFM 1039.
FIG. 6: relative abundance of Acinetobacter in faeces of CCFM1039 group of mice in example 9, where "×" indicates a significant difference from the blank group (P < 0.05).
Detailed Description
The invention is further illustrated with reference to specific examples.
Pepsin 1:10000U (product No. A600688, CAS: [9001-75-6]) referred to in the following examples was purchased from Biotechnology engineering (Shanghai) Ltd; trypsin 1:250 (product No. 64008867, CAS: [9002-07-7]) referred to in the following examples was purchased from national pharmaceutical group chemical Co., Ltd; bile salts referred to in the following examples were purchased from Shanghai Bayer Biotech Ltd; glucose referred to in the following examples was purchased from national pharmaceutical group chemical agents limited; fructooligosaccharides (FOS) and Galactooligosaccharides (GOS) referred to in the following examples were purchased from cheng bao bio-inc; xylo-oligosaccharides (XOS) referred to in the following examples were purchased from shanghai-sourced leaf biotechnology limited; the skim milk powders referred to in the examples below were purchased from illite.
The media involved in the following examples are as follows:
MRS liquidMedium (g/L): 10g/L of peptone, 5g/L of yeast extract, 20g/L of glucose, 2g/L of anhydrous sodium acetate and 2g/L, K of citric acid hydrogen diamine2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.5g/L、MnSO4·7H2O0.25 g/L, Tween-801 g/L and distilled water 1000 g/L.
MRS solid medium (g/L): 10g/L of peptone, 5g/L of yeast extract, 20g/L of glucose, 2g/L of anhydrous sodium acetate and 2g/L, K of citric acid hydrogen diamine2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.5g/L、MnSO4·7H2O0.25 g/L, Tween-801 g/L, agar 20g/L and distilled water 1000 g/L.
Example 1: screening and strain identification of lactobacillus rhamnosus
1. Screening
Using newborn feces from obstetrics and gynecology department in Tianjin Dagang Huaxing hospital as a sample, and performing gradient dilution by 10 times to 10 times by using sterile physiological saline-6Then 100. mu.L of each dilution gradient was set to 10-4、10-5、10-6The diluted solution is plated on an MRS solid culture medium, cultured for 48 hours at 37 ℃, and observed and recorded colony morphology; selecting colonies with different forms on an MRS solid culture medium for streaking separation, and after culturing for 48 hours at 37 ℃, selecting single colonies with different forms on the MRS solid culture medium again for streaking separation until obtaining pure single colonies with consistent forms; selecting pure colonies on an MRS solid culture medium, inoculating the pure colonies in 5mL of MRS liquid culture medium, and culturing at 37 ℃ for 18 h; and (3) taking 1mL of bacterial liquid in a sterile centrifuge tube, centrifuging for 3min at 8000r/min, removing an upper culture medium, resuspending bacterial sludge in a 30% glycerol solution, and preserving at-80 ℃ to obtain the bacterial strain.
2. Identification
The separated strain is subjected to PCR amplification of 16S rDNA, PCR products are sent to Huada gene sequencing limited company for sequencing (the 16S rDNA sequence of CCFM1039 is shown as SEQ ID NO. 1), the sequencing results are subjected to nucleic acid sequence comparison in NCBI, and finally 6 strains of Lactobacillus rhamnosus are obtained and are respectively named as Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039, Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR1, Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR2, Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR3, Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR4 and Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR 5.
Example 2: culture of Lactobacillus rhamnosus
After Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 is inoculated on an MRS solid culture medium and cultured for 48 hours at 37 ℃, the colony is observed and is found to be round, white, opaque and smooth.
Example 3: tolerance of different lactobacillus rhamnosus to simulated gastrointestinal fluids
The method comprises the following specific steps:
1. tolerance of different lactobacillus rhamnosus to simulated gastric juice
The Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1039, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR1, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR2, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR3, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR4 and the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR5 obtained in example 1 are respectively inoculated into an MRS liquid culture medium and cultured for 18 hours at 37 ℃, the cells are collected by centrifugation, the collected cells are washed by physiological saline, the cells are collected by centrifugation again after the washing is finished, the collected cells are respectively resuspended in physiological saline containing 3g/L pepsin with pH being 3 (the pH is adjusted by HCl) to OD600And 5.0, taking 1mL of bacterial liquid to perform flat viable count to serve as the original viable count of the rhamnose lactobacillus in the bacterial liquid, placing the residual bacterial liquid at 37 ℃ for culturing for 3 hours, and taking 1mL of bacterial liquid to perform flat viable count to serve as the viable count of the rhamnose lactobacillus in the bacterial liquid after tolerating simulated gastric juice.
Wherein the survival rate (%) after the gastric juice tolerance is 100% (viable count of lactobacillus rhamnosus in the bacterial liquid/original viable count of lactobacillus rhamnosus in the bacterial liquid after the gastric juice tolerance simulation).
The detection results are as follows: after 3h of culture in physiological saline containing pepsin and having a pH of 3, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 is 103.19 + -2.01%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR1 is 89.82 + -3.00%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR2 is 102.05 + -10.27%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR3 is 10.32 + -0.33%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR4 is 61.85 + -0.81%, and the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) 5 is 85.59 + -5.35%.
As can be seen, Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 and LR2 have strong tolerance to simulated gastric juice.
2. Tolerance of different lactobacillus rhamnosus to simulated intestinal fluid
The Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1039, Lactobacillus rhamnosus (Lactobacillus rhamnous) LR1, Lactobacillus rhamnosus (Lactobacillus rhamnous) LR2, Lactobacillus rhamnosus (Lactobacillus rhamnous) LR3, Lactobacillus rhamnosus (Lactobacillus rhamnous) LR4 and Lactobacillus rhamnosus (Lactobacillus rhamnous) LR5 obtained in example 1 were inoculated into an MRS liquid medium, respectively, cultured at 37 ℃ for 18 hours, centrifuged to collect cells, the collected cells were washed with physiological saline, after washing, centrifuged to collect cells again, the collected cells were resuspended in physiological saline having pH of 8(pH adjusted by NaOH) containing 1g/L trypsin and 0.3% (m/v) bile salt, respectively, to OD600And 5.0, taking 1mL of bacterial liquid to perform flat viable count to serve as the original viable count of the rhamnose lactobacillus in the bacterial liquid, placing the residual bacterial liquid at 37 ℃ for culturing for 4 hours, and taking 1mL of bacterial liquid to perform flat viable count to serve as the viable count of the rhamnose lactobacillus in the bacterial liquid after the intestinal juice tolerance simulation.
Wherein the survival rate (%) after the intestinal fluid tolerance is 100% (viable count of lactobacillus rhamnosus in the bacterial liquid/original viable count of lactobacillus rhamnosus in the bacterial liquid after the intestinal fluid tolerance simulation).
The detection results are as follows: after 4 hours of culture in physiological saline containing trypsin and bile salts at pH 8, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 was 12.79 + -0.14%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR1 was 7.23 + -0.34%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR2 was 10.03 + -0.75%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR3 was 5.73 + -0.31%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnos) LR4 was 14.45 + -0.37%, and the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnous LR) 5 was 10.46 + -0.83%.
As can be seen, Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 and LR4 have strong tolerance to simulated intestinal fluid.
3. Tolerance of different lactobacillus rhamnosus to simulated gastrointestinal fluids
The Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1039, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR1, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR2, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR3, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR4 and the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR5 obtained in example 1 are respectively inoculated into an MRS liquid culture medium and cultured for 18 hours at 37 ℃, the cells are collected by centrifugation, the collected cells are washed by physiological saline, the cells are collected by centrifugation again after the washing is finished, the collected cells are respectively resuspended in physiological saline containing 3g/L pepsin with pH being 3 (the pH is adjusted by HCl) to OD6005.0, taking 1mL of bacterial liquid to perform plate viable count to obtain the original viable count of the rhamnose lactobacillus in the bacterial liquid, placing the residual bacterial liquid in 37 ℃ for culturing for 3h, centrifugally collecting cells from the cultured bacterial liquid, and re-suspending the collected cells in physiological saline with pH of 8 (the pH is adjusted by NaOH) and containing 1g/L trypsin and 0.3% (m/v) bile salt to the OD of the bacterial liquid600And 5.0, culturing at 37 ℃ for 4h, and taking 1mL of bacterial liquid to perform plate viable count to serve as the viable count of the lactobacillus rhamnosus in the bacterial liquid after the bacterial liquid tolerates simulated gastrointestinal fluid.
Wherein the survival rate (%) after the gastrointestinal fluid tolerance is 100% (viable count of lactobacillus rhamnosus in the bacterial liquid after the gastrointestinal fluid tolerance simulation/original viable count of lactobacillus rhamnosus in the bacterial liquid).
The detection results are as follows: as can be seen from FIG. 1, after culturing for 3 hours in physiological saline containing pepsin at pH 3, and continuing to culture for 4 hours in physiological saline containing trypsin and bile salts at pH 8, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039 was 13.19. + -. 0.40%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR1 was 6.5. + -. 0.09%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR2 was 10.24. + -. 0.27%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR3 was 0.59. + -. 0.01%, the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR4 was 8.94. + -. 0.11%, and the survival rate of Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR5 was 8.95. + -. 0.95%.
As can be seen, Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039, LR2 are more tolerant to simulated gastrointestinal fluids.
Example 4: oligosaccharide utilization capacity of different lactobacillus rhamnosus
The method comprises the following specific steps:
(1) on the basis of MRS solid culture medium, removing glucose and beef extract in the formula, taking no sugar as a blank control, respectively adding 0.5% (m/v) of glucose, fructo-oligosaccharide, xylo-oligosaccharide and galacto-oligosaccharide as carbon sources, and adding bromocresol purple as an acid-base indicator to obtain a solid culture medium plate which is sugar-free, contains glucose, fructo-oligosaccharide, xylo-oligosaccharide or galacto-oligosaccharide;
(2) the Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1039, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR1, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR2, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR3, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR4 and the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR5 obtained in example 1 are respectively inoculated into an MRS liquid culture medium and cultured for 18 hours at 37 ℃, the cells are collected by centrifugation, and the cells are respectively resuspended in physiological saline to OD after being washed by the physiological saline600Obtaining bacterial liquid when the concentration is 0.5;
(3) and (3) sucking 10 mu L of bacterial liquid, respectively dropping the bacterial liquid on the solid culture medium plate obtained in the step (1), after the bacterial liquid is completely absorbed, carrying out inverted culture at 37 ℃, after 12h, observing whether a bromocresol purple indicator in the solid culture medium plate turns yellow or not, wherein if the bromocresol purple indicator turns yellow, the carbon source is utilized, and if the bromocresol purple indicator does not turn yellow, the carbon source is not utilized (the result is shown in table 1).
As can be seen from table 1, Lactobacillus rhamnosus can utilize xylose oligosaccharide and galactose oligosaccharide generally, Lactobacillus rhamnosus LR1 and Lactobacillus rhamnosus LR3 cannot utilize fructose oligosaccharide, and Lactobacillus rhamnosus CCFM1039 can utilize fructose oligosaccharide, xylose oligosaccharide and galactose oligosaccharide to generate acid, and has good ability of utilizing oligosaccharide.
TABLE 1 ability of different Lactobacillus rhamnosus to utilize different carbon sources
| Type of carbon source | CCFM1039 | LR1 | LR2 | LR3 | LR4 | LR5 |
| Sugar-free | - | - | - | - | - | - |
| Glucose | + | + | + | + | + | + |
| Fructo-oligosaccharide | + | - | + | - | + | + |
| Xylo-oligosaccharide | + | + | + | + | + | + |
| Galacto-oligosaccharides | + | + | + | + | + | + |
Wherein, + indicates that the Lactobacillus can utilize the carbon source, -indicates that it cannot.
Example 5: short chain fatty acid producing ability of different lactobacillus rhamnosus
The method comprises the following specific steps:
the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039, Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR1, and Lactobacillus rhamnosus (Lactobacillus rhamnosus) obtained in example 1 were mixed with each other) LR2, Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR3, Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR4 and Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR5 are respectively inoculated into an MRS liquid culture medium and cultured for 18h at 37 ℃, cells are centrifugally collected, washed by physiological saline and then respectively resuspended in physiological saline until the bacterial concentration is 2 multiplied by 108CFU/mL, obtaining bacterial liquid.
Mixing and acidifying the bacterial liquid and 10% sulfuric acid at a volume ratio of 25:1, adding diethyl ether with the volume 4 times that of the 10% sulfuric acid, oscillating and mixing uniformly to extract fatty acid to obtain an extracting solution; centrifuging the extractive solution at 18000g for 15min, separating upper diethyl ether phase, and passing the upper diethyl ether phase over anhydrous Na2SO4Drying, standing for 30min, centrifuging at 18000g for 5min, and analyzing each short chain fatty acid in the upper ether phase by GC-MS (see FIGS. 2-5).
As shown in FIGS. 2 to 5, the total short-chain fatty acid yield of Lactobacillus rhamnosus CCFM1039 is 2.893 + -0.05 mu mol/mL, wherein the acetic acid yield is 2.873 + -0.05 mu mol/mL, which is significantly superior to that of the other Lactobacillus rhamnosus.
From examples 3 to 5, it is known that Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1039, Lactobacillus rhamnosus (Lactobacillus rhamnous) LR2 and Lactobacillus rhamnosus (Lactobacillus rhamnous) LR5 are better in the ability to tolerate simulated gastrointestinal fluids, oligosaccharide utilization and in vitro production of short chain fatty acids, and can survive and function in the human gastrointestinal environment, and therefore Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1039, Lactobacillus rhamnosus (Lactobacillus rhamnous) LR2 and Lactobacillus rhamnosus (Lactobacillus rhamnous) LR5 were selected below for further experiments to study their probiotic functions.
Example 6: influence of different lactobacillus rhamnosus on health index of mouse, water content of feces and concentration of short-chain fatty acid in feces
The method comprises the following specific steps:
the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1039, Lactobacillus rhamnosus (Lactobacillus rhamnosus) LR2 and Lactobacillus rhamnosus (Lactobacillus rhamnosus) obtained in example 1 were mixed togetherrhamnosus) LR5 were inoculated into MRS liquid medium, cultured at 37 deg.C for 18h, centrifuged to collect cells, washed with physiological saline, resuspended in 12% (m/v) skimmed milk powder solution to bacterial concentration of 5 × 109CFU/mL to obtain bacterial suspension, and storing the bacterial suspension at-80 ℃ for later use.
Taking 40 SPF-grade BALB/c male mice with the weight of 20-22 g, randomly dividing the mice into 4 groups, wherein each group comprises 10 mice, and the 4 groups respectively comprise: the kit comprises a blank group, a CCFM1039 group of lactobacillus rhamnosus perfusion suspension, an LR2 group of lactobacillus rhamnosus perfusion LR2 suspension and an LR5 group of lactobacillus rhamnosus perfusion LR5 suspension, wherein the CCFM1039 group, the LR2 group and the LR5 group are experimental groups.
The experiment took 22 days: the first week (7 days) is the mouse adaptation period; beginning intragastric administration on day 8 until the experiment is finished, performing intragastric administration on the experimental groups of lactobacillus rhamnosus CCFM1039, LR2 and LR5 bacterial suspensions respectively at a dose of 0.2mL bacterial suspension/mouse/day, and performing intragastric administration on a blank group of the experimental groups only by taking an equivalent amount of skim milk powder solution as a control; gavage for 15 days.
After 15 days, the fecal content of short-chain fatty acids was measured in each mouse (see Table 2 for the results, and example 5 for the method of measuring the short-chain fatty acid content).
As can be seen from Table 2, compared with the blank group, the yields of total short-chain fatty acids, acetic acid, propionic acid, isobutyric acid and n-butyric acid in the mouse feces of the lactobacillus rhamnosus CCFM1039 are generally improved, wherein propionic acid is the most significant; the four short chain fatty acids and the total acid in the mouse excrement of the intragastric lactobacillus rhamnosus LR2 and LR5 are improved compared with the blank group, but the increase is relatively lower than that of the rest CCFM1039 group.
Therefore, the lactobacillus rhamnosus CCFM1039 has obvious advantages in improving the yield of short-chain fatty acids in intestinal tracts of mice.
TABLE 2 types and contents of short-chain fatty acids in feces of mice of different groups
Example 7: effect of different Lactobacillus rhamnosus on the fecal flora of mice
The method comprises the following specific steps:
the Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1039, the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR2 and the Lactobacillus rhamnosus (Lactobacillus rhamnous) LR5 obtained in example 1 are respectively inoculated into an MRS liquid culture medium to be cultured for 18 hours at 37 ℃, then the cells are collected by centrifugation, washed by physiological saline and then respectively resuspended in 12 percent (m/v) skim milk powder solution until the bacteria concentration is 5 multiplied by 109CFU/mL to obtain bacterial suspension, and storing the bacterial suspension at-80 ℃ for later use.
Taking 40 SPF-grade BALB/c male mice with the weight of 20-22 g, randomly dividing the mice into 4 groups, wherein each group comprises 10 mice, and the 4 groups respectively comprise: the kit comprises a blank group, a CCFM1039 group of lactobacillus rhamnosus perfusion suspension, an LR2 group of lactobacillus rhamnosus perfusion LR2 suspension and an LR5 group of lactobacillus rhamnosus perfusion LR5 suspension, wherein the CCFM1039 group, the LR2 group and the LR5 group are experimental groups.
The experiment took 22 days: the first week (7 days) is the mouse adaptation period; beginning intragastric administration on day 8 until the experiment is finished, performing intragastric administration on the experimental groups of lactobacillus rhamnosus CCFM1039, LR2 and LR5 bacterial suspensions respectively at a dose of 0.2mL bacterial suspension/mouse/day, and performing intragastric administration on a blank group of the experimental groups only by taking an equivalent amount of skim milk powder solution as a control; gavage for 15 days.
Collecting feces of each mouse in the morning of 15 days after gavage, extracting bacterial genome in a feces sample of the mouse by using a feces sample genome extraction Kit purchased from American MP company, amplifying a V4 region of 16S rDNA by using the bacterial genome as a template, performing gel cutting recovery after PCR verification, and quantifying (ng/uL) the recovered product according to the instruction of a QuantTM dsDNABR Assay Kit purchased from American Life Technologies company; according to the quantitative result of the PicoGreen fluorescent dye, samples are mixed according to equal mass concentration, and the barcode between the samples is not repeated; constructing a library according to the instruction of TurSeq DNALT Sample Preparation Kit purchased from Life Technologies, USA, and mainly comprising the steps of end repair, 3' end adding A, linker connection, PCR amplification and the like; performing on-machine sequencing on the library; and (3) carrying out offline treatment according to the sequencing result to obtain the distribution condition of the flora in the mouse feces of different experimental groups on the phylum level, the distribution condition of the flora in the mouse feces of different experimental groups on the genus level and the relative abundance of Acinetobacter in the mouse feces of different experimental groups (the detection result is shown in figure 6).
As can be seen from FIG. 6, the relative abundance of Acinetobacter in the feces of mice in the blank group is higher, about 0.016 + -0.004%, the relative abundance of Acinetobacter in the feces of mice with Lactobacillus rhamnosus L2 and L5 in the stomach is not significantly changed compared with the blank group, and the relative abundance of Acinetobacter in the feces of mice with Lactobacillus rhamnosus CCFM1039 in the stomach is only 0.003 + -0.005%, which is significantly reduced compared with the blank group.
Therefore, the lactobacillus rhamnosus CCFM1039 has obvious advantages in adjusting the relative abundance of Acinetobacter in intestinal tracts of mice, and has the potential of preventing and/or treating the infection of the Acinetobacter in the mice.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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<120> Lactobacillus rhamnosus capable of regulating and controlling relative abundance of acinetobacter in intestinal tract
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gctcgctccc taaaagggtt acgccaccgg cttcgggtgt tacaaactct catggtgtga 60
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agcgattccg acttcgtgta ggcgagttgc agcctacagt ccgaactgag aatggcttta 180
agagattagc ttgacctcgc ggtctcgcaa ctcgttgtac catccattgt agcacgtgtg 240
tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc 300
accggcagtc ttactagagt gcccaactaa atgctggcaa ctagtcataa gggttgcgct 360
cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg 420
tcattttgcc cccgaagggg aaacctgatc tctcaggtga tcaaaagatg tcaagacctg 480
gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc 540
gtcaattcct ttgagtttca accttgcggt cgtactcccc aggcggaatg cttaatgcgt 600
tagctgcggc actgaagggc ggaaaccctc caacacctag cattcatcgt ttacggcatg 660
gactaccagg gtatctaatc ctgttcgcta cccatgcttt cgagcctcag cgtcagttac 720
agaccagaca gccgccttcg ccactggtgt tcttccatat atctacgcat ttcaccgcta 780
cacatggagt tccactgtcc tcttctgcac tcaagtttcc cagtttccga tgcacttcct 840
cggttaagcc gagggctttc acatcagact taaaaaaccg cctgcgctcg ctttacgccc 900
aataaatccg gataacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc 960
cgtggctttc tggttggata ccgtcacgcc gacaacagtt actctgccga ccattcttct 1020
ccaacaacag agttttacga cccgaaagcc ttcttcactc acgcggcgtt gctccatcag 1080
acttgcgtcc attgtggaag attccctact gctgcctccc gtaggagttt gggccgtgtc 1140
tcagtcccaa tgtggccgat caacctctca gttcggctac gtatcattgc cttggtgagc 1200
cgttacctca ccaactagct aatacgccgc gggtccatcc aaaagcgata gcttacgcca 1260
tctttcagcc aagaaccatg cggttcttgg atttatgcgg tattagcatc tgtttccaaa 1320
tgttatcccc cacttaaggg caggttaccc acgtgttact cacccgtccg ccactcg 1377
Claims (10)
1. The Lactobacillus rhamnosus (Lactobacillus rhamnous) is characterized by being preserved in Guangdong province microorganism strain preservation center in 2018, 11 months and 20 days, wherein the preservation number is GDMCC No.60490, and the preservation address is No. 59 building 5 of Michelia furiosa No. 100 of Guangzhou city.
2. Use of a strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) according to claim 1 for the preparation of a product for improving intestinal health.
3. Use of a strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) according to claim 2 for the preparation of a product for improving intestinal health, wherein the Lactobacillus rhamnosus (Lactobacillus rhamnosus) has a viable count of not less than 1 x 106CFU/mL or 1X 106CFU/g。
4. Use of a strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) according to claim 2 or 3 for the preparation of a product for improving intestinal health, wherein the product comprises a food, a pharmaceutical or a nutraceutical product.
5. The use of a strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) according to claim 4 for the preparation of a product for improving intestinal health, wherein the product comprises Lactobacillus rhamnosus (Lactobacillus rhamnosus), a pharmaceutical carrier and/or a pharmaceutical excipient.
6. A product for improving gut health comprising Lactobacillus rhamnosus (Lactobacillus rhamnosus) according to claim 1.
7. The product of claim 6, wherein the product is effective for improving gut health, and wherein the product comprises Rhamnus frangulaThe viable count of Lactobacillus saccharosus (Lactobacillus rhamnosus) is not less than 1 × 106CFU/mL or 1X 106CFU/g。
8. The product for improving intestinal health according to claim 6 or 7, wherein the viable count of Lactobacillus rhamnosus is not less than 1 x 108CFU/mL or 1X 108CFU/g。
9. The product for improving gut health of any one of claims 6 to 8, wherein the product comprises a food, pharmaceutical or nutraceutical product.
10. The product for improving intestinal health of claim 9, wherein the product comprises Lactobacillus rhamnosus (Lactobacillus rhamnosus), a pharmaceutical carrier and/or a pharmaceutical excipient.
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Application publication date: 20200414 |