CN116144541A - Lactobacillus plantarum strain with triglyceride reducing, cholesterol reducing and lipid reducing functions and application thereof - Google Patents
Lactobacillus plantarum strain with triglyceride reducing, cholesterol reducing and lipid reducing functions and application thereof Download PDFInfo
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- CN116144541A CN116144541A CN202211732333.4A CN202211732333A CN116144541A CN 116144541 A CN116144541 A CN 116144541A CN 202211732333 A CN202211732333 A CN 202211732333A CN 116144541 A CN116144541 A CN 116144541A
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- 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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- 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
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- 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
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a lactobacillus plantarum strain with triglyceride reducing, cholesterol reducing and lipid reducing functions and application thereof, belonging to the technical field of functional microorganisms. The lactobacillus plantarum A21094 provided by the invention is separated from faeces of elderly people with hundred years old, and the deposit number is GDMCCNo 62961. Experiments show that the lactobacillus plantarum strain A21094 has the effects of reducing triglyceride, cholesterol and fat simultaneously, meanwhile, the lactobacillus plantarum strain A21094 has good strong acid and alkali tolerance, stronger self-hydrophobicity and cell adhesiveness, and meanwhile, the lactobacillus plantarum strain A21094 also has the biological properties of inhibiting main intestinal pathogenic bacteria such as escherichia coli, escherichia coli and the like, so that a foundation is provided for preparing oral medicines for preventing and/or treating hyperlipidemia or cardiovascular and cerebrovascular diseases.
Description
Technical Field
The invention belongs to the technical field of functional microorganisms, and particularly relates to a lactobacillus plantarum (Lactobacillus plantarum) strain with the functions of reducing triglyceride, cholesterol and fat and application thereof.
Background
Triglycerides (TG) are fat molecules synthesized from food fat and liver, and are formed from long chain fatty acids and glycerol. The lipid is the most important lipid in blood, and is the most abundant lipid in human body, most tissues can supply energy by utilizing triglyceride decomposition products, and tissues such as liver, fat and the like can synthesize the triglyceride and store the triglyceride in the adipose tissues. However, if the triglyceride is excessive, the body is obese when the triglyceride is accumulated under the skin, arteriosclerosis is caused when the triglyceride is accumulated on the wall of a blood vessel, cardiac hypertrophy is caused when the triglyceride is accumulated on the heart, and fatty liver is caused when the triglyceride is accumulated on the liver. At present, nearly 1/3 of the adults in China have high blood fat, and the hyperlipidemia is now the first invisible killer for human health.
Cholesterol in a human body is usually in the form of free cholesterol ester and cholesterol ester existing in cells and tissues, is highly distributed in brain and nerve tissues, kidneys, skin, livers and bile, is an important component in the structure of the internal membrane, participates in forming a biological membrane, plays an important role in the synthesis of plasma lipoproteins, lipid metabolism and the conduction of nerve excitation, and is also a synthetic raw material of bile acid, vitamin D and various hormones, so that cholesterol is an essential material for maintaining the physiological functions of the human body, plays an important physiological function in the human body, however, the cholesterol content in serum of the human body is often too high, and a series of cardiovascular diseases caused by the excessive serum cholesterol are commonly existing. Modern research has found that atherosclerosis, venous thrombosis, has a close correlation with hypercholesterolemia. WTO has predicted that cardiovascular disease will be the leading cause of death by 2030, affecting the health of about 2.36 million people worldwide.
With the improvement of living standard, the diet structure and the living style of people are continuously changed. In recent years, the incidence of cardiovascular and cerebrovascular diseases such as atherosclerosis and coronary heart disease and hyperlipidemia has been increasing. The incidence of "rich and expensive diseases" such as obesity and hypertension, hyperlipidemia and hyperglycemia is also continuously rising. Because of high price and great side effect of the therapeutic drugs, the effects of prevention and alleviation can be achieved only by establishing a healthy life style, especially following healthy diet standards. Therefore, it is necessary to regulate the environmental balance and circadian rhythm in the human body by targeted supplementation of foods rich in probiotics. In recent years, researchers at home and abroad have proved that some lactobacillus such as lactobacillus, bifidobacterium and the like have the probiotic function of reducing cholesterol, the lactobacillus with the function of reducing triglyceride is reported, and the lactobacillus with the functions of reducing cholesterol, reducing triglyceride and reducing lipid at the same time is not reported.
Disclosure of Invention
Therefore, the invention aims to provide the lactobacillus plantarum strain A21094 which has the effects of reducing triglyceride, cholesterol and fat simultaneously, and provides a new means for developing and preparing medicines or health-care products, foods and the like for hyperlipidemia.
The invention provides a lactobacillus plantarum strain A21094 with the functions of reducing triglyceride, cholesterol and fat, and the preservation number is GDMCC No. 62961.
The invention provides a probiotic agent, which comprises lactobacillus plantarum strain A21094 and auxiliary materials.
Preferably, the viable count of the lactobacillus plantarum strain A21094 is (1-1000) multiplied by 10 9 CFU/ml。
The invention provides a weight-losing medicine which comprises lactobacillus plantarum strain A21094 and medical acceptable auxiliary materials.
Preferably, the viable count of the lactobacillus plantarum strain A21094 is (1-1000) multiplied by 10 9 CFU/ml。
Preferably, the weight loss agent comprises an oral formulation.
The invention provides application of lactobacillus plantarum strain A21094 in preparation of a blood fat reducing probiotic or a weight reducing medicament.
The invention provides application of lactobacillus plantarum strain A21094 or the probiotics in preparing medicines for preventing and/or treating hyperlipidemia or cardiovascular and cerebrovascular diseases.
The invention provides a starter, which comprises lactobacillus plantarum strain A21094 and a carrier medium.
The invention provides application of lactobacillus plantarum strain A21094 or the leavening agent in preparing a fermented product or food for reducing oil triester, cholesterol and fat.
The invention provides a lactobacillus plantarum strain A21094 with the functions of reducing triglyceride, cholesterol and fat, and the preservation number is GDMCC No. 62961. The lactobacillus plantarum strain A21094 is obtained by separating and purifying the lactobacillus plantarum strain A21094 from the intestinal tracts of the aged with a long life, and comprehensive morphological and molecular biological identification shows that the lactobacillus plantarum strain A21094 is lactobacillus plantarum. Experiments show that the lactobacillus plantarum strain A21094 has the effects of reducing triglyceride, cholesterol and fat simultaneously, wherein the degradation rate of the triglyceride is more than 58.10 percent, the cholesterol reducing capacity is more than 60.10 percent, and the in vitro fat inhibition rate and the in vivo fat inhibition rate are more than 40 percent and 73.75 percent respectively. Meanwhile, the lactobacillus plantarum strain A21094 has good strong acid and alkali tolerance, strong self hydrophobicity and strong cell adhesiveness, and meanwhile, the lactobacillus plantarum strain A21094 also has biological properties of inhibiting main intestinal pathogenic bacteria such as escherichia coli and the like, so that a foundation is provided for preparing probiotics or oral medicines.
Drawings
FIG. 1 shows the colony morphology and gram staining results of the A21094 strain plates;
FIG. 2 shows cholesterol standard curve determination results;
FIG. 3 is a graph showing the effect of A21094 on fat accumulation in 3T3-L1 cells;
FIG. 4 shows the result of nematode fat particle oil red O staining;
FIG. 5 is a graph of A21094 simulating the growth of artificial gastrointestinal fluids;
FIG. 6 shows the viable count (lgCFU/ml) of A21094 for different bile salt concentrations;
FIG. 7 is the adhesion results of A21094 to NCM460 cells.
Biological material preservation information
Lactobacillus plantarum A21094 has a preservation date of 2022, 11 months and 11 days, and has a preservation unit of Guangdong microbiological bacterial culture collection center, GDMCC, and a location of Guangzhou Mitsui 100 th university building 59, guangdong province microbiological institute, and a preservation number of GDMCC No. 62961.
Detailed Description
The invention provides a lactobacillus plantarum strain A21094 with the functions of reducing triglyceride, cholesterol and fat, and the preservation number is GDMCC No. 62961.
In the invention, the lactobacillus plantarum strain A21094 is obtained by separating and purifying the lactobacillus plantarum strain A21094 from the intestinal tracts of the old with the hundred years old, has the typical characteristics of lactobacillus plantarum, is gram positive, facultative anaerobic, free of spores, multi-colony, white or light yellow, smooth in surface, raised in the middle, neat in edge and 2-4 mm in diameter; the cells are rod-shaped. The MRS culture medium is subjected to separation culture, the bacterial colony of the pure bacterial strain is off-white, the diameter is about 2-4 mm, the surface is smooth, the edge is neat, the middle is raised, and the microscopic examination form is short rod shape. The molecular identification of the isolated pure strains was carried out using the 16S rDNA method. The 16S rDNA sequence was aligned in NCBI (National Centerfor Biotechnology Information ) database, and as a result, the gene sequence of the strain was found to have 99.86% homology with Lactobacillus plantarum in all similar sequences. The comprehensive physiological and biochemical identification result shows that the strain A21094 is lactobacillus plantarum (Lactobacillus plantarum).
In the invention, the pure bacterial strain is subjected to experiments such as bacterial strain hydrophobicity, cell adhesion test, artificial gastrointestinal fluid resistance test, in-vitro bacteriostasis and the like. The result shows that the lactobacillus plantarum strain A21094 has good strong acid and alkali tolerance, strong self hydrophobicity and strong cell adhesion, and meanwhile, the lactobacillus plantarum strain A21094 also has the biological property of inhibiting main intestinal pathogenic bacteria such as escherichia coli and the like, thereby providing a foundation for preparing probiotics or oral medicines.
In addition, the in vitro degradation experiments of triglyceride and cholesterol and the in vitro lipid-lowering experiments show that the lactobacillus plantarum strain A21094 has the effects of simultaneously lowering triglyceride, lowering cholesterol and lowering lipid, wherein the degradation rate of triglyceride is more than 58.10 percent, the cholesterol-lowering capacity is more than 60.10 percent, and the in vitro fat inhibition rate and the in vivo fat inhibition rate are more than 40 percent and 73.75 percent respectively.
The invention provides a probiotic agent, which comprises lactobacillus plantarum strain A21094 and auxiliary materials.
In the present invention, the viable count of Lactobacillus plantarum strain A21094 is (1-1000). Times.10 9 CFU/ml, preferably 1X 10 10 CFU/ml. The preparation method of the probiotic is not particularly limited, and the probiotic is prepared by the preparation method well known in the art. The types of the auxiliary materials of the probiotics are related to the dosage form, and are not particularly limited herein.
Based on the lactobacillus plantarum strain A21094 has the effects of reducing triglyceride, cholesterol and fat, the invention provides a weight-losing medicament, which comprises the lactobacillus plantarum strain A21094 and medical acceptable auxiliary materials.
In the present invention, the viable count of Lactobacillus plantarum strain A21094 is (1-1000). Times.10 9 CFU/ml, preferably 1X 10 10 CFU/ml. The preparation method of the weight-losing medicine is not particularly limited, and the weight-losing medicine is prepared by adopting the preparation method of the weight-losing medicine which is well known in the art. The types of the auxiliary materials of the weight-reducing medicine are related to the dosage form, and are not particularly limited herein.
In the present invention, the weight loss agent preferably comprises an oral preparation. Experiments show that the lactobacillus plantarum strain A21094 has good strong acid and alkali tolerance, strong self hydrophobicity and strong cell adhesion, and meanwhile, the lactobacillus plantarum strain A21094 also has biological properties of inhibiting main intestinal pathogenic bacteria such as escherichia coli, escherichia coli and the like, so that the lactobacillus plantarum strain A21094 is suitable for preparing oral preparations. The type of the oral preparation is not particularly limited, and the type of the oral preparation known in the art, such as tablets, oral liquids, capsules, powders, and the like, may be used.
The invention provides application of lactobacillus plantarum strain A21094 in preparation of a blood fat reducing probiotic or a weight reducing medicament.
The invention provides application of lactobacillus plantarum strain A21094 or the probiotics in preparing medicines for preventing and/or treating hyperlipidemia or cardiovascular and cerebrovascular diseases.
In the present invention, the high content of fat in blood is generally represented by high content of triglyceride and cholesterol, and the lactobacillus plantarum strain a21094 has the property of degrading triglyceride, cholesterol and blood lipid in vitro based on the lactobacillus plantarum strain a21094, and therefore, the lactobacillus plantarum strain a21094 has the effect of preventing and/or treating hyperlipidemia. In addition, the lactobacillus plantarum strain A21094 is also applied to the preparation of medicines for preventing and/or treating cardiovascular and cerebrovascular diseases caused by excessive cholesterol and triglyceride.
The invention provides a starter, which comprises lactobacillus plantarum strain A21094 and a carrier medium.
The invention provides application of lactobacillus plantarum strain A21094 or the leavening agent in preparing a fermented product or food for reducing oil triester, cholesterol and fat.
In the present invention, the fermented product preferably comprises fermented milk. The preparation method of the fermented milk is that freeze-dried bacterial powder of lactobacillus plantarum A21094 is preferably inoculated into a sterile skim milk culture medium, the culture is carried out at the constant temperature of 37 ℃, and after full curding, the passage is carried out for 2 times, the inoculum size of each time is 3 percent, and the viable count reaches 10 percent 9 CFU/mL or more; inoculating lactobacillus plantarum to cooled soybean milk according to an inoculum size of 5%, mixing uniformly, fermenting at 37deg.C for 4 hr, and storing at 4deg.C.
In the present invention, the food preferably includes a solid beverage. The solid beverage comprises a blueberry solid beverage or a cranberry solid beverage. The blueberry solid beverage preferably comprises the following components in parts by mass: 1 part of lactobacillus fermentum freeze-dried powder, 0.5 to 1.0 part of xylo-oligosaccharide, 0.5 to 1.0 part of galacto-oligosaccharide, 0.5 to 1.0 part of soybean oligosaccharide and 0.5 to 1.0 part of blueberry fruit powder. The cranberry-flavored solid beverage preferably comprises the following components in parts by mass: 1 part of lactobacillus fermentum freeze-dried powder, 0.5 to 1.0 part of xylo-oligosaccharide, 0.5 to 1.0 part of galacto-oligosaccharide, 0.5 to 1.0 part of soybean oligosaccharide and 0.5 to 1.0 part of cranberry fruit powder. The xylooligosaccharide, galactooligosaccharide and soybean oligosaccharide are purchased from Jiangsu Rui Kang Lai technology Co., ltd, and the blueberry fruit powder is purchased from Sian Hengji chemical Co., ltd; cranberry fruit powder was purchased from Shaanxi Tex Biotechnology Inc. The preparation method of the lactobacillus fermentum freeze-dried powder preferably comprises the steps of fermenting and culturing lactobacillus fermentum strains, separating thalli from the obtained fermentation liquor, and freeze-drying to obtain the freeze-dried powder. The fermentation culture method is preferably that single colony of the activated strain is inoculated to MRS liquid culture medium, and the strain is subjected to static culture for 12 hours under anaerobic condition at 37 ℃ to obtain seed liquid, and the seed liquid is subjected to amplification culture to obtain bacterial liquid. The method for separating the thalli is preferably to centrifuge the bacterial liquid at the temperature of 4 ℃ in a refrigerated centrifuge at 5000rpm for 5min, and discard the supernatant; the cells were washed with physiological saline 2 times, 4℃and 5000rpm, and centrifuged for 5min. The cells were weighed wet. The freeze drying condition is preferably that the thalli are mixed with the freeze-drying protective agent in advance, and then placed at the temperature of minus 20 ℃ for 30min and then placed at the temperature of minus 80 ℃ for 1h. And (3) putting the treated thallus EP pipe into a freeze-drying bottle for freeze-drying, and continuously freeze-drying for 30 hours to finally obtain freeze-dried bacterial powder. The technology is carried out by using a plate counting method, and the bacterial powder is placed at 4 ℃ and stored for standby.
The present invention provides a lactobacillus plantarum strain with triglyceride reducing, cholesterol reducing and lipid reducing functions and application thereof, which are described in detail below with reference to examples, but should not be construed as limiting the scope of the present invention.
Example 1
The invention provides screening and identification of lactobacillus plantarum A21094 with triglyceride reducing, cholesterol reducing and lipid reducing functions.
1. Strain isolation and purification
Fresh fecal samples from the centenarian were diluted 10-fold to 10 -5 Respectively take 10 -3 、10 -4 、10 -5 Three-tube dilutions, 0.1ml each, were plated onto MRS solid medium for separation using a plate plating method. After the plate is cultured for 48 hours at 37 ℃, single colony of suspected target strain is selected, streaked and transferred to a new MRS solid plate, streaked and purified for 2 generations, after the streaked and purified for 2 generations, the pure strain is confirmed by microscopic examination, inoculated to a slant culture medium for culturing for 24 hours, and then placed in a refrigerator at 4 ℃ for standby.
The MRS solid culture medium comprises the following formula and conditions: weighing 10.0g of peptone, 5.0g of sodium acetate, 5.0g of beef extract powder, 20.0g of Tween-801.0mL of glucose and K 2 HPO 4 2.0g, yeast extract 4.0g, mnSO 4 ·4H 2 O0.05 g, triammonium citrate 2.0g, mgSO 4 0.2 12.0g of agar, adding 800mL of distilled water, uniformly mixing until the solute is completely dissolved, and adjusting the pH to 6.2+/-0. 2, fixing the volume to 1L, sterilizing at 121 ℃ for 20min, pouring the hot plate, cooling and solidifying, and storing at 4 ℃ for later use.
2. Identification of strains
The isolated and purified lactobacillus is subjected to bacterial morphology observation and gram-stained cell morphology observation, and the lactobacillus is identified by referring to the "Bergey bacteriology handbook" and the "lactobacillus classification identification and experimental method". The separated and purified bacterial colony is light yellow or white, has smooth surface, raised middle, regular edge and rod-shaped cell with diameter of 2-4 mm. The strain has positive gram staining, no motility, facultative anaerobic type and good growth under anaerobic conditions.
3. Strain 16S rDNA
The molecular identification of the isolated pure strains was carried out using the 16S rDNA method. The 16S rDNA amplification and sequencing was performed by Biotechnology (Shanghai) Inc. The 16S rDNA sequences of the submitted strain A21094 (Table 1) were aligned in the NCBI (National Center forBiotechnology Information ) database and, as a result, the gene sequence of strain A21094 (CGGGAGGGTGCCTATACATGCAAGTCGAACGAACTCTGGTAT TGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCTTAAGGGGCCAGAAT, SEQ ID NO: 1) was found to be 99.86% homologous to Lactobacillus plantarum in all similar sequences, thus, in combination with physiological and biochemical characteristics, strain A21094 was identified as Lactobacillus plantarum. The Lactobacillus plantarum (Lactobacillus plantarum) isolated from the intestinal tract of the elderly with a long life span has been deposited at the Cantonese microorganism strain collection at 11/2022 under the accession number GDMCCNo 62961.
Example 2
In vitro triglyceride reducing effect of lactobacillus plantarum A21094
1 preparation of the experimental Strain
First activation culture: the strain frozen tube preserved at the temperature of minus 80 ℃ is taken out to ice, and the inoculating loop is used for dipping the bacterial liquid on a MRS solid culture medium flat plate for streaking, and the strain frozen tube is subjected to stationary culture at the temperature of 37 ℃ for 24 to 48 hours.
Second activation culture: single colony is picked up in 5mL MRS liquid culture medium, and is subjected to stationary culture at 37 ℃ for 18-20 h.
The bacterial liquid obtained by the third activation culture was inoculated into 5mL of triglyceride-containing MRS liquid medium at an inoculum size of 3%, and the triglyceride-containing MRS liquid medium without the bacterial liquid inoculated was used as a control, and was subjected to stationary culture at 37℃for 24 hours.
Preparation and conditions of MRS liquid culture medium: weighing 2.0g of peptone, 5.0g of sodium acetate, 40g of beef extract powder, 20.0g of Tween-801.0mL of glucose and K 2 HPO 4 2.0g, yeast extract 4.0g, mnSO 4 ·4H 2 O0.04 g, triammonium citrate 2.0g, mgSO 4 0.2 And g, adding 800mL of distilled water, uniformly mixing until the solute is completely dissolved, regulating the pH value to 5.7, fixing the volume to 1L, sterilizing at 121 ℃ under high pressure, and cooling to room temperature for later use.
Triglyceride-containing MRS liquid culture medium preparation and conditions: mixing 2% polyvinyl alcohol aqueous solution with vegetable oil according to a volume ratio of 3:1, uniformly mixing the mixture and the mixture by ultrasonic treatment (controlling parameters of 5s each time, 5s interval and 12min total ultrasonic treatment) to prepare vegetable oil emulsion which is used as a source of triglyceride. Adding the above vegetable oil emulsion into MRS liquid culture medium at a ratio of 5%, adjusting pH to 6.5+ -0.2, sterilizing at 115deg.C for 30min, and making into triglyceride culture medium and storing at 4deg.C in refrigerator for use.
2 Triglycerides assay (Single reagent GPO-PAP method)
And (3) respectively taking 1mL of the cultured bacterial liquid and the triglyceride culture medium without the inoculated bacterial liquid at 4 ℃ and centrifuging at 8000r/min for 10min. The supernatant was taken and processed according to the Triglyceride (TG) test kit instructions (Nanjing built Bioreagent company A110-1-1). The operation is shown in Table 1:
TABLE 1 addition of reagents to wells
Triglyceride (TG) content (mmol/L) = (a sample well-a blank well)/(a standard well-a blank well) ×c standard formula I
Triglyceride (TG) degradation rate (%) = (total TG content-residual TG content)/total TG content 100% formula II
Wherein, the total TG content is the Triglyceride (TG) content in the triglyceride culture medium without inoculating the bacterial liquid; residual TG content-Triglyceride (TG) content in triglyceride medium after 24h incubation with inoculum.
Lactobacillus plantarum A21094 has a triglyceride degradation rate of 58.10%.
Example 3
In vitro cholesterol-lowering effect of lactobacillus plantarum A21094 with triglyceride-lowering, cholesterol-lowering and lipid-lowering functions
1 preparation of the experimental Strain
(1) First activation culture: the strain frozen tube preserved at the temperature of minus 80 ℃ is taken out to ice, and the inoculating loop is used for dipping the bacterial liquid on a MRS solid culture medium flat plate for streaking, and the strain frozen tube is subjected to stationary culture at the temperature of 37 ℃ for 24 to 48 hours.
(2) Second activation culture: single colony is picked in 5mLMRS liquid culture medium and is kept stand at 37 ℃ for 18-20 h.
The bacterial liquid obtained by the third activation culture was inoculated into 5mL of MRS (MRS-CHOL) liquid medium containing high cholesterol at an inoculum size of 5%, and the MRS-CHOL liquid medium without the bacterial liquid inoculated was used as a control, and was subjected to stationary culture at 37℃for 24 hours.
Preparation of high cholesterol MRS (MRS-CHOL) liquid culture medium and conditions: weighing 54g of MRS culture medium, 1.0g of cholesterol, 20mL of Tween 80 and 3.0g of bovine bile salt, dissolving cholesterol in Tween, heating with a water bath, pouring into the culture medium while the cholesterol is hot, fixing the volume to 1L, sterilizing at 121 ℃ under high pressure for 20min, cooling, and preserving for later use.
Determination of cholesterol content by 2 o-phthalaldehyde method
Inoculating activated strain into MRS high cholesterol liquid culture medium, anaerobic culturing at 37deg.C for 24 hr, centrifuging at 10000r/min for 10min, collecting supernatant, measuring cholesterol content by o-phthalaldehyde method (100 μl fermentation supernatant, 750 μl color-developing agent, 250 μl glacial acetic acid and 1mL concentrated sulfuric acid, dark reacting for 10 min), taking unvaccinated MRS-CHOL culture medium as control group, and adding water to obtain final product 550nm Where the absorbance is measured. And the cholesterol removal rate was calculated according to the following formula.
Cholesterol removal rate (%) = (A1-A2)/a1×100% formula V
Wherein A1 is the cholesterol content of the supernatant of the unvaccinated MRS-CHOL culture medium, and A2 is the cholesterol content of the fermentation supernatant after inoculation.
O-phthalaldehyde color developing agent: 10mg of phthalic dicarboxaldehyde is weighed, glacial acetic acid is fixed to 100ml, and the mixture is stored in a dark place.
Determination of cholesterol standard curve
A cholesterol standard solution was prepared at a concentration of 0. Mu.g/ml, 10. Mu.g/ml, 20. Mu.g/ml, 30. Mu.g/ml, 40. Mu.g/ml, and 50. Mu.g/ml, and the concentration of cholesterol was measured with the cholesterol content on the horizontal axis and the absorbance value on the vertical axis as a standard curve.
The cholesterol-lowering ability of strain A21094 was found to be 60.10% by experiment.
Example 4
In vitro lipid-reducing effect of lactobacillus plantarum A21094
1.3T3-L1 cell culture and treatment
(1) 3T3-L1 mouse preadipocytes (purchased from Shanghai cell Bank). Cells were seeded in 96-well plates at a density of 2 x 104 cells/well. Use of high sugar DMEM medium (DMEM, solarbio, beijing)), 10% new fetal bovine serum (Sijiqing) and 1% penicillin and streptomycin (DMEM, solarbio, beijing)) were added. The cells were incubated at 37℃with 5% CO 2 Is cultured in an incubator in a wet environment for 2 days. The induced differentiation liquid I is used for inducing the differentiation of the adiposity cell for 2 days, and the induced differentiation liquid II is used for differentiating for 2 days. Cells were then maintained in DMEM containing 10% fetal bovine serum and 1 μg/mL insulin for 6 days to allow intracellular lipid droplets to settle, with medium changed every other day. At differentiation stage I and differentiation stage II, pasteurized strains were used at 1X 10 8 Density of CFU/well cells were treated.
2.3T3-L1 cell oil red O staining
(1) On day 10 after bacterial treatment, adipocytes were stained with oil red O. Cells were washed twice with cold Phosphate Buffered Saline (PBS), pH 7.4, and fixed with 4% paraformaldehyde solution for 1 hour at room temperature without agitation. Cells were then gently washed twice with PBS, then with 60% isopropanol, followed by staining with oil red O solution (Sigma-Aldrich, st.Louis, MO, USA) containing 60% isopropanol for 15 minutes. The cells were washed repeatedly with cold distilled water and then visualized (200-fold) with an inverted microscope (Leica, germany). The stained intracellular lipid droplets were dissolved with 100% isopropyl alcohol for 10 minutes, and the resulting solution was transferred to a 96-well plate. The absorbance of the stain was read at 500nm using a microplate reader (FIG. 3).
DMEM medium of 10% neonatal bovine serum: a50 ml centrifuge tube was taken, 45ml of DMEM medium was added, 5ml of fresh bovine serum (NCBS) was then added, and the mixture was thoroughly blown and mixed well and stored at 4 ℃.
100mM IBMX stock: sterile ddH at 0.9ml 2 Adding 20mg IBMX into O to dissolve, adding 1M NaOH, blowing to dissolve completely, packaging, and preserving at-20deg.C.
1mM DEX stock: 5mg of DEX was added to 4ml of absolute ethanol and the mixture was blown up and homogenized, and 8.7ml of sterile ddH was added thereto 2 O makes it fully dissolved, and the product is packaged for standby and preserved at-20 ℃.
1mg/ml INS mother liquor: 10mg of INS is carefully weighed in an ultra-clean workbench, a trace amount of acetic acid is added to fully dissolve the INS, sterilized double distilled water is added to supplement to 10ml, and the mixture is gently blown and uniformly stirred and stored in a refrigerator at the temperature of minus 20 ℃ for standby.
Inducing differentiation liquid I: a10 ml centrifuge tube was added with 9.93ml DMEM (10% NBCS), and 50. Mu.l IBMX mother liquor, 10. Mu.l DEA and 10. Mu.l INS mother liquor were added to the centrifuge tube, and the mixture was thoroughly blown to homogeneity and ready for use.
Inducing differentiation liquid II: a10 ml centrifuge tube was taken, 10ml DMEM (10% NBCS) was added thereto, and 10. Mu.l INS mother liquor was added thereto, and the mixture was thoroughly blown to homogenize the mixture, and the mixture was ready for use.
4% paraformaldehyde: 2g of paraformaldehyde was dissolved in 50ml of PBS, and after stirring to dissolve it sufficiently, it was sterilized by filtration and stored at 4 ℃.
0.5% oil red O: 50mg of oil red O powder was added to 50ml of isopropanol, heated and stirred sufficiently to dissolve completely, and then filtered and sterilized for storage at 4 ℃.
Pasteurizing strain: a21094 is inoculated into MRS liquid culture medium after two times of activation, and is cultivated for 18 to 20 hours at the constant temperature of 37 ℃. Collecting thallus, washing twice with PBS, and adjusting the concentration of bacterial suspension to 1×10 8 CFU/ml. Inactivating the bacterial suspension in a water bath kettle at 70 ℃ for 30min.
Blank, untreated 3T3-L1 cells;
control, namely, inducing differentiation liquid to treat 3T3-L1 cells and sterilizing the cells;
a21094: inducing differentiation liquid to treat 3T3-L1 cells and adding A21094 to inactivate thallus for intervention.
Example 5
In vivo lipid-reducing effect of lactobacillus plantarum a21094
1. Culture of strains and nematodes
E.coli OP50 was cultured in LB medium, after overnight culture, a proper amount of plating was applied to nematode medium (nematode growth medium, NGM), and cultured overnight. The thawed nematodes were centrifuged and added to the OP50 NGM medium, and incubated in an incubator at 20 ℃. A21094 is inoculated into MRS liquid culture medium after two times of activation, and is cultivated for 18 to 20 hours at the constant temperature of 37 ℃.
2 nematode synchronization treatment
Suspending nematodes with M9buffer solution, sucking into culture tube, adding lysis solution (5N NaOH and 5% sodium hypochlorite solution) into each pipeline of worms, lysing for 6min, centrifuging for 1min at speed of 3500r/min, discarding supernatant, repeatedly cleaning for 4 times with M9buffer, discarding supernatant, transferring onto NGM culture medium, and culturing overnight at 20deg.C to obtain L1 stage larva; and then washing and centrifuging the mixture once by using an M9buffer, adding the nematodes on an NGM flat plate with OP50, and culturing the mixture at 20 ℃ for 28 to 30 hours to obtain the synchronized nematodes.
3 nematode fat particle experiment
Respectively picking 100 synchronized nematodes into the plates coated with OP50 and A21094, and changing the plates every other day; after feeding on day 5, after washing three times with cooled M9buffer, the nematodes were resuspended in 4% paraformaldehyde, each sample was gently shaken at room temperature for 1h, centrifuged at 3000-4000 rpm for 1min, and after removal of the supernatant, washed twice with M9 buffer; the nematodes were then resuspended in 60% isopropanol and 0.01% Triton X-100 in PBS and incubated for 15min; after the nematodes settle, removing isopropanol, adding 1mL of 40% oil red O coloring agent, and incubating the animals for 1-2 h at 25 ℃ in a shaking table; after the nematodes subside, the dye was removed, washed twice with M9buffer, 200 μ L M buffer was added, the nematodes were photographed one by one under an inverted fluorescence microscope, and the data of the images J were used to count the data of the fat granule degradation with GraphPad Prism 5.
Wherein M9buffer is 2.2mM KH 2 PO 4 ，4.2mM Na 2 HPO 4 8.55mM NaCl, constant volume to 1L, and autoclaving.
NGM agar medium: 3g NaCl,2.5g bactopeptone, 17g agar, 975mL sterile water, 121℃sterilization, cooling to 55deg.C, adding 1mL 5mg/mL cholesterol, 1mL 1M NaCl in sequence 2 、1mL 1M MgSO 4 And 25mL of 1M phosphate buffer.
The results are shown in FIG. 4. The test shows that after the nematode fed for 5d is dyed by oil red O, compared with the nematode fed with OP50, the nematode fat particles fed with A21094 are obviously reduced, and the inhibition rate is 73.75 percent (P is less than 0.05 or P is less than 0.01).
Example 6
Artificial gastric juice resistance test of Lactobacillus plantarum A21094
1 Strain simulation Artificial gastric juice experiment
Shaking the activated bacterial liquid for three times, shaking uniformly, taking 1ml of bacterial suspension in 9ml of artificial gastric juice (pH=3.0), standing and culturing for 3 hours at 37 ℃, and respectively taking the artificial gastric juice cultured for 0 hour and 3 hours to count the live bacteria on a flat plate. Survival was calculated according to formula III using 0h as a control.
Simulated artificial gastric fluid survival (%) =n t /N 0 *100% formula III
In N t Represents the number of viable bacteria after culturing t h, N 0 The viable count at 0h is shown (Table 1, FIG. 5).
2 Strain simulation Artificial intestinal juice experiment
1ml of artificial gastric juice after 3 hours of culture was taken and cultured in 9ml of artificial intestinal juice (ph=8.0) at 37℃for 2 hours, 4 hours, 6 hours and 8 hours. The artificial intestinal juice is respectively taken for culturing for 0h,2h,4h,6h and 8h for plate viable count. Survival was calculated with 0h as a control.
Simulated artificial intestinal juice survival rate (%) =nt/N0 ×100% formula IV
Wherein Nt represents the number of viable bacteria after culturing t h, and N0 represents the number of viable bacteria at 0h.
The results are shown in Table 2 and FIG. 5.
Table 2A21094 simulates the tolerance of artificial gastrointestinal fluids
As can be seen from Table 2 and FIG. 5, the survival rate of the strain A21094 reaches 171.43% and the viable count reaches 10 after the strain A21094 is subjected to gastric juice simulation treatment for 3 hours 8 CFU/ml; after simulated intestinal juice is treated for 8 hours, the survival rate of the strain reaches 112.71 percent, after the simulated intestinal juice is treated for 16 hours, the survival rate of the strain also reaches 74.59 percent, and the viable count reaches 10 percent 7 CFU/ml, demonstrating that strain A21094 has very strong viability in simulated gastrointestinal fluids,
in conclusion, the strain can effectively resist the influence of gastrointestinal fluid, so that high activity can be maintained after the strain passes through the digestive tract.
Example 7
Bile salt tolerance experiment of lactobacillus plantarum A21094
The twice activated strain was inoculated in 5% (v/v) to MRS liquid medium containing 0.1%,0.2% and 0.3% (m/v) bovine bile salt, and anaerobically cultured at 37℃for 0h,1h,2h,3h and 4h. Control group was 0h. The number of viable bacteria in the samples was then calculated using MRS solid medium and pour plate method. The poured plate was anaerobically incubated at 37℃for 48h. The survival rate of the strain was calculated according to formula V.
Strain survival (%) =c t /C 0 *100% formula V
C in the formula t Represents the number of viable bacteria after culturing t h, C 0 The viable count at 0h is shown.
The results are shown in Table 3 and FIG. 6.
Table 3a21094 tolerance to different bile salt concentrations
As can be seen from Table 3 and FIG. 6, the survival rates of strain A21094 were 65.74%,54.82% and 11.04% after 4 different bile salt concentrations (0.1%, 0.2% and 0.3%), and the viable count reached 10, respectively 8 CFU/ml，10 7 CFU/ml and 10 6 CFU/ml, demonstrating that strain A21094 has a strong bile salt tolerance.
Example 8
Antibacterial Activity test of Lactobacillus plantarum A21094
(1) Preparation of pathogenic bacteria escherichia coli: the pathogenic strain is frozen, streaked and activated, single colony is selected and cultured in 5ml of LB liquid culture medium, and the escherichia coli/escherichia coli is cultured at the constant temperature of 37 ℃ overnight.
(2) Shaking the activated experimental bacterial liquid for three times, taking 1ml of culture liquid, centrifuging at the temperature of 4 ℃ and at the speed of 8000rpm/min for 10min, taking the supernatant, and filtering and sterilizing.
(3) Preparation of solid medium: 100. Mu.L of the bacterial suspension cultured to logarithmic phase was uniformly spread on LB solid medium, sterile oxford cups each containing Lactobacillus plantarum P-8 (isolated from a probiotic powder product purchased from Taobao), LGG (isolated from a Phragmitis-known LGG yogurt purchased from Today's convenience store), lactobacillus plantarum A21094 were placed on a dish coated with bacterial liquid with forceps, and a negative control (MRS liquid culture medium) was also set. The plates were placed in a refrigerator at 4℃for 12 hours, placed in a thermostatic incubator at 37℃and incubated for 24 hours, photographed under observation, and the diameter of the zone of inhibition was measured with a vernier caliper (Table 4).
Table 4A21094 antibacterial action against Escherichia coli and Escherichia coli
The test result shows that the A21094 has obvious antibacterial effect on main pathogenic bacteria in gastrointestinal tract.
LB medium was prepared under the conditions of 3g of peptone, 1.2g of yeast extract powder, 0.5g of dipotassium hydrogen phosphate, 3g of maltose, 0.9g of anhydrous sodium acetate, 0.6g of ammonium citrate, 0.06g of magnesium sulfate heptahydrate, 0.03g of manganese chloride, 4.5g of agar and 300. Mu.l of Tween 80, and sterilized with ultra-pure water 300ml of high-pressure steam at 121℃for 15 minutes.
Example 9
Self hydrophobicity experiment of Lactobacillus plantarum A21094
Inoculating 5% of the secondary activated strain into a sterile MRS culture medium, culturing for 16-18 h at 37 ℃, centrifuging (8000 r/min,4 ℃ and 10 min), collecting thalli, centrifugally washing for 2 times with 50mmol/L phosphate buffer solution, and suspending the precipitate with the buffer solution. The buffer solution is used as a blank control, and the concentration of the bacterial cells is adjusted to make the initial concentration of the bacterial cells have an absorbance value of about 1.0 at a wavelength of 600nm (A) 0 ). Taking 4mL of bacteria liquid with turbidity adjusted, adding 0.8mL of dimethylbenzene, swirling at high speed for 2min, standing for 10min for layering, and taking the lower water phase to measure the absorbance at 600nm wavelength. The hydrophobicity of the cell surface of the lactic acid bacteria was calculated according to formula VII:
hydrophobicity (%) = (a) 0 -A)/A0×100% formula VI
Wherein A is 0 And A is the absorbance value measured at 600nm of the bacterial liquid before and after being uniformly mixed with dimethylbenzene.
The results showed that lactobacillus plantarum a21094 was 92.77% hydrophobic in itself.
Example 10
Cell adhesion ability test of Lactobacillus plantarum A21094
(1) NCM460 normal intestinal epithelial cell culture and plating: digesting the recovered NCM460, seeding a 6-well plate (containing cell climbing sheet) 3×10 5 Well, 37 ℃,5% CO 2 Culturing in a constant temperature incubator overnight.
Preparing a bacterial suspension: shaking the activated experimental bacterial liquid for three times, collecting bacterial liquid, washing with PBS for 2 times, adding high sugar DMEM culture medium to resuspension bacterial body, and adjusting bacterial concentration to 1×10 8 CFU/ml.
Bacterial fluids were added to 6-well plates, 1 ml/well, and three wells per sample were repeated. The bacterial solution and the cells are incubated for 2 hours. The 6-well plate was removed, washed 5 times with PBS, and the non-adherent cells were washed off as much as possible. Then, the cell slide was fixed with an alcohol lamp and gram staining was performed. Multiple fields of view the cell adhesion of the experimental strain was observed, one of which is shown in FIG. 7.
In summary, the number of adhering a21094 on each adhered NCM460 cell was >100, the adhesion rate was 90%, indicating that a21094 had good adhesion to NCM460 cells.
Example 11
Application of lactobacillus plantarum A21094 in preparation of fermented milk
(1) Preparation of soybean milk: according to the soybean: pure water = 1:12 water soak for 10h. Pulping with a soymilk grinder at 10000r/min for 1min, pulping, filtering, boiling for 5min, filtering with filter cloth, and cooling the obtained soymilk;
(2) Activation of the strain: inoculating lyophilized powder of Lactobacillus plantarum A21094 into sterile skimmed milk culture medium, culturing at 37deg.C, and passaging for 2 times after full curding, wherein the inoculum size is 3% each time, and the viable count is 10 9 CFU/mL or more;
(3) Preparation of fermented soybean milk: respectively inoculating lactobacillus plantarum to cooled soybean milk according to an inoculum size of 5%, mixing uniformly, fermenting at 37deg.C for 4 hr, coagulating, and storing at 4deg.C.
Example 12
Application of lactobacillus plantarum A21094 in preparation of solid beverage
1 preparation of bacterial powder
(1) Bacterial strain fermentation culture
Primary fermentation: and (3) picking single colony of the experimental strain after 2 times of activation to 15ml of MRS liquid culture medium, and standing and culturing for 12 hours at 37 ℃ under anaerobic condition to obtain seed liquid 1.
Secondary fermentation: inoculating the seed liquid 1 to 150ml of MRS liquid culture medium according to the inoculation amount of 3%, and standing and culturing for 20-22 h under the anaerobic condition at 37 ℃ to obtain the seed liquid 2.
And (3) amplifying culture: inoculating the seed solution to 300ml of MRS liquid culture medium according to the inoculation amount of 3%, and performing stationary culture for 20-24 h under the anaerobic condition at 37 ℃ to obtain the bacterial solution after the amplification culture.
(2) And (3) fermentation liquid treatment: collecting bacterial liquid, centrifuging at 4 ℃ and 5000rpm for 5min, and discarding supernatant. The cells were washed with physiological saline 2 times, 4℃and 5000rpm, and centrifuged for 5min. The cells were weighed wet.
(3) Freeze drying
Lyoprotectant: cell wet weight = 1: re-suspending at 1 volume ratio, sub-packaging with EP tube, standing at-20deg.C for 30min, and standing at-80deg.C for 1 hr. And (3) putting the packaged EP pipe into a freeze-drying bottle, putting the freeze-drying bottle into a freeze dryer for freeze-drying, and continuously freeze-drying for 30 hours to finally obtain the freeze-dried bacterial powder. The technology is carried out by using a plate counting method, and the bacterial powder is placed at 4 ℃ and stored for standby.
2 preparation of probiotic solid beverage
Probiotic solid beverage (3 g) formulation:
blueberry flavored solid beverage: 1g of lactobacillus fermentum freeze-dried powder, 0.5-1.0 g of xylooligosaccharide, 0.5-1.0 g of galactooligosaccharide, 0.5-1.0 g of soybean oligosaccharide and 0.5-1.0 g of blueberry fruit powder.
Cranberry flavored solid beverage: 1g of lactobacillus fermentum freeze-dried powder, 0.5-1.0 g of xylooligosaccharide, 0.5-1.0 g of galactooligosaccharide, 0.5-1.0 g of soybean oligosaccharide and 0.5-1.0 g of cranberry fruit powder.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A lactobacillus plantarum (Lactobacillus plantarum) strain A21094 with triglyceride reducing, cholesterol reducing and lipid reducing functions is characterized by being deposited with the accession number GDMCCNo 62961.
2. A probiotic comprising lactobacillus plantarum strain a21094 according to claim 1 and an adjunct.
3. The probiotic agent according to claim 2, characterized in that the viable count of lactobacillus plantarum strain a21094 is (1-1000) x 10 9 CFU/ml。
4. A weight loss agent comprising lactobacillus plantarum strain a21094 of claim 1 and a pharmaceutically acceptable adjuvant.
5. The weight loss agent according to claim 4, wherein the viable count of Lactobacillus plantarum strain A21094 is (1-1000) x 10 9 CFU/ml。
6. The weight loss medication of claim 4 wherein the weight loss medication comprises an oral formulation.
7. Use of lactobacillus plantarum strain a21094 according to claim 1 for the preparation of a hypolipidemic probiotic or a slimming medicament.
8. Use of lactobacillus plantarum strain a21094 according to claim 1 or a probiotic according to claim 2 or 3 for the preparation of a medicament for the prevention and/or treatment of hyperlipidemia or cardiovascular and cerebrovascular diseases.
9. A starter culture comprising lactobacillus plantarum strain a21094 of claim 1 and a carrier medium.
10. Use of lactobacillus plantarum strain a21094 according to claim 1 or a starter culture according to claim 9 for the preparation of a reduced-fat, cholesterol-reducing and lipid-lowering fermented product or food product.
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