CN113930367B - Lactic acid bacteria with cholesterol reducing performance and application thereof - Google Patents
Lactic acid bacteria with cholesterol reducing performance and application thereof Download PDFInfo
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- CN113930367B CN113930367B CN202111342225.1A CN202111342225A CN113930367B CN 113930367 B CN113930367 B CN 113930367B CN 202111342225 A CN202111342225 A CN 202111342225A CN 113930367 B CN113930367 B CN 113930367B
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- weissella
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- cholesterol
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 title claims description 67
- 235000012000 cholesterol Nutrition 0.000 title claims description 32
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title description 12
- 241000894006 Bacteria Species 0.000 title description 10
- 239000004310 lactic acid Substances 0.000 title description 6
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Classifications
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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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
-
- 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
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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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
- A61P3/04—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
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- 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
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- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
-
- 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
-
- 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
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
Abstract
The invention provides a lactobacillus with cholesterol-reducing performance and application thereof. The Weissella food provided by the invention is Weissella food LMLC4560-1 strain (Weissella cibaria LMLC 4560-1), which is preserved in China Center for Type Culture Collection (CCTCC) for 7 months and 2 days in 2021, and the preservation number is CCTCC NO: M2021811. The Weissella antrum LMLC4560-1 strain provided by the invention has high-efficiency cholesterol-reducing capability and strong antioxidant capability, and simultaneously has good gastrointestinal adverse-resistant environmental performance.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to lactic acid bacteria with cholesterol reducing performance and application thereof.
Background
Weissella is a lactic acid bacteria, and is widely distributed, and various Weissella are contained in the environments of food, soil, gastrointestinal tract, respiratory tract and the like, wherein the fermented food is a rich source of Weissella. Some Weissella from fermented food sources can significantly reduce biogenic amines in the fermented food due to the inclusion of the sufI gene encoding copper oxidase in the genome. The Weissella metabolism can produce bacteriocin to prevent the growth of perishable microorganisms and prolong the shelf life of food. Weissella also has the capacity of synthesizing oligosaccharides, and the products can be used as flavoring agents in foods for diabetics and obese patients, and can also be used in the industries of cosmetics and feed. Studies have shown that Weissella antrum has anticancer, immunomodulating, anti-inflammatory levels and antioxidant activity.
Hypercholesterolemia in humans has been shown to be a major cause of atherosclerosis, leading to cardiovascular and cerebrovascular diseases. Hypercholesterolemia is associated with many metabolic syndromes, including obesity, hyperlipidemia, hyperglycemia, and hypertension, etc. When the concentration of total cholesterol in serum in human body exceeds 7.28mmol/L, the incidence risk degree of acute coronary heart disease is greatly increased. Thus, reducing cholesterol intake by dietary containment and developing foods with regulated body cholesterol levels is a healthier and effective approach, while probiotics with cholesterol-lowering efficacy are functional ingredients of great development prospects.
At present, cholesterol is mainly reduced by drug treatment, and statin drugs reduce serum cholesterol, but also cause adverse reactions such as muscle spasm and rhabdomyolysis of organisms. The other main way is to obtain a certain effect through diet therapy, but the effect is greatly weakened along with the time extension, and meanwhile, long-term diet therapy is difficult to persist. Therefore, functional foods have been developed, and have no side effects and long-term adherence, and people have been paying more attention to the products, which are not only high in nutritive value, but also meet the public health demands and development requirements of the food industry. Therefore, research and development of functional foods and microecologics with cholesterol-reducing probiotics are of great significance in improving the health level of people.
Disclosure of Invention
The invention aims to provide a Weissella strain with high cholesterol reducing capacity.
The Weissella antrum strain provided by the invention also has strong antioxidant capacity and good gastrointestinal tract reverse environment resistance.
The Weissella strain provided by the invention is separated from Tibetan yak milk and has food property.
The invention provides a Weissella antrum strain, which is a Weissella antrum LMLC4560-1 strain (Weissella cibaria LMLC 4560-1), and the Weissella antrum LMLC4560-1 strain (Weissella cibaria LMLC 4560-1) is preserved in China Center for Type Culture Collection (CCTCC) at 7 and 2 days of 2021, and the preservation number is CCTCC NO: M2021811.
Preferably, the 16S rDNA gene sequence of the Weissella antrum LMLC4560-1 strain is shown in SEQ ID NO. 2.
The invention also provides a fermentation preparation method of the Weissella shirtula bacterial agent, which comprises the following steps: culturing said strain of Weissella food.
Preferably, the preparation method comprises the following steps:
(1) Amplifying and culturing the Weissella strain;
(2) And (3) adding the product obtained in the step (1) into a liquid culture medium, and fermenting and culturing at 26-32 ℃.
The invention also provides a microbial inoculum, which is obtained by the preparation method.
The invention also provides a fermentation product obtained by fermenting the antrum Weissella strain or the microbial inoculum.
Preferably, the fermentation product has cholesterol lowering properties;
preferably, the fermentation product has free radical scavenging capacity.
Preferably, the fermentation product acts on cholesterol with a cholesterol reduction rate of greater than 87%;
alternatively, preferably, the fermentation product has a DPPH radical scavenging rate of greater than 90%.
The invention also provides a preparation method of the fermentation product, which is prepared by culturing the Weissella antrum strain or the microbial inoculum.
Preferably, the preparation method comprises the following steps:
(1) Culturing said strain of Weissella antrum or said microbial inoculum in an expanded manner;
(2) And (3) adding the product obtained in the step (1) into a liquid culture medium, and fermenting and culturing at 26-32 ℃ to obtain the fermentation product.
The invention also provides application of the Weissella antrum strain or the microbial inoculum in products for reducing cholesterol, reducing fat, losing weight, resisting oxidation or resisting aging.
Preferably, the product is a food, cosmetic, health product or pharmaceutical product.
The Weissella antrum LMLC4560-1 strain provided by the invention belongs to lactic acid bacteria, and is derived from herdsman yak milk in Tibet areas, and has food properties. The Weissella antrum LMLC4560-1 strain provided by the invention has high-efficiency cholesterol-reducing capability and strong oxidation resistance, and simultaneously has good gastrointestinal adverse-resistant environmental performance, and strain resources of probiotics with cholesterol-reducing function are supplemented. The antral Weissella fermented food provided by the invention can enrich the nutritional ingredients of the food, and also has the effects of improving the palatability, the digestibility and the like of the food. After the food raw material is fermented by the Weissella food provided by the invention, proteins, fats and saccharides can be decomposed into a predigested state which is easier to be absorbed by human bodies, and meanwhile, the contents of soluble calcium, phosphorus, iron and certain B vitamins can be increased, so that the digestion and absorption performance and the nutritional value are improved.
Information on preservation of strains
The invention provides a Weissella food strain which is a Weissella food strain LMLC4560-1 (Weissella cibaria LMLC 4560-1) and is preserved in China Center for Type Culture Collection (CCTCC) for 7 months and 2 days in 2021, wherein the preservation number is CCTCC NO: M2021811, and the preservation address is: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: 027-68754052.
Lactobacillus plantarum LP-23 (Lactobacillus plantarum LP-23) in the embodiment of the invention is preserved in China Center for Type Culture Collection (CCTCC) on the 6 th month and 2 nd day of 2020, and the preservation number is CCTCC NO: M2020175, and the preservation address is: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: 027-68754052.
Drawings
FIG. 1 shows a cholesterol standard curve as described in example 3;
FIG. 2 is a diagram showing colony morphology of Weissella antrum LMLC4560-1 strain;
FIG. 3 shows a morphological characterization of Weissella antrum LMLC4560-1 strain.
Detailed Description
The Weissella antrum LMLC4560-1 strain provided by the invention is separated from Laseiulus yak milk in Shanshan, shanxi. The invention provides a Weissella antrum LMLC4560-1 strain which is a gram positive strain with acid production performance, wherein the strain is obtained by enriching Tibetan yak milk and is obtained by screening cycloheximide and colistin sulfate. The Weissella antrum LMLC4560-1 strain provided by the invention has food attribute, high-efficiency cholesterol-lowering capability and strong antioxidation capability, and simultaneously has good gastrointestinal tract reverse environment resistance. The fermented food can enrich the nutritional ingredients of the food and improve the palatability and digestibility of the food.
The specific sources of reagents used in the examples are listed in table 1 below.
TABLE 1 Source information of reagents and instruments used in the present invention
| Reagents/apparatus | Purity/model | Manufacturer(s) |
| Yeast extract powder | 99％ | Angel Yeast Co.,Ltd. |
| Glucose | 99％ | Yishui Daida pharmaceutical Co Ltd |
| Litmus cow milk culture medium | BR | Beijing Soilebao Biotech Co.Ltd |
| MRS broth | BR | Beijing Obo Star Biotechnology Co., ltd |
| BL agar medium | BR | Beijing Soilebao Biotech Co.Ltd |
| Gram staining solution | 99％ | Beijing Soilebao Biotech Co.Ltd |
| Super PCR Mix | 99.8％ | BEIJING TSINGKE XINYE BIOLOGICAL TECHNOLOGY Co.,Ltd. |
| Cycloheximide | ＞94％ | Hunan He Zhong Biotech Co.Ltd |
| Colistin sulfate | BR | Hunan He Zhong Biotech Co.Ltd |
| Cholesterol | AR | Sigma-Aldrich |
| Phthalic dicarboxaldehyde | AR | Microphone Lin Shi reagent net |
| Ultraviolet spectrophotometer | SP-754 | SHANGHAI SPECTRUM INSTRUMENTS Co.,Ltd. |
| High-speed centrifuge | Minispin | eppendorf |
| PCR instrument | C1000Touch | Bio-rad |
| Biochemical incubator | SPX250BSH-II | Shanghai CIMO Medical Instrument Co.,Ltd. |
EXAMPLE 1 enrichment, isolation and purification of gram-Positive bacilli
(1) Enrichment of strains: taking 1ml of a sample of Shangxian yak milk of Shangxian county in Shangnan city of Tibet, and carrying out gradient dilution to 10 -3 2ml of the diluted solution is added into 20ml of the litmus milk culture medium, then the mixture is placed into a 37 ℃ incubator for culture, and the culture medium is taken out when the culture medium is acidified and solidified and turns pink.
(2) Culturing single colonies: diluting the bacterial liquid of the litmus milk culture medium to 10 -5 -10 -6 Inoculating to BL agar medium containing 10ppm cycloheximide and 10ppm colistin sulfate, culturing at 37 deg.C for 24-48 hr, and forming colony.
(3) Screening acid-producing strains: single colony obtained by preliminary screening was inoculated with 0.2% CaCO 3 The BL agar medium is streaked for 24-48 h, and the transparent circle is observed after the culture is finished.
(4) Purifying acid-producing strains: single colonies with large transparent circles were selected and streaked for purification. Purification was repeated 3 times.
(5) Gram stain preliminary identification: and (3) carrying out gram staining on the purified acid-producing strain, and screening gram-positive bacillus with purple thalli for subsequent research.
EXAMPLE 2 preparation of gram-Positive bacilli fermentation broths
(1) Activating the strain: the bacterial liquid of the gram-positive bacillus stored in the glycerol pipe of 1ml is sucked by a liquid-transferring gun, inoculated into 9ml of MRS liquid culture medium, and activated and cultured for 24 hours at 37 ℃.
(2) Culturing seed liquid: 1ml of gram-positive bacilli activation culture solution was pipetted into 9ml of MRS liquid medium and incubated at 37℃for 24h.
(3) Fermentation: 5ml of gram-positive bacillus seed culture solution was aspirated with a pipette and inoculated into 45ml of MRS-CHOL (cholesterol MRS medium) liquid medium, and cultured at 37℃for 48 hours.
(4) Centrifuging fermentation liquor: the fermentation broth was centrifuged at 12000rpm for 48 hours for 10 minutes, and the supernatant was collected. Thus obtaining the supernatant of the fermentation liquor of gram-positive bacillus.
Example 3 determination of the ability of gram-Positive bacilli to reduce cholesterol
Drawing of cholesterol standard curve
Taking 0,0.1,0.2,0.3,0.4 and 0.5mL of cholesterol storage solution with the concentration of 1mg/mL respectively in a 10mL clean test tube, carrying out water bath at 60 ℃, adding 2mL of freshly prepared phthalaldehyde color-developing agent after ethanol volatilizes cleanly, standing at room temperature for 10min after uniform oscillation, slowly adding 1mL of concentrated sulfuric acid, uniformly mixing, standing for 20min, and carrying out color comparison under the wavelength of 560 nm. The horizontal axis represents cholesterol concentration, the vertical axis represents absorbance, and a standard curve was drawn. The standard curve obtained is shown in figure 1.
The gram-positive bacilli prepared in example 2 were assayed for their ability to lower cholesterol and the content of cholesterol in the broth was determined by the o-phthalaldehyde method.
A blank control group was prepared from cholesterol medium not inoculated with gram-positive bacilli. The cholesterol reduction rate of gram-positive bacilli was then calculated according to the following formula. The results are shown in Table 2.
X＝(1-A/B)*100％；
Wherein: cholesterol lowering rate of X-gram-positive bacilli;
cholesterol content in the supernatant of fermentation broth of A-gram-positive bacilli;
cholesterol content in the supernatant of the B-blank.
TABLE 2 reduction rate of cholesterol by gram-Positive bacilli
| Strain numbering | Cholesterol reduction rate | Strain numbering | Cholesterol reduction rate |
| LMLC4560-1 | 87.3％ | LMLC4560-11 | 41.3％ |
| LMLC4560-2 | 18.0％ | LMLC4560-12 | 39.0％ |
| LMLC4560-3 | 22.7％ | LMLC4560-13 | 40.6％ |
| LMLC4560-4 | 20.1％ | LMLC4560-14 | 20.6％ |
| LMLC4560-5 | 20.8％ | LMLC4560-15 | 35.2％ |
| LMLC4560-6 | 5.6％ | LMLC4560-16 | 30.5％ |
| LMLC4560-7 | 32.3％ | LMLC4560-17 | 23.5％ |
| LMLC4560-8 | 29.6％ | LMLC4560-18 | 35.2％ |
| LMLC4560-9 | 18.0％ | LMLC4560-19 | 48.7％ |
| LMLC4560-10 | 22.7％ | LP-23 | 36.5％ |
The results as shown in table 2: 19 gram-positive bacilli separated, screened and purified in yak milk and lactobacillus plantarum LP-23 in the laboratory, 10 strains with stronger cholesterol reducing capability in 20 strains are respectively: LMLC4560-1, LMLC4560-7, LMLC4560-11, LMLC4560-12, LMLC4560-13, LMLC4560-15, LMLC4560-16, LMLC4560-18, LMLC4560-19, LP-23.
The LP-23, namely lactobacillus plantarum LP-23 (Lactobacillus plantarum LP-23), is preserved in China Center for Type Culture Collection (CCTCC) on the 6 th month and 2 th day of 2020, and the preservation number is CCTCC NO: M2020175.
The lactobacillus plantarum LP-23 is obtained by separation and purification in the following way:
(1) enrichment of strains: taking 1ml of milk sample from a certain milk factory in Yichang city of Hubei province in China, and carrying out gradient dilution to 10 -3 Adding 2ml of the diluted solution into 20ml of litmus cow milk culture medium, culturing in a 37 ℃ incubator until the culture medium is acidified and coagulatedTaking out when the color is pink;
(2) culturing single colonies: diluting the bacterial liquid of the litmus milk culture medium to 10 -5 -10 -6 Inoculating to BL agar medium containing 10ppm cycloheximide and 10ppm colistin sulfate, culturing at 37deg.C for 24-48 hr, and forming colony;
(3) screening acid-producing strains: single colony obtained by preliminary screening was inoculated with 0.2% CaCO 3 Culturing in BL agar medium for 24-48 h, and observing whether transparent ring exists after culturing;
(4) purifying acid-producing strains: the largest single colony of the transparent circle, lactobacillus plantarum LP-23 strain, was selected.
The lactobacillus plantarum LP-23 strain was subjected to 16SrDNA sequencing and strain identification. The adopted primers are as follows: forward primer 27F:5'-AGAGTTTGATCATGGCTCAG-3'; reverse primer 1492R:5'-ACGGCTACCTTGTTACGACTT-3'. The PCR reaction system is as follows: 25 mu.l mix;1 μl sample; 20 μl ddH 2 O;2 μl 27F;2 μl 1492R; PCR reaction temperature: 98 ℃ for 2min;98 ℃,10s,58 ℃,10s,72 ℃,7s,35x;72 ℃ for 2min; preserving at 4 ℃. The PCR product preparation was sequenced and the gene sequence SEQ ID NO.1 of the 16SrDNA gene of the strain LP-23 of Lactobacillus plantarum was determined as follows:
EXAMPLE 4 determination of tolerance of artificial digestive juice of gram-Positive bacilli
The tolerance of 10 gram-positive bacilli screened in example 3, which had a large cholesterol lowering capacity, to artificial digestive juice was measured.
(1) Preparation of artificial digestive juice
Artificial gastric juice: naCl 0.20g/100mL, pepsin 0.35g/mL, and pH 2.5 with 1mol/L HCl, filtering and sterilizing.
Artificial intestinal juice: the solution A and the solution B are mixed according to the ratio of 2:1 to obtain the artificial intestinal juice. A. Pancreatic juice: 1.1g/100mL of sodium bicarbonate, 0.2g/100mL of NaCl and 0.1g/100mL of trypsin, adjusting the pH value to 8.0, and filtering and sterilizing for later use. B. Bile juice: bile acid salt 1.8g/100mL, pH value is adjusted to 6.8, and filtration and sterilization are carried out for standby.
(2) Determination of tolerance to artificial digestive juice
After single colony of gram positive bacillus is selected and cultured in MRS liquid culture medium for overnight, 1mL of culture solution is respectively inoculated into 9mL of artificial gastric juice or artificial intestinal juice, and the culture is carried out at 37 ℃ for 2 hours, so as to determine the number of viable bacteria. Gram-positive bacilli inoculated in 9mL of MRS broth served as positive control.
Survival (%) = (number of viable bacteria in artificial digest/number of viable bacteria in control) ×100%
TABLE 3 survival of gram-positive bacilli after treatment with artificial gastric juice and artificial intestinal juice
As shown in Table 3 above, gram positive bacilli LMLC4560-1 had higher survival rates in both artificial gastric juice and artificial intestinal juice, reaching 96.35% and 92.36%, respectively.
EXAMPLE 5 determination of the clearance of DPPH free radical from the supernatant of gram-Positive Bacillus fermentation broth
The supernatant of 10 gram-positive bacilli fermentation broth screened in example 3, which had a large cholesterol lowering capacity, was assayed for DPPH radical scavenging rate. The measurement method is as follows:
(1) activating the strain: the gram-positive bacillus liquid stored in the glycerol pipe of 1ml is sucked by a pipette, inoculated into 9ml of MRS liquid culture medium, and activated and cultured for 24 hours at 37 ℃.
(2) Culturing seed liquid: 1ml of the activated culture broth was aspirated with a pipette and inoculated into 9ml of MRS broth, and incubated at 37℃for 24h.
(3) Fermentation: 5ml of seed culture solution was aspirated by a pipette and inoculated into 45ml of MRS liquid medium and cultured at 37℃for 24 hours.
(4) Preparing a fermentation broth supernatant: the fermentation broth was centrifuged at 5000rpm for 24 hours, and the supernatant was collected. Thus obtaining the supernatant of the fermentation liquor of gram-positive bacillus.
(5) 3mL of fermentation broth to be tested is taken and evenly mixed with an equal volume of DPPH solution with the concentration of 0.16mmol/L (A1 pipe); mixing the same volume of absolute ethyl alcohol with 0.16mmol/L DPPH solution uniformly (A2 tube); uniformly mixing the absolute ethyl alcohol with the same volume with the liquid to be detected (A3 pipe); after light-shielding reaction at 30 ℃ for 30min, absorbance values of the A1, A2 and A3 tubes at 519nm are measured by taking distilled water as a blank group and are respectively recorded as ODA1, ODA2 and ODA3. The DPPH radical scavenging rate was calculated according to the following formula:
DPPH clearance= (OD) A2 +OD A3 -OD A1 )/OD A2 ×100％。
(6) The experimental results are shown in table 4.
TABLE 4 clearance of gram-Positive bacilli fermentation broths to DPPH free radical
As shown in the above Table 4, the supernatant obtained by fermenting gram-positive bacillus LMLC4560-1 has a higher clearance rate to DPPH free radical, reaching 90.35%.
Example 6 identification of strains
The obtained gram positive bacterium LMLC4560-1 strain is inoculated on BL solid culture medium for streak culture, and colony morphology is observed. On BL medium, colony is yellowish, flat circular, edge-trim, opaque, 1.5 mm+ -0.3 mm. Colony morphology is shown in FIG. 2.
Single colonies were picked up and dissolved in 1ml of sterile water and their cell morphology was observed under a microscope. The identification result shows that: the obtained gram positive strain LMLC4560-1 has no movement, no spore, no flagellum and no capsule, and the strain is in the shape of short rod with one blunt end and one smaller end, and exists in pairs. The morphological characteristics of the strain are shown in FIG. 3.
The LMLC4560-1 strain was subjected to 16SrDNA sequencing and strain identification. The adopted primers are as follows: forward primer 27F:5'-AGAGTTTGATCATGGCTCAG-3'; reverse primer 1492R:5'-ACGGCTACCTTGTTACGACTT-3'. The PCR reaction system is as follows: 25 mu.l mix;1 μl sample; 20 μl ddH2O;2 μl 27F;2 μl 1492R; PCR reaction temperature: 98 ℃ for 2min;98 ℃,10s,58 ℃,10s,72 ℃,7s,35x;72 ℃ for 2min; preserving at 4 ℃. The PCR product formulation was sequenced and the gene sequence SEQ ID NO.2 of the LMLC4560-1 strain 16SrDNA was as follows:
the LMLC4560-1 strain was named as Weissella antrum LMLC4560-1 strain (Weissella cibaria LMLC 4560-1) and was preserved, and the Weissella antrum LMLC4560-1 strain (Weissella cibaria LMLC 4560-1) was preserved in China Center for Type Culture Collection (CCTCC) at 7 and 2 days of 2021 with a preservation number of CCTCC NO: M2021811.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Hubei Angel biological group Co., ltd
<120> lactic acid bacterium having cholesterol-lowering property and use thereof
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ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac aggattagat accctggtag 780
tccataccgt aaacgatgaa tgctaagtgt tggagggttt ccgcccttca gtgctgcagc 840
taacgcatta agcattccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg 900
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Claims (16)
1. A strain of Weissella food is Weissella food LMLC4560-1 strainWeissella cibariaLMLC 4560-1) which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M2021811 at the month of 2021 and the day of 7.
2. The strain of weissella antrum according to claim 1, wherein the 16S rDNA gene sequence of the strain weissella antrum LMLC4560-1 is shown in SEQ ID No. 2.
3. A method for preparing a Weissella sinus inoculant by fermentation, which is characterized by comprising the following steps: culturing the strain of weissella antrum of claim 1 or 2.
4. A method of preparation according to claim 3, characterized in that the method of preparation comprises the steps of:
(1) Culturing the strain of weissella antrum according to claim 1 or 2 in an enlarged scale;
(2) And (3) adding the product obtained in the step (1) into a liquid culture medium, and fermenting and culturing at 26-32 ℃.
5. A microbial agent, characterized in that it is obtained by the fermentation preparation method of claim 3 or 4.
6. A fermentation product obtained by fermentation of the antral Weissella strain of claim 1 or 2 or the microbial agent of claim 5.
7. The fermentation product of claim 6, wherein the fermentation product has cholesterol lowering properties.
8. The fermentation product of claim 6, wherein the fermentation product has free radical scavenging capability.
9. The fermentation product of claim 7, wherein the fermentation product has free radical scavenging capability.
10. The fermentation product of claim 6, wherein the fermentation product acts on cholesterol with a cholesterol reduction rate of greater than 87%.
11. The fermentation product of claim 6, wherein the fermentation product has a DPPH-radical scavenging rate of greater than 90%.
12. The fermentation product of claim 10, wherein the fermentation product has a DPPH-radical scavenging rate of greater than 90%.
13. A process for the preparation of a fermentation product according to any one of claims 6 to 12, which comprises culturing a strain of westernum as defined in claim 1 or 2 or a microbial inoculum as defined in claim 5.
14. The preparation method according to claim 13, characterized in that the preparation method comprises the steps of:
(1) Culturing the strain of antral Weissella according to claim 1 or 2 or the microbial inoculum according to claim 5 in an enlarged scale;
(2) And (3) adding the product obtained in the step (1) into a liquid culture medium, and fermenting and culturing at 26-32 ℃ to obtain the fermentation product.
15. Use of a strain of weissella antrum as claimed in claim 1 or 2 or a microbial agent as claimed in claim 5 for the preparation of a cholesterol-lowering, lipid-lowering, weight-reducing, antioxidant or anti-ageing product.
16. The use according to claim 15, wherein the product is a food, a cosmetic, a health product or a pharmaceutical product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111342225.1A CN113930367B (en) | 2021-11-12 | 2021-11-12 | Lactic acid bacteria with cholesterol reducing performance and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202111342225.1A CN113930367B (en) | 2021-11-12 | 2021-11-12 | Lactic acid bacteria with cholesterol reducing performance and application thereof |
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| KR20150032380A (en) * | 2013-09-16 | 2015-03-26 | (사)광주명품김치산업화사업단 | Weissella cibaria 12-13 having mannitol production capacity and methods for preparing Kimchi comprising the same |
| CN113164528A (en) * | 2018-12-10 | 2021-07-23 | 韩国食品研究院 | Pharmaceutical composition for preventing or treating cancer comprising Weissella cibaria WIKIM28 as an active ingredient |
| KR20210108781A (en) * | 2020-02-26 | 2021-09-03 | 경일대학교산학협력단 | Weissella cibaria JSKIU 18-18 (KCTC18806P) from fermented local food in Jeju and composition with the function of anti-aging, anti-cancer and anti-dental caries thereof |
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| KR20150032380A (en) * | 2013-09-16 | 2015-03-26 | (사)광주명품김치산업화사업단 | Weissella cibaria 12-13 having mannitol production capacity and methods for preparing Kimchi comprising the same |
| CN113164528A (en) * | 2018-12-10 | 2021-07-23 | 韩国食品研究院 | Pharmaceutical composition for preventing or treating cancer comprising Weissella cibaria WIKIM28 as an active ingredient |
| KR20210108781A (en) * | 2020-02-26 | 2021-09-03 | 경일대학교산학협력단 | Weissella cibaria JSKIU 18-18 (KCTC18806P) from fermented local food in Jeju and composition with the function of anti-aging, anti-cancer and anti-dental caries thereof |
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