CN111849836B - Lactobacillus rhamnosus with antioxidant function and application thereof - Google Patents
Lactobacillus rhamnosus with antioxidant function and application thereof Download PDFInfo
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- CN111849836B CN111849836B CN202010792212.3A CN202010792212A CN111849836B CN 111849836 B CN111849836 B CN 111849836B CN 202010792212 A CN202010792212 A CN 202010792212A CN 111849836 B CN111849836 B CN 111849836B
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
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Classifications
-
- 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
-
- 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
- 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
- A23V2400/175—Rhamnosus
-
- 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
- C12R2001/225—Lactobacillus
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- 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 belongs to the technical field of microorganisms, and particularly relates to lactobacillus rhamnosus with oxidation resistance and application thereof. The lactobacillus rhamnosus is [ ]Lactobacillus rhamnosus) The strain is a probiotic strain with acid resistance and bile salt resistance, which is separated from Xinjiang natural fermented old yoghourt; the strain is preserved in China general microbiological culture Collection center, and the preservation number is: CGMCC No.20012. The lactobacillus rhamnosus not only has the probiotic effect of common lactobacillus, but also has antioxidant activity, and can improve the service life of nematodes.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus rhamnosus with antioxidation and nematode life prolonging functions, which is separated from Xinjiang natural fermentation old yoghourt, and application of lactobacillus rhamnosus in antioxidation foods and health care products.
Background
Aging refers to the occurrence of degenerative changes of tissue morphology, decrease in physiological functions and deterioration of mental functions, which are accompanied by heart disease, hypertension, hyperlipidemia, etc. during life. Currently, the global population is 60 hundred million, and the global population is predicted to reach 80 hundred million in 2025, and the world will walk into the aging society at that time, and people will face two major health problems, namely aging and diseases. "people's condition, morbid death and happiness" ("Ling Shu. Master" for Master), so longevity is a wish of everyone.
With the continuous development of population aging trend in the world, especially in China, the world medical subject of anti-aging is also receiving attention in China. In recent years, research on aging and research on anti-aging drugs and foods according to the mechanism of aging have become hot spots in the current life science and medical research fields. Oxidation is the biggest threat of skin aging, and unhealthy diet, sun exposure, pressure, environmental pollution and the like can cause free radical flooding of skin, so that oxidation phenomena such as dark complexion, water shortage and the like are generated, and are all "culprit and culprit" of body oxidation. Under normal conditions, free radicals in the human body are in dynamic equilibrium with constant production and elimination. The free radical is produced too much or eliminated too slowly, and it attacks life macromolecular substances and various cells to cause various damages of organism at molecular level, cell level and tissue organ level, accelerate aging process of organism and induce various diseases. The human body inevitably generates free radicals and simultaneously naturally generates antioxidant substances resisting the free radicals so as to counteract the oxidative attack of the free radicals on human cells. Research shows that the antioxidant system of human body is one system with perfect and complex functions, which is comparable to the immune system, and the stronger the antioxidant capacity of human body, the healthier and longer the life. The antioxidant effect of organism can keep balance of free radical generation and elimination, and has important significance for preventing diseases and resisting aging.
The most accepted theory of aging is that the aging process of organisms is that cells of tissues of the organisms continuously produce free radical accumulation results, and free radicals are normal metabolic intermediates and have strong reaction capacity, so that various substances in the cells are oxidized, and the biological membranes are damaged. And can crosslink macromolecules such as protein, nucleic acid and the like, influence normal biological functions, increase decay, reduce the activity of antioxidant enzymes in the body, reduce the antioxidant capacity and finally cause the aging of organisms. Antioxidant effect is an important index and approach for anti-aging.
Along with the deep research of lactic acid bacteria, some lactic acid bacteria are found to have the functions of regulating the stable state of intestinal flora, reducing the cholesterol content in serum, resisting bacteria and enhancing immunity, and more researches indicate that the lactic acid bacteria are more natural antioxidants, and some lactic acid bacteria have the functions of resisting oxidation, can remove free radicals in intestinal tracts and help organisms maintain the free radicals in a relatively stable state. Lactic acid bacteria can inhibit lipid peroxidation and chelate metal ions by generating antioxidant active substances, and can regulate and control cell pathways related to host cell oxidative stress and the oxidative damage repair system can play an antioxidant role.
The ideal strain that can be used as a probiotic should have the following properties: the microbial inoculum is harmless to a host, is not easy to hybridize with pathogenic bacteria and is not easy to mutate, can resist acid and bile salt, does not have drug resistance genes, can be planted in intestinal tracts, can be stored in products and the like.
Aiming at the problems, the invention separates the strain which is tolerant to acid and bile salt and can survive in intestinal tracts and has the functions of resisting oxidation and prolonging the service life of nematodes from the traditional fermented dairy product, and applies the strain to foods and health care products which are resisting oxidation and prolonging the service life of nematodes.
Disclosure of Invention
The invention aims to provide a lactobacillus rhamnosus strain which is an acid-resistant and bile-salt-resistant strain with probiotic characteristics, is separated from Xinjiang natural fermentation old yoghourt, and has the characteristics of resisting oxidation and prolonging nematode life.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) is preserved in China general microbiological culture Collection center, north Xielu No. 1, 3, the Korean area of Beijing, with a preservation time of 2020.6.3, and a preservation number: CGMCC No.20012.
Another object of the invention is the action of said Lactobacillus rhamnosus in resisting oxidation and prolonging nematode life.
The lactobacillus rhamnosus is applied to preparing food and health care products for resisting oxidation or prolonging the service life of nematodes.
Drawings
FIG. 1 colony morphology of Lactobacillus rhamnosus of the present invention on MRS plates;
FIG. 2 is a mass spectrum identification chart of lactobacillus rhamnosus of the present invention;
FIG. 3 Lactobacillus rhamnosus of the present invention increases nematode longevity.
Detailed Description
The present invention will be described in further detail with reference to the following examples, in order to make the objects, technical solutions and advantages of the present invention more apparent, and the present invention is not limited by these.
Example 1: lactobacillus rhamnosus (Lactobacillus rhamnosus) is preserved in China general microbiological culture Collection center, north Xielu No. 1, 3, the Korean area of Beijing, with a preservation time of 2020.6.3, and a preservation number: CGMCC No.20012.
1. Sample collection and isolation and identification of lactic acid bacteria
The sample is old yoghurt naturally fermented by Xinjiang herdsman by a traditional method, and is collected in Yili state of Xinjiang China, and the sample is placed in a foam box with an ice bag and transported to a laboratory for lactobacillus separation.
Samples were diluted in a multiple ratio and coated onto MRS plates containing calcium carbonate. Placing in an anaerobic bag, culturing at 37deg.C for 48-72 hr, and collecting apparent calcium carbonate-dissolved colony with inoculating loop, transferring onto new MRS plate, and obtaining pure culture.
Inoculating the obtained pure culture into MRS liquid culture medium to prepare test bacterial liquid, sucking 10 mu L of bacterial suspension, inoculating into 5ml of MRS liquid culture medium with pH value of 3.5, and culturing at 37 ℃ for 24h; 1.0mL of culture solution and 9.0mL of artificial gastric juice with pH value of 3.0 are sucked [ preparation method: naCl0.2%, pepsin (sigma) 0.35%, adjusting pH to 3.0 with 1mol/L HCl, filtering with 0.45um filter membrane, sterilizing, mixing, culturing at 37deg.C, sampling after starting (0 h) and 3h, measuring viable count with MRS agar medium, and calculating strain survival rate. Lactobacillus strains with high acid resistance and gastric acid resistance are screened out by the method.
The obtained pure culture is subjected to biochemical identification by a lactobacillus biochemical identification box (Guangdong Crohn microorganism technology Co., ltd.) and identified by mass spectrum to obtain a lactobacillus rhamnosus strain.
Identification of lactobacillus: the separated and purified lactobacillus is identified by biochemical characteristic analysis and mass spectrometry, the colony morphology of the lactobacillus rhamnosus on an MRS plate is shown in figure 1, the biochemical identification of the lactobacillus is shown in table 1, the fingerprint of the protein generated by mass spectrometry detection is shown in figure 2, the Score Value of the result compared with the spectrum of the lactobacillus rhamnosus is 1.85, and the separated strain can be determined to be the lactobacillus rhamnosus.
The lactobacillus rhamnosus (Lactobacillus rhamnosus) has the functions of resisting oxidation and prolonging the service life of nematodes besides the functions of common lactobacillus on human health, and can be used for producing antioxidant foods and health care products.
Table 1 Biochemical identification results of isolated Lactobacillus
Example 2: lactobacillus rhamnosus antioxidation and nematode life prolonging effects
A fresh culture of the lactic acid bacteria to be tested (obtained in example 1) was inoculated into a liquid medium and incubated overnight at 37 ℃. Centrifuging the bacteria-containing liquid culture medium at 12000r/min for 10min, and filtering the supernatant with a 0.22 μm filter membrane to obtain cell-free supernatant.
DPPH radical scavenging Activity
0.4mL of the cell-free supernatant was mixed with 1.6mL of a freshly prepared 0.2mM DPPH absolute ethanol solution and reacted in the dark for 25 minutes. The control group replaced cell-free supernatant with an equivalent amount of MRS medium. After the reaction, OD was measured 517 Is a value of (2). Control group is marked as A 0 The experimental group is marked as A 1 . The DPPH radical scavenging rate is:
Scavenging rate＝(A 0 -A 1 )/A 0 ×100％
2. hydroxyl radical scavenging Activity
The reaction system included an ethanol solution of salicylic acid (5 mM), a ferrous sulfate solution (5 mM), and hydrogen peroxide (3 mM). 1.4mL of each of the above solutions was mixed with 100. Mu.L of the cell-free supernatant. The control group replaced cell-free supernatant with an equivalent amount of MRS medium. After 50min reaction at room temperature and 1min centrifugation at 8000r/min, the OD was measured by UV spectrophotometer 510 Is a value of (2). Control group is marked as A 0 The experimental group is marked as A 1 . The hydroxyl radical removal rate is:
Scavenging rate＝(A 0 -A 1 )/A 0 ×100％
3. superoxide anion scavenging Activity
Taking 1mL of cell-free supernatant2.8mL Tris-HCl buffer (0.1M, pH 8.0) was mixed, and then 0.1mL pyrogallol (0.05M) was added thereto, followed by a reaction at room temperature in the absence of light for 4min. The control group replaced cell-free supernatant with an equivalent amount of MRS medium. The reaction was terminated by adding 80. Mu.L of hydrochloric acid solution (0.1M), and OD was measured 325 Is a value of (2). Control group is marked as A 0 The experimental group is marked as A 1 . The superoxide anion clearance is then:
Scavenging rate＝(A 0 -A 1 )/A 0 ×100％
the results are shown in Table 2:
TABLE 2 free radical scavenging ability of Lactobacillus rhamnosus
Measurement of the ability of lactobacillus rhamnosus to prolong nematode longevity: the caenorhabditis elegans was used as an animal model for life extension assays. The synchronized nematodes were incubated on NGM plates at 20 ℃ for 2 days to stage L4. 50. Mu.L of each of the cultured OP50 and lactobacillus solution (each having a concentration of 5X 10 8 CFU/mL) was plated onto NGM plates (4 cm) supplemented with 5-fluorodeoxyuridine (70 mM final concentration) and incubated overnight at 37 ℃. 10-20 nematodes were transferred to each plate, and the number of nematodes surviving was counted under a microscope every 24 hours until all the nematodes died. Each set of experiments was performed in triplicate. The results show that lactobacillus rhamnosus can significantly improve nematode longevity, and the results are shown in table 3 and fig. 3.
TABLE 3 Effect of Lactobacillus rhamnosus on nematode longevity
Conclusion: lactobacillus rhamnosus (Lactobacillus rhamnosus) CGMCC No.20012 has antioxidant activity and can improve nematode life.
Example 3: preparation of food and health product containing lactobacillus rhamnosus CGMCC No.20012 obtained by separating in example 1 is inoculated in MRS culture medium for fermentation, and is centrifugated to obtain bacterial mud, and is subjected to vacuum freeze drying to obtain freeze-dried bacterial powder, wherein the bacterial powder is prepared into food additive (w/w) according to the following formula: lactobacillus rhamnosus CGMCC No.20012 lyophilized powder: 50%, maltodextrin: 20% of inulin 30%, and the food additive containing lactobacillus rhamnosus CGMCC No.20012 is added into various foods and health care products according to the addition amount of 5-10%, so as to obtain the foods and health care products for resisting aging and prolonging the service life.
Claims (2)
1. Lactobacillus rhamnosus, characterized in that: the lactobacillus rhamnosus isLactobacillus rhamnosus)The microbial strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection) at 6/3/2020, and the preservation address is Hospital No. 3, north Chen West Lu 1, the Korean region of Beijing, and the preservation number is: CGMCC No.20012.
2. The use of lactobacillus rhamnosus according to claim 1 for preparing a food or health care product for resisting oxidation or prolonging nematode life.
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