CN117264839A - Application of saliva combined with lactobacillus MB1 in preparation of food and medicine for whitening and relieving gout - Google Patents

Application of saliva combined with lactobacillus MB1 in preparation of food and medicine for whitening and relieving gout Download PDF

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CN117264839A
CN117264839A CN202311304811.6A CN202311304811A CN117264839A CN 117264839 A CN117264839 A CN 117264839A CN 202311304811 A CN202311304811 A CN 202311304811A CN 117264839 A CN117264839 A CN 117264839A
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ligilactobacillus
saliva
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陆勇军
葛振煌
许敏青
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Guangdong Yuechuang Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of probiotics and application thereof, and particularly relates to application of saliva combined with lactobacillus MB1 in preparation of food and medicines for whitening and relieving gout. To further develop and utilize the probiotic functions of Lactobacillus salivarius, the invention isolates a Lactobacillus salivarius (Ligilactobacillus salivarius) MB1 strain from the feces of a healthy adult, and the strain MB1 has various probiotic effects including Glutathione (GSH) production and secretion, and xanthine oxidase activity inhibition. Therefore, the saliva combined lactobacillus MB1 strain has the functions of resisting aging, whitening and resisting oxidization, preventing and relieving hyperuricemia, gout and the like, and has important application value and economic value.

Description

Application of saliva combined with lactobacillus MB1 in preparation of food and medicine for whitening and relieving gout
Technical Field
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to application of saliva combined with lactobacillus MB1 in preparation of food and medicines for whitening and relieving gout.
Background
The saliva combines lactobacillus (Ligilactobacillus salivarius), also called lactobacillus salivarius, is gram positive bacillus, belongs to the lactobacillus family, the genus lactobacillus, the size is generally 0.6-0.9 μm multiplied by 1.5-5 μm, the two ends are round, and the saliva has single and double chains with different length, does not move and has no flagellum. The saliva combined lactobacillus is named after being separated in saliva of the oral cavity of a human body for the first time, has no special requirement on oxygen, is suitable for growing at 37 ℃, can be detected in almost all secretions of the human body, is thus widely existing lactobacillus in the human body, and also indicates that the saliva combined lactobacillus is a normal strain existing in the human body. In addition, the saliva combined lactobacillus has good acid resistance and bile salt resistance, and can survive in intestines and stomach of human and animals.
Saliva is widely present in the intestinal tract of humans and animals in combination with lactobacillus and is excreted with faeces. As part of the normal flora of the human body, lactobacillus salivarius is one of the important components of the normal microbial system of the intestinal tract and is associated with a host for life, and has important significance for maintaining the microecological balance of the intestinal tract. As probiotics in intestinal flora, the lactobacillus is also edible lactobacillus, and the saliva and lactobacillus can play a role in helping intestinal peristalsis, and can also enhance the digestion capacity of human bodies, improve immunity and the like. In recent years, lactobacillus salivarius has become a hotspot of research as a probiotic lactobacillus with great potential and is being continuously used to make probiotic preparations suitable for humans and animals.
Different strains of lactobacillus salivarius have been reported to have different probiotic functions, such as: (1) antibacterial action: the research shows that oral administration of the tablet containing the saliva combined with lactobacillus WB21 can reduce the number of pathogenic bacteria of periodontitis, regulate the microecological balance of the oral cavity and have a certain protection effect on periodontal environment. (2) The saliva combined lactobacillus has a certain adsorption effect, and can well colonize and grow on the surface of the intestinal tract so as to play a role in regulating immunity. The study shows that the saliva combined with the lactobacillus B1 can improve the intestinal microorganisms of the piglets and increase the number of immunocompetent cells in the intestinal tracts, and is beneficial to perfecting the immune system of the piglets and activating the local immunocompetence. (3) anti-aging effect: studies show that the average life of nematodes can be prolonged by feeding the nematodes with saliva from the faeces of the elderly, guangxi Ba Ma Baisui, in combination with lactobacillus FDB89, and the anti-aging effect is achieved. (4) cholesterol-lowering effect: along with the continuous improvement of the living standard of people, the more abundant the ingested nutrition is, the higher the content of serum cholesterol in the body is, thereby causing a series of diseases such as hypertension, coronary heart disease, arteriosclerosis and the like. Studies have shown that saliva in combination with Lactobacillus FBC05 has a degrading effect on cholesterol in vitro.
Saliva combines with the diversity of lactobacillus sources to give it genetic and functional diversity. Although a certain research progress has been made on the function and application of lactobacillus salivarius, the potential efficacy of lactobacillus salivarius is still far from being fully developed, so that the function of lactobacillus salivarius needs to be further mined according to different sources of lactobacillus salivarius so as to better play a role. In conclusion, the application of the probiotic saliva combined lactobacillus is intensively studied, and has a relatively wide development space.
Disclosure of Invention
In order to overcome the defects in the prior art, the saliva combined lactobacillus Ligilactobacillus salivarius MB strain is separated from the excrement of a healthy adult, and the strain MB1 has various probiotics effects, mainly comprising the production and secretion of Glutathione (GSH), the inhibition of xanthine oxidase activity and the like, and has important potential application values.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides the use of a strain of lactobacillus salivarius (Ligilactobacillus salivarius) MB1 for the production of Glutathione (GSH), said strain of lactobacillus salivarius (Ligilactobacillus salivarius) MB1 having the accession number: the complete sequence of the CCTCC NO: M20221502 and 16S rDNA is shown as SEQ ID No: 1.
Through researches, the probiotic saliva combined lactobacillus (Ligilactobacillus salivarius) MB1 strain can produce and secrete Glutathione (GSH), which suggests that the saliva combined lactobacillus MB1 strain is expected to be used for producing GSH, and is used in the fields of antioxidation, whitening, aging delay, immunity enhancement, anti-tumor, antiallergic and the like through the characteristic of GSH production.
In a second aspect, the invention provides the use of a strain of lactobacillus salivarius (Ligilactobacillus salivarius) MB1 as a xanthine oxidase inhibitor, said strain of lactobacillus salivarius (Ligilactobacillus salivarius) MB1 having the accession number: the complete sequence of the CCTCC NO: M20221502 and 16S rDNA is shown as SEQ ID No: 1.
Through researches, the probiotic bacteria saliva combined lactobacillus (Ligilactobacillus salivarius) MB1 strain can inhibit Xanthine Oxidase (XOD) activity, which suggests that the saliva combined lactobacillus MB1 strain is expected to reduce purine in vivo and uric acid generation by inhibiting the activity of xanthine oxidase, thereby controlling uric acid level and preventing gout attack.
The third aspect of the invention provides a method for preparing glutathione, which comprises inoculating saliva combined lactobacillus (Ligilactobacillus salivarius) MB1 strain into MRS liquid culture medium, culturing to obtain bacterial suspension after stationary phase, and separating and purifying glutathione in the bacterial suspension; the saliva combined lactobacillus (Ligilactobacillus salivarius) MB1 strain has a deposit number of: the complete sequence of the CCTCC NO: M20221502 and 16S rDNA is shown as SEQ ID No: 1.
Preferably, the conditions of the culture are: anaerobic at a constant temperature of 37 ℃.
Preferably, the Lactobacillus salivarius (Ligilactobacillus salivarius) MB1 strain is first cultured in MRS broth to stationary phase and then expanded to a new MRS broth at a dilution of 1:20-1000.
Compared with the prior art, the invention has the beneficial effects that:
the invention separates a saliva combined lactobacillus (Ligilactobacillus salivarius) MB1 strain from the feces of a healthy adult, and the strain MB1 has various probiotics effects including Glutathione (GSH) production and secretion, and xanthine oxidase activity inhibition. Therefore, the saliva combined lactobacillus MB1 strain has the functions of resisting aging, whitening and resisting oxidization, and can prevent and relieve hyperuricemia, gout and the like. Therefore, the saliva combined lactobacillus strain MB1 has various probiotics effects, can be used in the fields of whitening and relieving gout and the like, for example, can be prepared into medicines for whitening and relieving gout, and has important application value and economic value.
Drawings
FIG. 1 is a phylogenetic tree of Lactobacillus salivarius MB1 in combination with other homologous lactic acid bacteria;
FIG. 2 shows that Lactobacillus salivarius MB1 may produce and secrete GSH;
FIG. 3 shows that Lactobacillus salivarius MB1 may inhibit XOD activity.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
The following examples relate to the following experimental materials:
(1) Strains: saliva is combined with lactobacillus (Ligilactobacillus salivarius) MB1 strain, separated from the feces of a healthy adult in Guangdong, china, and stored in glycerol tubes at-80 ℃ in a low-temperature freezing way. Typically, it is inoculated onto the surface of an MRS solid medium plate and cultured upside down in a thermostatic anaerobic incubator at 37℃for 24 hours to obtain colonies, or cultured in an MRS liquid medium with shaking in a thermostatic anaerobic incubator at 37℃for 24-48 hours to obtain a fermentation broth.
(2) The kit comprises: a micro-reduced Glutathione (GSH) assay kit (Nanjing built, cat: A006-2-1), a xanthine oxidase activity assay kit (Box manufacturer, cat: AKAO 006M).
(3) MRS plate: 10g of beef extract, 10g of peptone, 5g of yeast extract, 2g of triammonium citrate, 5g of sodium acetate, 20g of glucose, 2g of dipotassium hydrogen phosphate, 1mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 15g of agar and ddH 2 Filling O to 1L, adjusting pH to 6.2-6.6, and autoclaving at 121deg.C for 20min to obtain MRS plate.
(4) MRS liquid medium: 10g of beef extract, 10g of peptone, 5g of yeast extract, 2g of triammonium citrate, 5g of sodium acetate, 20g of glucose, 2g of dipotassium hydrogen phosphate, 1mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate and ddH 2 Adding O to 1L, adjusting pH to 6.2-6.6, and autoclaving at 121deg.C for 20min to obtain MRS liquid culture medium.
Example 1 isolation and characterization of Lactobacillus salivarius (Ligilactobacillus salivarius) MB1
Saliva is isolated from faeces of a healthy adult (male, 34 years, bmi=24.4) in the sea ball region of guangdong, cantonese, the city of saliva is in combination with lactobacillus Ligilactobacillus salivarius MB, specifically as follows:
the fecal sample was repeatedly washed 3 times with sterile water, placed in a mortar, 500uL of sterile water was added per 100mg of fecal sample, thoroughly ground to a homogenate, and then an appropriate amount of the grinding fluid was pipetted, spread on an MRS plate, and incubated at room temperature for 3 days. The colonies to be streaked and purified in the separation experiment plates were numbered with a marker and strain numbers were marked on the plates accordingly. Colonies were picked and inoculated onto MRS plates and the strains were purified by plate streaking. If the strain cannot be separated by the method, colonies need to be picked from the enrichment plate, and the colonies are coated on the MRS plate after being subjected to gradient dilution by the MRS liquid culture medium. Reference is made to the "Berger's Manual of bacteria identification" (eighth edition) and the "manual of fungus classification identification", which identify strains belonging to bacteria first. The primary separation is carried out to obtain a purified strain, the strain is MB1, and after 24 hours of culture, the colony of the strain is observed to be smaller, colorless, smooth in surface, opaque, convex and neat in edge.
Next, after molecular identification by 16S rDNA universal primer (27F: AGAGTTTGATCCTGGCTCAG,1492R: TACGGCTACCTTGTTACGACCTT), the isolated L.salivarius MB1 strain was subjected to whole genome sequencing by Beijing Baimeike Biotechnology Co. The resulting sequence 16S rDNA sequence (SEQ ID No: 1) was subjected to BLAST alignment at NCBI' S Genome database. The results showed that the homology of strain MB1 with the known 16S rDNA sequence of Lactobacillus salivarius was >99%, and the analysis of the phylogenetic tree with the homologous strain was performed (FIG. 1), confirming that strain MB1 is a homologous, different strain of Lactobacillus salivarius.
Finally, the strain MB1 is preserved, and the preservation information is as follows: preservation time: 2022, 9, 27; preservation unit name: china Center for Type Culture Collection (CCTCC); deposit number: cctccc No. M20221502; deposit unit address: chinese university of Wuhan; classification naming: ligilactobacillus salivarius.
The saliva combined lactobacillus is a probiotic bacterial strain which is approved by the nation and can be used for food, has wide probiotic effects, such as anti-inflammatory, anti-aging, cholesterol reducing and the like, but different bacterial strains from different sources have different effects, which indicates that the novel saliva combined lactobacillus MB1 separated from human excrement can be used as probiotic bacteria, and possibly has novel effects and functions.
L.salivarius MB1 16S rDNA sequence(1436bp，SEQ ID No：1)：
ATACTGCAGTCGACGAAACTTTCTTACACCGAATGCTTGCATTCACCGTAAGAAGTTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTAAAAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATATCTCTAAGGATCGCATGATCCTTAGATGAAAGATGGTTCTGCTATCGCTTTTAGATGGACCCGCGGCGTATTAACTAGTTGGTGGGGTAACGGCCTACCAAGGTGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACACGAGTGAGAGTAACTGTTCATTCGATGACGGTATCTAACCAGCAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAACGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGTAGTGCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGTTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCTAAGAGATTAGGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTGTCAGTTGCCAGCATTAAGTTGGGCACTCTGGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAGACCGCGAGGTTTAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCGCAAGGAGCCAGCCGTCTAA。
Example 2 function of Lactobacillus salivarius (Ligilactobacillus salivarius) MB1 Strain and use thereof
(1) Saliva in combination with Lactobacillus MB1 strain can produce and secrete Glutathione (GSH)
Saliva cultured in MRS liquid medium to stationary phase is expanded into new MRS liquid medium at a ratio of 1:30, bacterial suspension is collected at the time of culturing to stationary phase for 24 hours, fermentation broth supernatant is collected after centrifugation at 10,000Xg and 4 ℃ for 10 minutes, and concentration of GSH in the fermentation broth supernatant is determined by a reduced Glutathione (GSH) determination kit (Cat: A006-2-1). The results show (fig. 2) that GSH concentration in the fermentation supernatant of MB1 was 271.9 μmol/L compared to the MRS control, suggesting a significant increase in GSH concentration after fermentation with MB1 (< 0.001), indicating that lactobacillus salivarius MB1 can produce and secrete Glutathione (GSH) during stationary phase.
Glutathione (GSH) is a tripeptide consisting of glutamic acid, cysteine and glycine, and containing gamma-amide bond and mercapto group, and has antioxidant effect and integrated detoxification effect. Wherein, the sulfhydryl group on the cysteine is the active group of glutathione (so glutathione is often abbreviated as GSH). Glutathione helps to maintain normal immune system function, has antioxidant effect, integrates detoxification effect, and plays an important role in various cell biochemical processes, such as free radical neutralization, detoxification, cysteine transport and storage, maintenance of cell redox effect, regeneration of ascorbic acid and vitamin E, etc.:
(1) detoxification: combined with poison or medicine to eliminate its toxic action;
(2) participate in the oxidation-reduction reaction: as an important reducing agent, participate in various oxidation-reduction reactions in the body;
(3) protection of thiol enzyme activity: maintaining the active group (-SH) of the sulfhydryl enzyme in a reduced state;
(4) maintenance of the stabilization of erythrocyte membrane structure: eliminating the damage of oxidant to erythrocyte membrane structure.
The multiple biological functions of GSH confer multiple functions and uses:
1) Antioxidant: scavenging free radicals in human bodies, protecting sulfhydryl groups in molecules such as a plurality of proteins, enzymes and the like from being oxidized by harmful substances, thereby ensuring that the molecules such as the proteins, the enzymes and the like exert normal physiological functions; the content of glutathione in human erythrocytes is great, which has important significance for protecting the sulfhydryl group of protein on erythrocyte membrane in a reduced state and preventing hemolysis; it also has effects in preventing skin aging and pigmentation, reducing melanin formation, improving skin antioxidant capacity, and making skin luster.
2) Clinical medicine: the sulfhydryl is utilized to chelate toxins such as heavy metals, fluoride, mustard gas and the like to avoid poisoning; also used as a medicament for the treatment or adjuvant therapy of hepatitis, hemolytic diseases, keratitis, cataract, retinal diseases and the like; correcting imbalance of acetylcholinesterase and cholinesterase, and resisting allergy.
3) Food additives: strengthening food nutrition, stabilizing vitamin C, and strengthening flavor.
In conclusion, the glutathione can be used as a medicament and a functional base material, and has wide application in the aspects of antioxidant whitening, anti-aging, immunity enhancing, anti-tumor, antiallergic and other functional medicaments. Thus, the probiotic saliva combines the functions of lactobacillus MB1 strain through the produced GSH, thereby exerting several of the above effects.
(2) Saliva in combination with Lactobacillus MB1 strain inhibits Xanthine Oxidase (XOD) activity
Saliva cultured in MRS liquid medium to stationary phase is expanded into new MRS liquid medium at a ratio of 1:30, bacterial suspension is collected at the time of culturing to stationary phase for 24 hours, fermentation broth supernatant is collected after centrifugation at 10,000Xg 4 ℃ for 10 minutes, and xanthine oxidase activity in the fermentation broth supernatant is measured by a xanthine oxidase activity measurement kit (Cat: AKAO 006M). The results showed that the fermentation supernatant of MB1 had a significant inhibition of xanthine oxidase activity compared to the blank medium MRS, at 60.0% (< 0.05), indicating that lactobacillus salivarius MB1 can produce and secrete metabolites during stationary phase to inhibit Xanthine Oxidase (XOD) activity (fig. 3).
Xanthine oxidase is a key enzyme in the catabolism of purines, and can catalyze the direct production of uric acid from hypoxanthine and xanthine. Thus, when xanthine oxidase activity is abnormally active in the body, it leads to the production of a large amount of uric acid, thereby causing hyperuricemia or gout.
Xanthine oxidase inhibitors such as allopurinol inhibit xanthine oxidase activity and prevent the metabolism of hypoxanthine and xanthine into uric acid, thereby reducing uric acid production and improving gout and hyperuricemia. Xanthine oxidase inhibitors can also reduce stress response and damage to tissues caused by free radicals, and are expected to be clinically used for treating gout and diseases caused by peroxide free radicals. Allopurinol is one of the main medicines for treating hyperuricemia and gout, and is the only chemical medicine for inhibiting uric acid generation clinically, however, the medicine has a plurality of side effects, can cause fever, allergic rash abdominal pain, diarrhea, leucocyte and thrombocytopenia and multiple organ damage, even has reports of death, has questioned safety, and has long been used until now because of good inhibition effect on xanthine oxidase. Therefore, the research of new low-toxicity high-efficiency xanthine oxidase inhibitors has very important significance.
It can be seen that the probiotic saliva combines with lactobacillus MB1 strain to reduce purine in vivo and uric acid generation by inhibiting xanthine oxidase activity, thereby controlling uric acid level and preventing gout attack.
Taken together, the saliva of the present invention combines the various probiotic effects of lactobacillus strain MB 1: (1) Glutathione (GSH) can be produced and secreted; (2) can inhibit xanthine oxidase activity. Therefore, the saliva combined lactobacillus strain MB1 has important application value and economic value.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.

Claims (5)

1. Use of a lactobacillus salivarius (Ligilactobacillus salivarius) MB1 strain for the production of Glutathione (GSH), characterized in that said lactobacillus salivarius (Ligilactobacillus salivarius) MB1 strain has the accession number: the complete sequence of the CCTCCNO: M20221502 and 16S rDNA is shown as SEQ ID No: 1.
2. Use of a lactobacillus salivarius (Ligilactobacillus salivarius) MB1 strain for the preparation of a xanthine oxidase inhibitor, wherein the lactobacillus salivarius (Ligilactobacillus salivarius) MB1 strain has the accession number: the complete sequence of the CCTCC NO: M20221502 and 16S rDNA is shown as SEQ ID No: 1.
3. A method for preparing glutathione is characterized in that saliva combined lactobacillus (Ligilactobacillus salivarius) MB1 strain is inoculated in MRS liquid culture medium, bacterial suspension is collected after the stationary phase is cultivated, and finally the glutathione in the bacterial suspension is separated and purified to obtain the glutathione; the saliva combined lactobacillus (Ligilactobacillus salivarius) MB1 strain has a deposit number of: the complete sequence of the CCTCC NO: M20221502 and 16S rDNA is shown as SEQ ID No: 1.
4. A method for preparing glutathione according to claim 3, wherein the conditions of the culture are: anaerobic at a constant temperature of 37 ℃.
5. A method of preparing glutathione according to claim 3, characterized in that the lactobacillus salivarius (Ligilactobacillus salivarius) MB1 strain is first cultivated to stationary phase with MRS liquid medium and then expanded into new MRS liquid medium at a dilution factor of 1:20-1000.
CN202311304811.6A 2023-10-10 2023-10-10 Application of saliva combined with lactobacillus MB1 in preparation of food and medicine for whitening and relieving gout Pending CN117264839A (en)

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CN118064333A (en) * 2024-04-18 2024-05-24 青岛诺和诺康生物科技有限公司 Saliva combined lactobacillus and application thereof in preparation of skin color improving products

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118064333A (en) * 2024-04-18 2024-05-24 青岛诺和诺康生物科技有限公司 Saliva combined lactobacillus and application thereof in preparation of skin color improving products

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