CN101333505A - Applications of lactobacillus casei for antioxidation - Google Patents
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Abstract
The invention relates to the preparation of a Lactobacillus casei Zhang strain and an application of the strain at the aspect of antioxidant products. The Lactobacillus casei Zhang strain is an acid- and bile acid-resistant probiotics strain separated from Lactobacillus casei Zhang of fermented mare milk which is the traditional dairy product of the inner Mongolia nationality; and the strain has been stored in the China General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and the Collection No. is: CGMCC No.1697. Besides the beneficial function to body health possessed by common lactobacillus, the Lactobacillus casei Zhang of the invention can also be used for the production of anti-oxidation functional products, such as: the production of various dairy products with antioxidant activity; and the Lactobacillus casei Zhang can be present in specific products by mixing with other materials, and can also be produced in the form of live bacteria preparation.
Description
Technical field
The present invention relates to a kind of separation from Inner Mongol national tradition milk-product---the preparation of lactobacterium casei (the Lactobacillus casei Zhang) bacterial strain in the koumiss and this bacterial strain are in the application aspect the oxidation resistant product.
Technical background
Oxygenizement is necessary for many life entities, the energy that it produces is used to keep normal vital movement, yet the carrying out that is accompanied by oxygenizement also can produce some biomolecules is had the active oxygen of destruction, it is the free radical at center that these active oxygens not only comprise with oxygen, as O
2 -Reach OH, also comprise the non-free radical derivative of some oxygen, as H
2O
2Have close related Deng the generation of the aging of, these active oxygens and organism and disease.Interior free yl can cause the peroxidation of the unsaturated fatty acids in the microbial film, and (Maleic dialdehyde MDA) waits product to generate lipid peroxide and mda.
Anti-oxidative defense system as body can make these active oxygen inactivations to a certain extent and repair the destruction that biomolecules is produced by it.The anti-oxidative defense system of body comprises enzyme and non-enzyme two big classes, and the former mainly is meant superoxide-dismutase (SOD), catalase (CAT), Selenoperoxidase (GSH-Px) etc.; The latter then is meant some micromolecular organic substances, also is referred to as natural antioxidant in the body, as vitamin-E, vitamins C, ubiquinone, halfcystine, gsh and trace element-selenium (Se) and uric acid (UA) etc.Yet, constantly produce because active oxygen is a body, so defense system can not be eliminated fully owing to active oxygen metabolic and that produced under extraneous conditioned stimulus.In the last few years, various synthetic or natural antioxidant are used to help human body to alleviate the destruction to body that oxygenizement is brought, yet the synthetic antioxidant is to the security of health and long-lastingly also have many disputes, therefore, provide and come from natural antioxidant and seem to have superiority and application prospect more.
Along with people are familiar with more to milk-acid bacteria, find some milk-acid bacteria have regulate that intestinal microflora is kept microecological balance, improves intestinal function, had the serum cholesterol concentration of reduction, hypotensive, antitumor, strengthening immunity and prebiotic effect such as anti-oxidant.It is found that by research, most lactic acid bacteria is removed hydroxy radical qiao and hydrogen peroxide by producing superoxide-dismutase (SOD) and Selenoperoxidase (GSH-Px), the cell-free extract of milk-acid bacteria has the ability of removing lipid peroxide product MDA simultaneously, thus the antioxygenation of playing; Therefore, utilize the anti-oxidant activity of milk-acid bacteria to reach to remove that deleterious lipid peroxide is the simple and effective method that a kind of prevention with disease is melted into daily life in the body.
The bacterial strain of ideal probiotics should have following characteristics: (1) must be harmless to the host, do not hybridize with pathogenic micro-organism; (2) bile and strong acid had strong tolerance; (3) can produce metabolism products such as antibacterial substance and lactic acid in the fermenting process; (4) in cultivation and in vivo, breed easily; (5) high survival rate is still arranged after the processing treatment; (6), also can survive at ambient temperature and for a long time wait even be blended in the feed.
The inventor is at above-mentioned problem, with the traditional zymotic milk-product---isolating milk-acid bacteria is the target flora in the koumiss, filter out the stronger potential probiotic bacterium of viability in intestinal environment through external acid resisting test and cholate tolerance test, and further studied its function and effect aspect antioxygenation.
Summary of the invention
The object of the present invention is to provide a kind of lactobacillus casei bacterial strain, described lactobacterium casei (Lactobacillus casei Zhang) bacterial strain is the probiotic strain of isolating acidproof and anti-bile acide from koumiss; This bacterial strain is on April 21st, 2006, (address is No. 13, one in Zhong Guan-cun, Haidian District, BeiJing, China city north in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, State Patent Office specifies patent microbial preservation center) preservation, preserving number: CGMCC No.1697.
Another object of the present invention is to the application of described lactobacterium casei aspect antioxygenation, particularly in cultured milk prod, improve the application of the antioxygenation of fermented-milk.
The objective of the invention is to be achieved through the following technical solutions: a kind of lactobacterium casei, described lactobacterium casei (Lactobacillus casei Zhang) bacterial strain are the probiotic strains of isolating acidproof and anti-bile acide from koumiss; This bacterial strain has been preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number: CGMCC No.1697.
The application of described lactobacterium casei aspect antioxygenation.
Described lactobacterium casei improves the application of the antioxygenation of fermented-milk in cultured milk prod.
Description of drawings
Fig. 1 is separation, purifying and the preservation operational flowchart of milk-acid bacteria of the present invention;
Fig. 2 is the present invention's superoxide-dismutase (SOD) vitality test result schematic diagram in the rat liver tissue homogenate;
Fig. 3 is the present invention's superoxide-dismutase (SOD) vitality test result schematic diagram in the rat red corpuscle;
Fig. 4 is the present invention's Selenoperoxidase (GSH-Px) vitality test result schematic diagram in the rat liver tissue homogenate;
Fig. 5 is the present invention's Selenoperoxidase (GSH-Px) vitality test result schematic diagram in rat blood serum;
Fig. 6 is the present invention's mda (MDA) concentration determination result schematic diagram in the rat liver tissue homogenate;
Fig. 7 is the present invention's mda (MDA) concentration determination result schematic diagram in rat blood.
Embodiment
Below in conjunction with drawings and Examples the present invention is further described, following examples only have illustrative, and the present invention is not subjected to the restriction of these embodiment.
Embodiment 1: a kind of lactobacterium casei, described lactobacterium casei (Lactobacillus casei Zhang) bacterial strain are the probiotic strains of isolating acidproof and anti-bile acide from koumiss; This bacterial strain has been preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number: CGMCCNo.1697.
1. the isolation identification of sample collecting and Bacterium lacticum
Sample is: the koumiss that traditional method is made, sample collecting is in inner mongolia Xilinguole League Zhenglan Banner.
2. the isolation and purification of Bacterium lacticum in the sample: referring to Fig. 1, the separation and purification process of lactobacterium casei among the present invention (Lactobacillus casei Zhang) is as follows:
With Inner Mongol traditional function fermented-milk---koumiss is the Bacterium lacticum sample separation, koumiss with herdsman family spontaneous fermentation making, after in its fermenting container, fully stirring, with the pipettor that has the suction nozzle of sterilizing, absorption 2mL puts into the sterilization small test tube that has plug, and (15mm * 150mm) seal draws 1mL again and puts into the band screw cap and sterilization CaCO is housed
3Aseptic, the volume of powder are in the sampling bottle of 2mL, tighten screw cap as standby sample.The sample of collecting is put into the ice chest internal cooling, and certain hour is changed ice bag at interval, keeps taking back the laboratory at a lower temperature and being positioned in 4 ℃ of refrigerators, as early as possible milk-acid bacteria is separated.
When Bacterium lacticum is separated, drawing 1mL koumiss sample with the sterilization suction pipe is inoculated in the 10mL sterilization litmus milk substratum, place and increase bacterium in 30 ℃ of thermostat containers and be cultured to acidifying and solidify, become when pink in the substratum color it is taken out,, on the BL nutrient agar that contains 10ppm cycloheximide and 10ppm colistin sulfate (Japanese Rong Yan Co., Ltd. goods), put into again with the streak inoculation of disinfection inoculation ring
In the gaspak anaerobic jar, place under 30 ℃ of conditions, cultivate 48h～72h.After treating that bacterium colony forms,, be inoculated in the TPY liquid, place under 30 ℃ of conditions, cultivate 24h～48h with transfering loop or inoculating needle picking colony.After treating that strain growth is good, streak inoculation places under 30 ℃ of conditions in the BL nutrient agar once more, cultivates 24h～48h, examines record colonial morphology and gramstaining cell morphological characteristic, and carries out catalase test simultaneously.The bacillus of Gram-positive, catalase test feminine gender is tentative for Bacterium lacticum and be further purified cultivation, and adopt refrigeration ,-85 ℃ to freeze to preserve or the freezing preservation of cryogenic vacuum.
Will be from the koumiss sample isolating lactobacterium strain, learning a series of analysis of experimentss such as test, lactic acid optical rotation determining through Physiology and biochemistry identifies, and the bacterial strain after will identifying is made respectively for examination bacterium liquid, drawing bacteria suspension 10 μ L, to be inoculated in the 5mL pH value be 3.5 MRS liquid nutrient medium, places to carry out 24h under 37 ℃ of conditions and cultivate; Draw bacteria suspension 1.0mL and 9.0mL pH value simultaneously and be 3.0 simulated gastric fluid [preparation method: NaCl 0.2%, stomach en-(pepsin, sigma) 0.35%, after adjusting pH value and be 3.0 with 1mol/L HCl, filtration sterilization is standby] mix after, place under 37 ℃ of conditions and cultivate, sampling is measured viable count with BCP nutrient agar (Japanese Rong Yan Co., Ltd. goods) behind beginning (0h) and 3h respectively, and calculating bacterial strain survival rate.Filter out lactobacterium strain with highly-acidproof growth and stomach juice-resistant by above method, the pacing of going forward side by side fixed its in different simulated gastric fluids and intestinal juice survival ability and test by external bile tolerance test and reducing cholesterol, obtain a strain lactobacterium casei (Lactobacillus caseiZhang) bacterial strain.
The evaluation of Bacterium lacticum: the Bacterium lacticum of separation and purification (Lactobacillus casei Zhang), identify that through physiological biochemical character analysis and 16SrDNA sequencing technique its Microbiological Characteristics is as shown in table 1.
The biological characteristics of table 1Lactobacillus casei Zhang
Lactobacterium casei of the present invention (Lactobacillus casei Zhang) except have that general Bacterium lacticum possesses to the useful effect of HUMAN HEALTH, this bacterial strain can also be used for the production of anti-oxidant function product, for example: the production with various milk-product of anti-oxidant activity; It both can be present in the certain products with the blended form with other raw material, form production that also can active bacteria formulation.
Embodiment 2: a kind of application as embodiment 1 described lactobacterium casei, the application of described lactobacterium casei aspect antioxygenation; Especially in cultured milk prod, improve the application of the antioxygenation of fermented-milk.
Tolerance analysis in the acid resistance of lactobacterium casei of the present invention (Lactobacillus casei Zhang) and the artificial gastrointestinal fluid thereof:
From the koumiss sample in the 50 strain Bacterium lacticum of isolation identification, through pH value is growth test and be that survival rate in 3.0 the simulated gastric fluid is measured at pH value in 3.5 the MRS liquid nutrient medium, screening obtains the extremely strong Bacterium lacticum of 1 strain acid resistance, i.e. lactobacterium casei (Lactobacillus casei Zhang).
Lactobacillus casei Zhang bacterial strain is a growth test and be that survival rate in 3.0 the simulated gastric fluid is measured at pH value in 3.5 the MRS liquid nutrient medium through pH value, the results are shown in Table 2.
Concrete steps: the Bacterium lacticum of freezing preservation is inoculated in the MRS liquid nutrient medium, place under 37 ℃ of conditions and cultivate 24h, through the MRS nutrient solution go down to posterity cultivate 2～3 times after, get 5mL Bacterium lacticum nutrient solution, through 3, the centrifugal 10min of 000rpm, collect thalline, add 5mL sterile saline mixing and make bacteria suspension, drawing bacteria suspension 10 μ L, to be inoculated in the 5mL pH value be in 3.5 the MRS liquid nutrient medium, place under 37 ℃ of conditions and cultivate 24h, observe its growing state.
The tolerance pH value is 3.0 simulated gastric fluids [NaCl 0.2%, stomach en-(pepsin, sigma) 0.35%, after adjusting pH value and be 3.0 with 1N HCl, filtration sterilization is standby] the screening of lactobacterium strain: get the 1.0mL Bacterium lacticum thalline suspension for preparing in the aforesaid method, with the 9.0mL pH value is after 3.0 simulated gastric fluid mixes, place under 37 ℃ of conditions and cultivate, respectively behind beginning (0h) and 3h, sampling is poured into cultivation with BCP nutrient agar (Plate Count Agar with Brom Cresol Purple), and measures viable count.
Table 2Lactobacillus casei Zhang growing state in pH3.5 and pH3.0 simulated gastric fluid
Annotate: the bacterium number average is the mean value of twice experiment in the table.
The result shows, lactobacterium casei of the present invention (Lactobacillus casei Zhang) has stronger patience and acidproof growth characteristics, can tolerate artificial gastro-intestinal digestion liquid, especially can tolerate pH value and be 2.0 simulated gastric fluid in separated milk, tolerant gallbladder salinity reaches 0.6%;
Because this specific character of lactobacterium casei of the present invention (Lactobacillus casei Zhang), the application of described lactobacterium casei in milk-product has its significance; The acidic metabolite that milk-acid bacteria produces makes the intestinal environment slant acidity, and the optimum pH of general digestive ferment is slant acidity (amylase 6.5, a saccharifying enzyme 4.4), so just help digesting and assimilating of nutrient substance, organic acid produces wriggling and the secretion that also can strengthen enteron aisle, also but promoting digestion absorbs nutrient, studies show that the utilization ratio ratio edible milk height that contain identical lactose concn of the crowd of edible sour milk, thereby alleviate the intolerance shape of lactose lactose.
In addition, the antitumous effect of milk-acid bacteria also has many reports.The more important thing is, since to milk-acid bacteria metabolism and hereditary property understand deeply and to the understanding of its mechanism, the result proof that also has a large amount of clinical trials with it as the health care co-adjuvant, the feasibility of fodder additives, people have accepted extensively this class health-care products of milk-acid bacteria.In addition, oral is the main path that probiotic bacterium enters enteron aisle, so probiotics must stand the influence of hydrochloric acid and small intestine middle and high concentration cholate in the stomach before entering large intestine, only the bacterial strain that surviving rate is high in hydrochloric acid and cholate just can play a role; Simultaneously, in the product in stable viable count or the probiotics amount of viable bacteria be its key that plays a role, so the application requiring unit product of active bacteria formulation reaches certain viable count, to meet the dose therapeutically effective standard.
Therefore, this bacterial strain is applicable to the production of functional cultured milk prod, also can be used as all respects that functional food additives or health-care preparation are applied to regulate immunologic function.
Application Example: lactobacterium casei (Lactobacillus casei Zhang) is to the influence of activities of antioxidant enzymes in rat liver tissue and the blood
1. experimental technique
1.1 the preparation of experimental strain preparation:
(Lactobacillus casei Zhang) (CGMCC No.1697) is inoculated in the MRS nutrient solution with lactobacterium casei, under 37 ℃ of conditions, cultivate 18h, through centrifugal (3000r/min, 10min) collect thalline, behind the sterile saline centrifuge washing, on bacterial sediment, add 10% sterilization separated milk, and to adjust its bacterium number be 2.0 * 10
9Cfu/mL (the bacterial flora sum that contains in every ml sample), pour into cultivation with the BCP nutrient agar behind the mixing, the meter viable count is confirmed, and the skimming milk bacteria suspension is divided into two equal portions: aly be viable bacteria body separated milk, another part places and transfers under 100 ℃ of conditions heating 10min under 50～55 ℃ of conditions behind the self-digestion 2h to and carry out deactivation, makes the dead thalline separated milk of thermic; With viable bacteria body skimming milk suspension and the dead thallus suspension liquid of thermic, by every day usage quantity be sub-packed in the freezing bottle, place preserve in-85 ℃ the refrigerator-freezer standby.
1.2 laboratory animal:
Body weight be about 100～120g 5 the week age male Wistar be 24 of big white mouse, provide by University of the Inner Mongol's Experimental Animal Center; The rat nutrient fodder is purchased the Animal Experimental Study center feed chamber fitted in University of the Inner Mongol.
1.3 experiment main agents box:
Superoxide-dismutase (SOD) testing cassete, Selenoperoxidase (GSH-Px) vigor testing cassete, mda (MAD) is measured test kit, and more than the mensuration used kit builds up Graduate School of Engineering by Nanjing provides.
1.4 laboratory animal and grouping feeding manner:
With Wistar is after big white mouse is bought, and water and nutrient fodder free choice feeding were fed to the 7th day.Weighed at the 7th day, make each experimental group mean body weight equal approximately.Since the 8th day test group, only irritate to feed the viable bacteria separated milk that prepared and the dead bacterium separated milk of thermic 2mL/ respectively, the grouping situation is specifically as shown in table 1.Weighing and tail vein blood in each test group the 28th day after official test, stops eating before the blood sampling and spend the night.Measure rat liver tissue homogenate, red corpuscle and liver homogenate SOD enzyme activity, liver and serum glutathione peroxidase (GSH-Px) vigor and liver and blood mda (MAD) content.
Table 1 animal experiment grouping situation
1.5 analysis of experimental data and statistics
Use SPSS 12.0 system statistics softwares each experimental data is carried out variance analysis and test of significance.
2. result and analysis
2.1 the active measurement result of rat liver tissue homogenate and erythrocyte sod
L.casei Zhang is gavaged behind the rat the 28th day with active bacteria formulation and the dead thalline preparation of thermic respectively, measured its hepatic homogenate and erythrocyte superoxide dismutase (SOD) activity respectively.Gavage the rat of L.casei Zhang viable bacteria body preparation, its hepatic homogenate SOD vigor and the apparent in view raising of control group that does not gavage Lemonal, reach utmost point significant difference level (P＜0.01), though and the dead thalline group of thermic SOD vigor is higher than control group but does not present the significant difference level, the result as shown in Figure 2.The result who measures SOD vigor in the red corpuscle shows that L.casei Zhang viable bacteria body preparation group is higher than control group, but does not reach the significant difference level as yet, dead thalline group of thermic and control group indifference, and the result is as shown in Figure 3.
2.2 the measurement result of rat liver tissue homogenate and serum GSH-Px vigor
Improve for two rats in test groups liver glutathione peroxidase vigor of rats gavaged L.casei Zhang bacterium liquid, but do not reach the significant difference level, referring to Fig. 4.
In addition, gavage the L.casei Zhang living bacterial liquid group rat blood serum GSH-Px vigor utmost point and be higher than control group (P＜0.01) significantly, the dead thalline liquid of its thermic group rat blood serum GSH-Px vigor also significantly improves (P＜0.05), as shown in Figure 5.
2.3 rat liver tissue homogenate and blood MDA assay result
As can be known from Fig. 6, mda (MDA) content obviously reduces in the rat group liver organization through gavaging L.casei Zhang viable bacteria body preparation, relatively reaches utmost point significant difference level (P＜0.01) with control group.But the dead thalline preparation of thermic gavages MDA content and control group no significant difference in rat group blood and the liver organization.This shows that Bacterium lacticum L.casei Zhang active bacteria formulation increases to antioxidase activity in the rat liver tissue, have certain effect reducing the lipid peroxide tool; But L.caseiZhang strain bacterium viable bacteria body preparation is lost the effect to rat anti oxydase effect of vigor after thermic is extremely handled.Measure mda (MDA) concentration results demonstration in the rat blood, L.casei Zhang viable bacteria body preparation group is lower than control group, but does not reach the significant difference level as yet, dead thalline group of thermic and control group indifference, and the result is as shown in Figure 7.
3. conclusion:
After lactobacterium casei L.casei Zhang (CGMCC No.1697) active bacteria formulation gavages experimental rat, can the utmost point improve in the rat liver tissue homogenate GSH-Px vigor in the SOD vigor and serum significantly, and significantly reduce mda in the rat liver tissue homogenate (MDA) concentration, thereby can play antioxygenation effectively.
Claims (3)
1, a kind of lactobacterium casei is characterized in that: described lactobacterium casei (Lactobacillus caseiZhang) bacterial strain is the probiotic strain of isolating acidproof and anti-bile acide from koumiss; This bacterial strain has been preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number: CGMCCNo.1697.
2, a kind of application of lactobacterium casei as claimed in claim 1 is characterized in that: the application of described lactobacterium casei aspect antioxygenation.
3, the application of lactobacterium casei according to claim 2 is characterized in that: described lactobacterium casei improves the application of the antioxygenation of fermented-milk in cultured milk prod.
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