CN101144065A - Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof - Google Patents

Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof Download PDF

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CN101144065A
CN101144065A CNA2007101459912A CN200710145991A CN101144065A CN 101144065 A CN101144065 A CN 101144065A CN A2007101459912 A CNA2007101459912 A CN A2007101459912A CN 200710145991 A CN200710145991 A CN 200710145991A CN 101144065 A CN101144065 A CN 101144065A
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lactobacterium casei
milk
casei
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CN101144065B (en
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田丰伟
陈卫
赵建新
张灏
张根义
张凤敏
陈海琴
戴小军
丁虎生
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Jiangnan University
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Abstract

The present invention relates to the technology field of the microorganism, in particular to antioxidation lactobadllus casei CN1566 which is hydrogen peroxide proof, and in which a free radical is removed, and the purpose in the fermentation food.

Description

Oxidation resistant Lactobacillus casei capable of a kind of resisting hydrogen peroxide, removing free radical and uses thereof
[technical field]
The present invention relates to microbial technology field, more specifically, the present invention relates to a kind of resisting hydrogen peroxide, remove oxidation resistant Lactobacillus casei capable of free radical and uses thereof.
[background technology]
(Lactic acid bacteria is the general designation that a class can fermentable carbohydrates produces the bacterium of a large amount of lactic acid LAB) to milk-acid bacteria, extensively is present in spontaneous fermentation milk-product, fermenting plant food such as pickles, sauerkraut, silage and the people's enteron aisle.Milk-acid bacteria is the bacterial classification commonly used in the foodstuffs industry, and is in close relations with the mankind.Milk-acid bacteria not only can be improved food value, improves flavour of food products, prolongs the shelf time, can also improve the functional property of food.In recent years, milk-acid bacteria becomes the main source of probiotic bacterium with its special physiologically active and trophic function, positive paid more and more attention.Milk-acid bacteria can be regulated the body gastrointestinal tract normal microflora, keep microecological balance, improve food digestion rate and biological value, reduce serum cholesterol, the control intracellular toxin, suppress the generation of corrupt bacteria growing breeding and spoilage product in the enteron aisle, thereby to nutritional status, physiological function, cell infection, drug effect, toxic reaction, immune response, tumour generation, aging course and the unexpected generation wholesome effects such as stress reaction of body.
Bio-oxidation is an important physiological process of life, can provide energy for life by the life oxidation.But in the life oxidising process, can produce free radical such as hydroxy radical qiao, ultra-oxygen anion free radical isoreactivity molecule, act on physiologically active substances such as intravital enzyme, protein, thereby cause multiple disease and cause aging.Studies show that, human some diseases such as cancer, pulmonary emphysema, atherosclerosis, liver cirrhosis, sacroiliitis, cardiovascular disorder etc. are all relevant with the generation of vivo oxidation and free radical, although people's body has the anti-oxidative defense system, these systems can not resist or repair the damage that oxygenizement is brought completely effectively.Therefore, the antioxidant of exploitation different sources is a research topic highly significant to removing oxyradical, protecting cell and tissue to avoid oxidative damage.
Physiological function to milk-acid bacteria has a large amount of research reports both at home and abroad, but the anti-oxidant activity of milk-acid bacteria is reported seldom.Caused the extensive attention of many subjects such as Food science, preventive medicine and microecology about the milk-acid bacteria Study on oxidation resistance.Cultured milk prod is the important channel that people take in milk-acid bacteria, screens good lactic bacterium strains with anti-oxidant activity and safety, for development functionality milk-product, abundant existing milk-product kind, improve the added value of dairy products with significant.Therefore, very big research and the using value of the milk-acid bacteria tool of tool anti-oxidant activity.
In disclosed document and patent or patent application at present, for example CN 1276077C discloses a kind of short lactobacillus that produces γ-An Jidingsuan and uses thereof, is used to produce γ-An Jidingsuan.CN1796540A discloses bifidus bacillus with anti-enterovirus and anti-oxidation characteristics and uses thereof, is used to prepare food compositions or pharmaceutical composition, kills or suppresses Hp or intestinal bacteria, and treatment is by its various diseases that causes.CN 1982437A discloses a kind of acidproof bile tolerance, anti-enterovirus and resistance of oxidation lactobacillus rhamnosus strain and meta-bolites and purposes of novelty, is used to prepare food or the medicine that suppresses Hp, intestinal bacteria or other pathogen enterobacteria.But these patents or patent application do not relate to the microorganism of performances such as having resisting hydrogen peroxide, remove free radical, be anti-oxidant fully.Therefore, also need a kind of food that has resisting hydrogen peroxide, removing free radical, oxidation resistant microorganism and contain them at present.
[summary of the invention]
[technical problem that will solve]
The object of the present invention is to provide a kind of oxidation resistant Lactobacillus casei capable bacterial strain that can tolerate hydrogen peroxide, can remove free radical.
Another object of the present invention is to provide the purposes of a kind of described lactobacterium casei in leavened food.
[technical scheme]
The inventor filters out a strains of lactic acid bacteria CN1566 from the fermentation fermented milk, utilize Microbiological Characteristics such as morphological specificity, cultivation proterties and physiological and biochemical property that this milk-acid bacteria CN1566 is accredited as lactobacterium casei (Lactobacillus casei) CN1566, this bacterial strain has been preserved in Chinese typical culture collection center (being called for short CCTCC), deposit number CCTCC No:M207107 on July 29th, 2007.
The morphological feature of lactobacterium casei CN1566 CCTCC No:M207107:
The thalline feature: be Gram-positive, cell is shaft-like, and the about 0.5-1.0 μ of thalline m is wide, and 2-4 μ m is long, and Cheng Dan, paired or chaining do not form gemma, the two ends circle.
Colony characteristics: on the MRS substratum, form tangible bacterium colony, diameter between 0.3-2.0mm, circle, neat in edge, oyster white, transparent, surface wettability is smooth, chromogenesis not is referring to attached Fig. 1 and 2.
The cultural characteristic of lactobacterium casei CN1566 CCTCC No:M207107 of the present invention:
The minimum growth temperature of lactobacterium casei CN1 566 is 20 ℃, and maximum growth temperature is 40 ℃, and 35-37 ℃ of growth temperature the best, the highest and minimum initial growth pH is 9.0 and 4.0, and the initial pH of the suitableeest growth is 6.0; The lag period of CN1566 bacterial strain is shorter relatively, and 4h enters logarithmic phase, and 12h reaches stationary phase.
The liquid culture feature of lactobacterium casei CN1566 CCTCC No:M207107 of the present invention:
Lactobacterium casei CN1566 is well-grown in the MRS liquid nutrient medium, 4h left and right sides nutrient solution begins muddiness, begins to have the somatic cells precipitation about 10h, and shaking does not gently have bubble to produce, a large amount of bacterial sediments are arranged behind the 15h, and cultivating 24h oyster white bacterial sediment obviously increases.
Lactobacterium casei CN1566 of the present invention derives from traditional fermented food, and (Generally Recognized As Safe, GRAS) bacterial classification can be used in the leavened food to belong to generally recognized as safe.
Described leavened food is lactic acid bacteria milk beverage, milk powder, capsule product or fermented-milk.
The preparation method of described leavened food is described respectively below.
Described lactic acid bacteria milk beverage prepares according to following step:
At first, described lactobacterium casei CN1566 original strain is preserved with 30 weight % glycerine suspensions under temperature-75 ℃, perhaps preserves standby with the form of lyophilize bacterium powder under 4 ℃ of temperature.
Then, can adopt two kinds of methods to prepare lactobacterium casei CN1566 working stock culture of the present invention:
First method is that above-mentioned lactobacterium casei CN1566 original strain is inoculated in 12 weight % in the skimming milk of 110 ℃ of sterilization 10min, cultivates 14-16h to curdled milk under 37 ℃ of conditions, and cultured continuously activated for two generations, as mother starter; Described mother starter is inoculated in the sterile milk by 3-5 volume %, cultivates 14-16h, at this moment the viable count about 10 in this curdled milk to curdled milk 9Cfu/mL obtains described working stock culture, can directly this working stock culture be added in the food, perhaps uses the preparation fermented-milk with commercial starter that can symbiotic preparation fermented-milk such as lactobacillus bulgaricus and thermophilus streptococcus.
Second method is that above-mentioned lactobacterium casei CN1566 original strain is inoculated in the MRS liquid nutrient medium, cultivating 12-16h under 37 ℃ of conditions activates, activated for two generations continuously, then the activation culture thing is inoculated in the MRS substratum by 2-4 volume %, cultivates 16-18h, the centrifugal 15min of 4000r/min under 4 ℃ of conditions, remove supernatant, obtain cell precipitation, will precipitate with a certain amount of aseptic skimming milk and make suspension, it is standby to obtain working stock culture.
Then, raw dairy is cooled to 4 ℃ then at 95 ℃ of following heat-sterilization 20min or at 140 ℃ of following elevated temperature heat sterilization 2s, adds foregoing lactobacterium casei CN1566 working stock culture again, makes its concentration reach 10 6More than the cfu/ml, promptly obtain containing the lactic acid bacteria milk beverage of lactobacterium casei CN1566 viable bacteria 4 ℃ of stored refrigerated.
In the present invention, described MRS liquid nutrient medium is that those skilled in the art know, and is that BD Difco company is with trade(brand)name
Figure A20071014599100071
Substratum (the reference: deMan that is used for the Bacterium lacticum cultivation that Lactobacilli MRS Broth sells, J.c., M.Rogosa, and M.E.Sharpe.1960.A medium for the cultivation of lactobacilli.J.Appl.Bacteriol.23: 130.).
Described heat-sterilization is that the 145C type sterilization Machine that for example uses Singapore APV company to sell carries out.
Described elevated temperature heat sterilization is that the PT-20C-R type tube-sheet type combined super high temperature sterilization Machine that for example uses Japanese Powerpoint International company limited to sell carries out.
In addition, the described milk powder that contains lactobacterium casei CN1566 prepares according to following step:
Preparation method's preparation according to above-mentioned lactic acid bacteria milk beverage, just raw dairy is at 95 ℃ of following heat-sterilization 20min or at 140 ℃ of following elevated temperature heat sterilization 2s, be cooled to 37 ℃ then, connect the foregoing lactobacterium casei CN1566 working stock culture of bacterium amount inoculation with 4% of raw dairy volume again, at 37 ℃ of bottom fermentation 16h, obtain lactobacterium casei CN1566 fermented-milk again; Described then lactobacterium casei CN1566 fermented-milk is added in the above-mentioned sterilization raw dairy according to 1: 3 (V/V), carries out homogeneous, and vacuum concentration, spraying drying obtain containing the milk powder of lactobacterium casei CN1566.
Described homogeneous is that the GYB40-10S type high pressure homogenizer that for example uses east, Shanghai magnificent high pressure homogenizer factory to sell carries out.
Described concentrating is that the vacuum concentration pan that for example uses Yangzhou food machinery factory to sell carries out.
Described spraying drying is that the experiment type spray drier that for example uses Shanghai Triowin Tech. Co., Ltd. to sell carries out.
In addition, described capsule product prepares according to following step:
According to the above-mentioned preparation method's preparation that contains the milk powder of lactobacterium casei CN1566, just this milk powder is incapsulated, make capsule product.
In addition, described fermented-milk prepares according to following step:
Preparation method's preparation according to above-mentioned lactic acid bacteria milk beverage, just raw dairy is at 95 ℃ of following heat-sterilization 20min or at 140 ℃ of following elevated temperature heat sterilization 2s, be cooled to 37 ℃ then, add foregoing lactobacterium casei CN1566 working stock culture according to 3-5 volume % again, adding 3-5 volume % again can symbiotic preparation fermented-milk commodity starter, behind the mixing 37 ℃ of following mixed fungus fermentations to titration acidity in lactic acid 0.6-0.7%, be cooled to 4 ℃ then, carry out stored refrigerated again and obtain described fermented-milk.
Described commodity starter is lactobacillus bulgaricus or thermophilus streptococcus preferably.
On meaning of the present invention, described raw dairy is one or more raw dairy that are selected from skimmed milk, fresh milk, recovery milk.
[beneficial effect]
Lactobacterium casei CN1566 can tolerate the hydrogen peroxide of 1.2mmol/L initial concentration; Cell concn is 10 10The lactobacterium casei CN1566 intact cell of cfu/mL and cell-free extract are respectively 88.2% and 86.6% to the clearance rate of phenylbenzene picryl phenylhydrazine (DPPH) free radical; Cell concn is 10 10The lactobacterium casei CN1566 intact cell of cfu/ml and cell-free extract are respectively 90.6% and 92.5% to the clearance rate of hydroxy radical qiao; Lactobacterium casei CN1566 intact cell and cell-free extract all have the ability of certain inhibition linolic acid oxidation, and linolic acid inhibition of oxidation rate is reached 80%.The concentration that lactobacterium casei CN1566 can tolerate cholate is 4%, and the sodium chloride concentration that can tolerate is 8%; The superoxide dismutase activity of lactobacterium casei CN1566 is 1.20 ± 0.26U/mL, and activity of glutathione peroxidase is 54.94 ± 12.54U/g albumen.
Experimentation on animals shows takes in the anti-oxidant index that lactobacterium casei CN1566 can significantly improve the mouse body.
[description of drawings]
The colonial morphology of Fig. 1 lactobacterium casei CN1566
The thalli morphology (1000x) of Fig. 2 lactobacterium casei CN1566
The suitableeest growth of Fig. 3 lactobacterium casei CN1566 pH
The growth curve of Fig. 4 lactobacterium casei CN1566
Fig. 5 lactobacterium casei CN1566 is to the effect of linolic acid inhibition of oxidation
Lactobacterium casei CN1566 of the present invention is preserved in Chinese typical culture collection center on July 29th, 2007, is numbered CCTCC No:M207107.
[embodiment]
The physiological and biochemical property of embodiment 1, lactobacterium casei CN1566 of the present invention and strain identification
Lactobacterium casei CN1566 is inoculated in MRS liquid nutrient medium (BD Dido company, trade(brand)name
Figure A20071014599100091
Lactobacilli MRS Broth), cultivate 18h 37 ℃ of anaerobism, the centrifuging and taking thalline, (for example go up the GNB10010 PBS damping fluid that the source of seawater bio tech ltd is produced, every liter contains O.144g KH with the PBS damping fluid 2P0 4, 9.0g NaCl, 0.795gNa 2HPO 47H 20, pH7.4) washing is 2 times, insert API 50 CHL liquid nutrient medium (Co., Ltd among the biological Mei Liai, API 50 CHL Medium) make bacteria suspension in, insert API 50CH indentifying substance bar (Co., Ltd among the biological Mei Liai), 37 ℃ of anaerobism are cultivated 24h-48h, and the record bacterial strain is to the fermentation result of 49 kinds of carbohydrate, with the evaluation software API LAB PLUS of its input Mei Liai company, these reaction results are as shown in table 1.Through data base querying, CN1566 of the present invention and lactobacterium casei Lactobacillus casei have 99.9% homology, therefore, with lactobacterium casei CN1566 identification of strains of the present invention is lactobacterium casei, called after Lactobacillus casei CN1566, be preserved in Chinese typical culture collection center on July 29th, 2007, be numbered CCTCC No:M207107.
The API 50 CHL reaction results of table 1 lactobacterium casei CN1566
The pipe number Carbohydrate Reaction result The pipe number Carbohydrate Reaction result
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Blank glycerine erythritol D-R Arabinose D-ribose D-wood sugar L-wood sugar D-adonite 1 methyl-β D-xylopyranose glucosides D-galactolipin D-Glucose D-Fructose D-MANNOSE L-sorbose L-rhamnose melampyrin inositol sweet mellow wine sorbierite methyl-α D-mannopyranose glycosides - - - - - + - - - - + + + + - - - - + + + 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 The sugar D-of the former xylitol D-of aesculin ironic citrate salicin D-cellobiose D-Maltose D-lactose D-melibiose D-sucrose D-trehalose inulin D-melezitose D-gossypose starch sugar gentiobiose D-Toulon lyxose D-Tag D-fucose L-fucose D-arabitol + + + + + - + + + - - - - - + - - - - - -
21 22 23 24 Methyl-α D-glucopyranoside N-acetyl grape amine amygdaloside ARBULIN + + + + 46 47 48 49 L-arabitol Potassium Gluconate 2-ketone group Potassium Gluconate 5-ketone group Potassium Gluconate - + - -
Annotate :+be fermentation ,-be nonfermented
The Microbiological Characteristics of embodiment 2, lactobacterium casei CN1566
Lactobacterium casei CN1566 is inserted the MRS liquid nutrient medium by 1% (V/V), leave standstill cultivation 24h and 48h under 10,15,20,25,30,40,45 and 50 ℃, observe upgrowth situation, the results are shown in Table 2 for it.The minimum growth temperature of lactobacterium casei CN1566 is 20 ℃, and maximum growth temperature is 40 ℃, can grow when room temperature, does not grow under 15 ℃.
The growing state of table 2 lactobacterium casei CN1566 bacterial strain under differing temps
Time (h) Growth temperature (℃)
10 15 20 25 30 37 40 45 50
24h 48h - - - - + + + + ++ ++ ++ ++ + + - - - -
Annotate :+be growth ,-for not growing
Insert lactobacterium casei CN1566 bacterial strain in the MRS liquid nutrient medium of different pH values by 1% (V/V), leave standstill cultivation 12h at 37 ℃, the UV2100 ultraviolet spectrophotometer that use is produced by UNICO(Shanghai) Instruments Co., Ltd. is measured the OD value (optical density value) of bacterium liquid at 600nm, observe the suitableeest growth pH of bacterial strain, its result as shown in Figure 3.Lactobacterium casei CN1566 grows when pH 3.0 hardly, all can grow in the scope of pH 4.0-9.0, and optimal pH is 5.0-7.0, and it is more vigorous that lactobacterium casei CN1566 grows in the MRS liquid nutrient medium of initial pH about 6.0.
Lactobacterium casei CN1566 is inserted in the MRS liquid nutrient medium by 1% (V/V) inoculum size, be adjusted to optimum pH value, measure the OD value and the pH value of a nutrient solution then every 2h at 600nm.With OD value, pH the time mapping is obtained the growth curve of lactobacterium casei CN1566 in the MRS substratum, its result as shown in Figure 4.In the MRS substratum, lactobacterium casei CN1566 enters logarithmic phase at 4h, and 12h enters stationary phase.Along with the increase of incubation time, pH constantly descends.After entering stationary phase, pH remains unchanged substantially.After cultivating 24h, pH is 3.96.Lactobacterium casei CN1566 viable count is 2.3 * 109cfu/mL in the 24h nutrient solution.
Embodiment 3, lactobacterium casei CN1566 are to the tolerance test of the initial concentration of hydrogen peroxide of difference
Hydrogen peroxide is a relative more weak oxygenant, has very high diffustivity and long action time, can directly cause the body oxidative damage.Therefore, lactobacterium casei CN1566 is an external evaluation index of its resistance of oxidation to the tolerance of the initial concentration of hydrogen peroxide of difference.The MRS substratum that in 100mL went out the triangular flask of bacterium, adds 60mL, add hydrogen peroxide, make initial concentration of hydrogen peroxide in the substratum be respectively 0.4,0.8,1.0,1.2 and 1.4mmol/L respectively, and by volume the inoculum size of mark 1% inserts lactobacterium casei CN1566 and control strain (plant lactobacillus ZH008, CCTCC No.M206033, the resisting hydrogen peroxide ability), place 37 ℃ of constant incubators to cultivate 24h, the upgrowth situation of observation under the initial concentration of hydrogen peroxide of difference, the results are shown in Table 3 for it.When initial concentration of hydrogen peroxide reached 1.2mmol/L, lactobacterium casei CN1566 still can grow, and the control strain of resisting hydrogen peroxide can not grown when concentration of hydrogen peroxide is 0.8mmol/L.
Table 3 CN1566 is at different initial concentration hydrogen peroxide
Upgrowth situation in the MRS liquid nutrient medium
Bacterial strain 0.4mmol/L 0.8mmol/L 1.0mmol/L 1.2mmol/L 1.4mmol/L
CN1566 control strain plant lactobacillus ZH008 CCTCC No.M206033 ++ ++ ++ - ++ - + - - -
Annotate :+, growth is arranged; ++, well-grown;-, can not grow
Embodiment 4, lactobacterium casei CN1566 remove the test of free radical ability
The intact cell and the cell-free extract that at first prepare lactobacterium casei CN1566
To be inoculated in the MRS liquid nutrient medium after the lactobacterium casei CN1566 activation culture, cultivate 24h at 37 ℃, culture is through 4000r/min, 4 ℃ of centrifugal 10min, obtain culture supernatant and bacterial sediment, bacterial sediment is after aseptic PBS damping fluid (pH7.4) washed twice, thalline is suspended again with stroke-physiological saline solution, adjusting cell concn respectively is 109, two groups of 1010cfu/mL, each is organized the gained bacteria suspension and is further divided into two groups, one group is intact cell group (IC), and another group is used for the preparation of cell-free extract (CFE), with cell suspending liquid ultrasonic disruption instrument (Sonics﹠amp; Materials company, VCX500 type), under 280W, the 4 ℃ of conditions, every processing 5s, 5s at interval, ultrasonication 10min, test under microscope do not have complete thalline, and then with 4 ℃, the centrifugal 10min of 6000r/min collects supernatant liquor, is cell-free extract.
Then, carry out lactobacterium casei CN1566 and remove phenylbenzene picryl phenylhydrazine (DPPH) free radical ability test
The DPPH free radical is a kind of stable organic free radical that single electronics is arranged.DPPH spectral photometry method is a kind of common screening and the effective ways of evaluation resistance of oxidation.Get different sample 2mL, add 0.2mmol/L DPPH ethanol solution 1mL, the 30min of lucifuge reaction at room temperature behind the mixing, and under 6000r/min centrifugal 10min, get supernatant liquor, the UV2100 ultraviolet spectrophotometer that uses UNICO(Shanghai) Instruments Co., Ltd. to produce is measured absorbancy, and its mean value is got in parallel survey 3 times.Blank group replaces DPPH solution with the equal-volume dehydrated alcohol, and control group replaces sample solution with equal-volume distilled water, and with equal-volume distilled water and the blank zeroing of dehydrated alcohol mixed solution.Calculate the clearance rate of DPPH free radical according to following formula:
Figure A20071014599100131
Ao is the control group absorbancy in the formula, and Ai is the sample sets absorbancy, and Aj is blank group absorbancy.Its measurement result is listed in the table below in 4.
Table 4 lactobacterium casei CN1566 cell-free extract,
Cell-free extract is to the clearance rate of DPPH free radical
Bacterial strain DPPH clearance rate %
Cell-free extract (cell concn 10 9cfu/mL) 36.2
Intact cell (cell concn 10 9cfu/mL) 37.9
Cell-free extract (cell concn 10 10cfu/mL) 86.6
Intact cell (cell concn 10 10cfu/mL) 88.2
Record lactobacterium casei CN1566 cell-free extract, viable cell is as shown in table 4 to DPPH free radical scavenging activity result.By 4 tables as can be seen, when cell concentration is 10 10During cfu/mL, lactobacterium casei CN1566 intact cell and cell-free extract reach 88.2%, 86.6% respectively to the clearance rate of DPPH free radical.
In addition, also carried out the test that lactobacterium casei CN1566 removes the hydroxy radical qiao ability.
Utilize hydrogen peroxide and Fe 2+Mix and produce hydroxy radical qiao, in reaction system, add Whitfield's ointment, can effectively catch hydroxy radical qiao and produce red material, this product has strong absorption at the 510nm place, has the measured matter of removing the hydroxy radical qiao function if add, and just can compete with Whitfield's ointment, thereby the growing amount of red product is reduced, survey absorbance at the 510nm place with spectrophotometer, and contrast, just can measure the scavenging(action) of analyte hydroxy radical qiao with blank solution.According to people such as Li Zhiying (brewing science and technology, 2006,142 (4): method 26-29), make positive control hydroxy radical qiao there is the xitix of scavenging(action), measure lactobacterium casei CN1566 viable cell, cell-free extract, the xitix clearance rate to hydroxyl radical free radical, the results are shown in Table 5 for these.
Table 5 milk-acid bacteria CN1566 living cell body and acellular
Extract is removed the hydroxy radical qiao ability relatively
The test group Hydroxy radical qiao clearance rate/%
Xitix control group (1mmol/L) 15.6
Cell-free extract (cell concn 10 9cfu/mL) 21.2
Intact cell (cell concn 10 9cfu/mL) 21.3
Cell-free extract (cell concn 10 10cfu/mL) 92.5
Intact cell (cell concn 10 10cfu/mL) 90.6
Lactobacterium casei CN1566 intact cell and cell-free extract all show significant removing hydroxy radical qiao ability, and cell concn is 10 9During cfu/ml, lactobacterium casei CN1566 intact cell and cell-free extract reach 21.3% and 21.2% respectively to the clearance rate of hydroxy radical qiao; Cell concn is 10 10During cfu/ml, lactobacterium casei CN1566 intact cell and cell-free extract reach 90.6% and 92.5% respectively to the clearance rate of hydroxy radical qiao, only are 15.6% and concentration is the xitix of 1mmol/L to the clearance rate of hydroxy radical qiao.
Embodiment 5, lactobacterium casei CN1566 suppress the test of linolic acid oxidation capacity
Suppressing the linolic acid oxidation capacity is to weigh a common counter of anti-oxidant activity.Linoleic oxidation meeting produces superoxide, can be with Fe 2+Be oxidized to Fe 3+, and Fe 3+With SCN -Form network and thing, maximum light absorption value is arranged in the 500nm place.Therefore, light absorption value is high more, shows that the linolic acid degree of oxidation is high more.With the blank group is contrast, method (Joumalof Agriculture and Food Chemistry 2000 according to people such as Than and Francois, 48:3033-3038) measure the ability of the inhibition linolic acid oxidation of lactobacterium casei CN1566 intact cell and cell-free extract respectively, these the results are shown among Fig. 5.By this figure as can be seen, along with the increase in reaction times, light absorption value also increases, the degree that therefore the shows the linolic acid oxidation cumulative height that continues.Compared with the control, cell-free extract and intact cell extract all have effective restraining effect, 10 to the linolic acid oxidation 9The ability that the oxidation inhibition ability of cfu/mL cell-free extract group and the xitix of 1mmol/L suppress the linolic acid oxidation is similar.Cell-free extract reaches 80% to linolic acid inhibition of oxidation rate during 48h.
Embodiment 6, lactobacterium casei CN1566 are to biliary tolerance test
In the MRS liquid nutrient medium, add oxgall salt, make its massfraction be respectively 0.0,0.10,0.20,0.30,0.35,0.40 and 0.45%, the inoculation of sterilization back, 37 ℃ of anaerobism are cultivated, with the purpurum bromocresolis is indicator, observe colour-change and thalli growth, the growing state of lactobacterium casei CN1566 in the MRS of various biliary juice concentration substratum sees the following form 6.As can be seen from Table 6, lactobacterium casei CN1566 can also grow in bile salt concentration is up to 0.4% substratum.Therefore lactobacterium casei CN1566 has certain anti-bile ability.
Cheese in the substratum of table 6 various biliary salt concn
The growing state of Bacterium lacticum CN1566
Gallbladder salinity (%) Growing state
0.0 ++
0.1 ++
0.2 ++
0.3 ++
0.4 +
0.5 -
1.0 -
Annotate: ++ be substratum variable color within 24h ,+be substratum variable color within 48h ,-be the substratum nondiscoloration
Embodiment 7, lactobacterium casei CN1566 are to the tolerance test of NaCl
In the MRS liquid nutrient medium, add NaCl, make its massfraction be respectively 0,2,4,6,7,8,9%, the inoculation of sterilization back, cultivating 37 ℃ of following anaerobism, is indicator with bromine potassium phenol violet, inoculation lactobacterium casei CN1566, observation is to the tolerance of NaCl, and the results are shown in Table 7 for these.Lactobacterium casei CN1566 is well-grown under 7% NaCl concentration, and poor growth under 8% NaCl concentration is not grown more than 9%, illustrates that CN1566 can tolerate the NaCl of 8% concentration.
Table 7 lactobacterium casei CN1566 is to the tolerance of NaCl
NaCl(％) 24h 48h
0 + +
2 + +
4 + +
6 + +
7 + +
8 - +
9 - -
Annotate :+be growth ,-for not growing
The antibiotic susceptibility test of embodiment 8, lactobacterium casei CN1566
Bacterial strain is inserted the MRS liquid nutrient medium by 1% (V/V) inoculum size, be cultured to logarithmic phase at 37 ℃, (for example go up the GNB10010 PBS damping fluid that the source of seawater bio tech ltd is produced, every liter contains 0.144g KH to the PBS buffered soln of usefulness pH7.4 2PO4,9.0g NaCl, 0.795g Na 2HPO 47H 2O) carry out gradient dilution (0-10 -2), obtain the bacterium liquid of different bacteria concentrations.Aseptic absorption nutrient solution 0.1mL is applied to the MRS flat board, and coating evenly selects for use 24 kinds of common microbiotic to carry out drug sensitive experiment.Aseptic nipper puts drug sensitive test paper to the MRS flat board of coating bacterium liquid, cultivates 48h 37 ℃ of anaerobism, and these experimental results are listed in the table 8.
Table 8 lactobacterium casei CN1566 is to common antibiotic susceptibility
Title Letter Content/sheet Inhibition zone (mm) Title Letter Content/sheet Inhibition zone (mm)
Gentamicin furadantin Norxin rocephin erythromycin tsiklomitsin paraxin penbritin Ciprofloxacin Amikacin Sulphate Prostaphlin penicillin The red tetrachloro ammonia of celebrating furan fluorine bacterium ring butylbenzene green grass or young crops 10ug 300ug 10ug 30ug 15ug 30ug 30ug 10ug 5ug 30ug 1ug 10IU 6 0 0 26 18 6 12 0 0 0 0 0 The compound sulfanilamide (SN) Kefzol of cefoperazone tobramycin Streptomycin sulphate cefotaxime Rifampin cefuroxime Pipril ceftazime vancomycin kantlex Must the sharp western oxygen glad ten thousand multiple V cards of appropriate chain oxime 75ug 10ug 10ug 30ug 5ug 30ug 100ug 30ug 30ug 1.25ug 30ug 30ug 20 0 5 22 14 19 21 0 0 17 21 10
In 24 kinds of microbiotic, lactobacterium casei CN1566 is for erythromycin, rocephin, paraxin, cefoperazone, cefotaxime, cefuroxime, Kefzol, Rifampin, Pipril, compound sulfanilamide (SN), 11 kinds of antibiotic sensitive of kantlex, to gentamicin, tsiklomitsin, 3 kinds of microbiotic of Streptomycin sulphate have tolerance, furadantin, Norxin, penbritin, Ciprofloxacin, penicillin, Amikacin Sulphate, ceftazime, Prostaphlin, tobramycin, 10 kinds of microbiotic of vancomycin to the growth of lactobacterium casei CN1566 without any influence.
The superoxide-dismutase of embodiment 9, lactobacterium casei CN1566 and activity of glutathione peroxidase test
Utilize Nanjing build up bio-engineering corporation kit measurement total superoxide-dismutase (SOD) and Selenoperoxidase (GSH-PX) vigor of lactobacterium casei CN1566, experimental result is listed in the table below in 9.The SOD activity of the cell-free extract of lactobacterium casei CN1566 is 1.20 ± 0.26U/mL, and glutathione peroxidase activity is 54.94 ± 12.54U/g albumen.
The SOD of table 9 lactobacterium casei CN1566 and GSH-PX activity
Testing index Measurement result
Protein concn (mg/mL) 0.065±0.028
SOD vigor (U/mL) 1.20±0.26
GSH-PX vigor (U/g albumen) 54.94±12.54
Oxidation resistant experimentation on animals in embodiment 10, the lactobacterium casei CN1566 body
High lipid oxidation Stress model is adopted in the anti-oxidant function experiment in the body.Laboratory animal is used 40 of Kunming kind cleaning level mouse, male and female half and half, and body weight 20 ± 2g is provided by laboratory animal field, the jiangsu wuxi Hui Shan south of the River.Mouse is at room temperature raised, free choice feeding drinking-water, after the adaptability of 3d is raised, be divided into 4 groups at random, 10 every group, the basal feed (prescription: 89.5% normal diet, 10% lard, 0.5% cholesterol) of feeding, control group is irritated stomach 0.2mL physiological saline every day, and Senior Three dosage was irritated stomach different concns lactobacterium casei CN1566 during its excess-three component was low, and is as shown in table 10 below, 0.2mL/ of every day, continuous 4 weeks.Last is to fasting 24h behind the sample, gather blood rapidly, separating red corpuscle and serum, detect erythrocyte superoxide dismutase (SOD) activity, whole blood Selenoperoxidase (GSH-Px) activity, active, blood plasma mda (MDA) content of blood plasma catalase (CAT), all data are all carried out statistical procedures with the DPS of statistical software, every index result represents with x ± s, with t check carrying out test of significance.The mensuration test kit is purchased in Nanjing and is built up bio-engineering corporation.
The experimentation on animals that table 10 lactobacterium casei CN1566 anti-oxidant function is estimated
Group The mode of feeding
Control group 0.2mL physiological saline/only+basal feed
Low dosage 0.2mL bacterium liquid (cell concn 0.01g/mL)/only+high lipid food
Middle dosage 0.2mL bacterium liquid (cell concn 0.02g/mL)/only+high lipid food
High dosage 0.2mL bacterium liquid (cell concn 0.04g/mL)/only+high lipid food
Irritate stomach lactobacterium casei CN1566 to active influence of mouse red blood cell superoxide-dismutase (SOD) such as table 12.With respect to control group, the erythrocyte sod vigor of irritating the mouse experiment group of stomach CN1566 improves, and shows certain dose-effect relationship, and there were significant differences (P＜0.05) for middle and high dosage filling stomach group and control group.
Table 11 lactobacterium casei CN1566 is in the mouse red blood cell
The active influence of SOD (x ± s, n=10)
Group SOD activity (U/mgHb)
Control group 169.72±18.20
Low dosage 229.51±18.53
Middle dosage 240.43±24.32 *
High dosage 287.71±11.52 *
*P<0.05
Selenoperoxidase (GSH-Px) is the enzyme that the extensive a kind of important catalyzing hydrogen peroxide that exists decomposes in the body, and catalytic reduction type gsh plays the effect of protection membrane structure and function to the reduction reaction of hydrogen peroxide.Irritate influence such as the table 12 of stomach lactobacterium casei CN1566 to mouse whole blood activity of glutathione peroxidase.With respect to control group, the GSH-Px vigor of irritating the mouse experiment group whole blood of stomach lactobacterium casei CN1566 raises to some extent, and tangible dose-effect relationship is arranged.There were significant differences for high dose group and control group (P＜0.05).
Table 12 lactobacterium casei CN1566 is in the mouse whole blood
The active influence of GSH-Px (x ± s, n=10)
Group GSH-PX (enzyme activity unit)
Control group 89.26±11.95
Low dosage 93.94±15.63
Middle dosage 109.37±17.78
High dosage 114.60±12.68 *
*P<0.05
The mda level has reflected the snperoxiaized degree of body inner lipid, reflects the degree of cellular oxidation damage indirectly.Filling stomach L.casei CN1566 sees Table 13 to the influence of mda in the mice plasma (MDA) level.Along with the increase of irritating stomach dosage, the MDA level of irritating stomach lactobacterium casei CN1566 experimental group is and reduces trend gradually, has tangible dose-effect relationship, and under the high dosage level, the mda level significantly reduces (P＜0.05) with respect to control group.
Table 13 lactobacterium casei CN1566 is in the mice plasma
The influence of mda level (x ± s, n=10)
Group MDA content (nmol/ml)
Control group 3.34±0.70
Low dosage 3.01±0.59
Middle dosage 2.86±0.84
High dosage 1.74±0.70 *
*P<0.05
Catalase is ubiquity in body, catalysis H 2O 2Be decomposed into H 2O and O 2H 2O 2Be the toxic byproduct that has of organism metabolism,, prevent that body from sustaining damage through changing into the chemicals of other low toxicity after the hydrogen peroxide enzyme catalysis.Irritate influence such as the table 14 of stomach lactobacterium casei CN1566 to the mice plasma catalase activity.With respect to control group, the blood plasma catalase activity that lactobacterium casei CN1566 irritates the middle and high dosage group of stomach obviously raise (P＜0.05); The blood plasma catalase activity of irritating the mouse experiment group of stomach low dosage lactobacterium casei CN1566 is higher than control group a little.
Table 15 is irritated stomach lactobacterium casei CN1566 to mice plasma
The influence of catalase activity (x ± s, n=10)
Group CAT activity (U/ml)
Control group 1.52±0.44
Low dosage 1.75±0.66
Middle dosage 2.36±0.67 *
High dosage 2.74±0.52 *
*P<0.05
Above experimentation on animals result shows that lactobacterium casei CN1566 can significantly improve the anti-oxidant index of mouse, has interior antioxidation action.
Application Example 1: utilize lactobacterium casei CN1566 to make the lactic acid bacteria milk beverage
The raw dairy skimmed milk at 95 ℃ of heat kill bacterium 20min, is cooled to 4 ℃ then, adds lactobacterium casei CN1566 working stock culture of the present invention again, make its concentration reach 10 6More than the cfu/ml, promptly obtain containing the lactic acid bacteria milk beverage of lactobacterium casei CN1566 viable bacteria 4 ℃ of stored refrigerated.
Application Example 2: utilize lactobacterium casei CN1566 to make milk powder
Raw dairy at 140 ℃ of elevated temperature heat sterilization 2s, is cooled to 37 ℃ then, connects bacterium amount inoculation lactobacterium casei CN1566 working stock culture of the present invention,, obtain lactobacterium casei CN1566 fermented-milk at 37 ℃ of fermentation 16h with 4% of raw dairy volume.Lactobacterium casei CN1566 fermented-milk is added homogeneous in the raw dairy after the sterilization with 1: 3 (V/V), after vacuum concentration, spray drying treatment, obtain milk powder, maybe the milk powder that obtains is installed to and make capsule product in the capsule.
Application Example 3: utilize lactobacterium casei CN1566 to make capsule product
Raw dairy at 140 ℃ of elevated temperature heat sterilization 2s, is cooled to 37 ℃ then, connects bacterium amount inoculation lactobacterium casei CN1566 working stock culture of the present invention,, obtain lactobacterium casei CN1566 fermented-milk at 37 ℃ of fermentation 16h with 4% of raw dairy volume.Lactobacterium casei CN1566 fermented-milk is added homogeneous in the raw dairy after the sterilization with 1: 3 (V/V), after vacuum concentration, spray drying treatment, obtain milk powder, the milk powder that obtains is installed to make capsule product in the capsule.
Application Example 4: utilize lactobacterium casei CN1566 to prepare fermented-milk
With raw dairy at 95 ℃ of heat kill bacterium 20min, be cooled to 37 ℃ then, amount with 4 volume % adds lactobacterium casei CN1566 working stock culture of the present invention, and add the commercial starter lactobacillus bulgaricus that 4 volume % can symbiotic preparation fermented-milk, at 37 ℃ of following mixed fungus fermentations to titration acidity is 0.6% (in lactic acid), and refrigeration to 4 ℃ and stored refrigerated promptly obtains fermented-milk.

Claims (10)

  1. A resisting hydrogen peroxide, remove oxidation resistant Lactobacillus casei capable (Lactobacilluscasei) CN1566 of free radical, its deposit number CCTCC NO:M207107.
  2. 2. according to the purposes of the described lactobacterium casei CN1566 of claim 1 in leavened food.
  3. 3. according to the purposes of the described lactobacterium casei CN1566 of claim 2, it is characterized in that described leavened food is lactic acid bacteria milk beverage, milk powder, capsule product or fermented-milk.
  4. 4. according to the purposes of the described lactobacterium casei CN1566 of claim 3, it is characterized in that described lactic acid bacteria milk beverage prepares according to following step:
    At first, prepare described lactobacterium casei CN1566 working stock culture,
    Then, raw dairy is cooled to 4 ℃ then at 95 ℃ of following heat-sterilization 20min or at 140 ℃ of following elevated temperature heat sterilization 2s, adds described lactobacterium casei CN1566 working stock culture again, makes its concentration reach 10 6More than the cfu/ml, promptly obtain containing the lactic acid bacteria milk beverage of lactobacterium casei CN1566 4 ℃ of stored refrigerated.
  5. 5. according to the purposes of the described lactobacterium casei CN1566 of claim 3, it is characterized in that described milk powder prepares according to following step:
    At first, prepare described lactobacterium casei CN1566 working stock culture,
    Then, raw dairy is cooled to 37 ℃ then at 95 ℃ of following heat-sterilization 20min or at 140 ℃ of following elevated temperature heat sterilization 2s, connects the described lactobacterium casei CN1566 working stock culture of bacterium amount inoculation with 4% of raw dairy volume again, at 37 ℃ of bottom fermentation 16h, obtain lactobacterium casei CN1566 fermented-milk again; Described then lactobacterium casei CN1566 fermented-milk is added in the above-mentioned sterilization raw dairy according to 1: 3 (V/V), carries out homogeneous, and vacuum concentration, spraying drying obtain containing the milk powder of lactobacterium casei CN1566.
  6. 6. according to the purposes of the described lactobacterium casei CN1566 of claim 3, it is characterized in that described capsule product prepares according to following step:
    At first, prepare described lactobacterium casei CN1566 working stock culture,
    Then, raw dairy is cooled to 37 ℃ then at 95 ℃ of following heat-sterilization 20min or at 140 ℃ of following elevated temperature heat sterilization 2s, connects the described lactobacterium casei CN1566 working stock culture of bacterium amount inoculation with 4% of raw dairy volume again, at 37 ℃ of bottom fermentation 16h, obtain lactobacterium casei CN1566 fermented-milk again; Described then lactobacterium casei CN1566 fermented-milk is added in the above-mentioned sterilization raw dairy according to 1: 3 (V/V), carries out homogeneous, and vacuum concentration, spraying drying obtain containing the milk powder of lactobacterium casei CN1566, this milk powder are incapsulated again, and make capsule product.
  7. 7. according to the purposes of the described lactobacterium casei CN1566 of claim 3, it is characterized in that described fermented-milk prepares according to following step:
    At first, prepare described lactobacterium casei CN1566 working stock culture,
    Then, raw dairy is at 95 ℃ of following heat-sterilization 20min or at 140 ℃ of following elevated temperature heat sterilization 2s, be cooled to 37 ℃ then, add described lactobacterium casei CN1566 working stock culture according to 3-5 volume % again, add the commodity starter that 3-5 volume % can symbiotic preparation fermented-milk again, behind the mixing 37 ℃ of following mixed fungus fermentations to titration acidity in lactic acid 0.6-0.7%, be cooled to 4 ℃ then, carry out the fermented-milk that stored refrigerated obtains containing lactobacterium casei CN1566 again.
  8. 8. according to the purposes of the described lactobacterium casei CN1566 of claim 7, it is characterized in that described commodity starter is lactobacillus bulgaricus and thermophilus streptococcus.
  9. 9. according to the purposes of claim 4,5,6,7 described cheese bacillus CN1566, it is characterized in that described lactobacterium casei CN1566 working stock culture prepares according to following preparation method:
    Lactobacterium casei CN1566 original strain is inoculated in 12 weight % in the skimming milk of 110 ℃ of sterilization 10min, cultivates 14-16h to curdled milk under 37 ℃ of conditions, cultured continuously activated for two generations, used as mother starter; Mother starter is inoculated in the described sterile milk by 3-5 volume %, cultivates 14-16h to curdled milk, viable count is about 10 in this moment curdled milk 9Cfu/mL obtains described working stock culture; Perhaps
    Lactobacterium casei CN1566 original strain is inoculated in the MRS liquid nutrient medium, cultivating 12-16h under 37 ℃ of conditions activates, activated for two generations continuously, then the activation culture thing is inoculated in the MRS substratum by 2-4 volume %, cultivate 16-18h, the centrifugal 15min of 4000r/min under 4 ℃ of conditions, remove supernatant, obtain cell precipitation, then, this precipitation suspends with aseptic skimming milk, obtains described working stock culture.
  10. 10. according to the described purposes of each claim in the claim 4,5,6,7, it is characterized in that described raw dairy is one or more raw dairy that are selected from skimmed milk, fresh milk, recovery milk.
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