CN103343107A - Human lactobacillus casei gr x 12 with antioxidant function and application thereof - Google Patents
Human lactobacillus casei gr x 12 with antioxidant function and application thereof Download PDFInfo
- Publication number
- CN103343107A CN103343107A CN2013103127847A CN201310312784A CN103343107A CN 103343107 A CN103343107 A CN 103343107A CN 2013103127847 A CN2013103127847 A CN 2013103127847A CN 201310312784 A CN201310312784 A CN 201310312784A CN 103343107 A CN103343107 A CN 103343107A
- Authority
- CN
- China
- Prior art keywords
- grx12
- lactobacillus casei
- strain
- activity
- relatively high
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to lactobacillus casei gr x 12 with antioxidant function and application of the lactobacillus casei gr x 12 in fermented milk. The lactobacillus casei gr x 12 is a probiotic strain which is separated from intestinal canals of the crowd in Changshou Village, Rugao City, Jiangsu, has remarkable oxidation resistance and shows high resistance to artificial gastric juice, artificial intestinal juice and cholate. The strain is stored with a number in General Microbiological Culture Collection Center of China Committee for Culture Collection of Microorganisms. The lactobacillus casei gr x 12 shows relatively high antioxidant activity, is relatively high in hydrogen peroxide resistance, can obviously remove DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical and hydroxyl radical, is relatively high in reducing capacity, has a certain anti-lipid peroxidation capacity, and also has relatively high activity on fermenting supernatant and intracellular superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The lactobacillus casei gr x 12 has the effects of general probiotics beneficial for human body health, and also can be used for producing products with the antioxidant function, such as the fermented milk with antioxidant activity.
Description
Technical field
The present invention relates to microbial technology field, particularly have anti-oxidant function lactobacterium casei grx12 bacterial strain and the application of this bacterial strain aspect oxidation resistant product.
Background technology
Bio-oxidation is the metabolic important physiological process of body, but meeting produces various active oxygen species (ROS) such as free radical in this process.Although under the normal circumstances, body exists the free radical scavenging system, if but body is subjected to some factor affecting, the free radical scavenging system breaks down and causes interior free yl excessively to produce, then can act on protein, nucleic acid equimolecular in the body, cause cell injury, bring very big threat to body health, cause as malignant diseases such as cancer, cataract, liver cirrhosis, atherosclerosiss.By replenishing the resistance of oxidation that antioxidant can enhancing body in the meals, but there is safety problem in the polyphenoils of many chemosynthesis, in addition, the antioxidant that replenishes from meals is easily become the material that other do not possess resistance of oxidation by digest and decompose because of it in the process that absorbs, or is difficult for being absorbed by body and directly excreting.Therefore, exploitation has the active and safe health diet supplement of high anti-oxidation becomes a research topic highly significant.
(Lactic Acid Bacteria LAB) as the resident flora of enteron aisle, can keep enteron aisle and be in the redox equilibrium state directly in keypoint part---the enteron aisle performance antioxygenation of body oxidative damage milk-acid bacteria.And milk-acid bacteria can also constantly breed after the enteron aisle field planting, produces the milk-acid bacteria that multipotency is more enough brought into play antioxygenation, plays the part of ROS street cleaner's role continuously at enteron aisle.In addition, the security of milk-acid bacteria is fully verified in a large amount of research.Therefore the antioxygenation of milk-acid bacteria has been compared more superiority with traditional antioxidant, screens the popular research topic that the milk-acid bacteria with good resistance oxidation capacity has also become current probiotic bacterium.
The bacterial strain of desirable probiotics preparation should have following attribute: (1) source (preferably deriving from human body); (2) security; (3) vigor and the activity in carrier (as food); (4) resistance of hydrochloric acid in gastric juice and bile; (5) in digestive tube, retain ability; (6) generation of antimicrobial substance etc.
The research bacterial strain uses therefor of screening high anti-oxidation living lactic acid bacteria separates acquisition mostly from fermented foodstuff or animal intestinal at present, and is then less to the milk-acid bacteria anti-oxidant activity research report of long-lived crowd's enteron aisle.And the important attribute of probiotic bacterium is preferably to derive from human body itself.
The inventor is at above-mentioned problem, be the target flora with Rugao, Jiangsu Changshou village crowd enteron aisle milk-acid bacteria, go out to have the probiotic bacterium of better anti-oxidant activity through the antioxidation in vitro experiment sieving, and studied its to the tolerance of simulated gastric fluid, simulated intestinal fluid and cholate with its survival ability in enteron aisle of further checking.
Summary of the invention
The object of the present invention is to provide a probiotics strain.
Bacterial strain of the present invention is lactobacterium casei grx12, and preserving number is CGMCC No:7696.Described lactobacterium casei grx12 is having remarkable resistance of oxidation and simulated gastric fluid, simulated intestinal fluid and cholate being had the probiotic strain of better tolerance of separating from the Changshou village crowd enteron aisle of Rugao, Jiangsu.
Of the present invention functional foodstuff or the application in medicine, the particularly application that in preparation cultured milk prod improve the antioxygenation of fermented-milk of described lactobacterium casei aspect preparation has antioxygenation also disclosed.
Description of drawings
Fig. 1 is the colony characteristics figure of lactobacterium casei grx12 of the present invention;
Fig. 2 is the thalline characteristic pattern of lactobacterium casei grx12 of the present invention;
Fig. 3 is the growth curve chart of lactobacterium casei grx12 of the present invention;
Fig. 4 is the 16S rDNA sequence pcr amplification of lactobacterium casei grx12 of the present invention figure as a result.
Bacterial strain grx12 of the present invention is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 9th, 2013, and (address is the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preserving number is CGMCC No:7696, classification name: lactobacterium casei Lactobacillus casei.
Embodiment
1. sample collecting
Sample is: the yellow soft stool of not taking Rugao, antibiotic Jiangsu Changshou village crowd in fresh, week.Put into the transportation substratum with sterilization bamboo let picking ight soil inside center faecal samples, add the sealing of 1mL sterilising liq paraffin again after, place rapidly in the ice chest, take back the laboratory and carry out the microbial bacteria cluster analysis.
2. the isolation and purification of Bacterium lacticum
With the sterile sampling diluent diluted sample of gathering is got the 0.5mL diluent at the aseptic technique platform to corresponding extent of dilution from test tube, be inoculated in the sterilization culture dish of modified MRS culture medium, place anaerobic jar to cultivate 48h in 37 ℃.Observe and record colonial morphology and gramstaining morphological features, and carry out catalase test simultaneously.Tentative the going forward side by side for Bacterium lacticum of the bacillus of Gram-positive, catalase test feminine gender one gone on foot and carries out the plate streaking purifying, obtain 75 strain Bacterium lacticum altogether, carry out vacuum lyophilization with skimming milk as protective material after, place-80 ℃ of preservations.
3. the contrary environment tolerance test of Bacterium lacticum digestive tube
The 75 strain Bacterium lacticum that will separate from the Changshou village crowd enteron aisle of Rugao, Jiangsu are made for examination bacterium liquid, and being inoculated in pH value by 1% inoculum size is in 3.0 the MRS liquid nutrient medium, places 37 ℃ of cultivation 24h down; Be inoculated in by 1% inoculum size that to add taurocholate concentration be in 0.2% the MRS liquid nutrient medium, place 37 ℃ to cultivate 24h down.Be inoculated in the MRS liquid nutrient medium of not doing any processing by 1% inoculum size simultaneously, cultivate 24h in contrast for 37 ℃.Measure bacterium liquid OD under the different treatment factor respectively with spectrophotometer
600The value, and with the contrast OD
600Value is compared.Filter out the lactobacterium strain that 12 strains have higher acidproof and bile tolerance ability by this method.This 12 strain Bacterium lacticum is as shown in table 1 to the tolerance of acid and cholate.
Acidproof and the bile tolerance ability of table 112 strain Bacterium lacticum
4.12 the strain Bacterium lacticum is to the tolerance test of the initial concentration of hydrogen peroxide of difference
Hydrogen peroxide is a kind of more weak oxygenant, but has very high diffustivity and long action time, can cause direct destruction to cell and tissue, or participate in oxidising process indirectly as hydroxy radical qiao (OH) precursor.Therefore measured the tolerance of 12 strain Bacterium lacticum to different initial concentration hydrogen peroxide.
In the MRS liquid nutrient medium, add H respectively
2O
2Solution makes the initial H in the substratum
2O
2Concentration is respectively 0.4,0.7,1.0mmol/L, and inserts Bacterium lacticum by 1% inoculum size, places 37 ℃ of constant incubators to cultivate 24h, with its OD of spectrophotometric determination
600Value is observed its growing state under the initial concentration of hydrogen peroxide of difference.The results are shown in Table 2.
Table 212 strain Bacterium lacticum is to the tolerance of different concns hydrogen peroxide
As shown in Table 2, all 12 strain Bacterium lacticum are containing 0.4mM H
2O
2The measured OD of growth in the substratum
600Value with do not contain H
2O
2Substratum in OD
600Value does not all have too big variation, shows that these bacterial strains are to 0.4mM H
2O
2Tolerance is preferably arranged.Wherein, the H of bacterial strain L3, L4, L7, L8, L9 and L11
2O
2Tolerance is stronger, and they are containing 0.7mM H
2O
2, 1.0mM H
2O
2OD in the substratum
600Value with do not contain H
2O
2Substratum in OD
600Value there are no significant difference (P〉0.05).And bacterial strain L5, L6, L10 and L12 are then to 1.0mM H
2O
2Comparatively responsive, its OD
600Value has been compared significant difference (P＜0.05) with blank.Therefore, select different concns H
2O
2Have better tolerance 8 strain Bacterium lacticum and further measure its different components resistance of oxidation
5.8 strain Bacterium lacticum different components resistance of oxidation test
Specimen preparation: 8 strain Bacterium lacticum passed for 2 generations in the MRS liquid nutrient medium after, cultivate 24h for 37 ℃, and under 4 ℃, the centrifugal 10min of 6000r/min collects supernatant liquor and is fermented supernatant fluid.
Thalline adds PBS damping fluid (pH7.4) in the centrifugal 5min of 6000r/min, washs 3 times.Thalline is resuspended in the PBS damping fluid, and adjusting cell concentration is 1.0 * 10
9ML
-1, be the thalline suspension.
Behind thalline suspension ice-bath ultrasonic ripple smudge cells 10min, at 4 ℃, the centrifugal 15min of 10000r/min, collect supernatant liquor, be extract in the born of the same parents.
(1) 8 strain Bacterium lacticum is removed the hydroxy radical qiao ability test
Hydroxy radical qiao is the very active and very strong free radical of oxidation capacity in the body, can damage the biomass cells macromole and influences the normal function of cell.Therefore, be to estimate a leading indicator of antioxidant property to hydroxyl radical free radical removing ability.
Get neighbour-F beautiful jade (2.5mmol/L) 1mL, (distilled water 1mL fully behind the mixing, adds FeS0 for 0.02mol/L, pH=7.4) 1mL to add PBS
4(2.5mmol/L) 1mL, mixing adds H
2O
2(20mmol/L) 1mL, measuring its absorbancy at the 536nm place behind 37 ℃ of water-bath 1.5h is A
1Replace 1mL H with 1mL distilled water
2O
2Be A
0Replacing the distilled water of 1mL with the 1mL sample is A
2
In the formula: A
0For not containing sample and H
2O
2A
1For not containing sample, contain H
2O
2A
2For containing sample and H
2O
2
Measurement result sees Table 3.
Table 38 strain Bacterium lacticum is to the clearance rate of OH
As shown in Table 3, each component of 8 strain Bacterium lacticum all shows certain hydroxy radical qiao and removes ability.It is higher that the hydroxy radical qiao of each strain fermentation supernatant liquor is removed ability, all surpassed 42.5%, and wherein that the highest is grx12, has reached 64.8%.A little less than the relative fermented supernatant fluid of the hydroxy radical qiao of extract removing ability is wanted in thalline and the born of the same parents, and differ greatly between each bacterial strain.Wherein, the hydroxy radical qiao of extract removing ability is stronger in the grx12, the thalline of this 3 strain Bacterium lacticum of L2 and L8 and born of the same parents.
(2) 8 strain Bacterium lacticum are removed DPPH free radical ability test
DPPH spectral photometry method is a kind of common screening and the effective ways of estimating certain material antioxidant property.8 strain Bacterium lacticum different componentss are removed DPPH free radical ability to be measured.
Get different sample 1mL, add the DPPH ethanol solution of 1mL0.2mmol/L again, shake up, lucifuge reaction 30min, the centrifugal 10min of 6000r/min gets supernatant liquor and measures absorbance Ai in the 517nm place; Blank group replaces the DPPH ethanol solution with the equal-volume dehydrated alcohol, and control group replaces sample solution with the equal-volume blank solvent, and with equal-volume distilled water and the blank zeroing of alcohol mixeding liquid.
In the formula: Ac is the control group absorbancy; Ai is the sample sets absorbancy; Aj is blank group absorbancy.
Measurement result sees Table 4.
Table 48 strain Bacterium lacticum is to the clearance rate of DPPH free radical
As shown in Table 4,8 each component of strain Bacterium lacticum all show certain DPPH radical scavenging activity, but its removing capacity variance is bigger.The DPPH radical scavenging activity of grx12 fermented supernatant fluid is the strongest, has reached 53.4%.In the thalline group, the DPPH radical scavenging activity of grx12 is the strongest.Compare with fermented supernatant fluid and thalline, in the born of the same parents extract DPPH radical scavenging activity a little less than, and difference is less, wherein extract DPPH radical scavenging activity is the strongest in the L2 born of the same parents.
The test of (3) 8 strain Bacterium lacticum reducing powers
Reducing power mainly refers to some enzymes (catalase, nadh oxidase, NADH peroxidase) and non-enzyme complex (V
C, V
E, gsh) have and reduce oxyradical and chelating Fe
2+Ability, and then reduce the generation of oxidizing reaction.Generally speaking, reducing power and the resistance of oxidation of sample are proportionate, and measure its absorbancy in the 700nm place, and absorbancy is more big, shows that the reducing power of sample is more strong.
Get the PBS(0.2mol/L of different sample 0.5mL and 0.5mL, pH6.6) mix, add 1% Tripotassium iron hexacyanide 0.5mL again.Mixture adds 0.5mL10%TCA in 50 ℃ of water bath with thermostatic control 20min after the cooling, 3000r/min centrifugation 10min gets supernatant liquor 1mL, adding distil water 1mL and 0.1%FeCl
31mL, reaction 10min measures light absorption value at the 700nm place.The result is as shown in table 5.
The reducing power of table 58 strain Bacterium lacticum
As shown in Table 5,8 each component of strain Bacterium lacticum all present certain reducing power.The reducing power of each strain fermentation supernatant liquor is stronger, and wherein grx12 is the highest, is 2.540.It is much lower that the reducing power of extract is compared fermented supernatant fluid in thalline and the born of the same parents.In the thalline group, the grx12 reducing power is the strongest; And in the interior extract group of born of the same parents, the L2 reducing power is the strongest.
(4) 8 strain Bacterium lacticum anti peroxidation of lipid abilities
The anti peroxidation of lipid test also is a kind of common method of estimating certain material antioxidant property.The unsaturated system of linolic acid is by Fe in the test
2+Generate superoxide behind the catalyzed oxidation, it continues to decompose the secondary products mda (MDA) that produces and generate red condenses with thiobarbituricacid (TBA) reaction under acidic conditions, and at the 532nm place strong absorption being arranged, the adding of oxidation-resistance material then can suppress the generation of red product.
0.5mL PBS solution (0.02mol/L, pH7.4) add the linoleic emulsion of 1mL in, FeSO4 (1%) 1mL adds the 0.5mL sample again, 37 ℃ of water-baths 1.5 hours, mixed solution adds 0.2mL TCA (4%), the TBA of 2mL (0.8%), 100 ℃ of water-bath 30min of reaction solution, cooling rapidly, the centrifugal 15min of 4000r/min, the collection supernatant liquor is surveyed absorbancy and is A under 532nm; Control group replaces sample to be A with 0.5mL distilled water
0, return to zero after adding isopyknic sample liquid centrifuging with PBS liquid.
In the formula: A is the sample sets absorbancy; A
0Be the control group absorbancy.
Measurement result sees Table 6.
Table 68 strain Bacterium lacticum is to the inhibiting rate of anti peroxidation of lipid
As shown in Table 6,8 each component of strain Bacterium lacticum all show certain anti peroxidation of lipid ability.In the fermented supernatant fluid group, grx12 anti peroxidation of lipid ability is the strongest, is 19.9%, with other bacterial strain significant differences (P＜0.05).In the thalline group, L2 anti peroxidation of lipid ability is the strongest, is 8.1%.In the extract group, grx12 anti peroxidation of lipid ability is the strongest, has reached 14.7% in the born of the same parents.
Resistance of oxidation to blank substratum is also measured.By table 3～table 6 data as can be known, the influence of blank substratum antagonism oxidation value is very small, so, can get rid of substratum substantially to the influence of experimental result.
Comprehensive above-mentioned experimental result is screened each component antioxidant property all 4 strain Bacterium lacticum grx12, L2, L3 and L8 preferably, further measures the activity of extract antioxidase in its fermented supernatant fluid and the born of the same parents.
The T-SOD of (5) 4 strain Bacterium lacticum and GSH-Px activity
SOD removes the main enzyme of superoxide anion in the body, the existence of SOD makes superoxide radical (O
2-) speed of cancellation disproportionation reaction improved 10
10Times, and stop active oxygen radical to the transformation of hydroxy radical qiao.Therefore, the SOD that keeps certain level in body is very important.GSH-Px also is very important antioxidase in the cell defense system.Because antioxidase mainly is present in lactobacillus ferment supernatant liquor and the born of the same parents, therefore adopt Nanjing build up bio-engineering research T-SOD and GSH-Px test kit SOD and the GSH-Px activity of extract in 4 strain lactobacillus ferment supernatant liquors and the born of the same parents are measured.Measuring method carries out in strict accordance with the test kit specification sheets.Measurement result sees Table 7 and table 8.
Table 74 strain Bacterium lacticum T-SOD activity
Table 84 strain Bacterium lacticum GSH-Px activity
| Bacterial strain | GSH-Px/(U/mL) | |
| Fermented supernatant fluid | Extract in the born of the same parents | |
| grx12 | 45.012±0.006 | 24.091±0.004 |
| L2 | 37.273±0.003 | 18.182±0.004 |
| L3 | 27.273±0.005 | 6.364±0.008 |
| L8 | 36.818±0.007 | 10.909±0.002 |
As shown in Table 7, extract all has certain SOD activity in 4 strain bacterium fermented supernatant fluids and the born of the same parents, and same component S OD activity difference is less.Wherein, extract SOD activity is the highest in grx12 fermented supernatant fluid and the born of the same parents, but in the born of the same parents extract SOD activity a little less than, only be equivalent to 13.2% of its fermented supernatant fluid; And extract SOD activity is minimum in L8 fermented supernatant fluid and the born of the same parents.
As shown in Table 8, extract all has certain GSH-Px activity in 4 strain bacterium fermented supernatant fluids and the born of the same parents, but differs greatly between different strains and the different components.The GSH-Px activity of extract is the highest in grx12 fermented supernatant fluid and the born of the same parents, is significantly higher than other bacterial strains (P＜0.05), and extract GSH-Px activity is equivalent to 53.5% of its fermented supernatant fluid in its born of the same parents.And the GSH-Px activity of extract is minimum in L3 fermented supernatant fluid and the born of the same parents, in its born of the same parents extract GSH-Px activity a little less than, only be equivalent to its fermented supernatant fluid 23.3%.
Comprehensive above test-results, Bacterium lacticum grx12 has tolerance preferably to acid and cholate, hydrogen peroxide also had better tolerance, and it is totally higher that each component is removed free radical ability, reducing power and anti peroxidation of lipid ability, the activities of antioxidant enzymes of extract is totally also higher in fermented supernatant fluid and the born of the same parents, and it is identified and its Microbiological Characteristics is studied.
6. the Microbiological Characteristics of Bacterium lacticum grx12 and evaluation
The colony characteristics of Bacterium lacticum grx12: form tangible bacterium colony at the MRS substratum, diameter between 0.2-2.0 μ m, circle, neat in edge, oyster white, surface wettability is smooth, not chromogenesis.Referring to Fig. 1.
The thalline feature of Bacterium lacticum grx12: Gram-positive, cell is shaft-like, and Cheng Dan, paired or chaining do not form gemma, the two ends circle.Referring to Fig. 2.
The growth characteristics of Bacterium lacticum grx12: insert in the MRS liquid nutrient medium by 3% inoculum size, measure it at 37 ℃ of growing states of cultivating 48h down, with OD with the growth curve determinator
600Value is ordinate zou, and the time is X-coordinate, makes the growth curve chart of Bacterium lacticum grx12, referring to Fig. 3.As shown in Figure 3, Bacterium lacticum grx12 cultivates 3h down at 37 ℃ and namely enters logarithmic phase, and 14h enters stationary phase.
Reference literature (Zhao Bin, He Shaojiang. Experiment on Microbiology handbook [M]. Beijing: Science Press, 2002:251-255) method is carried out sugar fermentating test, physiological biochemical character test to the Bacterium lacticum grx12 of separation and purification, the results are shown in Table 9 and table 10.According to table 9 and table 10, can tentatively Bacterium lacticum grx12 be accredited as lactobacterium casei.
Reference literature (Zhang Jie, Xu Guihua, You Liqin .16S rDNA sequence analysis identify milk-acid bacteria [J]. processing of farm products innovation version, 2009,4:47-49,69) and method carries out 16S rDNA sequential analysis to the Bacterium lacticum grx12 of separation and purification and identifies.
Total DNA with Bacterium lacticum grx12 is template, adopt universal primer to carry out pcr amplification, obtain the specific amplification products (see figure 4) of an about 1500bp of length, and the PCR product is served the sea give birth to the order-checking of worker bio-engineering corporation, sequencing result is submitted to NCBI website gene pool by the Blast program and carries out the homology search.Search Results shows that the 16S rDNA sequence homology of the 16S rDNA sequence of Bacterium lacticum grx12 and many strains lactobacterium casei (Lactobacillus casei) is up to 99%.Therefore, in conjunction with the microbiology form, sugar-fermenting, Physiology and biochemistry experiment and 16S rDNA sequencing are accredited as lactobacterium casei with Bacterium lacticum grx12 of the present invention, and called after Lactobacillus casei grx12.。
The experiment of table 9grx12 sugar-fermenting
| Carbohydrate | The result | Carbohydrate | The result |
| Amygdaloside | ＋ | Sucrose | ＋ |
| Pectinose | － | Sunmorl N 60S | ＋ |
| Fructose | ＋ | Trehalose | ＋ |
| N.F,USP MANNITOL | ＋ | Maltose | ＋ |
| Raffinose | － | Melizitose | ＋ |
| Rhamnosyl | － | Melibiose | － |
| Wood sugar | － | Vitamin C2 | ＋ |
The experiment of table 10grx12 Physiology and biochemistry
| Testing index | The result |
| Gelatine liquefication | － |
| Produce indoles | － |
| Produce hydrogen sulfide | － |
| Catalase | － |
7. the tolerance of the simulated gastric fluid of lactobacterium casei grx12, intestinal juice and cholate
As the probiotic bacterium of excellent property, play a role at human body, must tolerate gastric juice and the intestinal juice of human body, and after by gi tract, can guarantee that still viable count is 1 * 10
6Competence exertion biological activity when cfu/mL is above.Gastric juice pH is generally about 3.0, and normal human's small intestine bile salt levels is between 0.03%-0.30%; Food was generally about 2h by the GI time.
Aforementioned acidproof and bile tolerance test-results tentatively shows lactobacterium casei grx12 at pH3.0 and to contain in the MRS liquid nutrient medium of 0.2% cholate tolerance better, but its cholate tolerance to simulated gastric fluid, intestinal juice and different concns is not known as yet.Therefore measured the tolerance of the simulated gastric fluid of the pH2.0 of lactobacterium casei grx12 and pH3.0; To the tolerance of simulated intestinal fluid and to the tolerance of different concns cholate.
The preparation of simulated gastric fluid: the pH that regulates sterile distilled water with the hydrochloric acid of 1mol/L is respectively 2.0 and 3.0, add 0.2%NaCl respectively, 1% stomach en-is fully after the dissolving, with aperture 0.20 μ m millipore filtration filtration sterilization in the aseptic technique platform, it is standby to change in the aseptic reagent bottle cryopreservation over to.
The preparation of simulated intestinal fluid: get KH
2PO
43.4g, add the 250mL dissolved in distilled water, regulate pH to 6.8 with 0.4%NaOH solution, adding distil water is to 500mL, add 0.2%NaCl respectively, 1% trypsinase is fully after the dissolving, with aperture 0.20 μ m millipore filtration filtration sterilization in the aseptic technique platform, it is standby to change in the aseptic reagent bottle cryopreservation over to.
(1) the simulated gastric fluid tolerance test of lactobacterium casei grx12.
The lactobacterium casei grx12 in two generations of activation is inoculated in the pH value by 10% inoculum size to be respectively in 2.0 and 3.0 the simulated gastric fluid.Mixing, 37 ℃ of cultivations respectively at 0,1,2 and 3h sampling, are adopted MRS solid medium plate count.The results are shown in Table 11.
The tolerance of the simulated gastric fluid of table 11grx12
As shown in Table 11, the simulated gastric fluid of the pH2.0 of lactobacterium casei grx12 and pH3.0 has good tolerance, and behind the cultivation 3h, survival rate is 52.75% in the simulated gastric fluid of pH3.0, and viable count is 2 * 10
9More than the fu/mL.Viable count still has 10 cultivate 3h in the simulated gastric fluid of pH2.0 after
9More than the fu/mL.
(2) the simulated intestinal fluid tolerance test of lactobacterium casei grx12.
The lactobacterium casei grx12 in two generations of activation is inoculated in simulated intestinal fluid by 10% inoculum size, mixing, 37 ℃ of cultivations respectively at 0,1,2 and 3h sampling, are adopted MRS solid medium plate count.The results are shown in Table 12.
The tolerance of the simulated intestinal fluid of table 12grx12
As shown in Table 12, the simulated intestinal fluid of lactobacterium casei grx12 has good tolerance, and behind the cultivation 3h, survival rate is 50.51%, and viable count is also far away more than 1 * 10
6Cfu/mL.
(3) the cholate tolerance test of lactobacterium casei grx12
In the MRS liquid nutrient medium, add bovine bile, making its mass concentration is 0.30,0.50 and 1.00g/100mL, 121 ℃ of sterilization 30min, cool off standby, lactobacterium casei grx12 after the activation is seeded in the substratum after the above-mentioned processing by 5% inoculum size, behind 37 ℃ of cultivation 24h, measure the OD of above-mentioned different concns substratum respectively
600nmValue, and be contrast with the substratum that does not contain cholate, the tolerance of the cholate of lactobacterium casei grx12 calculated.The results are shown in Table 13.
The tolerance of the cholate of table 13grx12
As shown in Table 13, the certain tolerance of the cholate tool of lactobacterium casei grx12 is cultivated 24h in the MRS of 0.3% cholate substratum, and tolerance is 25.34%; Still having tolerance preferably and cultivate 24h in the MRS of 1% cholate substratum, is 11.09%.
At present, oral is the main path that probiotic bacterium enters human intestinal.And probiotic bacterium will be brought into play physiologically active in enteron aisle, must stand the influence of gastric juice, intestinal juice and small intestine middle and high concentration cholate.Only surviving rate surpasses 10 in gastric juice, intestinal juice and cholate
6The effect of cfu/mL competence exertion; Simultaneously, in the product in stable viable count or the probiotics amount of viable bacteria how much 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.
The data of consolidated statement 11～table 13 as can be known, the contrary environment of the digestive tube of lactobacterium casei grx12 of the present invention has good tolerance, survival rate is higher therein.Therefore, this bacterial strain can be used for the production of functional cultured milk prod, also can be used as functional food additives or probiotics for all respects such as adjusting functions of human body.
Application Example 1: utilize lactobacterium casei grx12 to prepare fermented-milk
At first, preparation lactobacterium casei grx12 starter.
The lactobacterium casei grx12 culture of activation after 2 generations is inoculated in the heat treated recovery skimming milk of 95 ℃/5min (10%) by 3% inoculum size, cultivates 16h in 37 ℃, be cooled to 4 ℃ for the preparation of starter, the viable count of this starter is about 10
9Cfu/mL.
Secondly, will restore whole-milk (11.5%) and be heated to about 50 ℃, add 6% sucrose, after fully dissolving, will restore whole-milk and be preheating to about 60 ℃, under 20MPa pressure, carry out homogeneous.To restore whole-milk thermal treatment under 95 ℃/5min condition then, to be cooled to 38 ℃.Insert lactobacterium casei grx12 starter by 5% inoculum size, to curdled milk, the cooling back namely obtains having the lactobacterium casei grx12 fermented-milk of anti-oxidant function in 4 ℃ of refrigerations at 37 ℃ of bottom fermentation 14-18h.
Application Example 2: utilize lactobacterium casei grx12 and streptococcus thermophilus grx 02 mixed fermentation to prepare fermented-milk
Streptococcus thermophilus grx 02 has higher anti-oxidant activity and has the patented strain of alcoholic liver damage protection function for what the inventor obtained, and the patent No. is ZL200810023012.0, and the bacterial strain preserving number is CGMCC No:2525.
Prepare lactobacterium casei grx12 and streptococcus thermophilus grx 02 starter at first, respectively
Lactobacterium casei grx12 and the streptococcus thermophilus grx 02 culture that will activate respectively after 2 generations are inoculated in the heat treated recovery skimming milk of 95 ℃/5min (10%) by 3% inoculum size, be cultured to curdled milk respectively at 37 ℃ and 42 ℃, be cooled to 4 ℃ for the preparation of starter.
Secondly, will restore whole-milk (11.5%) and be heated to about 50 ℃, add 6% sucrose, after fully dissolving, will restore whole-milk and be preheating to about 60 ℃, under 20MPa pressure, carry out homogeneous.To restore whole-milk thermal treatment under 95 ℃/5min condition then, to be cooled to about 42 ℃.Insert lactobacterium casei grx12 starter by 2.5% inoculum size, and add 2.5% streptococcus thermophilus grx 02 starter, to curdled milk, the cooling back namely obtains having lactobacterium casei grx12 and the streptococcus thermophilus grx 02 mixing cultured milk of anti-oxidant function in 4 ℃ of refrigerations at 42 ℃ of bottom fermentations.
Claims (3)
1. a lactobacterium casei (Lactobacillus casei) grx12, its preserving number is CGMCC No:7696.
2. the described lactobacterium casei grx12 of claim 1 has the functional food of antioxygenation or the application in the medicine in preparation.
3. the described lactobacterium casei grx12 of claim 1 has purposes in the cultured milk prod of anti-oxidant function in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013103127847A CN103343107A (en) | 2013-07-23 | 2013-07-23 | Human lactobacillus casei gr x 12 with antioxidant function and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013103127847A CN103343107A (en) | 2013-07-23 | 2013-07-23 | Human lactobacillus casei gr x 12 with antioxidant function and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103343107A true CN103343107A (en) | 2013-10-09 |
Family
ID=49277934
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013103127847A Pending CN103343107A (en) | 2013-07-23 | 2013-07-23 | Human lactobacillus casei gr x 12 with antioxidant function and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103343107A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103893215A (en) * | 2014-04-17 | 2014-07-02 | 扬州大学 | Application of lactobacillus casei grx12 in preparation of product for treating chronic alcoholic liver injury |
| CN106399162A (en) * | 2016-09-08 | 2017-02-15 | 济南康多宝生物技术有限公司 | Novel lactobacillus casei and application thereof |
| RU2617946C1 (en) * | 2015-11-24 | 2017-04-28 | Автономная Некоммерческая Организация "Научно-Исследовательский Центр Биотехнологии Антибиотиков И Других Биологически Активных Веществ "Биоан" | Lactobacillus brevis and lactobacillus rhamnosus strains with established genomic sequency synthesizing glutathion and intracellular antioxidants complex |
| CN106858606A (en) * | 2016-12-28 | 2017-06-20 | 石家庄君乐宝乳业有限公司 | Probiotic powder with anti-oxidation function and preparation method thereof |
| CN109430380A (en) * | 2018-12-10 | 2019-03-08 | 扬州大学 | A kind of source of people mixing lactic acid bacteria acidified milk and preparation method thereof reducing cholesterol effect with auxiliary |
| CN111297914A (en) * | 2020-02-24 | 2020-06-19 | 北京农学院 | Application of lactobacillus fermentation clear liquid in antioxidation or preparation of product for antioxidation and antioxidation product |
| CN111705025A (en) * | 2020-08-03 | 2020-09-25 | 江西善行生物科技有限公司 | Preparation of lactobacillus casei and application of lactobacillus casei in anti-aging aspect |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101144065A (en) * | 2007-09-03 | 2008-03-19 | 江南大学 | Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof |
| CN101333505A (en) * | 2007-06-26 | 2008-12-31 | 内蒙古农业大学 | Applications of lactobacillus casei for antioxidation |
-
2013
- 2013-07-23 CN CN2013103127847A patent/CN103343107A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333505A (en) * | 2007-06-26 | 2008-12-31 | 内蒙古农业大学 | Applications of lactobacillus casei for antioxidation |
| CN101144065A (en) * | 2007-09-03 | 2008-03-19 | 江南大学 | Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| YONG ZHANG ET AL.: "The antioxidative eVects of probiotic Lactobacillus casei Zhang on the hyperlipidemic rats", 《EUR FOOD RES TECHNOL》 * |
| 黄玉军等: "6株人源乳酸菌体外抗氧化活性的比较", 《现代食品科技》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103893215A (en) * | 2014-04-17 | 2014-07-02 | 扬州大学 | Application of lactobacillus casei grx12 in preparation of product for treating chronic alcoholic liver injury |
| RU2617946C1 (en) * | 2015-11-24 | 2017-04-28 | Автономная Некоммерческая Организация "Научно-Исследовательский Центр Биотехнологии Антибиотиков И Других Биологически Активных Веществ "Биоан" | Lactobacillus brevis and lactobacillus rhamnosus strains with established genomic sequency synthesizing glutathion and intracellular antioxidants complex |
| CN106399162A (en) * | 2016-09-08 | 2017-02-15 | 济南康多宝生物技术有限公司 | Novel lactobacillus casei and application thereof |
| CN106858606A (en) * | 2016-12-28 | 2017-06-20 | 石家庄君乐宝乳业有限公司 | Probiotic powder with anti-oxidation function and preparation method thereof |
| CN109430380A (en) * | 2018-12-10 | 2019-03-08 | 扬州大学 | A kind of source of people mixing lactic acid bacteria acidified milk and preparation method thereof reducing cholesterol effect with auxiliary |
| CN111297914A (en) * | 2020-02-24 | 2020-06-19 | 北京农学院 | Application of lactobacillus fermentation clear liquid in antioxidation or preparation of product for antioxidation and antioxidation product |
| CN111705025A (en) * | 2020-08-03 | 2020-09-25 | 江西善行生物科技有限公司 | Preparation of lactobacillus casei and application of lactobacillus casei in anti-aging aspect |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105132322B (en) | One lactobacillus plantarum and application thereof | |
| CN104845912B (en) | One lactobacillus plantarum | |
| CN105018379B (en) | One plant of active lactobacillus plantarum of tool high anti-oxidation and its application | |
| CN103343107A (en) | Human lactobacillus casei gr x 12 with antioxidant function and application thereof | |
| CN101144065B (en) | Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof | |
| CN103937709B (en) | Lactobacillus paraceasi FM-LP-4 with acid resistance and high reducing activity and uses thereof | |
| CN104651268B (en) | A kind of Lactobacillus plantarum and its application | |
| CN102533588B (en) | Lactobacillus brevis for producing extracellular exopolysaccharide and application thereof | |
| CN105132318A (en) | Lactobacillus plantarum grx16 and application thereof | |
| CN103013879A (en) | Bacterial strain having capacity of giving high yield of Gamma-aminobutyric acid | |
| CN103540545A (en) | Pediococcus pentosaceus and application thereof | |
| KR20210088408A (en) | Lactobacillus Plantarum and uses thereof | |
| CN105400727A (en) | Lactobacillus paracasei with antioxidant activity and application thereof | |
| CN107201322A (en) | Bacillus subtilis and its application for degrading aflatoxin B 1 | |
| CN106883995A (en) | Pediococcus acidilactici JQII-5 bacterial strains and application thereof | |
| CN105420150A (en) | Lactobacillus acidophilus and application thereof | |
| CN113980848A (en) | Pediococcus pentosaceus SBC5 and application thereof | |
| CN105132321A (en) | Enterococcus faecium, culture medium thereof for high-density solid-state fermentation, and high-density solid-state fermentation method | |
| CN110129219B (en) | Pediococcus pentosaceus and application thereof | |
| CN109022313A (en) | One lactobacillus plantarum | |
| CN108330086A (en) | A kind of extracellular polysaccharide spatial plant lactobacillus SS18-33 and its application in improving biological antioxidant activity | |
| CN104877940B (en) | One plant of streptococcus thermophilus | |
| CN108018248B (en) | Lactobacillus casei capable of regulating flora structural disorder caused by antibiotics | |
| CN105624071A (en) | Lactobacillus salivarius XJP2 and application thereof | |
| CN103966139A (en) | Lactobacillus brevis capable of producing gamma-aminobutyric acid at high yield in Sichuan pickle vegetables |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20131009 |