CN105420150A - Lactobacillus acidophilus and application thereof - Google Patents
Lactobacillus acidophilus and application thereof Download PDFInfo
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- CN105420150A CN105420150A CN201510895324.0A CN201510895324A CN105420150A CN 105420150 A CN105420150 A CN 105420150A CN 201510895324 A CN201510895324 A CN 201510895324A CN 105420150 A CN105420150 A CN 105420150A
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- CEQFOVLGLXCDCX-WUKNDPDISA-N Methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- ROWKJAVDOGWPAT-UHFFFAOYSA-N acetoin Chemical compound CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 description 2
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Abstract
The invention relates to lactobacillus acidophilus with high gastrointestinal tract stress resistance, wherein the preservation number of the lactobacillus acidophilus is CGMCC No.10436. The screened lactobacillus acidophilus has high gastrointestinal tract stress resistance, and can be applied to fields of food industry, medicine and the like, and thus a theoretical basis is laid for the further research on the gastrointestinal tract stress resisting mechanism of the lactobacillus acidophilus.
Description
Technical field
The present invention relates to a strain and there is the Lactobacterium acidophilum (Lactobacillusacidophilus) that high anti-gastrointestinal tract coerces, belong to microbial technology field.
Background technology
ExpertCommittee define probiotic bacterium be " live microorganism, with some amount absorption, benefit can be produced to health; and this benefit surmounts general intrinsic nutrition ", this shows: probiotic bacterium must be alive, will exist with high quantity simultaneously, and usual total daily dose is 10
9cell concentration.Most of bacterial strain of probiotic bacterium is lactobacillus and genus bifidobacterium, because these milk-acid bacterias produce the log history that cultured milk prod has a segment length, so be used always.
Probiotic bacterium enters body, needs tolerance gi tract sour environment and gi tract cholate environment, so how to tolerate hydrochloric acid in gastric juice, intestines acid and bile, becomes the prerequisite that probiotic bacterium plays a role.Therefore, filter out and there is the milk-acid bacteria that high anti-gastrointestinal tract coerces be very important.
The probiotic bacterium that Northeast Agricultural University's Master of engineering academic dissertation " anti-gastrointestinal tract coerces screening and the product development of probiotic bacterium " (Tian Fen an etc.) literary composition is coerced with regard to anti-gastrointestinal tract is screened, and utilize bacterial ecology, physiology and gene sequencing, filter out performance preferably 5 probiotics as research object, be respectively Lactobacterium acidophilum KLDSAD1 and AD2, animal bifidobacteria KLDS2.0001, 2.0002 with 2.0604, continue follow-up research, and show that Lactobacterium acidophilum and animal bifidobacteria have well acid and biliary salts tolerance, the acid resistance of animal bifidobacteria is comparatively better than the acid resistance of Lactobacterium acidophilum.
Although the hydrochloric acid in gastric juice tolerance that the equal tool of Lactobacterium acidophilum KLDSAD1 and AD2 that research draws is good, but the good gi tract that have under still can not reaching peracid condition coerce ability, therefore, on this basis, applicant carried out further research, the higher anti-gastrointestinal tract of screening coerces the Bacterium lacticum of ability further.
Summary of the invention
An object of the present invention filters out a kind of Lactobacterium acidophilum CGMCC10436, and it has higher gi tract and coerces ability.
Another object of the present invention is the application of openly this Lactobacterium acidophilum.
To achieve these goals, the present invention adopts following technical scheme:
One strain Lactobacterium acidophilum (Lactobacillusacidophilus) CGMCC10436, this bacterial strain filters out in the milk-acid bacteria be separated from traditional dairy products, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on 01 26th, 2015, microbial preservation number is CGMCCNo.10436, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Concrete, described Lactobacterium acidophilum comes from the rare cream of tradition or acidifying cream that gather traditional method making in the herdsman family in the areas such as In Xilingol League In Inner Mongolia, Hailar City and Ordos City, for the screening of milk-acid bacteria.Specific as follows:
(1) 25g sample is aseptically shredded, be dissolved in 225mL stroke-physiological saline solution, after gradient dilution, select suitable extent of dilution, get 1mL diluent and add sterilized petri dishes, be poured on 20mLMRS solid medium, cultivate 72h for 30 DEG C.
(2) choose colony counts 15 ~ 150 flat board, random selecting meets the bacterium colony of milk-acid bacteria colony morphology characteristic, and therefrom a small amount of bacterium of picking, is dissolved in MRS liquid nutrient medium, and carry out 30 DEG C cultivate 48h.
(3) by the bacterial strain be separated on MRS solid medium repeatedly line carry out purifying.
(4) bacterial strain good for purifying is carried out gramstaining and catalase experiment.
(5) all Gram-positives, negative catalase, the bacterial strain (being tentatively judged as milk-acid bacteria) that do not form gemma are elected, glycerine is added in its nutrient solution, make the ultimate density of glycerine be 30% (v/v), be stored in-80 DEG C stand-by.
(6) the above-mentioned bacterial strain of fixing tentatively as milk-acid bacteria that is separated to is activated the laggard row filter experiment of 3 generations in MRS liquid nutrient medium.
(7) with reference culture Lactobacterium acidophilum bacterial strain in contrast, carry out following physiological and biochemical test: H2S test, methyl red test, litmus milk tests, gelatin liquification test, Starch Hydrolysis test, Citrate trianion utilize test, urea decomposition test, nitrate Degrading experiment and acetoin to produce test, choose the milk-acid bacteria identical with reference culture Lactobacterium acidophilum physiological and biochemical test.
(8) strain gene group DNA is extracted, carry out 16SrDNA order-checking, the 16SrDNA sequence of bacterial strain of the present invention and the sequence of NCBI Lactobacterium acidophilum are compared, identify, this bacterial strain is Lactobacterium acidophilum (Lactobacillusacidophilus).
Then, Lactobacterium acidophilum of the present invention and Lactobacterium acidophilum (Lactobacterium acidophilum KLDSAD1, Lactobacillus acidophilus Fermented Milk bacillus KLDSAD2) are contrasted, the anti-gastrointestinal tract studying the Lactobacterium acidophilum this time filtered out coerces ability.Test-results shows, the Lactobacterium acidophilum that this research filters out all has good tolerance to acid solution, intestinal juice and artificial cholate, and successful is better than Lactobacterium acidophilum KLDSAD1, lactobacterium lactis KLDSAD2.
The Lactobacterium acidophilum screened has high anti-gastrointestinal tract and coerces ability.
And described Lactobacterium acidophilum can make cultured milk prod, these cultured milk prods are selected from yoghourt, whey lactobacillus drink, cheese, the processed food containing fermented yogurt, the protective foods etc. containing fermented yogurt.Described processed food or the raw material of protective foods are selected from animals milk, milk casein, cereal, corn protein, wheat, aleuronat, soybean, Soybean Milk, skimmed soy beans, soy-protein and composition thereof etc., and are not limited to this.The content of concrete Lactobacterium acidophilum in milk-product can add according to actual needs.
Further, described cultured milk prod also comprises bifidus bacillus or other all probiotic bacteriums beneficial to enteron aisle.
Described Lactobacterium acidophilum can also be made has the active bacteria formulation that high anti-gastrointestinal tract coerces function, and preparation comprises all acceptable dosage forms clinically such as capsule, tablet, oral liquid, pulvis.
Beneficial effect of the present invention:
The present invention filters out a strain by acidproof, bile tolerance and bacteriostatic test etc. to be had high anti-gastrointestinal tract and coerces active Lactobacterium acidophilum, and it effectively can be made milk-product or pharmaceutical preparation and keep active, is widely used in the field such as foodstuffs industry, medicine.
The Lactobacterium acidophilum that the present invention filters out has the ability that higher anti-gastrointestinal tract is coerced, for the anti-gastrointestinal tract studying Lactobacterium acidophilum further coerces mechanism based theoretical.
Accompanying drawing explanation
Fig. 1 is the phylogenetic tree of Lactobacterium acidophilum of the present invention based on 16SrDNA complete sequence.
Fig. 2 is the tolerance of Lactobacterium acidophilum of the present invention to simulated gastric fluid;
Fig. 3 is the tolerance of Lactobacterium acidophilum KLDSAD1 to simulated gastric fluid;
Fig. 4 is the tolerance of Lactobacterium acidophilum KLDSAD2 to simulated gastric fluid;
Fig. 5 is that three strain lactobacilluss are to the tolerance of simulated intestinal fluid;
Fig. 6 is the bile tolerance ability of Lactobacterium acidophilum KLDSAD1;
Fig. 7 is the bile tolerance ability of Lactobacterium acidophilum KLDSAD2;
Fig. 8 is the bile tolerance ability of Lactobacterium acidophilum of the present invention;
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
One, material and reagent
Bacterial strain: Lactobacterium acidophilum KLDSAD1 and Lactobacterium acidophilum KLDSAD2 are all separated from the Inner Mongol rare cream of tradition or acidifying cream, by DSMZ of key lab of the dairy science the Ministry of Education (KLDS-DICC) preservation, bacterial classification activates above bacterial strain with MRS substratum before using, at 37 DEG C, cultivate 14h with 2% inoculum size, use in order to experiment;
Substratum: MRS substratum.
MRS substratum is prepared: peptone 5g/L, extractum carnis 5g/L, Tryptones 10g/L, yeast powder 5g/L, glucose 20g/L, anhydrous sodium acetate 5g/L, citric acid hydrogen diamine 2g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.58g/L, manganous sulfate 0.25g/L, tween-80 1ml/L, pH6.3 ± 0.1, sterilizing in 121 DEG C, 15 minutes;
Main agents: dipotassium hydrogen phosphate: Tianjin recovery development in science and technology company limited; Manganous sulfate: the huge chemical apparatus factory in Dongli District, Tianjin; Citric acid hydrogen diamine: the huge chemical apparatus factory in Dongli District, Tianjin.
Key instrument: JY92-2D ultrasonication pulverizer: SCIENTZ company; Anaerobic box FormaAnaerobicsystem1029:THERMOELECTRONCORPORATION company; DU800 type ultraviolet spectrophotometer: BECKMANCOULTER; GL-21M type cryogenic freezing whizzer: ShangHai City centrifugal Machine Institute.
Two, the screening of lactobacillus acidophilus strains
The rare cream of tradition or the acidifying cream of traditional method making is gathered, for the screening of milk-acid bacteria from the herdsman family in the areas such as In Xilingol League In Inner Mongolia, Hailar City and Ordos City.Specific as follows:
(1) 25g sample is aseptically shredded, be dissolved in 225mL stroke-physiological saline solution, after gradient dilution, select suitable extent of dilution, get 1mL diluent and add sterilized petri dishes, be poured on 20mLMRS solid medium, cultivate 72h for 30 DEG C.
(2) choose colony counts 15 ~ 150 flat board, random selecting meets the bacterium colony of milk-acid bacteria colony morphology characteristic, and therefrom a small amount of bacterium of picking, is dissolved in MRS liquid nutrient medium, and carry out 30 DEG C cultivate 48h.
(3) by the bacterial strain be separated on MRS solid medium repeatedly line carry out purifying.
(4) bacterial strain good for purifying is all carried out gramstaining and catalase experiment.
(5) all Gram-positives, negative catalase, the bacterial strain (being tentatively judged as milk-acid bacteria) that do not form gemma are elected, glycerine is added in its nutrient solution, make the ultimate density of glycerine be 30% (v/v), be stored in-80 DEG C stand-by.
(6) above-mentioned being separated to after the bacterial strain of fixing tentatively as milk-acid bacteria activated for 3 generations in MRS liquid nutrient medium is carried out identification experiment.
Three, the qualification of lactobacillus acidophilus strains
1, Physiology and biochemistry qualification
With reference culture Lactobacterium acidophilum bacterial strain in contrast, carry out following physiological and biochemical test: H
2s test, methyl red test, litmus milk tests, gelatin liquification test, Starch Hydrolysis test, Citrate trianion utilize test, urea decomposition test, nitrate Degrading experiment and acetoin to produce test, choose the milk-acid bacteria identical with reference culture Lactobacterium acidophilum physiological and biochemical test.
2, Molecular Identification
Extract strain gene group DNA, carry out 16SrDNA order-checking, its nucleotide sequence is as shown in sequence table SeqIDNo1, the 16SrDNA sequence of bacterial strain of the present invention and the sequence of NCBI Lactobacterium acidophilum are compared, 16SrDNA sequence and the Lactobacillusacidophilus sequence similarity of bacterial strain of the present invention reach more than 95%, according to 16SrDNA sequential analysis, this bacterial strain is accredited as Lactobacterium acidophilum (Lactobacillusacidophilus), and its phylogenetic tree based on 16SrDNA complete sequence as shown in Figure 1.Identify, this bacterial strain is Lactobacterium acidophilum (Lactobacillusacidophilus).And be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 01 26th, 2015, microbial preservation number is CGMCCNo.10436, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Lactobacterium acidophilum of the present invention and Lactobacterium acidophilum KLDSAD1 and Lactobacterium acidophilum KLDSAD2 are tested as follows:
Test the tolerance test of 1 Lactobacterium acidophilum to gastric juice
First milk-acid bacteria by arriving stomach through esophagus behind oral cavity, acid environment in gastric juice and complicated digestive ferment, most of microorganism can be caused can not to survive at this, and lactobacillus must arrive the effect of enteron aisle competence exertion by stomach and with abundant quantity, therefore, be necessary that research lactobacillus is to the tolerance of gastric juice.The pH of gastric juice is generally about 3.0, and when empty stomach or feed acidic food, pH can reach 0.9-1.8, and during edible basic food, pH can reach 4-5, and food is approximately 1-2h by the time of stomach.The compound method of simulated gastric fluid, with reference to Pharmacopoeia of People's Republic of China, regulates pH to be 1.5,2.5,3.5,4.5 with the hydrochloric acid that concentration is 1mol/L respectively.By the three strain Lactobacterium acidophilums activated with 4% inoculum size access in the simulated gastric fluid of different pH value respectively, 37 DEG C of waters bath with thermostatic control are cultivated, respectively 0,0.5,1,1.5,2,3h sampling, spread plate method carries out live bacterial count, parallel laboratory test 3 times.The result of Lactobacterium acidophilum of the present invention, Lactobacterium acidophilum KLDSAD1 and Lactobacterium acidophilum KLDSAD2 is shown in Fig. 2, Fig. 3 and Fig. 4 respectively.
As shown in Figure 2, Lactobacterium acidophilum of the present invention is when pH is 1.5, viable count starts to decline, and reduce to 0 when 3h, when pH is 2.5-4.5, viable count logarithmic value remains on more than 7 always, and does not still have downward trend when 3h, illustrates that this bacterium all has good tolerance to the simulated gastric fluid of different pH.As shown in Figure 3, when pH is 1.5, Lactobacterium acidophilum KLDSAD1 does not just have viable bacteria when 0.5h; During pH2.5, viable count keeps stable, and has the trend of rising when 3h, illustrates that this bacterium has good adaptability to this pH bad border; When pH is 3.5 and 4.5, an order of magnitude though viable count declines, in 3h, still keep higher survival rate, therefore Lactobacterium acidophilum of the present invention and Lactobacterium acidophilum KLDSAD1 all have good tolerance to simulated gastric fluid.As shown in Figure 4, when pH is 1.5, KLDSAD2 viable count when 0.5h also reduces to 0; When pH is respectively 2.5,3.5,4.5, viable count starts to decline over time, drop to 1 × below 106CFU/mL when 2h, due to food through time of stomach generally at 1-2h, therefore we think that Lactobacterium acidophilum KLDSAD2 also has tolerance to simulated gastric fluid.It can thus be appreciated that the capacity of water of three strain Lactobacterium acidophilum tolerance simulated gastric fluids is: Lactobacterium acidophilum > Lactobacterium acidophilum KLDSAD1 > Lactobacterium acidophilum KLDSAD2 of the present invention.
Test the tolerance test of 2 Lactobacterium acidophilums to intestinal juice
Small intestine is the ground of living away from home of most of microbe in body, is also the main place that various probiotic oral plays prebiotic effect.Probiotic bacterium needs adapt to small intestine condition and keep maximum keep alive rate just likely in small intestine, to play prebiotic effect.The pH of intestinal fluid is approximately 7.6, and containing multiple digestive ferment and bile acide etc., wherein trypsinase is topmost digestive ferment.Food is about 1.5h through the time of small intestine.The compound method of simulated intestinal fluid is with reference to Pharmacopoeia of People's Republic of China.By the lactobacillus activated with 4% inoculum size be inoculated in the simulated intestinal fluid configured respectively, 37 DEG C of waters bath with thermostatic control are cultivated, respectively 0,0.5,1,1.5,2,3h sampling, spread plate method carries out live bacterial count, parallel laboratory test 3 times.The results are shown in Figure 5.
As shown in Figure 5, three strain Lactobacterium acidophilums all have good tolerance to simulated intestinal fluid, and during 0-2h, live lactobacillus acidophilus number of the present invention rises to some extent, illustrate that the tolerance of this bacterium and adaptive faculty are better than other two strains bacterium.When 1.5h, though Lactobacterium acidophilum KLDSAD2 and Lactobacterium acidophilum KLDS1015 declines to some extent, viable count logarithmic value still remains on more than 7.Therefore, in simulation simulated intestinal fluid environment, three strain Lactobacterium acidophilums all have good tolerance performance, and strong and weak size is Lactobacterium acidophilum > Lactobacterium acidophilum KLDSAD2 > Lactobacterium acidophilum KLDSAD1 of the present invention.
Test the tolerance test of 3 Lactobacterium acidophilums to cholate
Add oxgall powder at MRS liquid nutrient medium, regulate its concentration to be respectively 1,2,3,4g/kg, 121 DEG C, 15min autoclaving.By the lactobacillus activated with 4% inoculum size be inoculated in the substratum of different concns, 37 DEG C of constant temperature culture, respectively 0,0.5,1,1.5,2,3h sampling, spread plate method live bacterial count, parallel laboratory test 3 times.
In human intestinal, the gallbladder salinity of different sites is different, and wherein in duodenum, gallbladder salinity fluctuates within the scope of 0.3-3g/kg, and food is short by duodenal time antipode.Probiotic bacterium must can tolerate the cholate of different concns, and maintains certain survival rate, the performance of its prebiotic effect of guarantee, and therefore, it is very important for evaluating the tolerance of probiotic bacterium to cholate.Three strain Lactobacterium acidophilums are suspended in the MRS liquid nutrient medium containing different concns cholate, in 37 DEG C of constant temperature culture, 0,0.5,1,1.5,2,3h sampling and measuring viable count respectively.
Lactobacterium acidophilum KLDSAD1 and Lactobacterium acidophilum KLDSAD2 viable count in the cholate of surveyed different concns rises (seeing Fig. 6 and Fig. 7 respectively) all to some extent, illustrates that this two strains bacterium has good tolerance to the gallbladder salinity in body scale.As shown in Figure 8, when gallbladder salinity is within the scope of 1g/kg-3g/kg, Lactobacterium acidophilum of the present invention has good tolerance, and when gallbladder salinity is 4g/kg, this bacterium quantity after 1h declines gradually, but its viable count is still 1 × 10 when 3h
7more than CFU/mL, because food compole when duodenal is short, so Lactobacterium acidophilum also can tolerate the environment of gallbladder salinity well at this moment.
Test example 4
Fermented milk is produced with the above-mentioned Lactobacterium acidophilum filtered out.Lactobacillus acidophilus strains is 37 DEG C of cultivation 14h in 100g10% (weight percent) skimming milk, be blended in 37 DEG C mutually afterwards continue cultivation 14 hours with the fresh culture of 3.2kg.After fermentation, whole fermented milk (lactobacillus acidophilus cells's number 3.4 × 10
8individual/mL) as above-mentioned skimming milk starter again at 35 DEG C cultivate within 22 hours, obtain fermented milk.
Can also obtained yogurt drink further on this basis, the fermented milk that 44kg obtained as got and 2.75kg white sugar, 3kg water, the homogenate of 0.25kg high methoxyl pectin obtain 50kg yoghourt drink.Yoghourt beverage has good agreeable to the taste sense, records pH4.2, and viable count is 1.6 × 10
8individual/mL.
And be not limited to this, can also conventionally be processed into various milk-product, addition also can increase and decrease according to practical situation, and described milk-product can also add other bacterium, such as bifidus bacillus, to this not restriction.
Experimental example 5
Pharmaceutical preparation is made with the above-mentioned Lactobacterium acidophilum filtered out.By above-mentioned Lactobacterium acidophilum MRS culture medium culturing; Get the Lactobacterium acidophilum bacterium liquid 50ml cultivating 14h, centrifuging and taking bacterium mud, is then dissolved in the aseptic PBS of 1ml, obtains bacterium mud; Bacterium mud is stirred, then conventionally adds suitable auxiliary material and make clinical acceptable formulation.Formulation comprises the various formulations such as capsule, tablet, pulvis.
Claims (7)
1. a Lactobacterium acidophilum (Lactobacillusacidophilus), is characterized in that, described Lactobacterium acidophilum is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNo.10436.
2. a cultured milk prod, is characterized in that, comprises Lactobacterium acidophilum according to claim 1 in this cultured milk prod.
3. cultured milk prod according to claim 2, is characterized in that, also comprises bifidus bacillus in this cultured milk prod.
4. the cultured milk prod according to Claims 2 or 3, is characterized in that, described cultured milk prod is selected from yoghourt, whey lactobacillus drink, cheese, the processed food containing fermented yogurt or the protective foods containing fermented yogurt.
5. cultured milk prod according to claim 4, it is characterized in that, the raw material of processed food used or protective foods is selected from animals milk, milk casein, cereal, corn protein, wheat, aleuronat, soybean, Soybean Milk, skimmed soy beans, soy-protein and composition thereof.
6. have the active bacteria formulation that high anti-gastrointestinal tract coerces function, it is characterized in that, this active bacteria formulation comprises Lactobacterium acidophilum according to claim 1.
7. according to claim 6 have the active bacteria formulation that high anti-gastrointestinal tract coerces function, and it is characterized in that, described active bacteria formulation comprises capsule, tablet, oral liquid, pulvis.
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| CN109136129A (en) * | 2018-08-27 | 2019-01-04 | 南昌大学 | One plant of lactobacillus acidophilus NCU426 |
| CN109136129B (en) * | 2018-08-27 | 2021-08-20 | 南昌大学 | Lactobacillus acidophilus NCU426 |
| WO2020083119A1 (en) * | 2018-10-22 | 2020-04-30 | 华南理工大学 | Lactobacillus harbinensis and application thereof |
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