CN102399728B - Lactobacillus and fermentation method thereof - Google Patents
Lactobacillus and fermentation method thereof Download PDFInfo
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- CN102399728B CN102399728B CN201110394727.9A CN201110394727A CN102399728B CN 102399728 B CN102399728 B CN 102399728B CN 201110394727 A CN201110394727 A CN 201110394727A CN 102399728 B CN102399728 B CN 102399728B
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Abstract
The invention provides a lactobacillus and a fermentation method thereof, which belong to the technical field of biology. The lactobacillus is the lactobacillus sp. preserved in China General Microbiological Center Culture Collection Center on January 27th, 2010, and the accession number in the collection center is CGMCC No.3614, the lactobacillus belongs to the strain separated from the piglet excrement, the strain has the outstanding characteristics that the high-temperature-resistance and oxygen-resistance capability is high, and simultaneously, the strain has stable performance that the gastric acid and cholate resistance effects are realized, the lactic acid is produced, the Escherichia coli growth inhibiting effect is realized, and the like. The viable bacteria number per milliliter after the milk powder and bouillon culture medium fermentation can reach more than 6.2*10<10>.
Description
Technical field
The present invention relates to a kind of lactobacillus and fermentation process thereof, belong to biological technical field.
Background technology
The revolution of aquaculture and fodder industry has been created in the application of microbiotic in feed, breeding scale has obtained developing rapidly in the whole world, in animal and fowl fodder, add microbiotic for improving disease resistance of livestock and poultry, improve production performance and have good effect, but in feed, antibiotic life-time service, abuse exist serious negative effect.Main manifestations is antibiotic residual in the generation of pathogenic bacteria resistance to drugs and livestock product.To the mankind and livestock and poultry, treatment brings very big difficulty with prevention and control in the generation of pathogenic bacteria resistance to drugs, and antibioticly in livestock product residually people's health is produced to serious harm after entering human body by food chain.Many countries have all formulated corresponding rules and have limited use or cancel feeding antibiotic.In antibiotic substitute research, the application of new green feed additive is a kind of inexorable trend, probiotics is the Typical Representative of new generation product, they are mainly by regulating intestinal canal microecological balance in animal body, suppress pathogenic micro-organism in enteron aisle, help animal to set up be conducive to host Micro Ecosystem, improve human body immune function, improve food utilization efficiency etc., reach and promote growth of animal, prophylactic effect.Due to probiotics safety non-toxic, noresidue, do not develop immunity to drugs, overcome microbiotic and used the drawback existing, therefore, its research and application have been a great concern.Along with people are to the attention of food safety and healthy concern, the research of probiotics and application will be more and more deep, extensive.
Milk-acid bacteria is that a class can utilize fermentable carbohydrate to produce the general name of a large amount of lactic-acid-bacteriums.This class bacterium of having found at present is at least comprising 18 genus on systematic bacteriology, wherein, lactobacillus genus and genus bifidobacterium are the dominant microfloras in animal intestinal, the probiotic formulations applicating history of these Pseudomonas more early, type of preparation is more, comprising lactobacillus-fermented feed, lactobacillus powder and milk-acid bacteria extract.Can there be as the lactobacillus of fodder additives Lactobacillus acidophilus, lactobacillus of cheese, fermentation lactobacillus and lactobacillus plantarum, saliva lactobacillus, short and small lactobacillus etc.Bifidus bacillus is in many probiotics Product labellings, to indicate a kind of probiotic strain containing.Because bifidus bacillus is the dominant bacteria in human body and animal intestinal, an important indicator of its quantity Chang Zuowei intestinal microecology balance, the bifidobacterium bacterial strains often using in probiotics product has bifidobacterium infantis, bifidobacterium adolescentis, bifidus longum bb, bifidobacterium breve etc.Although milk-acid bacteria can be improved immunizing power and the production performance of animal, because milk-acid bacteria resistance is poor, hindered the application of this bacterioid in production practice, for example, to temperature sensitive, oxygen is very strong etc. to its lethality.
Although the development along with mechanical processing technique, utilize granulation, coated and cryodrying technology has made the application of lactobacillus on fodder industry become possibility, but from bacterial classification, solves the resistance problem of lactic acid bacteria formulation, is the basic method of producing this class probiotics.
Summary of the invention
First object of the present invention is to provide a kind of lactobacillus, and this lactobacillus can tolerate higher temperature and airborne oxygen, has stomach juice-resistant and cholate simultaneously, and produces lactic acid, can produce restraining effect to colibacillary Growth and reproduction.
Second object of the present invention is to provide a kind of fermentation process that contains above-mentioned lactobacillus.
The 3rd object of the present invention is to provide a kind of above-mentioned microecological preparation of lactobacillus that contains, and said preparation, to regulating animal intestine stomach microflora, maintains intestinal microflora balance, improves the aspects such as production performance and has good promoter action.
For realizing first object of the present invention, provide a kind of lactobacillus, protect a surname in this milk-acid bacteria on January 27th, 2010 at the lactobacillus Lactobacillus sp. of < < China Committee for Culture Collection of Microorganisms's common micro-organisms center > >, the preservation center numbering of registering on the books: CGMCC No.3614.
Described lactobacillus (Lactobacillus sp.) CGMCC No.3614, being a kind of piglet ight soil that is located away from, is Gram-positive, anaerobic bacillus(cillus anaerobicus), G+C content 37mol% in its DNA, form becomes elongated rod-shaped, two terminal circle, is 0.6 μ m * 1.5 μ m, with single, become two or chaining to occur, do not move, atrichia, without gemma, bacterium colony is coarse, adds tween 80 in substratum, and bacterium colony is smooth; Deep colony is the irregularly shaped of branch, non-pigment; Fermentable carbohydrates produces a large amount of lactic acid; Catalase is negative, does not produce cytopigment, and liquefy gelatin, does not decompose casein, edwardsiella hoshinae and hydrogen sulfide.
Lactobacillus provided by the invention (Lactobacillus sp.) CGMCC No.3614 is except having above-mentioned biological property, also have high temperature resistant strong with oxygen-resistant ability, there is stomach juice-resistant and cholate simultaneously, and suppress the bacterial classification of the stable performances such as Escherichia coli Growth:
1) this lactobacillus, in the environment of 80 degrees Celsius of temperature, reached more than 95% with interior survival rate at 2 minutes.Usually, lactobacillus 65-75 degree Celsius can be dead, and lactobacillus of the present invention is at this temperature, survival rate is very high.
2) this lactobacillus can normal growth and breeding under non-anaerobic condition, and under aerobic and anaerobic condition, thalline fermentation density is identical.Because milk-acid bacteria is anerobe or facultative anaerobe, oxygen has strong lethality to bacterium, and therefore, most lactic acid bacteria must under anaerobic could growth and breeding.
3) this lactobacillus, in the environment of pH=2, reached more than 93% with interior survival rate at 3 hours.Usually, the pH of animal gastric juice is 2～5, and this sour environment condition has very strong lethal effect to the lethality rate of microorganism, and lactobacillus of the present invention is under this acidic conditions, and survival rate is very high.
4) in the environment that this lactobacillus is 0.5% at pig cholate content, process 3 hours, survival rate is more than 97%.Cholate has sterilization and bacteriostasis effect, and the content in chyme is generally in 0.2-0.6% left and right, and therefore, bacterial classification of the present invention can tolerate cholate.
5) intestinal bacteria and Salmonellas bacterium are the major microorganisms that causes animal generation digestive tract diseases, so the growth that suppresses intestinal bacteria and Salmonellas bacterium can be controlled the generation of digestive tract diseases, bacterial classification of the present invention can produce restraining effect to the Growth and reproduction of intestinal bacteria and Salmonellas bacterium, and its antibacterial radius is respectively 1.3 centimetres and 1.5 centimetres of left and right.
Second object of the present invention is to provide a kind of fermentation process that contains above-mentioned lactobacillus.
Described lactobacillus fermentation method comprises the steps:
1) inoculum preparation
Lactobacillus (Lactobacillus sp.) CGMCC No.3614 is stored in seed culture medium, and utilizes this substratum to inoculate.
Described seed culture based formulas is: peptone, 10g; Beef extract, 10g; Yeast soaks powder, 5g; Dipotassium hydrogen phosphate, 2g; Citric acid two ammoniums, 2g; Glucose, 20g; Sodium-acetate, 5g; Tween 80,1g; Distilled water, 1000ml, regulates pH to 5.4 with Glacial acetic acid.
Lactobacillus is inoculated in to seed culture and based on anaerobism pipe, cultivates, the inoculum obtaining.
Described culturing process is microorganism culturing, the conventional method of fermenting.
It is preferred, and described cultivation is under temperature is the condition of 35～38 ℃, cultivates 10-30 hour.
It is preferred, and lactobacillus is inoculated in anaerobism pipe, is to cultivate 24 hours in 37 ℃ of thermostat containers, as inoculum in temperature.
2) diastatochromogenes preparation
Described shake-flask culture based formulas is: milk powder, 2g; Peptone, 1g; Extractum carnis, 0.5g; Lactose, 1.5g; Yeast extract paste, 0.5g; Distilled water, 1000ml, regulates pH to 5.4 with Glacial acetic acid.
By the inoculum in anaerobism pipe, be inoculated in shake-flask culture base, inoculative proportion is 1.0-20%.
Described inoculation method is microorganism culturing, the conventional method of fermenting.
Described culturing process is microorganism culturing, the conventional method of fermenting.
It is preferred, and described cultivation is under temperature is the condition of 35～38 ℃, cultivates 10-30 hour.
It is preferred, and lactobacillus is inoculated in shaking flask, and inoculative proportion is 10%, is to cultivate 12 hours in 37 ℃ of thermostat containers, as diastatochromogenes in temperature.
3) preparation of fermentor cultivation thing
Described fermentative medium formula is: milk powder, 0.2%; Peptone, 0.1%; Extractum carnis, 0.05%; Sucrose, 0.15%; Yeast extract paste, 0.05%, with Glacial acetic acid, regulate pH to 5.4.
By the culture in shaking flask, be inoculated in fermentation tank culture medium, inoculative proportion is 1.0-20%.
Described inoculation method is microorganism culturing, the conventional method of fermenting.
Described culturing process is microorganism culturing, the conventional method of fermenting.
It is preferred, and described cultivation is under temperature is the condition of 35～37 ℃, cultivates 10-30 hour.
It is preferred, and lactobacillus is inoculated in fermentor tank, and inoculative proportion is 10%, is to cultivate 20 hours in 37 ℃ of fermentor tanks, as large tank culture in temperature.
Preservation explanation
Bacterial strain: lactobacillus Lactobacillus sp.
Preservation date: on January 27th, 2010
Depositary institution: Chinese microorganism strain Bao Jian management committee's common micro-organisms center
Deposit number: CGMCC No.3614
Embodiment
Below in conjunction with embodiment, introduce in detail the present invention, described embodiment is used for understanding and also unrestricted the present invention.
Embodiment 1
The present embodiment is that preserving number is the screening process of the lactobacillus of CGMCC No.3614, and concrete steps are as follows:
(1), the primary dcreening operation of bacterial classification
Utilize the complete selective medium of Rogosa SL to select row filter to the microorganism of piglet stomach mucous membrane different positions, to obtain lactobacillus.
Wherein the complete selective medium formula of Rogosa SL is prior art, is specially: Tryptones 10g, and yeast soaks powder 5g, glucose 10g, pectinose 5g, sucrose 5g, sodium-acetate 15g, citric acid ammonium 2g, potassium primary phosphate 6g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, ferrous sulfate 0.03g, tween 80 1g, agar 13g, distilled water 1000mL, with vinegar acid for adjusting pH to 5.0.
(2), the stomach juice-resistant of lactobacillus sieves again
Utilizing Rogosa SL substratum to carry out anaerobically fermenting to the lactobacillus just sifting out, is 2 with vinegar acid for adjusting pH, ferments 3 hours, carries out multiple sieve;
(3), the bile tolerance of lactobacillus sieves again
Utilize Rogosa SL substratum to carry out anaerobically fermenting to the lactobacillus just sifting out, pig gallbladder salinity is 0.5%, ferments 3 hours, carries out multiple sieve;
(4), the heatproof characteristic of lactobacillus is sieved again
Utilizing Rogosa SL substratum to carry out anaerobically fermenting to the lactobacillus just sifting out, is 2 with vinegar acid for adjusting pH, ferments 12 hours, then puts into 80 degrees Celsius of thermostat containers, observes the tolerance situation of microorganism, carries out heatproof and sieves again;
(5), the oxytolerant characteristic of lactobacillus is sieved again
Utilizing Rogosa SL substratum to ferment to the lactobacillus just sifting out, is 2 with vinegar acid for adjusting pH, under non-anaerobic condition, ferments 12 hours, then observes microbial growth situation, carries out oxytolerant and sieves again;
(6), lactobacillus antagonistic property
Utilize the screening of Mai Kangkai substratum to pathogenic colon bacillus rejection, to the standard of the screening of curing the disease property intestinal bacteria and Salmonellas rejection, be that lactobacillus will reach 1.2cm left and right to the bacteriostatic diameter of pathogenic colon bacillus, to the bacteriostatic diameter of Salmonellas, will reach 1.3cm left and right.
Through screening, obtain a strain lactobacillus Lactobacillus sp., be preserved in < < Chinese microorganism strain Bao Zang management committee's common micro-organisms center > > on January 27th, 2010, preserving number is CGMCC No.3614.This bacterium characteristic is for being Gram-positive, anaerobic bacillus(cillus anaerobicus), G+C content 37mol% in its DNA, form becomes elongated rod-shaped, two terminal circle, is 0.6 μ m * 1.5 μ m, with single, become two or chaining to occur, do not move, atrichia, without gemma, bacterium colony is coarse, adds tween 80 in substratum, and bacterium colony is smooth; Deep colony is the irregularly shaped of branch, non-pigment; Fermentable carbohydrates produces a large amount of lactic acid; Catalase is negative, does not produce cytopigment, and liquefy gelatin, does not decompose casein, edwardsiella hoshinae and hydrogen sulfide.
Embodiment 2
The lactobacillus that embodiment 1 is obtained (Lactobacillus sp.) preserving number: CGMCC No.3614 is inoculated in anaerobism pipe, and seed culture based formulas used is: peptone, 10g; Beef extract, 10g; Yeast soaks powder, 5g; Dipotassium hydrogen phosphate, 2g; Citric acid two ammoniums, 2g; Glucose, 20g; Sodium-acetate, 5g; Tween 80,1g; Distilled water, 1000mL, regulates pH to 5.4 with Glacial acetic acid.In thermostat container, temperature is to cultivate 24 hours at the temperature of 37 ℃, obtains inoculum.This culture can freezingly be preserved, and also can directly inoculate.
Embodiment 3
The inoculum that embodiment 2 is obtained is inoculated in shake-flask culture base, and shake-flask culture based formulas used is: milk powder, 2g; Peptone, 1g; Extractum carnis, 0.5g; Lactose, 1.5g; Yeast extract paste, 0.5g; Distilled water, 1000mL, regulates pH to 5.4 with Glacial acetic acid.Inoculative proportion is 10%, is to cultivate 12 hours in 37 ℃ of thermostat containers, as diastatochromogenes in temperature.
Experimental example 4
The diastatochromogenes that embodiment 3 is obtained is inoculated in fermentation tank culture medium, and fermentor cultivation based formulas used is: milk powder, 0.2%; Peptone, 0.1%; Extractum carnis, 0.05%; Sucrose, 0.15%; Yeast extract paste, 0.05%, with Glacial acetic acid, regulate pH to 5.4.Be inoculated in fermentor tank, inoculative proportion is 10%, is to cultivate 20 hours in 37 ℃ of thermostat containers, as large tank culture in temperature.During fermentation ends, every milliliter of the cell density of large tank culture can reach 6.2 * 10
10with last viable bacteria.After this culture is dry, can be used as probiotics uses.
Claims (3)
1. a lactobacillus (Lactobacillus sp.), it is characterized in that, this lactobacillus is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 27th, 2010, the preservation center numbering of registering on the books: CGMCC No.3614.
2. lactobacillus according to claim 1, is characterized in that, the biological characteristics of described lactobacillus is as follows:
1) be a kind of piglet ight soil that is located away from, for Gram-positive, anaerobic bacillus(cillus anaerobicus), G+C content 37mol% in its DNA, form becomes elongated rod-shaped, two terminal circle, is 0.6 μ m * 1.5 μ m, with single, become two or chaining to occur, do not move, atrichia, without gemma, bacterium colony is coarse, in substratum, add tween 80, bacterium colony is smooth; Deep colony is the irregularly shaped of branch, non-pigment; Fermentable carbohydrates produces a large amount of lactic acid; Catalase is negative, does not produce cytopigment, and liquefy gelatin, does not decompose casein, edwardsiella hoshinae and hydrogen sulfide;
2) in the environment of 80 degrees Celsius of temperature, at 2 minutes, with interior survival rate, reach more than 95%;
3) can normal growth and breeding under non-anaerobic condition, under aerobic and anaerobic condition, thalline fermentation density is identical;
4), in the environment of pH=2, at 3 hours, with interior survival rate, reach more than 93%;
5) in the environment that is 0.5% at pig cholate content, process 3 hours, survival rate is more than 97%;
6) Growth and reproduction of intestinal bacteria and Salmonellas is produced to restraining effect, its antibacterial radius is respectively 1.3 centimetres and 1.5 centimetres.
3. a fermentation process that contains lactobacillus described in claim 1 or 2, is characterized in that being divided into following steps:
1) lactobacillus obtains inoculum in seed culture medium after abundant cultivation, fermentation;
2) make diastatochromogenes;
3) make fermentor cultivation thing.
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| CN105062922B (en) * | 2015-08-12 | 2018-10-16 | 华南农业大学 | It is a kind of inhibit uropathogenic Escherichia coli Bacillus acidi lactici and its application |
| CN107988094A (en) * | 2017-11-14 | 2018-05-04 | 北京好实沃生物技术有限公司 | One plant of lactobacillus fermenti HEW-A846 and its application |
| CN116784419A (en) * | 2023-02-27 | 2023-09-22 | 北京中科景明生物技术有限公司 | Feed metazoan preparation and preparation method and application thereof |
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| KR100593255B1 (en) * | 2004-05-11 | 2006-06-28 | 대한민국 | Novel Bacillus Strain and Its Use |
| CN101475919A (en) * | 2008-12-12 | 2009-07-08 | 北京巨润农业科技发展有限公司 | A strain of lactobacillus strain, preparation of micro-ecological formulation containing the same and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100593255B1 (en) * | 2004-05-11 | 2006-06-28 | 대한민국 | Novel Bacillus Strain and Its Use |
| CN101475919A (en) * | 2008-12-12 | 2009-07-08 | 北京巨润农业科技发展有限公司 | A strain of lactobacillus strain, preparation of micro-ecological formulation containing the same and use thereof |
Non-Patent Citations (2)
| Title |
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| 仔猪源罗伊氏乳酸杆菌生物学特性的研究;黄沧海等;《云南农业大学学报》;20041231;第19卷(第6期);全文 * |
| 黄沧海等.仔猪源罗伊氏乳酸杆菌生物学特性的研究.《云南农业大学学报》.2004,第19卷(第6期),722-726. |
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Inventor after: Ma Yongxi Inventor after: Jiang Sheng Inventor after: Wu Lianfu Inventor after: Xu Xuexin Inventor after: Bian Guiping Inventor before: Jiang Sheng Inventor before: Wu Lianfu Inventor before: Xu Xuexin Inventor before: Bian Guiping |
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Free format text: CORRECT: INVENTOR; FROM: JIANG SHENG WU LIANFU XU XUEXIN BIAN GUIPING TO: MA YONGXI JIANG SHENG WU LIANFU XU XUEXIN BIAN GUIPING |