CN104593294B - A kind of high bacteriocinogeny enterococcus faecalis and its application - Google Patents
A kind of high bacteriocinogeny enterococcus faecalis and its application Download PDFInfo
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- CN104593294B CN104593294B CN201410835326.6A CN201410835326A CN104593294B CN 104593294 B CN104593294 B CN 104593294B CN 201410835326 A CN201410835326 A CN 201410835326A CN 104593294 B CN104593294 B CN 104593294B
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- enterococcus faecalis
- bacteriocin
- enterococcus
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- sodium acetate
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/097—Preservation
- A23C19/10—Addition of preservatives
- A23C19/11—Addition of preservatives of antibiotics or bacteriocins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
Abstract
The invention discloses the enterococcus faecalis of a plant height bacteriocinogeny (Enterococcus faecalis) and its application.The present invention relates to the enterococcus faecalis for the plant height bacteriocinogeny that preserving number is CGMCC NO.9966, in traditional dairy products, its caused bacteriocin obtains the strain isolation through ammonium sulfate precipitation, a series of purifying of gel chromatography.It is demonstrated experimentally that bacteriocin can be easily degraded by proteases caused by the bacterial strain, there is very strong inhibitory action to listeria monocytogenes (listeria monolytogenes), and with good heat-resisting and acid-fast ability.The bacteriocin is added in fresh cheese, can effectively suppress the growth of listeria monocytogenes, therefore, a kind of high bacteriocinogeny enterococcus faecalis of the invention has the potentiality being applied to as natural additive for foodstuff in fresh cheese.
Description
Technical field
The present invention relates to a kind of high bacteriocinogeny enterococcus faecalis and its application, belong to biological technical field.
Background technology
With constantly improving for people's living standard, the consumption of dairy products also increasingly increases, particularly always not
The cheese being accepted very much is increasingly becoming a part for people's diet, and fresh cheese refers to raw milk, dilute cream, breast
Clear or fermented dose of fermentation of their mixture, rennet curdling, excludes the product that whey is formed.Fresh cheese is making work
The step for fermenting-ripening is eliminated in skill, so that its flavor is soft light, water content is more than 67%, and quality is between Yoghourt
It is in good taste between cheese, the degree of recognition highest in consumer at home.It is most micro- but nutriment enriches in cheese
The ecotopia of biological growth breeding, especially pathogenic bacteria and spoilage organisms.Frequently manual operations will also result in cheese production
The pollution of post-processing, field planting of the fresh cheese to Listeria (L.Monocytogenes) is particularly sensitive, is particularly susceptible to Lee
The pollution of this special bacterium (L.Monocytogenes), causes microbes to poison by food, and has a strong impact on the quality of life of people, institute
To ensure that the security of cheese is particularly important.
At this stage, people mainly control the growth of microorganism in food by adding chemical preservative, extend food
Shelf-life.But long-term the eating of these chemical addition agents has very bad influence to human heart, blood pressure and kidney, even
Some chemical addition agents have the ill-effect of carcinogenesis, mutagenesis and teratogenesis.Also usually lead in the production and processing of food
Thermally-sterilized method is crossed to control the growth of microorganism, but thermally-sterilized processing mode, there is also some drawbacks, it can shadow
The quality and nutritional ingredient for ringing food can be destroyed.Although as the method for ultraviolet disinfection, filtration sterilization these physical sterilizations
The quality of food can be avoided damage to, but is packed in the infection opened and be just highly susceptible to bacterium afterwards.So develop height
Effect, natural sandy gravel stably, safe have become the highly desirable of people.
Lactic acid bacteria is widely used in food industry, and has been the quasi-microorganism that people often consume, some lactic acid
Bacterium can produce bacteriocin growing into certain phase, and bacteriocin is a kind of polypeptide, protein or albumen with antibacterial activity
Matter compound material, it can effectively suppress the growth of most of gram-positive bacterias and pathogenic bacteria, have well acidproof
And temperature capacity, it can be digested in vivo by protease hydrolytic, be good a kind of biological preservative.By bacteriocin lab
In being processed applied to fresh cheese, the mouthfeel and taste of cheese can also effectively suppress Listeria all without being affected
(L.Monocytogenes) growth.
Bacteriocin has huge space in the application of cheese as a kind of biological preservative, at this stage, commercialized thin
Rhzomorph product is also very limited, and develop new bacteriocin has turned into the expectation of people.With the continuous improvement of technology, bacteriocin
A kind of safe natural sandy gravel will be eventually become to be widely used in cheese.
The content of the invention
First purpose of the present invention is to provide a kind of enterococcus faecalis (Enterococcus of high bacteriocinogeny
Faecalis), KLDS6001 is named as, Classification And Nomenclature is enterococcus faecalis (Enterococcus faecalis), is deposited in China
Microbiological Culture Collection administration committee common micro-organisms center, address is in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Chinese Academy of Sciences
Institute of microbiology, its culture presevation numbering are：CGMCC No.9966, preservation time are on November 13rd, 2014.
Enterococcus faecalis provided by the present invention is to separate and obtain from traditional dairy products, on MRS solid mediums
Colonial morphology is circular, projection, and color and luster is milky, opaque, and Gram's staining is the positive, and thalli morphology is circle, short chain shape
Arrangement, enterococcus faecalis is accredited as through 16SrDNA.
Second object of the present invention is to provide a kind of enterococcus faecalis bacteriocin and produces the enterococcus faecalis bacteriocin
Method.
Bacteriocin provided by the present invention be by the present invention enterococcus faecalis (Enterococcus faecalis) fermentation and
Obtain.Specifically, comprise the following steps：
(1) preparation of Enterococcus faecalis fermentation liquid
Enterococcus faecalis (Enterococcus faecalis) described in claim 1 is inoculated in MRS culture mediums, 37
DEG C, obtain Enterococcus faecalis fermentation liquid after cultivating 12-16h；
(2) preliminary purification of enterococcus faecalis bacteriocin
The zymotic fluid that step (1) is obtained carries out centrifugal treating, and draws supernatant liquor, and sulphur is added into the supernatant liquor
Sour ammonium is saltoutd, and centrifugation, is collected precipitation, is redissolved in sodium acetate buffer solution, that is, obtain the crude extract of bacteriocin；
(3) enterococcus faecalis bacteriocin is secondarily purified
The crude extract of bacteriocin is further isolated and purified using glucan G-25 gel chromatography columns, produced after purification
Bacteriocin.
In method of the present invention, it is preferred that step (1) is used to cultivate enterococcus faecalis (Enterococcus
Faecalis MRS medium components) are as follows：Peptone 10.0g, beef extract 10.0g, dusty yeast 5g, glucose 20.0g, tell
Warm 801.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, lemon acid diamine 2.0g, magnesium sulfate 0.6g, manganese sulfate 0.25g and
Distilled water 1000mL.
In method of the present invention, it is preferred that the cultivation temperature in step (1) is 37 DEG C, incubation time 14h.
In method of the present invention, it is preferred that step carries out preliminary purification in accordance with the following methods in (2)：By step
(1) zymotic fluid obtained carries out centrifugal treating, and draws supernatant liquor, and the sulphur that saturation degree is 70% is added into the supernatant liquor
Sour ammonium is saltoutd, 10000 × g, 4 DEG C of centrifugation 30min, is collected precipitation, is redissolved the 0.02mol/L acetate buffers in pH6.0
In solution, that is, obtain the crude extract of bacteriocin.
In method of the present invention, it is preferred that separated in step (3) using glucan G-25 gel chromatography columns
Chromatography condition is used in purifying：Eluted with 50% (w/w) sodium chloride and the buffer solution of 50% (w/w) sodium acetate, flow velocity
For 1mL/min, sample applied sample amount is 400 μ L, and it is after purification to collect protein peak product of the retention time in 14-16min
Bacteriocin.
Research shows that the bacteriocin for obtaining methods described and the Enterococcus faecalis fermentation liquid containing bacteriocin add with 2%
It is added in the fresh cheese containing 1% Listeria, in 5 days of storage, can effectively suppresses the growth of Listeria, and newly
The moisture and protein content of fresh cheese are without significant change.
In addition, the bacteriocin that is prepared according to the method for the invention and the Enterococcus faecalis fermentation containing bacteriocin
Liquid, there is good temperature capacity, 121 DEG C of processing 30min still keep bacteriostatic activity, stable to acid, in the range of pH2.5-7.5
Keep stable Antibacterial Activity.Pepsin, Proteinase K, alpha-amylase can make bacteriocin partial inactivation.
Therefore, third object of the present invention is to provide described enterococcus faecalis bacteriocin or the excrement intestines containing the bacteriocin
Application of the coccus zymotic fluid in Listeria (L.Monocytogenes) growth is suppressed.And
Described enterococcus faecalis bacteriocin or the Enterococcus faecalis fermentation liquid containing the bacteriocin are preparing fresh cheese hair
Application in ferment agent.
Brief description of the drawings
Fig. 1 is suppression situation of the bacteriocin to Listeria in fresh cheese；
Fig. 2 is the manufacturing process of fresh cheese.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But embodiment is only exemplary, does not form any restrictions to the scope of the present invention.Those skilled in the art should
It should be appreciated that the details and form of technical solution of the present invention can be carried out without departing from the spirit and scope of the invention
Modifications or substitutions, but these modifications and replacement are each fallen within protection scope of the present invention.
The separation and identification of the enterococcus faecalis of embodiment 1
Enterococcus faecalis provided by the present invention is to separate and obtain from traditional dairy products, on MRS solid mediums
Colonial morphology is circular, projection, and color and luster is milky, opaque, and Gram's staining is the positive, and thalli morphology is circle, short chain shape
Arrangement.Enterococcus faecalis (Enterococcus faecalis) is accredited as through 16SrDNA.
Described enterococcus faecalis (Enterococcus faecalis), is named as KLDS6001, and Classification And Nomenclature is excrement intestines ball
Bacterium (Enterococcus faecalis), is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, ground
Location is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation numbering：CGMCC
No.9966, preservation time are on November 13rd, 2014.
The preparation of the enterococcus faecalis bacteriocin of embodiment 2
(1) preparation of Enterococcus faecalis fermentation liquid
Enterococcus faecalis KLDS6001 is inoculated in MRS fluid nutrient mediums with 3% inoculum concentration, its composition is：Peptone
10.0g, beef extract 10.0g, dusty yeast 5g, glucose 20.0g, Tween 80 1.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g,
Lemon acid diamine 2.0g, magnesium sulfate 0.6g, manganese sulfate 0.25g, distilled water 1000mL.37 DEG C culture 16h, 12000 × g, 4 DEG C from
Heart 15min, obtains fermented supernatant fluid；
(2) preliminary purification of enterococcus faecalis bacteriocin
The zymotic fluid that step (1) is obtained carries out centrifugal treating, and draws supernatant liquor, is added into the supernatant liquor full
Saltoutd with degree for 70% ammonium sulfate, 10000 × g, 4 DEG C of centrifugation 30min, collect precipitation, redissolve in pH6.0's
In 0.02mol/L sodium acetate buffer solutions, that is, obtain the crude extract of bacteriocin.
(3) enterococcus faecalis bacteriocin is secondarily purified
The crude extract of bacteriocin is further isolated and purified using glucan G-25 gel chromatography columns, chromatography strip used
Part is：Eluted with 50% (w/w) sodium chloride and the buffer solution of 50% (w/w) sodium acetate, flow velocity 1mL/min, sample
Applied sample amount is 400 μ L, and it is bacteriocin after purification to collect protein peak product of the retention time in 14-16min.
The bacteriostatic activity analysis of the enterococcus faecalis of embodiment 3 (Enterococcus faecalis)
1st, the analysis method of bacteriostatic activity
15mL Listerias (L.Monocytogenes) solid medium is toppled over into every flat board, takes 100 μ L indicator bacterias,
It is spread evenly across on flat board, after bacterium solution fully absorbs, 3 a diameter of 6mm Oxford cup is put into every flat board, draws 50 μ L
Fermented supernatant fluid is put into each Oxford cup, and room temperature places 1h, is put into 37 DEG C of constant incubators after 24h, observes and determine suppression
The diameter of bacterium circle.
Enterococcus faecalis is inoculated in MRS fluid nutrient mediums with 3% inoculum concentration, its composition is：Peptone 10.0g, ox
Meat extract 10.0g, dusty yeast 5g, glucose 20.0g, Tween 80 1.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, citric acid two
Amine 2.0g, magnesium sulfate 0.6g, manganese sulfate 0.25g, distilled water 1000mL.16h, 12000 × g, 4 DEG C of centrifugation 15min of 37 DEG C of cultures,
Fermented supernatant fluid is obtained, is done following experiment.
(1) influence of organic acid is excluded：The pH of fermented supernatant fluid is adjusted to neutrality, then determines the antibacterial work of fermented supernatant fluid
Property.
(2) influence of hydrogen peroxide is excluded：Catalase is dissolved in pH 7.0 phosphate buffer and is made into mother liquor, adds
The final concentration for entering to make in fermented supernatant fluid catalase is 5.0mg/mL, is taken out after 37 DEG C of water-bath 2h, detects catalase
Handle the bacteriostatic activity of after fermentation supernatant.
(3) determination of protide antibacterial substance：Proteinase K is made into mother liquor with sterile MilliQ water, adds acellular hair
In ferment supernatant, the final concentration for making Proteinase K is 1.0mg/mL, is taken out after mixing after 37 DEG C of water-bath 2h, detection process after fermentation
The bacteriostatic activity of liquid.
By the processing of (1), (2), the bacteriostatic activity of fermented supernatant fluid does not disappear, but passes through the processing of (3), in fermentation
Clear liquid loses bacteriostatic activity.Tentatively it can be determined that the antibacterial substance in fermented supernatant fluid is bacteriocin.
2nd, the fungistatic effect of bacteriocin
Fermented supernatant fluid has very strong fungistatic effect to Listeria (L.Monocytogenes), and bacteriostatic diameter can reach
To 16.11 ± 0.11mm.
3rd, the biological feature study of bacteriocin
(1) sensitiveness of the bacteriocin to temperature：
8 parts of equivalent 1mL fermented supernatant fluids are taken, keep 10min and 30min at 60 DEG C, 80 DEG C, 100 DEG C and 121 DEG C respectively,
Bacteriostatic experiment is done after being cooled with ice, as a result as shown in table 1：Bacteriocin shows very strong temperature capacity, in 121 DEG C of processing
After 30min, bacteriostatic activity still remain.
Influence of the temperature of table 1 to bacteriocin Antibacterial Activity
(2) sensitiveness of the bacteriocin to pH：8 parts of equivalent fermented supernatant fluids are taken, pH value 2- is adjusted with 3mol/L HCl or NaOH
10,37 DEG C of incubation 2h, do bacteriostatic experiment, as a result as shown in table 2：Bacteriocin is active under conditions of pH2.5-6.5, and with
The increase reduced activity of pH value, in more than pH7.5, bacteriocin loses bacteriostatic activity.
Influences of the table 2pH to bacteriocin Antibacterial Activity
(3) sensitiveness of the bacteriocin to enzyme：Equivalent fermented supernatant fluid is taken, adjusts pH most suitable to following enzyme with HCl, NaOH
Action pH value.Pepsin, trypsase, Proteinase K, Chymetin, Papain are separately added into by final concentration 1mg/mL
Enzyme and alpha-amylase, 37 DEG C of incubation 2h.PH is adjusted back to the optimum pH of bacteriocin, does bacteriostatic experiment, as a result as shown in table 3：
Pepsin, Proteinase K, alpha-amylase can make bacteriocin partial inactivation.
Influence of the protease of table 3 to bacteriocin Antibacterial Activity
The bacteriocin of embodiment 4 and application of the Enterococcus faecalis fermentation liquid in fresh cheese containing bacteriocin
The manufacturing process of fresh cheese is as shown in Figure 2.
Indicator bacteria Listeria (L.Monocytogenes) is added while leavening is added after milk pasteurization
(1%), the Enterococcus faecalis fermentation liquid (2%) and bacteriocin (2%) containing bacteriocin that embodiment 2 is prepared, exclude in milk
Original pathogenic bacteria, compareed with the sample for only adding indicator bacteria, every other day to the Listeria in sample
(L.Monocytogenes) counted, suppression of the checking bacteriocin to Listeria in cheese (L.Monocytogenes) is made
With.As a result represent：In 5 days of storage, it with the addition of bacteriocin and the Enterococcus faecalis fermentation liquid containing bacteriocin can be effective
Antibacterial fresh cheese in Listeria growth, as a result as shown in Figure 1.
Claims (3)
- A kind of 1. method for producing enterococcus faecalis bacteriocin, it is characterised in that comprise the following steps：(1) preparation of Enterococcus faecalis fermentation liquidEnterococcus faecalis (Enterococcus faecium) is inoculated in MRS culture mediums, 37 DEG C, excrement is obtained after cultivating 12-16h Enterococcus zymotic fluid；Described enterococcus faecalis is the enterococcus faecalis of high bacteriocinogeny, is named as KLDS6001, is deposited in Chinese microorganism strain Preservation administration committee common micro-organisms center, its culture presevation numbering are：CGMCC No.9966；(2) preliminary purification of enterococcus faecalis bacteriocinThe zymotic fluid obtained by step (1) carries out centrifugal treating, and draws supernatant liquor, and saturation degree is added into the supernatant liquor Saltoutd for 70% ammonium sulfate, 10000 × g, 4 DEG C of centrifugation 30min, collect precipitation, redissolve the 0.02mol/L in pH6.0 In sodium acetate buffer solution, that is, obtain the crude extract of bacteriocin；(3) enterococcus faecalis bacteriocin is secondarily purifiedThe crude extract of bacteriocin is further isolated and purified using glucan G-25 gel chromatography columns, produces after purification thin Rhzomorph；Use glucan G-25 gel chromatography columns isolated and purified used in chromatography condition for：With 50% (w/w) sodium chloride and The buffer solution of 50% (w/w) sodium acetate is eluted, flow velocity 1mL/min, and sample applied sample amount is 400 μ L, collects retention time Protein peak product in 14-16min is bacteriocin after purification.
- 2. according to the method for claim 1, it is characterised in that：Step (1) is used to cultivate enterococcus faecalis (Enterococcus Faecium MRS medium components) are as follows：Peptone 10.0g, beef extract 10.0g, dusty yeast 5g, glucose 20.0g, tween 80 1.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, lemon acid diamine 2.0g, magnesium sulfate 0.6g, manganese sulfate 0.25g and steaming Distilled water 1000mL.
- 3. according to the method for claim 1, it is characterised in that：Cultivation temperature in step (1) is 37 DEG C, and incubation time is 14h。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107189968A (en) * | 2017-07-21 | 2017-09-22 | 湖北省新兴地生物技术有限公司 | A kind of enterococcus faecalis and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109805167A (en) * | 2019-01-10 | 2019-05-28 | 河南爱猪人生物科技股份有限公司 | A kind of environment-friendly high-efficiency liquid feed additive |
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Cited By (2)
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| CN107189968B (en) * | 2017-07-21 | 2020-06-30 | 湖北省新兴地生物技术有限公司 | Enterococcus faecalis and preparation method thereof |
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