CN104593294A - Enterococcus faecalis with high bacteriocin yield and application of enterococcus faecalis - Google Patents
Enterococcus faecalis with high bacteriocin yield and application of enterococcus faecalis Download PDFInfo
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- CN104593294A CN104593294A CN201410835326.6A CN201410835326A CN104593294A CN 104593294 A CN104593294 A CN 104593294A CN 201410835326 A CN201410835326 A CN 201410835326A CN 104593294 A CN104593294 A CN 104593294A
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Abstract
The invention discloses enterococcus faecalis with high bacteriocin yield and application thereof. The preservation number of the enterococcus faecalis with high bacteriocin yield is CGMCC NO.9966; the enterococcus faecalis is separated from traditional dairy products; the bacteriocin produced by the enterococcus faecalis is obtained through a series of purification such as ammonium sulfate salting out and gel chromatography. Experiments prove that the bacteriocin produced by the enterococcus faecalis can be degraded by protease, has strong inhibiting effect on listeria monocytogenes and has favorable heat resistance and acid resistance. When added into fresh cheese, the bacteriocin enterococcus faecalis can effectively inhibit the growth of the listeria monocytogenes, so that the enterococcus faecalis with high bacteriocin yield has the potential of being applied to fresh cheese as a natural food additive.
Description
Technical field
The present invention relates to a kind of high bacteriocinogeny enterococcus faecalis and application thereof, belong to biological technical field.
Background technology
Along with constantly improving of people's living standard, the consumption of milk-product also increases day by day, the cheese be particularly always not too accepted also becomes a part for people's diet gradually, fresh cheese refers to that raw dairy, rare cream, whey or their mixture are through ferment-fermented, rennet curdling, gets rid of the product that whey is formed.Fresh cheese eliminates this step of fermenting-ripening in manufacture craft, thus makes its local flavor soft light, and water content is greater than 67%, and quality is between Yoghourt and cheese, and mouthfeel is good, and the degree of recognition at home in human consumer is the highest.But cheese Middle nutrition material abundance is the ecotopia of most microbial growth, especially pathogenic bacterium and spoilage organism.Cheese is the pollution that also can cause post-treatment hand-manipulated frequently in producing, the field planting of fresh cheese to listeria bacteria (L.Monocytogenes) is especially responsive, especially easily be subject to the pollution of listeria bacteria (L.Monocytogenes), microbes is caused to poison by food, have a strong impact on the quality of life of people, so guarantee that the security of cheese is particularly important.
Present stage, people control the growth of microorganism in food mainly through adding Chemical Preservative, extend the quality guaranteed period of food.But long-term edible of these chemical additives has very bad impact to human heart, blood pressure and kidney, the chemical additive even had has the undesirable action of carcinogenesis, mutagenesis and teratogenecity.In the process for processing of food, also control growing of microorganism often through thermally-sterilized method, but thermally-sterilized processing mode also also exists some drawbacks, it can affect the quality of food and nutritive ingredient can be damaged.Although the method as uv sterilisation, these physical sterilizations of filtration sterilization can avoid the quality destroying food, packaging is just easy to the infection being subject to bacterium after opening.So develop efficient, stable, safe natural sandy gravel become the urgent hope of people.
Milk-acid bacteria is widely used in foodstuffs industry, and be the quasi-microorganism that people often consume, some milk-acid bacterias can produce bacteriocin growing into certain phase, bacteriocin is that a class has the polypeptide of anti-microbial activity, protein or protein complex material, it effectively can suppress the growth of most of gram-positive microorganism and pathogenic bacterium, having good acidproof and temperature capacity, can be digested in vivo by protease hydrolysis, is a good class biological preservative.Be applied to by bacteriocin lab in fresh cheese processing, the mouthfeel of cheese and taste all can not be affected, and effectively can also suppress the growth of listeria bacteria (L.Monocytogenes).
Bacteriocin has huge space in the application of cheese, present stage as a class biological preservative, and business-like bacteriocin product is also very limited, develops the expectation that novel bacteriocin has become people.Along with improving constantly of technology, bacteriocin is widely used in finally becoming a kind of safe natural sandy gravel in cheese.
Summary of the invention
First object of the present invention is to provide the enterococcus faecalis (Enterococcus faecalis) of a kind of high bacteriocinogeny, called after KLDS6001, Classification And Nomenclature is enterococcus faecalis (Enterococcus faecalis), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC No.9966, and the preservation time is on November 13rd, 2014.
Enterococcus faecalis provided by the present invention is separated from traditional milk-product and obtains, on MRS solid medium, colonial morphology is circular, projection, and color and luster is oyster white, opaque, and gramstaining be the positive, thalli morphology is circle, short catenation, is accredited as enterococcus faecalis through 16SrDNA.
Second object of the present invention is to provide a kind of enterococcus faecalis bacteriocin and produces the method for this enterococcus faecalis bacteriocin.
Bacteriocin provided by the present invention is fermented by enterococcus faecalis of the present invention (Enterococcus faecalis) and obtains.Concrete, comprise the following steps:
(1) preparation of Enterococcus faecalis fermentation liquid
Enterococcus faecalis according to claim 1 (Enterococcus faecalis) is inoculated in MRS substratum, 37 DEG C, after cultivating 12-16h, obtains Enterococcus faecalis fermentation liquid;
(2) preliminary purification of enterococcus faecalis bacteriocin
The fermented liquid that step (1) obtains is carried out centrifugal treating, and draws supernatant liquid, add ammonium sulfate and saltout in this supernatant liquid, centrifugal, collecting precipitation, redissolves in sodium acetate buffer solution, namely obtains the crude extract of bacteriocin;
(3) enterococcus faecalis bacteriocin is secondarily purified
Adopt dextran G-25 gel chromatography column to carry out separation and purification further the crude extract of bacteriocin, obtain the bacteriocin after purifying.
In method of the present invention, preferably, step (1) is as follows for the MRS medium component cultivating enterococcus faecalis (Enterococcus faecalis): peptone 10.0g, extractum carnis 10.0g, yeast powder 5g, glucose 20.0g, tween 80 1.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, citric acid diamines 2.0g, magnesium sulfate 0.6g, manganous sulfate 0.25g and distilled water 1000mL.
In method of the present invention, preferably, the culture temperature in step (1) is 37 DEG C, and incubation time is 14h.
In method of the present invention, preferably, step carries out preliminary purification in (2) in accordance with the following methods: the fermented liquid obtained by step (1) carries out centrifugal treating, and draw supernatant liquid, add in this supernatant liquid saturation ratio be 70% ammonium sulfate saltout, 10000 × g, 4 DEG C of centrifugal 30min, collecting precipitation, redissolves in the 0.02mol/L sodium acetate buffer solution of pH6.0, namely obtains the crude extract of bacteriocin.
In method of the present invention, preferably, step (3) middle employing dextran G-25 gel chromatography column carries out separation and purification chromatography condition used and is: carry out wash-out with the damping fluid of sodium-chlor and 50% (w/w) sodium acetate of 50% (w/w), flow velocity is 1mL/min, sample applied sample amount is 400 μ L, and the protein peak product of collection retention time in 14-16min is the bacteriocin after purifying.
Research shows, the bacteriocin obtain described method and the Enterococcus faecalis fermentation liquid containing bacteriocin add to containing in 1% listerial fresh cheese with 2%, in 5 days that store, can effectively suppress listerial growth, and the moisture of fresh cheese and protein content are without considerable change.
In addition, the bacteriocin prepared according to the method for the invention and the Enterococcus faecalis fermentation liquid containing bacteriocin, have good temperature capacity, and 121 DEG C of process 30min still keep bacteriostatic activity, acid is stablized, in the scope of pH2.5-7.5, keeps stable Antibacterial Activity.Stomach en-, Proteinase K, α-amylase all can make bacteriocin part inactivation.
Therefore, the 3rd object of the present invention is to provide described enterococcus faecalis bacteriocin or is suppressing the application in the growth of listeria bacteria (L.Monocytogenes) containing the Enterococcus faecalis fermentation liquid of this bacteriocin.And
Described enterococcus faecalis bacteriocin or the application of the Enterococcus faecalis fermentation liquid containing this bacteriocin in preparation fresh cheese starter.
Accompanying drawing explanation
Fig. 1 is that bacteriocin is to suppression situation listerial in fresh cheese;
Fig. 2 is the making processes of fresh cheese.
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.
The Isolation and Identification of embodiment 1 enterococcus faecalis
Enterococcus faecalis provided by the present invention is separated from traditional milk-product and obtains, and on MRS solid medium, colonial morphology be circular, projection, and color and luster is oyster white, opaque, and gramstaining be positive, and thalli morphology is round, short catenation.Enterococcus faecalis (Enterococcus faecalis) is accredited as through 16SrDNA.
Described enterococcus faecalis (Enterococcus faecalis), called after KLDS6001, Classification And Nomenclature is enterococcus faecalis (Enterococcus faecalis), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC No.9966, and the preservation time is on November 13rd, 2014.
The preparation of embodiment 2 enterococcus faecalis bacteriocin
(1) preparation of Enterococcus faecalis fermentation liquid
By enterococcus faecalis KLDS6001 with 3% inoculum size be inoculated in MRS liquid nutrient medium, its composition is: peptone 10.0g, extractum carnis 10.0g, yeast powder 5g, glucose 20.0g, tween 80 1.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, citric acid diamines 2.0g, magnesium sulfate 0.6g, manganous sulfate 0.25g, distilled water 1000mL.Cultivate 16h, 12000 × g for 37 DEG C, 4 DEG C of centrifugal 15min, obtain fermented supernatant fluid;
(2) preliminary purification of enterococcus faecalis bacteriocin
The fermented liquid that step (1) obtains is carried out centrifugal treating, and draw supernatant liquid, add in this supernatant liquid saturation ratio be 70% ammonium sulfate saltout, 10000 × g, 4 DEG C of centrifugal 30min, collecting precipitation, redissolves in the 0.02mol/L sodium acetate buffer solution of pH6.0, namely obtains the crude extract of bacteriocin.
(3) enterococcus faecalis bacteriocin is secondarily purified
Dextran G-25 gel chromatography column is adopted to carry out separation and purification further the crude extract of bacteriocin, chromatography condition used is: carry out wash-out with the damping fluid of sodium-chlor and 50% (w/w) sodium acetate of 50% (w/w), flow velocity is 1mL/min, sample applied sample amount is 400 μ L, and the protein peak product of collection retention time in 14-16min is the bacteriocin after purifying.
The bacteriostatic activity analysis of embodiment 3 enterococcus faecalis (Enterococcus faecalis)
1, the analytical procedure of bacteriostatic activity
15mL listeria bacteria (L.Monocytogenes) solid medium is toppled in every flat board, get 100 μ L indicators, be spread evenly across on flat board, after bacterium liquid absorbs completely, put into the Oxford cup that 3 diameters are 6mm in every flat board, draw 50 μ L fermented supernatant fluids and put into each Oxford cup, room temperature places 1h, after putting into 37 DEG C of constant incubator 24h, observe and measure the diameter of inhibition zone.
By enterococcus faecalis with 3% inoculum size be inoculated in MRS liquid nutrient medium, its composition is: peptone 10.0g, extractum carnis 10.0g, yeast powder 5g, glucose 20.0g, tween 80 1.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, citric acid diamines 2.0g, magnesium sulfate 0.6g, manganous sulfate 0.25g, distilled water 1000mL.Cultivate 16h, 12000 × g for 37 DEG C, 4 DEG C of centrifugal 15min, obtain fermented supernatant fluid, are done following experiment.
(1) organic acid impact is got rid of: the pH of fermented supernatant fluid is adjusted to neutrality, then measures the bacteriostatic activity of fermented supernatant fluid.
(2) impact of hydrogen peroxide is got rid of: catalase is dissolved in the phosphoric acid buffer of pH 7.0 and is made into mother liquor, add in fermented supernatant fluid and make catalatic final concentration be 5.0mg/mL, take out after 37 DEG C of water-bath 2h, be detected the bacteriostatic activity of hydrogen oxide ferment treatment secondary fermentation supernatant liquor.
(3) determination of protein antibacterial substance: the aseptic MilliQ water of Proteinase K is made into mother liquor, add in cell free fermentation supernatant liquor, make the final concentration of Proteinase K be 1.0mg/mL, take out after mixing rear 37 DEG C of water-bath 2h, the bacteriostatic activity of check processing secondary fermentation liquid.
Through the process of (1), (2), the bacteriostatic activity of fermented supernatant fluid does not disappear, but passes through the process of (3), and fermented supernatant fluid loses bacteriostatic activity.Tentatively can judge that antibacterial substance in fermented supernatant fluid is as bacteriocin.
2, the fungistatic effect of bacteriocin
Fermented supernatant fluid has very strong fungistatic effect to listeria bacteria (L.Monocytogenes), and bacteriostatic diameter can reach 16.11 ± 0.11mm.
3, the biological feature study of bacteriocin
(1) bacteriocin is to the susceptibility of temperature:
Get 8 parts of equivalent 1mL fermented supernatant fluids, keep 10min and 30min 60 DEG C, 80 DEG C, 100 DEG C and 121 DEG C respectively, after ice-cooled, do bacteriostatic experiment, result is as shown in table 1: bacteriocin shows very strong temperature capacity, after 121 DEG C of process 30min, still remain bacteriostatic activity.
Table 1 temperature is on the impact of bacteriocin Antibacterial Activity
(2) bacteriocin is to the susceptibility of pH: get 8 parts of equivalent fermented supernatant fluids, with 3mol/L HCl or NaOH adjust pH 2-10,37 DEG C of incubation 2h, do bacteriostatic experiment, result is as shown in table 2: bacteriocin all has activity under the condition of pH2.5-6.5, and along with the increase reduced activity of pH value, when more than pH7.5, bacteriocin loses bacteriostatic activity.
Table 2pH is on the impact of bacteriocin Antibacterial Activity
(3) bacteriocin is to the susceptibility of enzyme: get equivalent fermented supernatant fluid, adjusts pH to the Optimun pH of following enzyme with HCl, NaOH.Stomach en-, trypsinase, Proteinase K, Chymetin, papoid and α-amylase is added respectively, 37 DEG C of incubation 2h by final concentration 1mg/mL.PH is adjusted back to the optimum pH of bacteriocin, do bacteriostatic experiment, result is as shown in table 3: stomach en-, Proteinase K, α-amylase all can make bacteriocin part inactivation.
Table 3 proteolytic enzyme is on the impact of bacteriocin Antibacterial Activity
Embodiment 4 bacteriocin and the application of Enterococcus faecalis fermentation liquid in fresh cheese containing bacteriocin
The making processes of fresh cheese as shown in Figure 2.
The Enterococcus faecalis fermentation liquid (2%) containing bacteriocin that while adding starter after milk pasteurization, interpolation indicator listeria bacteria (L.Monocytogenes) (1%), embodiment 2 prepare and bacteriocin (2%), get rid of original pathogenic bacterium in milk, contrast with the sample only adding indicator, every other day count the listeria bacteria (L.Monocytogenes) in sample, checking bacteriocin is to the restraining effect of listeria bacteria in cheese (L.Monocytogenes).Result represents: in 5 days of storage, and with the addition of bacteriocin and the Enterococcus faecalis fermentation liquid containing bacteriocin can listerial growth in effective antibacterial fresh cheese, and result as shown in Figure 1.
Claims (10)
1. the enterococcus faecalis (Enterococcus faecalis) of a plant height bacteriocinogeny, called after KLDS6001, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC No.9966.
2. produce a method for enterococcus faecalis bacteriocin, it is characterized in that by the enterococcus faecalis according to claim 1 that ferments (Enterococcus faecalis) and obtain.
3. method according to claim 2, is characterized in that comprising the following steps:
(1) preparation of Enterococcus faecalis fermentation liquid
Enterococcus faecalis according to claim 1 (Enterococcus faecalis) is inoculated in MRS substratum, 37 DEG C, after cultivating 12-16h, obtains Enterococcus faecalis fermentation liquid;
(2) preliminary purification of enterococcus faecalis bacteriocin
The fermented liquid that step (1) obtains is carried out centrifugal treating, and draws supernatant liquid, add ammonium sulfate and saltout in this supernatant liquid, centrifugal, collecting precipitation, redissolves in sodium acetate buffer solution, namely obtains the crude extract of bacteriocin;
(3) enterococcus faecalis bacteriocin is secondarily purified
Adopt dextran G-25 gel chromatography column to carry out separation and purification further the crude extract of bacteriocin, obtain the bacteriocin after purifying.
4. method according to claim 2, is characterized in that: step (1) is as follows for the MRS medium component cultivating enterococcus faecalis (Enterococcus faecalis): peptone 10.0g, extractum carnis 10.0g, yeast powder 5g, glucose 20.0g, tween 80 1.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, citric acid diamines 2.0g, magnesium sulfate 0.6g, manganous sulfate 0.25g and distilled water 1000mL.
5. method according to claim 2, is characterized in that: the culture temperature in step (1) is 37 DEG C, and incubation time is 14h.
6. method according to claim 2, it is characterized in that: step carries out preliminary purification in (2) in accordance with the following methods: the fermented liquid obtained by step (1) carries out centrifugal treating, and draw supernatant liquid, add in this supernatant liquid saturation ratio be 70% ammonium sulfate saltout, 10000 × g, 4 DEG C of centrifugal 30min, collecting precipitation, redissolve in the 0.02mol/L sodium acetate buffer solution of pH6.0, namely obtain the crude extract of bacteriocin.
7. method according to claim 2, it is characterized in that: step (3) middle employing dextran G-25 gel chromatography column carries out separation and purification chromatography condition used and is: carry out wash-out with the damping fluid of sodium-chlor and 50% (w/w) sodium acetate of 50% (w/w), flow velocity is 1mL/min, sample applied sample amount is 400 μ L, and the protein peak product of collection retention time in 14-16min is the bacteriocin after purifying.
8. the enterococcus faecalis bacteriocin prepared by described method arbitrary in claim 2 to 7 or the Enterococcus faecalis fermentation liquid containing this bacteriocin.
9. enterococcus faecalis bacteriocin according to claim 8 or the Enterococcus faecalis fermentation liquid containing this bacteriocin are suppressing the application in the growth of listeria bacteria (L.Monocytogenes).
10. enterococcus faecalis bacteriocin according to claim 8 or the application of the Enterococcus faecalis fermentation liquid containing this bacteriocin in preparation fresh cheese starter.
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| 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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| 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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| CN101282743A (en) * | 2005-03-18 | 2008-10-08 | 由农业部部长代表的美利坚合众国 | Bacteriocin and novel bacterium mycopremna |
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| CN101282743A (en) * | 2005-03-18 | 2008-10-08 | 由农业部部长代表的美利坚合众国 | Bacteriocin and novel bacterium mycopremna |
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| 王雪芹等: "两株粪肠球菌的安全性评价", 《食品工业科技》 * |
Cited By (1)
| 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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