CN103966286A - Sinonovacula constricta-derived bacillus alvei protein, application thereof to aquatic product preservation and application method thereof - Google Patents
Sinonovacula constricta-derived bacillus alvei protein, application thereof to aquatic product preservation and application method thereof Download PDFInfo
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- CN103966286A CN103966286A CN201310034691.2A CN201310034691A CN103966286A CN 103966286 A CN103966286 A CN 103966286A CN 201310034691 A CN201310034691 A CN 201310034691A CN 103966286 A CN103966286 A CN 103966286A
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Abstract
A sinonovacula constricta-derived bacillus alvei protein and application thereof aquatic product preservation and an application method. The preparation method is as below: culturing sinonovacula constricta symbiotic bacillus alvei strains in a liquid medium comprising 40g / L glucose and 20g / L peptone and having pH value adjusted to 5; filling 150-250 mL of the liquid medium into each bottle and conducting shaking culture at the temperature of 30-35 DEG C and speed of 150-250 rpm for 4-6 days; centrifuging at 4000 r / min to remove thalli in the fermentation liquid, carrying out decompression concentration at 50 DEG C until 1/2 of the original volume, adding ammonium sulfate until the final saturation concentration reaching 90%, standing overnight at 4 DEG C, filtering, dissolving the precipitate with a PBS buffer liquid, conducting dialysis by using a dialysis bag with the cut-off molecular weight of 3600 Dalton for 4 days, conducting freeze-drying on the products in the dialysis bag, so as to obtain the sinonovacula constricta-derived bacillus alvei protein. The preparation process provided by the invention is simple and low in cost.
Description
Technical field
The present invention relates to food fresh keeping field, particularly a kind of Yi razor clam source bacillus alvei albumen and application and methods for using them thereof.
Background technology
Fishery products have the feature of lower fat, high protein, are indispensable important component in rational diet structure, have become people and absorb the important sources of animal protein.And the fishery products delicious meat such as fish, shrimp, crab, unique flavor, has the laudatory title of " seafood ", is deeply subject to consumers in general's favor.The freshness of fishery products is its topmost quality parameter, it is the principal element that determines its price, fishery products are easily putrid and deteriorated in addition, therefore strengthen the Study on preservation technique of fishery products, extend quality guaranteed period and the shelf-lives of fishery products, make the fresh water product can long distance transportation, meet the demand of non-coastal cities human consumer to fishery products, the development that drives culture fishery is significant.
Fishery products etc. at present conventional preservation agent point can be divided into chemical preservative and crude antistaling agent from originating, although chemical preservative Chemical Preservative effect is fast, easy to use, but there are some potential safety hazards, it is residual also can cause that chemical agent pollutes, and therefore sight is turned to exploitation little to side effect, natural antisepsis fresh-keeping agent that timeliness is long by people.In recent years, biological way of keeping fresh has had certain development, especially in bio-extract fresh-keeping aspect progress rapidly, but still has a lot of problems.The one, zymin etc. are expensive have been limited it and has applied, the 2nd, permitted the natural microbial antisepsis antistaling agent that uses, only have lactic acid coccus element and tennecetin etc. a few, the application of other biological technology is little, overall development is slow, kind is single.
Summary of the invention
The object of the invention is load provides a kind of Yi razor clam source bacillus alvei albumen simple to operate, with low cost and application and methods for using them thereof, overcomes the deficiencies in the prior art.
A kind of Yi razor clam of the present invention source bacillus alvei albumen, as follows preparation:
A. get bacterial strain and be the Yi razor clam bacillus alvei 110(that grows nonparasitically upon another plant altogether
bacillus alvei110), this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number: CGMCC No:7079 on January 10th, 2013;
B. the Yi razor clam bacillus alvei 110 of growing nonparasitically upon another plant is altogether cultivated, the liquid culture based formulas of bacillus alvei 110 is: glucose 40 g/L and peptone 20 g/L, and pH is deployed into 5;
C. bacillus alvei 110 fermentation conditions: every bottled 150-250 mL liquid nutrient medium, temperature 30-35 DEG C, rotating speed is 150-250 rpm, shaking table is cultivated 4-6 days;
D. the fermented liquid of centrifugal removal thalline under the condition of 4000 r/min, at 50 DEG C, be evaporated to 1/2nd of original volume, then add ammonium sulfate, the saturated final concentration of ammonium sulfate is 90%, 4 DEG C of hold over night, filter and will precipitate with after the dissolving of PBS damping fluid, then are that 3600 daltonian dialysis tubings are dialysed 4 days with molecular weight cut-off, get thing lyophilize in dialysis tubing, obtain Yi razor clam source bacillus alvei albumen.
A kind of Yi razor clam of the present invention source bacillus alvei albumen is in the application of fresh-keeping aspect.
A kind of Yi razor clam of the present invention source bacillus alvei albumen is at the application method of fresh-keeping aspect: Yi razor clam source bacillus alvei albumen is mixed with to the solution of 200 μ g/mL, and fresh-keeping for food, described food comprises fishery products.
Yi razor clam of the present invention source bacillus alvei albumen preparation process is simple, with low cost, the albumen extracting from Yi razor clam is grown nonparasitically upon another plant the fermented liquid of honeycomb spore bacillus strain 110 altogether has obvious bacteriostasis, preservation activity, can effectively suppress microbial reproduction, slow down the rising of pH value and TVB-N value, slow down the change of color and luster smell.Therefore the good refreshing effect that is applied to fishery products or other food etc., has industrial value.
Yi razor clam source bacillus alvei bacterial strain in the present invention has been preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number: CGMCC No:7079 on December 31st, 2012.
embodiment
A kind of Yi razor clam of the present invention source bacillus alvei albumen, preparation as follows:
A. the bacillus alvei 110 (Bacillus alvei 110) of production bacterial strain Yi razor clam being grown nonparasitically upon another plant altogether, be inoculated in glucose 40 g/L and peptone 20 g/L, pH is in 5 liquid nutrient medium, every bottled 150 mL or 200 mL or 250 mL liquid nutrient mediums, 30 DEG C or 35 DEG C of temperature, rotating speed is 150 rpm or 200rpm or 250rpm, and shaking table is cultivated 4 days or 5 days or 6 days.This bacillus alvei is preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number: CGMCC No:7079 on December 31st, 2012.
B. detect the bacteriostatic activity of fermented liquid by filter paper method.Intestinal bacteria Escherichia coli(EC), Bacillus proteus Proteus vulgaris(PV), streptococcus aureus Staphylococcus aureus (SA), gas bacillus Enterobacter aerogenes(EA) and subtilis Bacillus subtilis(BS) all adopt existing beef-protein medium.Above-mentioned Jun Shouzengyu Dalian Chemiclophysics Inst., Chinese Academy of Sciences.Each bacterial classification is inoculated in respectively in beef extract-peptone liquid nutrient medium, and shaking culture 24 h do indicator.
Get indicator liquid 100 μ L on beef extract-peptone solid medium with liquid-transfering gun respectively, being coated with rod with aseptic triangle smears evenly, then in the outside of culture dish bottom, beef extract-peptone solid medium culture dish is divided into San Ge district with marking pen, each district puts into respectively a filter paper (diameter 6 mm), move and get respectively grow nonparasitically upon another plant the altogether each 10 μ L of fermented product liquid, 75% alcohol and sterilized water of bacterial strain 110 of Yi razor clam and drip to respectively the filter paper central authorities in each district with liquid-transfering gun again, be placed in 37 DEG C of incubators and cultivate 2 days.Do 3 re-treatments, positive control is 75% alcohol, and negative control is sterilized water.Observe the growing state of indicator, measure the diameter of inhibition zone.Result shows that 110 bacterial strain fermentation liquors are respectively 11 mm to the bacteriostatic diameter of five kinds of indicators, 10 mm, 12 mm, 11.2 mm, 14.8 mm.
C. the fermented liquid of the centrifugal removal thalline of 4000 r/min is evaporated to 1/2nd of original volume at 50 DEG C, then add saturation ratio to be respectively 70%, 75%, 80%, 90% and 95% ammonium sulfate, 4 DEG C of hold over night, filter and will precipitate with after distilled water dissolving, be 3600 daltonian dialysis tubing dialysis 4 days with molecular weight cut-off again, get thing lyophilize in dialysis tubing, be mixed with the solution just as concentration, with filter paper method (the same) detection bacteriostatic activity, result shows that the throw out activity that the saturated final concentration of ammonium sulfate is 90% is best, yield is the highest, SDS-PAGE electrophoresis result shows that this throw out is protein.
D. fresh shrimp preserve test:
1) shrimp that market is bought covers with ice cube at once, behind laboratory, and picking freshness, the fresh prawns that size is consistent as far as possible.Clean up, decontamination, picks out damaged and the inadequate shrimp of freshness, the individuality that removal differs greatly, then by individual sample random packet, every group of 0.5 Kg, use respectively bacillus alvei 110 protein solutions of 200 mg/ L, immersion treatment 5 min, and compare with distilled water.Elimination soak solution, packs shrimp in crisper into, is placed in 4 DEG C of refrigerations.Observe every 24 h, observe altogether 10 days.Until pH >8.0 or the TVB-N>35 mg N per 100 g samples of shrimp.
2) mensuration of pH: take each group of sample 5 g, add 50 mL distilled water, grinding stirs, filters, and gets filtrate and measures with acidometer, directly reads pH value, and the sample pH value increase of result it is as shown in the table 110 albumen processing is starkly lower than contrast.
3) mensuration of total volatile basic nitrogen (TVB-N): specify by GB2741-94, (mg/ 100 g)≤25 is one-level freshness to TVB-N; (mg/ 100 g)≤30 is secondary freshness to TVB-N.The sample TVB-N of result it is as shown in the table 110 albumen processing increases and is starkly lower than contrast.
4
)the mensuration of total plate count: adopt GB plate count culture method, in the shrimp of 110 albumen processing, total number of bacterial colony is starkly lower than contrast, in 4 days of counting all lower than an order of magnitude of contrast.
5
)the external appearance characteristic of shrimp is observed: shrimp body integrity, body surface have between tarnish, carapace and body segment, is connected tight; Eyeball is full outstanding; Grain of meat is clear, flexible, and shell is difficult for peeling off; There is the intrinsic smell of prawn, without other peculiar smell.The shrimp outward appearance that result just can be found out the processing of bacterial strain DNN6 albumen from second day after treatment is obviously due to contrast, and therefore 110 albumen fresh-keeping effects are obvious.
E. bacillus alvei 110 bacterial strains are cultured to logarithmic phase in aforesaid liquid substratum, the centrifugal collection thalline of 4000 rpm, by thalline lyophilize, then with the thalline dosage of 5 g/kg and 15 g/kg, mouse is carried out to disposable gavage respectively, result mouse is without any abnormal; With the albumen of 5 g/kg dosage, mouse is carried out to disposable gavage, mouse is without any abnormal simultaneously.
Claims (3)
1. a Yi razor clam source bacillus alvei albumen, is characterized in that: preparation as follows:
A. get bacterial strain and be the Yi razor clam bacillus alvei 110(that grows nonparasitically upon another plant altogether
bacillus alvei110), this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number: CGMCC No:7079 on January 10th, 2013;
B. the Yi razor clam bacillus alvei 110 of growing nonparasitically upon another plant is altogether cultivated, the liquid culture based formulas of bacillus alvei 110 is: glucose 40 g/L and peptone 20 g/L, and pH is deployed into 5;
C. bacillus alvei 110 fermentation conditions: every bottled 150-250 mL liquid nutrient medium, temperature 30-35 DEG C, rotating speed is 150-250 rpm, shaking table is cultivated 4-6 days;
D. the fermented liquid of centrifugal removal thalline under the condition of 4000 r/min, at 50 DEG C, be evaporated to 1/2nd of original volume, then add ammonium sulfate, the saturated final concentration of ammonium sulfate is 90%, 4 DEG C of hold over night, filter and will precipitate with after the dissolving of PBS damping fluid, then are that 3600 daltonian dialysis tubings are dialysed 4 days with molecular weight cut-off, get thing lyophilize in dialysis tubing, obtain Yi razor clam source bacillus alvei albumen.
2. a Yi razor clam according to claim 1 source bacillus alvei albumen is in the application of fresh-keeping aspect.
3. a Yi razor clam according to claim 2 source bacillus alvei albumen is at the application method of fresh-keeping aspect: it is characterized in that: the solution that Yi razor clam source bacillus alvei albumen is mixed with to 200 μ g/mL, be used for the fresh-keeping of food, described food comprises fishery products.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006030698A1 (en) * | 2004-09-17 | 2006-03-23 | Sumitomo Chemical Company, Limited | Process for producing sulfurized hydroxycarboxylic acid |
| WO2008038810A1 (en) * | 2006-09-25 | 2008-04-03 | Sumitomo Chemical Company, Limited | Process for the preparation of 2-hydr0xy-4- (alkylthio or arylthio) butanoic acid by microbial conversion of 2-0x0-4- (alkylthio or arylthio) 1-butanol employing bacteria of the genus pseud0m0nas, rh0d0c0ccus, or bacillus |
| CN101744837A (en) * | 2009-12-29 | 2010-06-23 | 大连水产学院 | Preparation method for sinonovacula constricta enzymolysis liquid with diversified bioactivities |
| CN102093473A (en) * | 2010-12-16 | 2011-06-15 | 大连海洋大学 | Preparation method of bacteriostatic active protein of Sinonovacula constricta Lamarck epipsymbiosis strain |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006030698A1 (en) * | 2004-09-17 | 2006-03-23 | Sumitomo Chemical Company, Limited | Process for producing sulfurized hydroxycarboxylic acid |
| WO2008038810A1 (en) * | 2006-09-25 | 2008-04-03 | Sumitomo Chemical Company, Limited | Process for the preparation of 2-hydr0xy-4- (alkylthio or arylthio) butanoic acid by microbial conversion of 2-0x0-4- (alkylthio or arylthio) 1-butanol employing bacteria of the genus pseud0m0nas, rh0d0c0ccus, or bacillus |
| CN101744837A (en) * | 2009-12-29 | 2010-06-23 | 大连水产学院 | Preparation method for sinonovacula constricta enzymolysis liquid with diversified bioactivities |
| CN102093473A (en) * | 2010-12-16 | 2011-06-15 | 大连海洋大学 | Preparation method of bacteriostatic active protein of Sinonovacula constricta Lamarck epipsymbiosis strain |
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