CN105567768A - Novel natural polypeptide preservative for fresh shrimps and preparation method thereof - Google Patents

Novel natural polypeptide preservative for fresh shrimps and preparation method thereof Download PDF

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CN105567768A
CN105567768A CN201510937457.XA CN201510937457A CN105567768A CN 105567768 A CN105567768 A CN 105567768A CN 201510937457 A CN201510937457 A CN 201510937457A CN 105567768 A CN105567768 A CN 105567768A
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sanitas
novel polypeptide
fresh shrimp
natural novel
fresh
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曹庸
陈飞龙
彭勃
苗建银
陈咏春
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South China Agricultural University
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    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3526—Organic compounds containing nitrogen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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Abstract

The invention provides a novel natural polypeptide preservative for fresh shrimps and a preparation method. The method comprises: inoculating Tibet Kefir grains in the logarithmic later period into an MRS (Methicillin Resistant Staphylococcus) fluid medium, carrying out culturing on a swing bed, and activating bacterial strains; inoculating activated Tibet Kefir grain culture solution diluted by the MRS fluid medium into pure milk, adding a carbon source and a nitrogen source, and fermenting for 65 to 75 hours at a temperature of 32 to 38 DEG C; after completing fermentation, adding distilled water to stir in a water bath, and regulating pH of the obtained product to a range of 3 to 4.5; carrying out centrifugal separation on the obtained fermentation liquor to obtain supernate containing the novel natural polypeptide preservative, filtering, and drying filtrate to obtain the novel natural polypeptide preservative for fresh shrimps. The invention also provides a using method of the preservative. The product provided by the invention can effectively prolong the shelf life of fresh shrimps, not only can keep nutritional ingredients and taste of fresh shrimps, but also is safe, sanitary, natural, non-toxic, highly effective and broad-spectrum, and has stable performance.

Description

A kind of fresh shrimp natural novel polypeptide sanitas and preparation method thereof
Technical field
The present invention relates to a kind of preservative technology field, be specifically related to preparation technology and the using method thereof of the natural novel polypeptide sanitas of a kind of fresh shrimp.
Technical background
In processing, storage, transport and process of consumption, cause the reason of food spoilage more, have physical factor, chemical factor and biological factor, the food spoilage wherein caused by microbial contamination is the most general.While microbial growth causes food spoilage, carry out serious harm also to food securing band.And Chemical Preservative is due to the limitation of the aspects such as its security, be difficult to the demand meeting human development, naturalization of food preservatives oneself become development trend from now on.
The seashore line length of China, oceanic resources enrich, and coastland had formed complete industrial chain already as the shrimp aquaculture on the ground such as Guangdong, Fujian, support local Economic development.China is Xian Xia producing country maximum in the world, and the output of nearest fresh shrimp keeps great-jump-forward to increase always, and the cultivation amount of Penaeus vannamei in 2010, up to 134.8 ten thousand tons, accounts for 1/3 of global total amount, and fresh shrimp has become one of most important outlet fishery products of China.The developed countries such as the U.S., Japan, European Union are the major consumers market of fresh shrimp, and they are very high to the demand of fresh shrimp, but they are very strict to the specification of quality of import shrimps, and therefore the competition of fresh shrimp outlet is very fierce.The most important quality parameter of fresh shrimp is freshness, but the feature of its high protein high-moisture makes it easily putrid and deteriorated.Now, the antisepsis antistaling agent kind of fresh shrimp is a lot, but all there is toxic side effect in various degree, Food Quality changes, high in cost of production problem, the requirement of people to the fresh shrimp of high-quality can not be met, thus reduce the competitive edge in international trade.Therefore, now need to find the novel anticorrosion preservation agent of a kind of safe, natural, efficient, wide spectrum, stable performance, this sanitas needs the shelf-lives that effectively can extend fresh shrimp, can keep again nutritive ingredient and the mouthfeel of fresh shrimp to greatest extent.
Summary of the invention
The object of the present invention is to provide the natural novel polypeptide sanitas of a kind of fresh shrimp, it utilizes a kind of is raw material with pure milk, adds Tibet Kefir granule and ferments, then the natural novel polypeptide sanitas of fresh shrimp that obtains of separation and purification.It effectively can extend the shelf-lives of fresh shrimp, can not only keep nutritive ingredient and the mouthfeel of fresh shrimp, and safety and sanitation, Nantural non-toxic, high-efficiency broad spectrum, stable performance.
For achieving the above object, embodiment of the present invention are: the natural novel polypeptide sanitas of a kind of fresh shrimp, are made up of the method comprising the following steps:
(1) be seeded in MRS liquid nutrient medium by the Tibet Kefir granule of late log phase, cultivate in shaking table, activated strains, obtains the Tibet Kefir granule nutrient solution of activation;
(2) with the Tibet Kefir granule nutrient solution of MRS liquid nutrient medium dilution activation, its OD is adjusted 600=0.2 ~ 0.5;
(3) the Tibet Kefir granule nutrient solution after the activation of step (2) gained is seeded in pure milk, meanwhile, adds Carbon and nitrogen sources, fully stir and each material is mixed and dissolves, be placed on 32 ~ 38 ° of C fermentations 65 ~ 75 hours;
(4) after having fermented, add distilled water, water-bath limit, limit is stirred, and regulates its pH3 ~ 4.5;
(5) get the centrifugal 10 ~ 20min of step (4) gained fermented liquid, remove undissolved macro-molecular protein precipitation, obtain the supernatant liquor containing natural novel polypeptide sanitas;
(6) by the supernatant liquid filtering containing natural novel polypeptide sanitas, filtrate is got;
(7) by filter vacuum lyophilize, vacuum freezing Rotary drying or be spray dried to Powdered rear freezen protective, the natural novel polypeptide sanitas of fresh shrimp is obtained.
In step (1), described Tibet Kefir granule is 8 ~ 15%(v/v in the inoculum size of MRS liquid nutrient medium), culture temperature is 30 ~ 40 DEG C, and shaking speed is 100 ~ 200r/min.
In step (3), Tibet Kefir granule nutrient solution after described inoculation activation and the quality volume proportion of milk are 5 ~ 15:100, described carbon source is maltose, glucose, sucrose, quality proportioning is 1 ~ 2:3 ~ 7:0 ~ 3, carbon source and milk mass ratio be 3 ~ 7%(m/v), described nitrogenous source is peptone, yeast powder, fish meal, and ratio is 3 ~ 5:0 ~ 2:1 ~ 3, nitrogenous source and milk mass ratio be 2 ~ 5%(m/v).
In step (4), the distilled water added and the volume ratio of fermented-milk are 0.5 ~ 2:1, and bath temperature is 35 ~ 55 DEG C, and the time is 30 ~ 90min.
In step (5), described centrifugation rotating speed is 3000 ~ 5000r/min.
In step (6), described filter type is: utilize the ceramic membrane of 200 ~ 500nm to carry out membrane filtration or carry out suction filtration with the solvent micropore filtering film of 0.22 ~ 0.45 μm.
As optimization, in step (3), the Tibet Kefir granule after inoculation activation and the quality volume proportion of milk are 9:100, the mass volume ratio of described carbon source and milk is 3.44:100, the mass volume ratio of described nitrogenous source and milk is 2:100, leavening temperature 32 DEG C, fermentation time 72 hours.
As optimization, in step (4), the distilled water added and the volume ratio of fermented-milk are 1:1, and water bath time 40min, pH are 3.5.
As optimization, in step (5), described centrifugation rotating speed 4000r/min, time 20min.
As optimization, in step (6), described filter type selective solvent micropore filtering film carries out suction filtration.
As optimization, in step (7), adopt vacuum lyophilization, temperature is-40 ± 3 DEG C, and freezen protective temperature is-18 ± 2 DEG C.
The present invention also provides a kind of natural novel polypeptide sanitas of fresh shrimp prepared as aforesaid method.
The present invention also provides the using method in fresh shrimp of the natural novel polypeptide sanitas of a kind of fresh shrimp, comprise: the fresh shrimp freezed to death through frozen water is soaked in the natural novel polypeptide sanitas after thin up, the mass ratio of natural novel polypeptide sanitas and water is 0.1 ~ 2:100, pull out after first soaking 5 ~ 15min, then draining 5 ~ 10min.
The present invention confirms that the natural novel polypeptide aseptic active obtained detects through 96 orifice plate micro-dilution method experiments and analyzes, and finds that it has remarkable restraining effect to the common food-borne pathogens such as intestinal bacteria (Escherlchiacoli), streptococcus aureus (Staphylococcusaureus), Salmonellas (salmonella) and the aquatic pathogenic bacteria such as Aeromonas hydrophila (A.hydrophila), the lonely bacterium (Vibrioharveyi) of Kazakhstan Vickers.Meanwhile, it has preferably acid and enzymolysis tolerance, and to light, thermally-stabilised, multigelation does not affect its anti-microbial activity, and can keep stable anti-microbial activity under common physico chemical factor, for its application in fresh shrimp is had laid a good foundation.
The invention has the advantages that: the Tibet Kefir granule that (1) the present invention uses is the wild microorganism resource that can be used for yogurt production, Kefir grains or Kefir grains breast is called with the milk of Kefir granule fermentation, Kefir grains is that one is rich in viable bacteria, the sour healthy beverage of tool, the some areas such as Tibet, Qinghai have the making of several thousand and the history of edible Kefir grains, and edible safety is high; (2) it is low that base cost compare is trained in the fermentation that the present invention uses, and its main component is pure milk, cheap, will be greatly cost-saving in industrialization process, economical and practical; (3) the fresh shrimp of gained of the present invention is simple, easy and simple to handle by the preparation technology of natural novel polypeptide sanitas; (4) the natural novel polypeptide sanitas has a broad antifungal spectrum of fresh shrimp of gained of the present invention, active strong, to aquatic pathogenic bacterias such as food-borne pathogens, Aeromonas hydrophila such as intestinal bacteria, there is remarkable restraining effect.Meanwhile, it has very strong stability: good sour tolerance, and to light, thermally-stabilised, multigelation does not affect its anti-microbial activity, and metal ion does not affect its anti-microbial activity, and can preserve for a long time; (5) the fresh shrimp of gained of the present invention can preserve nutritive ingredient and the mouthfeel of fresh shrimp to greatest extent with natural novel polypeptide sanitas, and the shelf-lives extended, safety and sanitation, Nantural non-toxic, high-efficiency broad spectrum, stable performance, and containing multiple to human body beneficial physiological active substance.
Accompanying drawing explanation
Fig. 1 fresh shrimp triketohydrindene hydrate color reaction of natural novel polypeptide sanitas.In figure, A: the natural novel polypeptide sanitas of fresh shrimp; B: water; C: soybean peptides;
Fig. 2 is the HPLC color atlas of fresh shrimp with natural novel polypeptide sanitas;
Fig. 3 is the HPLC color atlas of fresh shrimp with natural novel polypeptide sanitas polypeptide relative molecular weight distribution;
Fig. 4 is that thermal treatment is on the impact of fresh shrimp with the bacteriostatic activity of natural novel polypeptide sanitas;
Fig. 5 is that different light process is on the impact of fresh shrimp with natural novel polypeptide sanitas anti-microbial activity;
Fig. 6 is the change of South America prawn pH in 4 DEG C of storages;
Fig. 7 is the change of South America prawn TVB-N in 4 DEG C of storages;
Fig. 8 is the change of South America prawn total plate count in 4 DEG C of storages;
Fig. 9 is the change of South America prawn organoleptic evaluation points in 4 DEG C of storages.
Embodiment
Provide embodiments of the invention below, specifically set forth the present invention.
Embodiment 1:
A kind of fresh shrimp preparation method of natural novel polypeptide sanitas:
200 μ L Tibet Kefir granule are seeded to 2mLMRS liquid nutrient medium, and in 37 DEG C of shaking tables, 150r/min cultivates 10 ~ 18h.Get the Kefir granule nutrient solution of 1mL activation, with MRS substratum with the dilution proportion of 1:13 (v/v), OD 600=0.3.4L milk is placed in fermentation flask, in 9%(m/v) ratio inoculation activation after Tibet Kefir granule, according to 3.44%(m/v) ratio add carbon source (maltose: glucose: sucrose=1 ~ 2:3 ~ 7:0 ~ 3), according to 2%(m/v) ratio nitrogenous source (peptone: yeast powder: fish meal=3 ~ 5:0 ~ 2:1 ~ 3).Material various in fermentor tank is thoroughly shaken up dissolving, is placed on 32 ° of C and ferments 72 hours.Take out after fermentation, add isopyknic distilled water, water-bath also stirs 40min, and to adjust its pH be 3.5, and under being then placed in 4000r/min, centrifugal 20min, gets supernatant liquor.The supernatant liquor filter paper in 0.45 μm of aperture is crossed film, and it is for subsequent use to get filtrate.Collect repeatedly filtrate-18 ± 2 DEG C of cryopreservation after-40 ± 3 DEG C of vacuum lyophilizations are powdered, obtain the natural novel polypeptide sanitas of a kind of fresh shrimp.
Embodiment 2:
A kind of fresh shrimp preparation method of natural novel polypeptide sanitas:
160 μ L Tibet Kefir granule are seeded to 2mLMRS liquid nutrient medium, and in 35 DEG C of shaking tables, 150r/min cultivates 10 ~ 18h.Get the Kefir granule nutrient solution of 1mL activation, with MRS substratum with the dilution proportion of 1:14, OD 600=0.5.4L milk is placed in fermentation flask, in 15%(m/v) ratio inoculation activation after Tibet Kefir granule, according to 3%(m/v) ratio add carbon source (maltose: glucose: sucrose=1 ~ 2:3 ~ 7:0 ~ 3), according to 3%(m/v) ratio add nitrogenous source (maltose: glucose: sucrose=1 ~ 2:3 ~ 7:0 ~ 3).Material various in fermentor tank is thoroughly shaken up dissolving, is placed on 35 DEG C of fermentations 72 hours.Take out after fermentation, add distilled water according to the ratio of volume ratio 1:2, water-bath also stirs 30min, and to adjust its pH be 4, fermented liquid is directly placed in centrifugal 20min under 4000r/min, gets supernatant liquor.Supernatant liquor utilizes the ceramic membrane of 150nm to carry out membrane filtration, and it is for subsequent use to get filtrate.Collect repeatedly filtrate-18 ± 2 DEG C of cryopreservation after-40 ± 3 DEG C of vacuum lyophilizations are powdered, obtain the natural novel polypeptide sanitas of a kind of fresh shrimp.
Embodiment 3:
A kind of fresh shrimp preparation method of natural novel polypeptide sanitas:
240 μ L Tibet Kefir granule are seeded to 2mLMRS liquid nutrient medium, cultivate 10 ~ 18h in shaking table 40 ° of C, 150r/min.Get the Tibet Kefir grains nutrient solution of 1mL activation, with MRS substratum with the dilution proportion of 1:14, OD 600=0.4.4L milk is placed in fermentation flask, in 5%(m/v) ratio inoculation activation after Tibet Kefir granule, according to 4%(m/v) ratio add carbon source (maltose: glucose: sucrose=1 ~ 2:3 ~ 7:0 ~ 3), according to 1%(m/v) ratio nitrogenous source (maltose: glucose: sucrose=1 ~ 2:3 ~ 7:0 ~ 3).Material various in fermentor tank is thoroughly shaken up dissolving, is placed on 32 ° of C and ferments 65 hours.Take out after fermentation, add isopyknic distilled water and stir, water-bath also stirs 60min, and adjusting its pH is 3.5, and under fermented liquid being placed in 4000r/min, centrifugal 15min, gets supernatant liquor.The supernatant liquor filter paper in 0.22 μm of aperture is crossed film, and it is for subsequent use to get filtrate.Collect repeatedly filtrate-18 ± 2 DEG C of cryopreservation after-40 ± 3 DEG C of vacuum lyophilizations are powdered, obtain the natural novel polypeptide sanitas of a kind of fresh shrimp.
In above embodiment, embodiment 1 is preferred version.
Embodiment 4:
The invention provides the analysis of fresh shrimp natural novel polypeptide sanitas minimum inhibitory concentration:
Adopt 96 orifice plate micro-dilution methods to analyze, indicator is TTC, and concrete steps are:
4.1 indicator activation
The single bacterium of the test tube slant indicator that picking 4 DEG C is preserved in Bechtop, in 8mLLB broth culture, is positioned over 37 DEG C of constant-temperature tables, 150r/min activated overnight.It is 1 × 10 that the indicator liquid of activation is diluted to bacteria concentration 8cFU/mL, for subsequent use.
The mensuration of 4.2 minimum inhibitory concentrations
First the fresh shrimp configured is transferred to 96 orifice plates the 11st with natural novel polypeptide sanitas mother liquor to arrange in (sample controls hole) plate hole, every hole adds 100 μ L.The bacterium liquid to be measured of taking the logarithm vegetative period, the centrifugal 5min of 4000r/min collects thalline, after PBS cleans 3 times, resuspended to 1 × 10 with the LB substratum of fresh sterile 8cFU/mL, then transferred in the 1 to 10 row (sample test hole) and the 12nd row (growth control hole) plate hole, every hole adds 100 μ L.Then, join in the plate hole of first row by 100 μ L testing sample mother liquors, transfer to the 2nd row, so analogize after mixing from the 1st row taking-up 100 μ L, until the 10th row, the 100 μ L that the 10th row take out abandon.Finally, every hole adds 0.5% TTC indicator 5 μ L and mixes, and whether is used to indicate bacterial growth.Often row can make a sample, adds a cover vibration 1min, cultivate 6 ~ 8h in 37 DEG C of incubators after application of sample, is activatedly masked as without colour-change, its color pinkiness of person that has bacteria growing.The minimum weaker concn that colour-change occurs is the minimum inhibitory concentration of test substance, meanwhile, replaces the natural novel polypeptide sanitas mother liquor of fresh shrimp to do negative control, positive control respectively with isopyknic sterilized water, sulfuric acid kalamycin.
Fresh shrimp the results are shown in Table 1 with natural novel polypeptide sanitas antimicrobial spectrum.
The fresh shrimp of table 1 minimum inhibitory concentration (X ± SD, n=3) of natural novel polypeptide sanitas to different microorganisms
Indicator MIC(mg/mL)
Streptococcus aureus 5.15±0.22
Intestinal bacteria 4.85±0.25
Salmonellas 5.00±0.18
Dysentery He Zhi bacillus 4.96±0.19
Bacillus thuringiensis 3.95±0.11 4 -->
Aeromonas hydrophila 6.25±0.22
Breathe out the lonely bacterium of Vickers 3.13±0.12
Pseudomonas aeruginosa 4.00±0.23
Can know from table 1, the natural novel polypeptide sanitas of fresh shrimp has very strong restraining effect to common food-borne pathogens (streptococcus aureus (Staphylococcusaureus), intestinal bacteria (Escherlchiacoli), Salmonellas (salmonella)) and common aquatic pathogenic bacteria (Aeromonas hydrophila (A.hydrophila), the lonely bacterium (Vibrioharveyi) of Kazakhstan Vickers).Therefore, fresh shrimp is very wide by the antimicrobial spectrum of natural novel polypeptide sanitas, active strong.
Embodiment 5
The present invention fresh shrimp component analysis of natural novel polypeptide sanitas and safety evaluation.
First utilize ninhydrin to carry out qualitative analysis to the natural novel polypeptide sanitas of fresh shrimp, as shown in Figure 1, the fresh shrimp of triketohydrindene hydrate color reaction test display is polypeptides matter by the main component of natural novel polypeptide sanitas to result.By quantitative analysis, the fresh shrimp content of peptides of natural novel polypeptide sanitas is greater than 72.3%, and other compositions are that lactic acid, active polysaccharide etc. are to human body beneficial physiological active substance.Fresh shrimp with the HPLC color atlas of natural novel polypeptide sanitas and polypeptide relative molecular weight distribution HPLC color atlas thereof respectively as shown in Figure 2 and Figure 3.
Measure the hemolytic activity of fresh shrimp under natural novel polypeptide sanitas different concns, result is as table 2.
The fresh shrimp hemolytic activity of natural novel polypeptide sanitas under table 2 different concns
Shrimp is with natural novel polypeptide concentration of preservatives (mg/mL) Result
0 Without haemolysis
5 Without haemolysis
10 Without haemolysis
20 Without haemolysis
30 Without haemolysis
As table 2, we can know that the natural novel polypeptide sanitas of fresh shrimp does not substantially have hemolytic under effective Mlc, namely fresh shrimp kills bacterium with natural novel polypeptide sanitas energy Selective depression bacterial growth, and mammiferous red corpuscle is not almost acted on, there is higher security.
Simultaneously, we know that Kefir grains is that one is rich in viable bacteria, the sour healthy beverage of tool, the some areas such as Tibet, Qinghai have the making of several thousand and the history of edible Kefir grains, and the invention provides the natural novel polypeptide sanitas of fresh shrimp be exactly by Tibet Kefir granule ferment get.Therefore, the natural novel polypeptide sanitas of shrimp has edible safety, safety non-toxic.
Embodiment 6
The invention provides the stability evaluation of fresh shrimp with natural novel polypeptide sanitas
6.1 fresh shrimps with natural novel polypeptide sanitas to the stability of heat
After thermal treatment fresh shrimp with the change of natural novel polypeptide sanitas anti-microbial activity as shown in Figure 4, after fresh shrimp processes through 0 DEG C, 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C, 110 DEG C with natural novel polypeptide sanitas, fresh shrimp not to have yet 5 kinds of indicator bacteriostatic action with natural novel polypeptide sanitas and obviously weakens, this explanation, the natural novel polypeptide sanitas energy higher temperature resistant of fresh shrimp is more stable to heat.
6.2 fresh shrimps with natural novel polypeptide sanitas to the stability of pH
Fresh shrimp is with natural novel polypeptide sanitas after different pH process, and its fungistatic effect is in table 3
The table 3 fresh shrimp pH stability of natural novel polypeptide sanitas bacteriostatic activity
As known from Table 3, fresh shrimp natural novel polypeptide sanitas bacteriostatic activity under acidity and neutrallty condition is stablized, and illustrates that the natural novel polypeptide sanitas of fresh shrimp has stronger acid acceptance.
6.3 fresh shrimps with natural novel polypeptide sanitas to the stability of illumination
After different light process, fresh shrimp with the anti-microbial activity of natural novel polypeptide sanitas as shown in Figure 5, fresh shrimp is more stable to illumination with natural novel polypeptide sanitas, different light medium is also less with the bacteriostatic activity impact of natural novel polypeptide sanitas on fresh shrimp, illustrates that the natural novel polypeptide sanitas of fresh shrimp has good light stability.
6.4 fresh shrimps with natural novel polypeptide sanitas to the stability of freeze thawing
As shown in table 4, fresh shrimp with natural novel polypeptide sanitas after multigelation 5 times, the bacteriostatic activity of indicator streptococcus aureus is not had a significant impact (P>0.05), show that the natural novel polypeptide sanitas of fresh shrimp still preserves good anti-microbial activity, good stability after multigelation.
Table 4 number of freezing and thawing is on the impact (X ± SD, n=3) of fresh shrimp with natural novel polypeptide sanitas anti-microbial activity
Number of freezing and thawing (secondary) 1 2 3 4 5 0(contrasts)
MIC（mg/mL） 5.16±0.12 5.15±0.08 5.14±0.17 5.21±0.23 5.13±0.13 5.15±0.11
6.5 deposit number of days to the impact of fresh shrimp with natural novel polypeptide sanitas anti-microbial activity
As can be seen from Table 5, after fresh shrimp preserves 30d with natural novel polypeptide sanitas under normal temperature condition, it does not weaken the bacteriostatic activity of streptococcus aureus, does not have significant difference (P>0.05), shows that fresh shrimp is better by the stability of natural novel polypeptide sanitas.Also find in an experiment, when cold preservation time is longer (-4 DEG C more than 15 months), after fresh shrimp is redissolved with natural novel polypeptide sanitas lyophilized powder, solution is clear the same as the fermented liquid before freeze-drying still, relatively itself and the natural novel polypeptide sanitas of fresh shrimp obtained at first are to the MIC of streptococcus aureus, without significant difference (P>0.05), show that the natural novel polypeptide sanitas of fresh shrimp can be preserved under refrigerated conditions for a long time.
Table 5 deposits number of days to the impact (X ± SD, n=3) of fresh shrimp with natural novel polypeptide sanitas anti-microbial activity
Deposit number of days (d) 5 10 15 20 30 0(contrasts)
MIC（mg/mL） 5.17±0.18 5.21±0.13 5.24±0.12 5.16±0.2 5.15±0.19 5.24±0.21
6.6 metal ions are on the impact of fresh shrimp with natural novel polypeptide sanitas anti-microbial activity
By table 6, we are known Fe 3+, Na +, Cu 2+, Ca 2+, K +, Ba 2+under high, medium and low concentration with fresh shrimp with the fungistatic effect after the effect of natural novel polypeptide sanitas with contrast (the natural novel polypeptide sanitas of fresh shrimp without the Action of Metal Ions) difference (P>0.05) that compares that there are no significant.Therefore, metal ion is very little with the impact of natural novel polypeptide sanitas anti-microbial activity on fresh shrimp.
Table 6 metal ion is on the impact (X ± SD, n=3) of fresh shrimp with natural novel polypeptide sanitas anti-microbial activity
(remarks: Ca 2+, Ba 2+, Fe 3+, Cu 2+high, medium and low concentration be respectively 50mmol/L, 5mmol/L, 1mmol/L; Na +, K +high, medium and low concentration be respectively 500mmol/L, 50mmol/L, 10mmol/L)
In sum, fresh shrimp is highly stable with the anti-microbial activity of natural novel polypeptide sanitas, and this is for having established solid basis in its application anti-corrosive fresh-keeping field.
Embodiment 7:
The invention provides the application of the natural novel polypeptide sanitas antibacterial peptide of bright fresh shrimp in fresh shrimp.
This embodiment is for illustration of the present invention, and concrete test method is as follows:
The process of 7.1 South America shrimp samples
Choose fresh and alive, complete, color is vivid, uniform South America prawn, rejects dead impaired individuals, South America prawn is cleaned with distilled water and drains, random packet after immersing and freezing to death in frozen water (1:2, w/v), and experiment particular case of dividing into groups is as shown in table 7.South America prawn after grouping is respectively charged in freshness protection package, then freshness protection package is placed in crisper, finally crisper is placed in 4 DEG C of refrigerators and stores.In 0,2,4,6,8,10d sampling, often organize sampling 10 at every turn, often organize sample parallel and measure 3 times.
Process grouping information slip tested by table 7
Group Processing mode
Blank group In sterilized water, soak 10 min, draining is to dry
Potassium sorbate group Be soak 10 min in 0.5 % potassium sorbate aqueous solution in concentration, draining is to dry
Nisin group Be soak 10 min in the 0.5 % nisin aqueous solution in concentration, draining is to dry
The natural novel polypeptide sanitas group of fresh shrimp Be that the fresh shrimp of 0.5 % soaks 10 min with in natural novel polypeptide aqueous preservative solution in concentration, draining is to dry
The physical and chemical index of 7.2 different time sections South America prawns measures
7.2.1 the mensuration of different time sections South America prawn pH value index
Get 5g shrimp to add 45mL distilled water and carry out homogenate, get supernatant liquor after the centrifugal 5min of 4000r/min, measure its pH with pH meter.
7.2.2 different time sections South America prawn total volatile basic nitrogen (TVB-N) measures
South America prawn sample is in storage process, and alkaline nitrogenous substances that breaks down proteins produces, as ammonia, primary amine, secondary amine and tertiary amine etc., after can volatilizing collected by absorbed liquid, and measures its content with standard acidometric titration in the alkali lye of 37 DEG C.Measuring method in the mensuration with reference to SC/T3032-2007 total volatile basic nitrogen in fishery products, namely microdiffusion measures the TVB-N content of different time sections South America prawn sample.Take 10g South America shrimp samples in homogeneous cup, then add 90mL perchloric acid solution, homogeneous 2min, with filter paper filtering or centrifugation, filtrate, in the storage under ambient conditions of 2 DEG C-6 DEG C, can preserve 2d.Draw 10mL boric acid absorption liquid to inject in Erlenmeyer flask, then add 2-3 mixture indicators, and Erlenmeyer flask is placed in semimicro and determine nitrogen device distillation prolong lower end, under making the liquid level of its lower end insertion boric acid absorption liquid.Accurate absorption 5.0mL sample filtrate is injected semimicro and is determined in nitrogen device reaction chamber, then adds 1-2 phenolphthalein indicator, 1-2 silicone oil foam preventers, 5mL sodium hydroxide respectively, then rapid capping plug, and adds water with anti-gas-leak.Pass into steam, after distillation 5min, prolong end is moved apart the liquid level of absorption liquid, redistillation 1min, rinse prolong end with a small amount of water, wash in Erlenmeyer flask.In Erlenmeyer flask, absorption liquid hydrochloric acid standard solution (0.01mol/L) is titrated to solution to show bluish voilet is terminal, uses 5.0mL perchloric acid solution (0.6mol/L) to replace sample filtrate to carry out blank test simultaneously.Reference SC3113-2002, TVB-N value >=25mg/100g is putrid and deteriorated judging criterion.The content of total volatile basic nitrogen in the prawn sample of South America is calculated according to formula 1.
In formula:
X: the content of total volatile basic nitrogen in the prawn sample of South America, unit is the every hectogram of milligram (mg/100g);
V 1: titration sample quota of expenditure acid solution volume, unit is milliliter (mL);
V 2: titration blank quota of expenditure acid solution volume, unit is milliliter (mL);
C: the actual concentrations of hydrochloric acid standard solution, unit is mole often liter (mol/L);
14: the quality of the nitrogen suitable with 1.00mL Hydrochloric Standard Titration [c(HCl)=1.000mol/L], unit is milligram (mg);
M: South America prawn sample quality, unit is gram (g).
The mensuration of 7.3 different time sections South America prawn microbiological indicators
The total plate count in the prawn sample of South America is measured with reference to GB4789.2-2010 method.Take 25g South America prawn sample in the Erlenmeyer flask of 225mL stroke-physiological saline solution, jolting 30min, makes the sample diluting liquid of 1:10, then carries out 10 times dilutions of sample with 1mL aseptic straw or micropipet successively.According to the estimation to sample contamination situation, select the sample diluting liquid of acceptable diluent degree in sterilized petri dishes, pour 15 ~ 20mL into and be cooled to the PCA of 46 DEG C and rotate plate mixing.Make blank simultaneously.After culture medium solidifying, by plate, 36 ° of C ± 1, ° C is inverted cultivation 48h ± 2h.Plate count, result is with logarithm log(CFU/g) represent.In general, South America prawn total plate count (CFU/g)≤10 5for one-level freshness ,≤5 × 10 5for secondary freshness.
The Oranoleptic indicator of 7.4 different time sections South America prawn samples analyzes
Carry out after Oranoleptic indicator's scoring criteria measured with reference to the measuring method in GB/T9959.2-2008 and description makes suitably amendment.South America prawn sample is taken out from refrigerator, after placement 15min divides, please 10 accepted strict subjective appreciation training and the personnel with subjective appreciation experience evaluate its smell, form, color and luster, muscle tissue etc. according to sensory evaluation scores table, and get often organize sense organ differentiate after sample carry out cooking test.The evaluation result of every Oranoleptic indicator all adopts marking system above, and population of samples scoring is the mean value of each metrics evaluation result summation.Sensory evaluation scores table is as shown in table 8.
Table 8 South America prawn sensory evaluation scores table
(remarks: total score is more than or equal to 24 points of in season shrimps and belongs to one-level freshness, total score is less than 24, is more than or equal to 18 timesharing is secondary freshness, then illustrates that fresh shrimp is putrid and deteriorated, not edible lower than 18 points.）
The change of South America prawn pH in 4 DEG C of storages as shown in Figure 6, in the process of South America prawn storage, along with the prolongation of cold preservation time, the pH of four groups of samples raises gradually, but blank group raises faster, there were significant differences (P<0.05) with other three groups, interpolation 0.5% potassium sorbate, 0.5% nisin are described, the natural novel polypeptide sanitas of 0.5% fresh shrimp can suppress microbial growth effectively, reduce the decomposition rate of protein, slow down the speed of the rising of pH, extend the shelf-lives of South America prawn.
The change of South America prawn TVB-N in 4 DEG C of storages as shown in Figure 7, when South America prawn refrigerates 0d, the content of the volatile alkali nitrogen of each group is more or less the same, but along with the prolongation of cold preservation time, the TVB-N value of four groups all constantly increases, and wherein blank group ramp-up rate is obviously fast and be greater than 0.5% potassium sorbate, 0.5% nisin, 0.5% fresh shrimp all the time with natural novel polypeptide sanitas group (P<0.05).Blank group is when 4d, and TVB-N value is 24.18mg/100g, and close to danger threshold, fresh shrimp is by putrid and deteriorated.During 6d, blank group TVB-N value is 32.48mg/100g, exceedes danger threshold, and fresh shrimp is putrid and deteriorated.Now, 0.5% potassium sorbate, 0.5% nisin, the 0.5% fresh shrimp TVB-N value of natural novel polypeptide sanitas group be respectively 23.24,22.96,19.6mg/100g, more than 25mg/100g, fresh shrimp is not putrid and deteriorated.During 8d, 0.5% potassium sorbate, the TVB-N value of 0.5% nisin group is all more than 25mg/100g, and 0.5% fresh shrimp is 24.08mg/100g by natural novel polypeptide sanitas group TVB-N value, within danger threshold, 0.5% potassium sorbate is described, 0.5% nisin, the natural novel polypeptide sanitas of 0.5% fresh shrimp suppresses the too fast breeding of microorganism enough preferably, the degraded of arrestin matter generates the alkaline nitrogenous substances such as ammonia and amine, thus the speed that TVB-N value rises reduces, but 0.5% fresh shrimp is better than the effect that with the addition of 0.5% potassium sorbate and 0.5% nisin by the effect of natural novel polypeptide sanitas.
As shown in Figure 8, compared with blank group, microorganism is (P<0.05) that be significantly inhibited in the growth of other experimental group in the change of South America prawn total plate count in 4 DEG C of storages.In general, fresh shrimp total plate count (CFU/g)≤10 5for one-level freshness ,≤5 × 10 5for secondary freshness, total plate count reaches 10 6time, usual shrimp is corrupt, can not eat, and now predicates shelf-lives terminal.It is 4.4 × 10 that the total plate count of blank group has increased when 6d 6cFU/g, far away higher than 10 6cFU/g, illustrates that the shelf-lives refrigerating fresh shrimp is only 4d.Time 8d, 0.5% potassium sorbate, 0.5% nisin, 0.5% fresh shrimp are 1.48 × 10 by the total plate count of natural novel polypeptide sanitas group respectively 6, 2.84 × 10 6, 6.00 × 10 5cFU/g, illustrate with the addition of 0.5% potassium sorbate, the fresh shrimp shelf-lives of 0.5% nisin can extend to 6d, and the fresh shrimp shelf-lives adding the natural novel polypeptide sanitas of 0.5% fresh shrimp can extend to 8d, therefore, the natural novel polypeptide sanitas of 0.5% fresh shrimp is obviously better than 0.5% potassium sorbate, 0.5% nisin to the fresh-keeping effect of fresh shrimp.
The change of South America prawn organoleptic evaluation points in 4 DEG C of storages as shown in Figure 9, in the refrigeration of South America prawn the most at first, the sensory evaluation scores of each group is basically identical, interpolation 0.5% potassium sorbate, 0.5% nisin is described, the natural novel polypeptide sanitas of 0.5% fresh shrimp all can not have a negative impact to the sense organ of fresh shrimp.Along with the prolongation of storage time, the sensory evaluation scores of four groups of samples is all more and more lower, and the speed that blank group declines is significantly higher than other test group (P<0.05).When refrigeration 6d, blank group sensory evaluation scores is 10.8 points, and 0.5% potassium sorbate, 0.5% nisin, 0.5% fresh shrimp are respectively 20,20.45,24 points by the sensory evaluation scores of natural novel polypeptide sanitas group, wherein fresh shrimp is also in one-level freshness with the fresh shrimp of natural novel polypeptide sanitas group.During to 8d, only have the natural novel polypeptide sanitas group of 0.5% fresh shrimp to be in secondary freshness, its sensory evaluation scores is 20.5 points, and 0.5% potassium sorbate, 0.5% nisin group are putrid and deteriorated.Refrigeration is to 10d, and the mark of four groups of samples is all down to less than 18 points, and illustrate that four groups of fresh shrimps are all corrupt, sensory evaluation scores terminates.Therefore, the fresh shrimp sense organ acceptable time can be made after 0.5% potassium sorbate, 0.5% nisin immersion treatment to extend 6d by 4d, and through 0.5% fresh shrimp with after natural novel polypeptide sanitas immersion treatment, the acceptable time lengthening of fresh shrimp sense organ is to 8d, and namely the fresh shrimp fresh-keeping effect of natural novel polypeptide sanitas group is better than other experimental group.
In sum, South America prawn is in cold storage procedure, and the variation tendency of sensory evaluation scores, pH, TVB-N and total number of bacterial colony is basically identical, TVB-N value for 25mg/100g and total number of bacterial colony be 10 6cFU/g is as the boundary judging shelf-lives terminal.From blank group, the shelf-lives of South America prawn is 4d, after with the addition of 0.5% potassium sorbate, 0.5% nisin, the shelf-lives of South America prawn can extend to 6d, but interpolation 0.5% fresh shrimp can by the shelf life extension of fresh shrimp to 8d with natural novel polypeptide sanitas.Therefore, the natural novel polypeptide sanitas of 0.5% fresh shrimp is obviously better than 0.5% potassium sorbate, 0.5% nisin to the fresh-keeping effect of South America prawn, has good application prospect.

Claims (11)

1. a fresh shrimp preparation method for natural novel polypeptide sanitas, is characterized in that, comprise the following steps:
(1) be seeded in MRS liquid nutrient medium by the Tibet Kefir granule of late log phase, cultivate in shaking table, activated strains, obtains the Tibet Kefir granule nutrient solution of activation;
(2) with the Tibet Kefir granule nutrient solution of MRS liquid nutrient medium dilution activation, its OD is adjusted 600=0.2 ~ 0.5;
(3) the Tibet Kefir granule nutrient solution after the activation of step (2) gained is seeded in pure milk, meanwhile, adds Carbon and nitrogen sources, fully stir and each material is mixed and dissolves, be placed on 32 ~ 38 ° of C fermentations 65 ~ 75 hours;
(4) after having fermented, add distilled water, water-bath limit, limit is stirred, and regulates its pH3 ~ 4.5;
(5) get the centrifugal 10 ~ 20min of step (4) gained fermented liquid, remove undissolved macro-molecular protein precipitation, obtain the supernatant liquor containing natural novel polypeptide sanitas;
(6) by the supernatant liquid filtering containing natural novel polypeptide sanitas, filtrate is got;
(7) by filter vacuum lyophilize, vacuum freezing Rotary drying or be spray dried to Powdered rear freezen protective, the natural novel polypeptide sanitas of fresh shrimp is obtained.
2. a kind of fresh shrimp according to claim 1 preparation method of natural novel polypeptide sanitas, it is characterized in that, in step (1), described Tibet Kefir granule is 8 ~ 15% in the inoculum size of MRS liquid nutrient medium, culture temperature is 30 ~ 40 DEG C, and shaking speed is 100 ~ 200r/min.
3. a kind of fresh shrimp according to claim 1 preparation method of natural novel polypeptide sanitas, it is characterized in that, in step (3), Tibet Kefir granule nutrient solution after described inoculation activation and the quality volume proportion of milk are 5 ~ 15:100, described carbon source is maltose, glucose, sucrose, quality proportioning is 1 ~ 2:3 ~ 7:0 ~ 3, carbon source and milk mass ratio be 3 ~ 7%, described nitrogenous source is peptone, yeast powder, fish meal, quality proportioning is 3 ~ 5:0 ~ 2:1 ~ 3, nitrogenous source and milk mass ratio be 2 ~ 5%.
4. a kind of fresh shrimp preparation method of natural novel polypeptide sanitas according to claim 1, is characterized in that, in step (4), the distilled water added and the volume ratio of fermented-milk are 0.5 ~ 2:1, and bath temperature is 35 ~ 55 DEG C, and the time is 30 ~ 90min.
5. a kind of fresh shrimp according to claim 1 preparation method of natural novel polypeptide sanitas, it is characterized in that, in step (6), described filter type is: utilize the ceramic membrane of 200 ~ 500nm to carry out membrane filtration or carry out suction filtration with the solvent micropore filtering film of 0.22 ~ 0.45 μm.
6. a kind of fresh shrimp according to claim 1 preparation method of natural novel polypeptide sanitas, it is characterized in that, in step (3), Tibet Kefir granule nutrient solution after inoculation activation and the quality volume proportion of milk are 9:100, the mass volume ratio of described carbon source and milk is 3.44:100, the mass volume ratio of described nitrogenous source and milk is 2:100, leavening temperature 32 DEG C, fermentation time 72 hours.
7. the preparation method of natural novel polypeptide sanitas of a kind of fresh shrimp according to claim 1 or 4, is characterized in that, in step (4), the distilled water added and the volume ratio of fermented-milk are 1:1, and water bath time 40min, pH are 3.5.
8. the preparation method of natural novel polypeptide sanitas of a kind of fresh shrimp according to claim 1 or 6, is characterized in that, in step (6), described filter type selective solvent micropore filtering film carries out suction filtration.
9. a kind of fresh shrimp preparation method of natural novel polypeptide sanitas according to claim 1, is characterized in that, in step (7), adopt vacuum lyophilization, temperature is-40 ± 3 DEG C, and freezen protective temperature is-18 ± 2 DEG C.
10. the natural novel polypeptide sanitas of fresh shrimp obtaining method and prepare as described in claim 1-10 any one.
The using method that 11. 1 kinds of fresh shrimps as claimed in claim 11 obtain with natural novel polypeptide sanitas, it is characterized in that, comprise: the fresh shrimp freezed to death through frozen water is soaked in the natural novel polypeptide sanitas after thin up, the mass ratio of natural novel polypeptide sanitas and water is 0.1 ~ 2:100, pull out after first soaking 5 ~ 15min, then draining 5 ~ 10min.
CN201510937457.XA 2015-12-16 2015-12-16 Novel natural polypeptide preservative for fresh shrimps and preparation method thereof Pending CN105567768A (en)

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CN1635138A (en) * 2004-11-23 2005-07-06 黑龙江大学 Fermentation method of small peptide natural microbiological antiseptic
CN104186681A (en) * 2014-07-25 2014-12-10 上海交通大学 Preparation method of Tibetan kefir semi-hard cheese
CN104855492A (en) * 2015-04-02 2015-08-26 曹庸 A natural preservative for meat products and a preparation and use method thereof
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