CN111280244B - Fresh-keeping agent for salmon and fresh-keeping method thereof - Google Patents
Fresh-keeping agent for salmon and fresh-keeping method thereof Download PDFInfo
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- CN111280244B CN111280244B CN202010152120.9A CN202010152120A CN111280244B CN 111280244 B CN111280244 B CN 111280244B CN 202010152120 A CN202010152120 A CN 202010152120A CN 111280244 B CN111280244 B CN 111280244B
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Images
Classifications
-
- 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
- A23B4/22—Microorganisms; Enzymes; Antibiotics
-
- 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
- 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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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
Abstract
Fresh-keeping method for raw salmonThe fresh salmon preservative is obtained by adding a modified polymer bacteriostatic agent A and nisin into distilled water according to the mass ratio of 2:3-3:2 and carrying out ultrasonic oscillation, wherein the structural formula of the modified polymer bacteriostatic agent A is as follows:
wherein n is a positive integer of 5 to 12. The method for preserving salmon by using the fresh salmon preservative comprises the following steps of: washing peeled salmon with purified water, draining, soaking in fresh salmon preservative at room temperature for 3min, draining again, sealing in a sealing bag, and storing at 4deg.C. The advantages are that: the fresh salmon preservative has antibacterial effect on gram-positive bacteria and gram-negative bacteria, has no adverse effect on low-temperature storage and preservation of salmon, has good antibacterial aging, and can effectively prolong the shelf life of salmon.
Description
Technical Field
The invention belongs to the field of aquatic product preservation, and particularly relates to a preservation method for raw salmon.
Background
Salmon (Salmon), also known as Salmon or Salmon, belongs to the order Salmon, class teleosts. Salmon is rich in vitamins A, B, D, E and various mineral elements (such as zinc, selenium and phosphorus), and contains a large amount of unsaturated fatty acids such as docosahexaenoic acid, eicosapentaenoic acid and docosapentaenoic acid which are the main components of brain gold and deep sea fish oil, and is very beneficial to human health. The fresh salmon meat is fine and smooth and has orange color, and is suitable for slicing and raw eating. The salmon has high nutrient and moisture content, and is easy to be polluted by microorganisms during storage to cause putrefaction and deterioration.
At present, the seafood fresh-keeping adopts a preservative to keep fresh, and the preservative is applied to fresh foods in a soaking, spraying or coating mode, so that the effects of corrosion prevention and fresh keeping are achieved. Because salmon mainly takes raw food, the requirements on the preservative are severe, natural and safe biological preservatives are generally selected, the best effect is achieved by using a small amount of the preservatives in the dosage, and the factors lead to relatively lag research on the preservation of salmon. Therefore, there is a need to develop novel antistaling agents to maintain the fresh state of salmon and maintain higher quality, thereby meeting the needs of different transportation or consumers.
Nisin (Nisin) is a light brown solid powder, a polypeptide compound composed of a plurality of amino acids, can be absorbed and utilized by human body as a nutrient substance, can be hydrolyzed by protease in human digestive tract to digest amino acids, is harmless to health and has biological activity at low concentration, is a safer preservative, does not change normal flora of intestinal tract, does not cause drug property, and does not generate cross resistance of other antibiotics. Nisin is a world-recognized and safe natural biological food preservative and antibacterial agent, and has the characteristics of unique high efficiency, no toxicity, no residue, no drug resistance and the like, so that Nisin is widely applied to the fields of meat products, dairy products, cans, beverages, fruit juice beverages, egg products, seasonings, brewing processes, baked foods, instant foods, aroma-based flavors, cosmetics and the like. However, researches also show that Nisin has limitation in bacteriostasis, can only inhibit gram-positive bacteria, has no obvious bacteriostasis effect on gram-negative bacteria, saccharomycetes and mould, and therefore limits the application range. As most of dominant spoilage bacteria causing spoilage of aquatic products are gram-negative bacteria, application reports of Nisin in aquatic products are few, and when Nisin is singly used in reported documents, a great amount of Nisin is often added to achieve a good fresh-keeping effect, and the application of Nisin in raw food products is more recently reported.
Disclosure of Invention
The technical problem to be solved by the invention is to provide the fresh salmon preservative and the fresh-keeping method thereof, wherein the fresh salmon preservative has an antibacterial effect on gram-positive bacteria and gram-negative bacteria, has no adverse effect on low-temperature storage and fresh-keeping of salmon, has good antibacterial aging, and can prolong the shelf life of salmon.
The technical scheme of the invention is as follows:
a fresh-keeping agent for salmon is characterized in that: the fresh salmon preservative is obtained by adding a modified polymer bacteriostatic agent A and nisin into distilled water according to the mass ratio of 2:3-3:2 and carrying out ultrasonic oscillation, wherein the structural formula of the modified polymer bacteriostatic agent A is as follows:
wherein n is a positive integer of 5 to 12.
Further, the mass ratio of the modified polymer bacteriostatic agent A to distilled water is 0.2:100-0.3:100.
Further, the preparation method of the fresh salmon preservative specifically comprises the following steps:
dissolving the modified polymer bacteriostatic agent A with ethanol of 5-10 times of mass, adding the dissolved bacteriostatic agent A into distilled water for dilution, adding nisin, and carrying out ultrasonic oscillation dissolution to obtain the fresh salmon preservative.
Further, the mass ratio of the modified polymer bacteriostatic agent A to distilled water is 0.3:100, and the mass ratio of the nisin to the modified polymer bacteriostatic agent A is 1:1.
Further, the synthesis steps of the modified polymer bacteriostat A are as follows:
ethanol is taken as solvent, N is added α -tert-butoxycarbonyl-L-lysine, 4-n-hexyloxybenzaldehyde and 4-diethylaminophenylisonitrile are fed according to the molar ratio of 1:2:2, stirred for 48-72 hours at room temperature, ethanol is unscrewed, petroleum ether is used for precipitation, and the precipitate is dried in vacuum to obtain an intermediate; and (3) dissolving the intermediate by using a solvent at the temperature of 0 ℃, adding trifluoroacetic acid according to the mass ratio of the intermediate to the trifluoroacetic acid of 1:0.2, stirring for 3-6 hours, then spinning out the solvent, precipitating by using petroleum ether, and drying the precipitate to obtain the modified polymer bacteriostatic agent A.
A method for preserving salmon by using fresh salmon preservative comprises the following specific steps:
washing peeled salmon with purified water, draining, soaking in fresh salmon preservative at room temperature for 3min, draining again, sealing in a sealing bag, and storing at 4deg.C.
The preparation method of the fresh salmon preservative is simple and the operation is easy; the raw salmon preservative is obtained by ultrasonic oscillation of the modified polymer bacteriostatic agent A and nisin, wherein the side chain of the modified polymer bacteriostatic agent A is provided with a plurality of N atoms and O atoms, and the molecule has strong proton receiving capability, so that the bacteriostatic performance is enhanced; meanwhile, the N atom and the O atom can interact with nisin through hydrogen bonds, so that structural stability can be better for bacteriostasis, and the nisin can not be replaced by simple mixing. The fresh salmon preservative has antibacterial effect on gram-positive bacteria and gram-negative bacteria, has no adverse effect on low-temperature storage and preservation of salmon, has good antibacterial aging, and can effectively prolong the shelf life of salmon.
Drawings
FIG. 1 is a scanning electron microscope image of Shewanella putrefying without any treatment;
FIG. 2 is a scanning electron microscope image of Shewanella putrefying bacteria treated with raw salmon preservative of the present invention (corresponding to example 6);
FIG. 3 is a transmission electron microscope image of Shewanella putrefying without any treatment;
FIG. 4 is a transmission electron microscopy image of Shewanella putrefying fresh food preservative treatment of the present invention (corresponding to example 6);
FIG. 5 is the effect of the raw salmon preservative of the invention (corresponding to example 6) on the integrity of the cell membrane of Shewanella putrefying;
FIG. 6 is a graph showing pH changes of salmon meat treated with different preservatives;
FIG. 7 shows TBA changes of salmon meat treated with different preservatives;
FIG. 8 is a TVB-N change of salmon meat treated with different preservatives;
FIG. 9 is a graph showing the total bacterial count change for salmon flesh treated with different preservatives;
FIG. 10 is a modified polymeric bacteriostat A of the present invention 1 H-NMR；
FIG. 11 is a mass spectrum of the modified polymer bacteriostatic agent A according to the invention.
Detailed Description
The invention will be further illustrated with reference to specific examples.
Example 1
Ethanol is taken as solvent, N is added α tert-Butoxycarbonyl-L-lysine, 4-n-hexyloxybenzaldehyde and 4-diethylaminophenylisonitrile in a molar ratio of 1:2:2, stirring at room temperature for 48h, eluting ethanol, and dissolving the crude product in CH 2 Cl 2 Adding petroleum ether for multiple precipitation, filtering, and drying the solid precipitate under vacuum to obtain an intermediate; at 0 ℃, adding trifluoroacetic acid according to the mass ratio of the intermediate to the trifluoroacetic acid of 1:0.2, stirring for 6 hours, and screwing out CH 2 Cl 2 Adding petroleum ether to precipitate for multiple times, filtering, and drying to obtain modified polymerAnd (3) a physical bacteriostat A. The synthetic modified polymer bacteriostat A was characterized by H-NMR (FIG. 10) and MALDI-TOF-Mass (FIG. 11), and has the structural formula
Wherein n is a positive integer of 5 to 12.
Example 2
Ethanol is taken as solvent, N is added α tert-Butoxycarbonyl-L-lysine, 4-n-hexyloxybenzaldehyde and 4-diethylaminophenylisonitrile in a molar ratio of 1:2:2, stirring at room temperature for 72h, eluting ethanol, and dissolving the crude product in CH 2 Cl 2 Adding petroleum ether for multiple precipitation, filtering, and drying the solid precipitate under vacuum to obtain an intermediate; at 0 ℃, adding trifluoroacetic acid according to the mass ratio of the intermediate to the trifluoroacetic acid of 1:0.2, stirring for 3 hours, and screwing out CH 2 Cl 2 And adding petroleum ether for precipitation for multiple times, filtering and drying to obtain the modified polymer bacteriostat A. The synthetic modified polymer bacteriostat A was characterized by H-NMR (FIG. 10) and MALDI-TOF-Mass (FIG. 11), and has the structural formula
Wherein n is a positive integer of 5 to 12.
Example 3
Ethanol is taken as solvent, N is added α tert-Butoxycarbonyl-L-lysine, 4-n-hexyloxybenzaldehyde and 4-diethylaminophenylisonitrile in a molar ratio of 1:2:2, stirring at room temperature for 60h, eluting ethanol, and dissolving the crude product in CH 2 Cl 2 Adding petroleum ether for multiple precipitation, filtering, and drying the solid precipitate under vacuum to obtain an intermediate; at 0 ℃, adding trifluoroacetic acid according to the mass ratio of the intermediate to the trifluoroacetic acid of 1:0.2, stirring for 5 hours, and screwing out CH 2 Cl 2 And adding petroleum ether for precipitation for multiple times, filtering and drying to obtain the modified polymer bacteriostat A. The synthetic modified polymer bacteriostat A was characterized by H-NMR (FIG. 10) and MALDI-TOF-Mass (FIG. 11), and has the structural formula
Wherein n is a positive integer of 5 to 12.
Example 4
(1) Fresh-keeping agent for making raw salmon
0.03g of the modified polymer bacteriostatic agent A synthesized in the example 3 is weighed, dissolved by 0.15g of ethanol, slowly dripped into 10mL of distilled water by a pipette, then added with 0.02g of Nisin, and vibrated by ultrasound until dissolved, thus obtaining the fresh salmon preservative for standby.
(2) Fresh-keeping treatment for salmon
Washing peeled salmon with purified water, draining, soaking in fresh salmon preservative at room temperature for 3min, draining again, sealing in a sealing bag, and storing at 4deg.C.
Example 5
(1) Fresh-keeping agent for making raw salmon
0.02g of the modified polymer bacteriostatic agent A synthesized in the example 3 is taken, dissolved by 0.15g of ethanol, slowly dripped into 10mL of distilled water by a liquid-transfering gun, then 0.03g of Nisin is added into the distilled water, and the solution is vibrated by ultrasound until the Nisin is dissolved, so as to obtain the fresh salmon preservative for standby.
(2) Fresh-keeping treatment for salmon
Washing peeled salmon with purified water, draining, soaking in fresh salmon preservative at room temperature for 3min, draining again, sealing in a sealing bag, and storing at 4deg.C.
Example 6
(1) Fresh-keeping agent for making raw salmon
0.03g of the modified polymer bacteriostatic agent A synthesized in the example 3 is weighed, dissolved by 0.3g of ethanol, slowly dripped into 10mL of distilled water by a pipette, then added with 0.03g of Nisin, and vibrated by ultrasound until dissolved, thus obtaining the fresh salmon preservative for standby.
(2) Fresh-keeping treatment for salmon
Washing peeled salmon with purified water, draining, soaking in fresh salmon preservative at room temperature for 3min, draining again, sealing in a sealing bag, and storing at 4deg.C.
Salmon fresh-keeping experiment
Salmon of examples 4, 5 and 6 were stored in a refrigerator at 4 c and meat was taken at intervals of days to test the corresponding freshness index. The preparation and soaking time of the control group solution are the same, and the soaked fish meat is treated according to the process and then subjected to the corresponding freshness index test.
Fresh salmon preservative bacteriostasis experiment
(1) Preparing LB broth culture medium: weighing 5g of LB broth, dissolving in 200mL of distilled water, and sterilizing at 121 ℃ for 15min to obtain LB broth culture medium for activating escherichia coli (gram-negative bacteria) and bacillus subtilis (gram-positive bacteria);
(2) Taking out Escherichia coli and Bacillus subtilis in a refrigerator at-80deg.C, adding 200 μL respectively into 10mL LB broth, and culturing in a shaker at 37deg.C for 12 hr;
(3) Preparing LB nutrition agar: weighing 32g of LB nutrient agar, dissolving in 800mL of distilled water, heating to boil for dissolution, and sterilizing at 121 ℃ for 15min for plating of escherichia coli;
(4) Preparing nutrient agar: weighing 26.4g of nutrient agar, adding into 800mL of distilled water, heating, boiling for dissolution, sterilizing at 121 ℃ for 15min, and placing the agar after high-temperature sterilization into a baking oven at 55-60 ℃ for standby application, wherein the agar is used for the plating of bacillus subtilis.
(5) E, adding the escherichia coli/bacillus subtilis cultured in the step (2) into respective agar media, and then pouring the agar media into a flat plate with oxford cups; after the agar is dried, the oxford cup is pulled out, 200 mu L of bacteriostat is pumped into the hole, and finally the mixture is put into a baking oven at 37 ℃ for culturing for 12 hours, and the size of a bacteriostasis ring is measured.
Analysis of bacteriostasis properties of flat plate
According to the bacteriostasis experimental method, the fresh salmon preservative of the example 4 is used as a bacteriostat, and a single bacteriostat is respectively selected as a control sample, (1) 0.3%wt epsilon-PL, (2) 0.2%wt Nisin and (3) 0.3%wt of the modified polymer bacteriostat A of the example 3; the results of measuring the size of the inhibition zone are as follows:
coli: 0.3%wt epsilon-PL sample antibacterial ring diameter is 11.65mm; the diameter of a bacteriostasis ring of a 0.2%wt Nisin sample is 11.20mm;0.3% wt. of the modified polymeric bacteriostatic agent A sample of example 3 had a zone of 13.80mm in diameter; the diameter of the antibacterial circle of the raw salmon preservative sample of example 4 is 15.08mm.
Bacillus subtilis: 0.3%wt epsilon-PL sample antibacterial ring diameter is 13.56mm; the diameter of a bacteriostasis ring of a 0.2%wt Nisin sample is 13.44mm;0.3% wt. of the modified polymeric bacteriostatic agent A sample of example 3 had a zone of inhibition of 14.94mm diameter; the diameter of the antibacterial circle of the fresh salmon preservative sample in example 4 is 15.47mm.
(II) according to the antibacterial experimental method, using the raw salmon preservative of example 5 as a bacteriostatic agent, and respectively selecting a single bacteriostatic agent as a control sample, (1) 0.2% wt epsilon-PL, (2) 0.3% wt Nisin and (3) 0.2% wt modified polymer bacteriostatic agent A of example 3; the results of measuring the size of the inhibition zone are as follows:
coli: 0.2%wt epsilon-PL sample antibacterial ring diameter is 11.38mm; the diameter of a bacteriostasis ring of a 0.3%wt Nisin sample is 12.00mm;0.2% wt. of the modified polymeric bacteriostatic agent A sample of example 3 had a zone of inhibition of 12.44mm diameter; the diameter of the antibacterial circle of the fresh salmon preservative sample in example 5 is 15.50mm.
Bacillus subtilis: 0.2%wt epsilon-PL sample antibacterial ring diameter is 13.46mm; the diameter of a bacteriostasis ring of a 0.3%wt Nisin sample is 13.82mm;0.2% wt. of the modified polymeric bacteriostatic agent A sample of example 3 had a zone of 13.96mm in diameter; the diameter of the antibacterial circle of the raw salmon preservative sample of example 5 is 15.75mm.
(III) according to the antibacterial experimental method, using the raw salmon preservative of the example 6 as a bacteriostatic agent, and respectively selecting a single bacteriostatic agent as a control sample, (1) 0.3%wt epsilon-PL, (2) 0.3%wt Nisin and (3) 0.3%wt of the modified polymer bacteriostatic agent A of the example 3; the results of measuring the size of the inhibition zone are as follows:
coli: 0.3%wt epsilon-PL sample antibacterial ring diameter is 11.65mm; the diameter of a bacteriostasis ring of a 0.3%wt Nisin sample is 12.00mm;0.3% wt. of the modified polymeric bacteriostatic agent A sample of example 3 had a zone of 13.80mm in diameter; the diameter of the antibacterial circle of the fresh salmon preservative sample in example 6 is 15.67mm.
Bacillus subtilis: 0.3%wt epsilon-PL sample antibacterial ring diameter is 13.56mm; the diameter of a bacteriostasis ring of a 0.3%wt Nisin sample is 13.82mm;0.3% wt. of the modified polymeric bacteriostatic agent A sample of example 3 had a zone of inhibition of 14.94mm diameter; the diameter of the antibacterial circle of the fresh salmon preservative sample in example 6 is 16.19mm.
Conclusion:
from the three groups of examples of example 4, example 5 and example 6, it is understood that: as can be seen from comparison of the size of the inhibition zone for the same test strain, the antibacterial capacity of the modified polymer bacteriostat A group of single example 3 is higher than that of the epsilon-PL group. The antibacterial circle of the raw salmon preservative in the embodiment 4, the embodiment 5 and the embodiment 6 is obviously larger than that of the modified polymer antibacterial agent A in the embodiment 3 and the nisin control group, so that the antibacterial performance of the compound preservative is higher than that of the single control group, and the antibacterial performance is improved. For the modified polymer bacteriostat A of the embodiment 3 with different concentrations, the content of the modified polymer bacteriostat A of the embodiment 3 is different, the bacteriostasis capacity is different, the inhibition effect is related to the concentration of the modified polymer bacteriostat A of the embodiment 3, and the inhibition effect of 0.3% concentration is optimal; nisin also has the same effect. 0.3% of the modified polymer bacteriostatic agent A+0.3% of Nisin (raw salmon preservative of example 6) of example 3 has the largest diameter of the inhibition zone and the best inhibition effect.
The antibacterial mechanism of the fresh salmon preservative in the embodiment 6 of the invention is as follows: morphology observations of Shewanella putrefying (gram negative bacteria)
Taking a certain amount of Shewanella putrefying suspension in a centrifuge tube, adding sterile PBS buffer solution, regulating the concentration of the Shewanella putrefying suspension to be OD600 = 0.5, respectively taking 20mL of PBS suspension, taking one group as a blank control group, adding the fresh salmon preservative of the raw food of the example 6 into the other group for treatment, carrying out shake culture at 30 ℃ for 12h, centrifuging at 3000r/min for 10min, pouring out supernatant, adding sterile water for cleaning for 3 times, and centrifuging for 10min at the speed of 3000r/min. Mixing cooled 2.5% glutaraldehyde 20mL with thallus, fixing for 2h, cleaning with sterile water for 2-3 times, respectively soaking with 50%, 70%, 80% and 90% ethanol for 30min, soaking with 100% absolute ethanol for 1h, performing gradual dehydration, taking bacterial suspension and cover glass with a pipette, drying at 37deg.C for 72h, and observing cell morphology with a scanning electron microscope as shown in FIG. 1 and FIG. 2, wherein untreated thallus is smooth into ellipsoid shape as shown in FIG. 1 and FIG. 2, and the Shewanella putrefying bacteria treated with the raw salmon preservative of example 6 destroys cell wall and cell membrane system of bacteria, increases bacterial permeability, and kills bacteria, thereby achieving antibacterial effect.
The culture, antibiosis and centrifugation processes of the thalli are the same as the above method, glutaraldehyde solution is mixed with thalli, the thalli are fixed and then scattered on a copper net, the thalli are dried at room temperature, the appearances of the thalli are observed by a transmission electron microscope, as shown in fig. 3 and 4, the inner structures of the thalli are destroyed after the treatment by the putrefying Shewanella of the raw salmon preservative of the embodiment 6 in fig. 3 and 4, and the cytoplasm loss is more, so that the thalli structure is obviously changed due to the destruction of the cytoplasm, the occurrence of holes, the leakage of intracellular proteins and the like, and the death of the thalli.
Example 6 determination of bacterial Membrane permeability of fresh salmon preservative
Fresh bacterial suspension is taken out in a centrifuge tube, centrifuged for 10min at 3000r/min, and after supernatant is removed, the bacterial suspension concentration is adjusted to OD600 apprxeq 0.5 by PBS. Respectively taking 10mL PBS bacterial suspension in a centrifuge tube, adding the fresh salmon preservative for raw food of the embodiment 6 into the centrifuge tube, uniformly mixing, and measuring the conductivity of each treatment group at intervals of 10min until the equilibrium value is reached. As a result, as shown in FIG. 5, the conductivity of the Shewanella putrefying suspension gradually increased with the increase of the preservative treatment time to reach a stable maximum at 420 min. The change of the conductivity value of the bacterial liquid after the composite preservative is treated is larger than that of the bacterial suspension of a blank group, which indicates that the preservative breaks cell membranes of bacteria to a certain extent and electrolytes in bacterial cells leak into the culture liquid. The fluidity of the cell membrane is deteriorated, the instability of the intracellular environment is increased, and the electrolyte in the cell is leaked, so that the conductivity of the bacterial liquid is remarkably increased. The effective components in the preservative are also shown to destroy cell membranes of the thalli, and the sterilization effect is good.
Experimental group: the embodiment 6 of the invention is a fresh-keeping agent for salmon; treatment group: (1) 0.3% wt ε -PL, (2) 0.3% wt Nisin, (3) 0.3% wt modified Polymer bacteriostatic agent A of example 3, pH change in preservative test
From the above, the fresh salmon preservative of the embodiment 6 of the invention has good antibacterial performance, and is used for salmon preservation test.
Firstly, testing pH change, weighing 5g of salmon meat of different treatment groups, adding 45mL of boiled and cooled distilled water into the fish meat, homogenizing, standing for 30min, filtering the homogenized liquid, and measuring the pH value of the filtrate. As can be seen from fig. 6, the pH of salmon meat showed a tendency of "V" shape with the extension of the storage time. Along with the continuous extension of the storage time, the pH value of the blank group is always obviously higher than that of the treatment group, which indicates that epsilon-PL, nisin and the modified polymer bacteriostat A can effectively inhibit the growth of microorganisms and enzymolysis, and reduce the decomposition of nitrogen substances in fish meat. In addition, the pH of the modified polymeric bacteriostat A is lower than epsilon-PL. The pH rise of the fish meat treated by the fresh salmon preservative is slightly lower than that of the fish meat treated by a single factor group, and the effect is obviously enhanced in the later period of preservation. The fresh salmon preservative has a good inhibition effect on the decomposition of protein, amino acid and other nitrogen-containing substances in the fish meat.
Experimental group: the embodiment 6 of the invention is a fresh-keeping agent for salmon; treatment group: (1) 0.3% wt ε -PL, (2) 0.3% wt Nisin, (3) 0.3% wt modified Polymer bacteriostatic agent A of example 3, change in thiobarbituric acid (TBA) value in fresh-keeping test
10g of minced salmon in different treatment groups are weighed and placed in a beaker, distilled water (25 mL) is added into the minced salmon, the mixture is homogenized, trichloroacetic acid (25 mL) with the mass concentration of 10% is added into the minced salmon, the homogenization is carried out uniformly, the mixture is kept stand for 30 minutes, filtration is carried out, 5mL of supernatant fluid is taken, 0.02mol/L of thiobarbituric acid (5 mL) is added into the supernatant fluid, the mixture is uniformly mixed, the mixture is heated in a constant-temperature water bath at 80 ℃ for 40 minutes for color development, then the mixture is immediately cooled to room temperature, and the absorbance is measured at the wavelength of 532 nm.
As shown in fig. 7, the absorbance value shows a trend of rising along with the extension of the storage time, and the absorbance value treated by epsilon-PL, nisin, the modified polymer antibacterial agent a and the fresh salmon preservative is lower than that of the blank group, which indicates that various antibacterial agents can block the salmon from contacting with external oxygen, thereby effectively slowing down the oxidation of fat in the fish body and reducing the absorbance value of the sample. In addition, the absorbance values of modified polymeric bacteriostat group A are lower than those of epsilon-PL group. In addition, the absorbance value of the modified polymer bacteriostatic agent A group is lower than that of the Nisin group in the early stage and higher than that of the Nisin group from the 9 th day, which shows that the capacity of preventing the fat oxidation of the fish body in the later stage of Nisin is stronger, and the fat oxidation rancidity can be effectively inhibited. Most importantly, the absorbance value of the fresh salmon preservative is the lowest during the whole storage period, which indicates that the preservative has the best preservation effect.
Experimental group: the embodiment 6 of the invention is a fresh-keeping agent for salmon; treatment group: (1) 0.3% wt ε -PL, (2) 0.3% wt Nisin, (3) 0.3% wt modified Polymer bacteriostatic agent A of example 3, determination of volatile basic Nitrogen (TVB-N) in fresh-keeping experiments
Accurately weighing 10.00g of fish meat in a beaker, homogenizing, pouring into a distillation tube, adding 1.00g of magnesium oxide powder, and measuring the TVB-N value of a fish slice sample by using a full-automatic Kjeldahl nitrogen determination instrument, wherein the unit is expressed by mg N/100 g. TVB-N changes were determined as described above for salmon flesh from different treatment groups. According to GB/T5009.45-2003, 30mg/100g is prescribed as the minimum limit for the quality of the seafood to be accepted by consumers. Primary freshness was found when the TVB-N value was below 13mg/100g, and secondary freshness was found when the TVB-N value was below 30mg/100 g.
As shown in fig. 8, the TVB-N value of salmon increases with the storage time, and the TVB-N value of the blank group is significantly higher than each of epsilon-PL, nisin, modified polymer bacteriostatic agent a and fresh salmon preservative, which shows that these bacteriostatic agents have good antibacterial properties, and can inhibit the growth of microorganisms, thereby reducing the decomposition of fish protein by microorganisms and reducing the TVB-N value. As can be seen from the graph, the TVB-N value of the modified polymer bacteriostat A group is lower than that of the epsilon-PL group. In addition, the TVB-N value of the early Nisin group is lower than that of the modified polymer bacteriostat A group, the TVB-N value increase rate at the 9 th day is higher than that of the modified polymer bacteriostat A group, and finally the early Nisin group overtakes, so that the later-stage fresh-keeping effect of the Nisin is better. Most importantly, the TVB-N value of the fresh salmon preservative group is always the lowest, which indicates that the fresh salmon preservative group has the optimal antibacterial effect, can prolong the shelf life of salmon meat, and has remarkable effect.
Experimental group: the embodiment 6 of the invention is a fresh-keeping agent for salmon; treatment group: (1) 0.3% wt ε -PL, (2) 0.3% wt Nisin, (3) 0.3% wt modified Polymer bacteriostatic agent A of example 3, variation of total bacterial count in fresh-keeping experiments
Placing the processed salmon meat on an ultra-clean workbench, taking 10g of the salmon meat, placing into a sterilized stewing bag, adding sterile physiological saline (90 mL) with the concentration of 0.85%, and beating and homogenizing for 120s. The homogenized solution was subjected to 10-fold gradient dilution. 2-3 dilutions were appropriately selected, 1mL each was added to the sterilized dishes, and then poured into about 20mL of sterilized plate count agar medium (PCA), shaken well, and three replicates were made for each dilution. After the agar solidified, the plate was placed upside down in a 30℃incubator for 48 hours. Finally, colony counting is performed.
The results of the measurement of the total number of microorganisms in salmon flesh of the different treatment groups were carried out in accordance with the above-described method, and are shown in FIG. 9. Microorganisms propagated in fish meat are one of the main factors responsible for spoilage thereof, and the total number of bacteria (CFU/g) is 10 or less as specified in GB2741-94 5 The primary freshness is less than or equal to 5×10 5 For the second grade freshness, when reaching 10 6 -10 7 Indicating that it is extremely spoiled and inedible. As can be seen from FIG. 7, the total number of bacteria increased with the increase in storage time. On day 5, the total bacterial count of the blank group reaches 5.12Lg (CFU/g), and the total bacterial count of the fish meat treated by the preservative is still within the primary freshness range, which indicates that epsilon-PL, the modified polymer bacteriostat A and Nisin can inhibit the growth of microorganisms and delay the spoilage of the fish meat. The value of the total colony count of the fresh salmon preservative is the lowest in the whole storage process, which shows that the antibacterial effect of the group is optimal, and the shelf life of salmon is prolonged to 9 days from blank 5 days as seen from the numerical value of fig. 9, so that the preservative effect is obvious.
The above is only a specific embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A fresh-keeping agent for salmon is characterized in that: the fresh salmon preservative is obtained by adding a modified polymer bacteriostatic agent A and nisin into distilled water according to the mass ratio of 2:3-3:2 and carrying out ultrasonic oscillation, wherein the structural formula of the modified polymer bacteriostatic agent A is as follows:
wherein n is a positive integer of 5 to 12.
2. The fresh-keeping agent for raw salmon according to claim 1, characterized in that: the mass ratio of the modified polymer bacteriostatic agent A to distilled water is 0.2:100-0.3:100.
3. The fresh-keeping agent for raw salmon according to claim 1, characterized in that: the preparation method of the fresh salmon preservative specifically comprises the following steps of:
dissolving the modified polymer bacteriostatic agent A with ethanol of 5-10 times of mass, adding the dissolved bacteriostatic agent A into distilled water for dilution, adding nisin, and carrying out ultrasonic oscillation dissolution to obtain the fresh salmon preservative.
4. The fresh-keeping agent for raw salmon according to claim 1, characterized in that: the mass ratio of the modified polymer bacteriostat A to distilled water is 0.3:100, and the mass ratio of the nisin to the modified polymer bacteriostat A is 1:1.
5. The fresh-keeping agent for raw salmon according to claim 1, characterized in that:
the synthesis steps of the modified polymer bacteriostat A are as follows:
ethanol is taken as solvent, N is added α -tert-butoxycarbonyl-L-lysine, 4-n-hexyloxybenzaldehyde and 4-diethylaminophenylisonitrile are fed according to the molar ratio of 1:2:2, stirred for 48-72 hours at room temperature, ethanol is unscrewed, petroleum ether is used for precipitation, and the precipitate is dried in vacuum to obtain an intermediate; and (3) dissolving the intermediate by using a solvent at the temperature of 0 ℃, adding trifluoroacetic acid according to the mass ratio of the intermediate to the trifluoroacetic acid of 1:0.2, stirring for 3-6 hours, then spinning out the solvent, precipitating by using petroleum ether, and drying the precipitate to obtain the modified polymer bacteriostatic agent A.
6. A method for preserving salmon by using the fresh salmon preservative according to claim 1, which comprises the following steps: washing peeled salmon with purified water, draining, soaking in fresh salmon preservative at room temperature for 3min, draining again, sealing in a sealing bag, and storing at 4deg.C.
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|---|---|---|---|---|
| EP0453860A1 (en) * | 1990-04-20 | 1991-10-30 | HAARMANN & REIMER CORP. | A method of killing gram negative bacteria |
| CN105669973A (en) * | 2016-01-06 | 2016-06-15 | 中国科学院海洋研究所 | Compound having antibacterial activity and preparation and application thereof |
| CN106117548A (en) * | 2016-06-27 | 2016-11-16 | 中国科学院长春应用化学研究所 | A kind of amino acidic group cluster peptide and preparation method thereof |
| CN110818895A (en) * | 2019-11-22 | 2020-02-21 | 渤海大学 | Modified epsilon-polylysine, preparation method and application thereof in preservation of turbot |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0453860A1 (en) * | 1990-04-20 | 1991-10-30 | HAARMANN & REIMER CORP. | A method of killing gram negative bacteria |
| CN105669973A (en) * | 2016-01-06 | 2016-06-15 | 中国科学院海洋研究所 | Compound having antibacterial activity and preparation and application thereof |
| CN106117548A (en) * | 2016-06-27 | 2016-11-16 | 中国科学院长春应用化学研究所 | A kind of amino acidic group cluster peptide and preparation method thereof |
| CN110818895A (en) * | 2019-11-22 | 2020-02-21 | 渤海大学 | Modified epsilon-polylysine, preparation method and application thereof in preservation of turbot |
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