CN111742978A - Preparation method of compound natural preservative for chilled donkey meat - Google Patents
Preparation method of compound natural preservative for chilled donkey meat Download PDFInfo
- Publication number
- CN111742978A CN111742978A CN202010776084.3A CN202010776084A CN111742978A CN 111742978 A CN111742978 A CN 111742978A CN 202010776084 A CN202010776084 A CN 202010776084A CN 111742978 A CN111742978 A CN 111742978A
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- alcohol extract
- donkey meat
- natural
- clove
- nisin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention relates to a preparation method of a compound preservative for chilled fresh donkey meat, which adopts a natural bacteriostatic agent and a natural antioxidant, takes ginger alcohol extract, clove alcohol extract and Nisin Z with better bacteriostatic effect as the natural bacteriostatic agent, carries out compound optimization and determines the optimal proportion; simultaneously, ascorbic acid, rosemary alcohol extract and resveratrol with good antioxidant effect are used as natural antioxidants for compounding optimization, and the optimal proportion is determined; the optimal compounding ratio of the preservative is comprehensively obtained. Modern food preservatives are typically synthetic chemicals such as potassium sorbate, benzoate, Butylated Hydroxyanisole (BHA) and sodium lactate, and the health risks of synthetic food additives are below regulatory agency-defined recommended limits. The natural preservative is more and more popular with consumers due to the bacteriostatic effect and better tolerance in human bodies. Many flavors such as clove and ginger have been experimentally demonstrated to have activity against pathogenic and spoilage bacteria.
Description
Technical Field
The invention relates to a preparation method of a compound natural preservative for chilled fresh donkey meat, belonging to the technical field of biological food preservation.
Background
The donkey meat is a nutritional food with high protein, low saturated fatty acid, and rich vitamins and minerals. Donkey meat products exhibit lower saturated fatty acids, higher polyunsaturated fatty acid content than traditional beef and pork products. From the perspective of nutrition and dietetics, donkey meat is a food material integrating medicine, nourishing and eating. In recent years, the microbial agent is popular with consumers, but is easily infected by microorganisms in the processes of division, transportation, storage, sale and the like. At present, the food industry usually adopts the method of adding a proper amount of chemical preservative to prolong the shelf life of meat. With the intensive research, almost all chemical preservatives present potential hazards to human health.
At present, some plants can be used as natural preservatives to prevent food from being rotten and deteriorated, and the natural preservatives have better tolerance in human bodies, are healthier compared with chemical synthetic products, and meet the requirements of consumers better. The theoretical basis of the compound bacteriostatic agent is based on the barrier effect, although a single preservative has good bacteriostatic performance, the bacteriostatic spectrum is respectively emphasized, different bacteriostatic agents are scientifically and reasonably compounded to carry out multi-target attack on different bacteria, the synergistic effect of the compound bacteriostatic agent is exerted, and the aim of keeping fresh is fulfilled. The compound natural antioxidant has excellent antioxidant performance in different meat products, effectively delays meat lipid and protein oxidation and improves the sensory characteristics of meat products.
Disclosure of Invention
The invention aims to develop the preservation technology of donkey meat, popularize the market of the chilled fresh donkey meat, overcome the defects and shortcomings of a chemically synthesized preservative, identify the spoilage bacteria of the chilled fresh donkey meat through 16S rDNA, and obtain a preparation method of the compound natural preservative for the chilled fresh donkey meat by combining methods such as a trace enzyme-linked immunosorbent assay, an antioxidant capacity single-factor experiment, sensory evaluation, response surface optimization and the like.
The invention aims to realize the purpose, and the preparation method of the compound natural preservative for the chilled fresh donkey meat is characterized by comprising the following steps:
step 1), identifying dominant spoilage bacteria of donkey meat: vacuum packaging the fresh donkey meat sample in a sterilized food-grade PE packaging bag, refrigerating and storing at 4 ℃ for 15 days, and after the donkey meat is rotted and deteriorated, determining that the putrefactive bacterial phase of the donkey meat contains lactic acid bacteria, pseudomonas, enterobacter and hot killed cyclosporine, wherein the lactic acid bacteria, the pseudomonas, the enterobacter and the hot killed cyclosporine respectively account for 54.81%, 34.07%, 5.19% and 4.81% of the putrefactive bacterial phase; separating and purifying lactobacillus, Pseudomonas, Enterobacter, and Thermomyces for 16S rDNA identification, wherein the putrefactive dominant bacteria of the chilled fresh ass meat is Lactobacillus sake (G)+) And Pseudomonas fluorescens (G)-) The 4 kinds of enterobacteria are Enterobacter halofani (G)-) Serratia liquefaciens (G)-) Hafnia alvei (G)-) Raoultella origani (G)-)；
Extracting spices: weighing 20.00g of 80-mesh spice, placing the 80-mesh spice into a 500mL round-bottom flask, adding a 95% ethanol solution according to the material-liquid ratio of 1:10(g/mL), refluxing the round-bottom flask filled with the spice and the ethanol in a water bath kettle at 85 ℃ for 3 hours, carrying out suction filtration, repeatedly extracting the spice for 3 times, and carrying out suction filtration; mixing filtrates, vacuum concentrating (50 deg.C and-0.1 MPa) filtrate with rotary evaporator, evaporating solvent, dissolving crude extract of spice with sterile distilled water, freezing and storing at-20 deg.C for 12 hr, and vacuum freeze drying for 48 hr to obtain ethanol extract of spice; the extracted ethanol extracts of the spices are respectively as follows: ginger alcohol extract, clove alcohol extract and rosemary alcohol extract; simultaneously, preparing food-grade Nisin Z, ascorbic acid and resveratrol; wherein, the ginger alcohol extract, the clove alcohol extract and Nisin Z are natural bacteriostats, and the ascorbic acid, the rosemary alcohol extract and the resveratrol are natural antioxidants;
step 2), the lactobacillus sake (G) identified by the step 1)+) Pseudomonas fluorescens (G)-) Serratia liquefaciens (G)-) Hafnia alvei (G)-) Raoultella origani (G)-) The strain to be tested; measuring the Minimum Inhibitory Concentration (MIC) of the ginger alcohol extract, the clove alcohol extract and Nisin Z to the tested strain by a 96-well plate micro enzyme-linked immunosorbent assay; treating cold fresh donkey meat with ginger alcohol extract, clove alcohol extract and Nisin Z with different concentrations, vacuum packaging, standing at 4 deg.C for 10 days, and performing sensory evaluation to obtain optimal sensory evaluation concentration; the method is characterized in that a result obtained by integrating a 96-pore plate microplate reader method and sensory evaluation is a single-factor experimental result, Design is carried out by applying Design expert8.0.6 on the basis of the single factor, a 3-factor 3 horizontal response surface experiment is designed, a ginger alcohol extract 41.07mg/mL, a clove alcohol extract 5.39mg/mL and Nisin Z56.28mg/mL are obtained by utilizing response surface optimization, and the reaction is corrected, detected and verified on the basis of the theoretical values to obtain the ginger alcohol extract 40.00g/mL, the clove alcohol extract 5.50mg/mL and the Nisin Z55.00mg/mL in consideration of the feasibility of actual operation; obtaining the best concentration of DPPH free radical clearance of three antioxidants through a single-factor test (DPPH free radical clearance measuring method) for the antioxidant capacity of ascorbic acid, rosemary alcohol extract and resveratrol; treating cold fresh donkey meat with ascorbic acid, rosemary alcohol extract and resveratrol with different concentrations, vacuum packaging, standing at 4 deg.C for 10 days, and performing sensory evaluation to obtain optimal sensory evaluation concentration; the results obtained by the comprehensive antioxidant capacity single-factor test and sensory evaluation are single-factor test results, Design is carried out on the basis of single factors by using Design expert8.0.6, 3-factor 3 horizontal response surface tests are designed, 30.64mg/mL of ascorbic acid, 0.68mg/mL of rosemary alcohol extract and 31.83mg/mL of resveratrol are obtained by using response surface optimization, and in consideration of feasibility of actual operation, the reaction is corrected, detected and verified on the basis of the theoretical values to obtain 30.00mg/mL of ascorbic acid, 0.65mg/mL of rosemary alcohol extract and 30.00mg/mL of resveratrol;
the optimal formula of the compound preservative finally obtained is 40.00mg/mL of ginger alcohol extract, 5.50mg/mL of clove alcohol extract, Nisin Z55.00mg/mL, 30.00mg/mL of ascorbic acid, 0.65mg/mL of rosemary alcohol extract and 30.00mg/mL of resveratrol.
When in use, the prepared compound preservative is sprayed on the chilled fresh donkey meat through the step 2), and the food-grade PE preservative bag is vacuum-packaged, sealed and refrigerated to finish the refrigeration of the chilled fresh donkey meat.
The natural composite preservative comprises the following substances in percentage by weight: alcohol extract of clove: nisin Z ═ 1:1:1, preparation of ascorbic acid: rosemary alcohol extract: 1, resveratrol: 1:1, preparation; uniformly spraying the mixture on the surface of donkey meat in a sterile environment, spraying 0.1mL for 1 time on the front side and the back side respectively, air drying for 5min, vacuum packaging, and placing in a refrigerator at 4 ℃.
The method is advanced and scientific, and the invention provides a preparation method of the compound natural preservative for the chilled fresh donkey meat, which comprises the following steps: step 1), bacterium identification: vacuum packaging the fresh ass meat in sterilized food-grade PE packaging bag, refrigerating at 4 deg.C, storing until the ass meat is putrefactive, and determining according to GB 4789.2-2016 "determination of total number of food microorganism colony". Separating and purifying putrefying bacteria: selecting typical colony from selective culture medium, streaking on corresponding plate, separating and purifying for 2-3 times, transplanting representative strain to slant, and storing for inspection. 16S rDNA identification was performed. The microbial culture medium and culture conditions used were:
TABLE 1 microbiological culture media and culture conditions
Table 1 Microbial culture medium and culture conditions
And 2) selecting the dominant putrefying bacteria identified in the step 1 as indicator bacteria, and extracting ginger alcohol extract, clove alcohol extract and rosemary alcohol extract. Measuring the Minimum Inhibitory Concentration (MIC) of the ginger alcohol extract, the clove alcohol extract and Nisin Z by a 96-hole method of a microaenzyme reader, and obtaining respective optimal concentrations by sensory evaluation, thereby obtaining the optimal natural composite bacteriostatic agent by response surface optimization; and simultaneously, the maximum concentrations of the antioxidant activities of the resveratrol, the rosemary alcohol extract and the ascorbic acid are measured by adopting a DPPH method, and the respective optimum concentrations are obtained by combining sensory evaluation of donkey meat, so that the optimum compound natural antioxidant is obtained by response surface optimization. Comprehensively obtain the optimal natural preservative formula.
And 3) obtaining a matching result of the composite natural preservative according to the step 2, and performing aseptic spraying treatment on the chilled donkey meat. And (3) packaging the food-grade PE freshness protection package in vacuum, sealing the freshness protection package for storage at 4 ℃, and detecting whether the total bacterial colony count and the TVB-N value meet the sanitary standard of cold fresh meat in GB/T9961-2008 to obtain the shelf life of the added compound natural freshness protection agent.
And 4) on the basis of determining the shelf life in the step 3, researching the influence of the compound natural preservative on the nutrition quality of the vacuum-packaged chilled donkey meat under the refrigeration condition of 4 ℃.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the ginger alcohol extract, the clove alcohol extract and Nisin Z with good bacteriostatic effect are used as natural bacteriostatic agents, and are compounded and optimized to determine the optimal proportion; simultaneously, ascorbic acid, rosemary alcohol extract and resveratrol with good antioxidant effect are used as natural antioxidants for compounding optimization, and the optimal proportion is determined; the optimal compounding ratio of the preservative is comprehensively obtained. Modern food preservatives are typically synthetic chemicals such as potassium sorbate, benzoate, Butylated Hydroxyanisole (BHA) and sodium lactate, and the health risks of synthetic food additives are below regulatory agency-defined recommended limits. The natural preservative is more and more popular with consumers due to the bacteriostatic effect and better tolerance in human bodies. Many flavors such as clove and ginger have been experimentally demonstrated to have activity against pathogenic and spoilage bacteria.
At present, research on donkey meat at home and abroad mainly focuses on the aspect of nutrition analysis of meat products, and the detection of spoilage bacteria of chilled donkey meat and the formula research of a compound natural preservative thereof are not reported. The invention provides a certain theoretical basis for transporting and storing the chilled donkey meat.
Production practices show that the compounding of a plurality of natural food additives can generate synergistic effect, not only can replace chemical synthetic additives, but also can further improve the quality of food and improve the edible safety of the food, and the economic significance and the social significance are self-evident.
The clove alcohol extract and the clove extract destroy the integrity of a cell membrane structure, so that some important ions in the clove extract are lost, so that corresponding important enzymes lose the action, the substance metabolism of thalli is influenced, and researches show that the bacteriostatic effect of the clove alcohol extract is obviously better than that of other spices.
The ginger alcohol extract has a long medicinal history in China, and can be used for treating diseases such as nausea, vomiting, diarrhea, dyspepsia, rheumatism, cold and the like. Some volatile compounds are responsible for the antibacterial activity of ginger, including alpha-pinene, borneol, camphene and linalool. The ginger alcohol extract has a better antibacterial effect than other solvent extracts, and has a good antibacterial effect on serotype escherichia coli, serotype salmonella and listeria monocytogenes.
Nisin (Nisin Z), a promising biological preservative in the dairy, meat, ready-to-eat vegetables and fruits markets, has been proposed by Nisin manufacturers. Streptococcus lactis destroys the integrity of the cell membrane by transient pores, resulting in a rapid efflux of bacterial substances (amino acids and adenosine triphosphate) from the bacteria, dissipation of the proton motive force, inhibition of the biosynthetic pathways of the cells, in particular the natural variant Z, with high solubility, stability and broad antimicrobial spectrum in different food systems.
The resveratrol applied to meat products does not influence the meat color and can obviously inhibit lipid oxidation.
The rosemary has good oxidation resistance and antibacterial potential, can stabilize free radicals, is more effective than BHT and BHA, has the oxidation resistance mainly related to myrcene, and has the antibacterial performance related to 1, 8-eucalyptol and alpha-pinene. The rosemary alcohol extract has good antioxidant effect, and can be used for meat products to improve the sensory properties of foods and prolong the shelf life.
Ascorbic acid, also known as vitaminBiotin, a reducing agent, with antioxidant action expressed in the ability to react with O2The rapid reaction of- (OH), HOO and OH generates semi-dehydroascorbic acid, and removes singlet oxygen and mercapto naphthenate free radicals, and the antioxidation is completed by reversible dehydrogenation. The ascorbic acid and the rosemary extract are sprayed on the surface of pork in a combined mode and stored in a vacuum package mode, so that the oxidation of pork lipid is effectively delayed, and the pH value, the color and the water content of the pork are not affected.
In conclusion, the invention aims to overcome the defects of a chemically synthesized preservative, adopts a natural bacteriostatic agent and natural antioxidation, and combines response surface optimization to obtain the optimal compound natural preservative proportion, the natural preservative has a bacteriostatic action on the dominant putrefying bacteria of the chilled fresh donkey meat, has no adverse effect on low-temperature storage and preservation of the chilled fresh donkey meat, has good bacteriostatic aging effect, and can effectively prolong the shelf life of the chilled fresh donkey meat.
Drawings
FIG. 1 shows the composition of putrefying bacteria phase of chilled fresh donkey meat.
FIG. 2 is a 16S rDNA PCR electrophoretogram of the strain;
note: j1 and J2 are 2 kinds of pseudomonas respectively, R is lactic acid bacteria, and C1, C2, C3 and C4 are 4 kinds of enterobacteria respectively.
FIG. 3 shows the effect of different concentrations of alcohol extracts of Zingiber officinale on 5 species of bacteria;
note: a. different letters of b and c indicate that the difference of 5 bacteria at the same concentration is significant (p < 0.05).
FIG. 4 shows the effect of different concentrations of syringa oleifera extracts on 5 bacteria;
note: a. different letters of b and c indicate that the difference of 5 bacteria at the same concentration is significant (p < 0.05).
FIG. 5 shows the effect of different concentrations of Nisin Z on Serratia liquefaciens;
note: a. different letters of b and c indicate that the difference of 5 bacteria at the same concentration is significant (p < 0.05).
FIG. 6 is a graph of the effect of different concentrations of ginger alcohol extract on the sensory score of donkey meat;
note: a. the different alphabets of b and c show significant difference (p < 0.05).
FIG. 7 is the effect of different concentrations of syringyl alcohol extract on the sensory score of donkey meat;
note: a. the different alphabets of b and c show significant difference (p < 0.05).
FIG. 8 is the effect of different concentrations of Nisin Z on the sensory score of donkey meat;
note: a. the different alphabets of b and c show significant difference (p < 0.05).
FIG. 9-1 is a three-dimensional response surface diagram of chilled fresh donkey meat with natural bacteriostatic agent.
Fig. 9-2 is a three-dimensional response surface diagram of the chilled fresh donkey meat with the natural bacteriostatic agent.
Fig. 9-3 is a three-dimensional response surface diagram of the chilled fresh donkey meat with the natural bacteriostatic agent.
Fig. 9-4 are three-dimensional response surface diagrams of the chilled fresh donkey meat with the natural bacteriostatic agent.
Fig. 9-5 are three-dimensional response surface diagrams of the chilled fresh donkey meat with the natural bacteriostatic agent.
Fig. 9-6 are three-dimensional response surface diagrams of the chilled fresh donkey meat with the natural bacteriostatic agent.
FIG. 10 is a graph showing the variation trend of DPPH values of ascorbic acid at different concentrations;
note: a. the different alphabets of b and c show significant difference (p < 0.05).
FIG. 11 shows the trend of DPPH variation of rosemary alcohol extracts at different concentrations;
note: a. the different alphabets of b and c show significant difference (p < 0.05).
FIG. 12 shows the variation trend of DPPH of resveratrol in different concentrations;
note: a. the different alphabets of b and c show significant difference (p < 0.05).
Figure 13 is a graph of the sensory score effect of different concentrations of ascorbic acid on donkey meat;
note: a. the different alphabets of b and c show significant difference (p < 0.05).
Figure 14 is a graph of the sensory score effect of various concentrations of rosemary extract on donkey meat;
note: a. the different alphabets of b and c show significant difference (p < 0.05).
Figure 15 is a graph of the effect of different concentrations of resveratrol on the sensory score of donkey meat;
note: a. the different alphabets of b and c show significant difference (p < 0.05).
FIG. 16-1 is a three-dimensional response surface diagram of the natural antioxidant chilled fresh donkey meat.
FIG. 16-2 is a three-dimensional response surface diagram of the natural antioxidant chilled fresh donkey meat.
Fig. 16-3 is a three-dimensional response surface diagram of the natural antioxidant chilled fresh donkey meat.
Fig. 16-4 is a three-dimensional response surface diagram of the natural antioxidant chilled fresh donkey meat.
Fig. 16-5 are three-dimensional response surface diagrams of the natural antioxidant chilled fresh donkey meat.
Fig. 16-6 are three-dimensional response surface diagrams of the natural antioxidant chilled fresh donkey meat.
FIG. 17 is the change in the log of the total number of donkey meat bacteria during preservation;
note: the difference in letters indicates significant differences between treatments at different times (p < 0.05);
indicates significant differences between different treatments at the same time (p < 0.05).
FIG. 18 is the change in TVB-N value of donkey meat during preservation;
note: the difference in letters indicates significant differences between treatments at different times (p < 0.05);
indicates significant differences between different treatments at the same time (p < 0.05);
indicates the most significant differences between the different treatments at the same time (p < 0.01).
FIG. 19 is the change of pH of donkey meat during preservation;
note: the difference in letters indicates significant differences between treatments at different times (p < 0.05);
indicates significant differences between different treatments at the same time (p < 0.05).
FIG. 20 is a change in cooking loss of donkey meat during preservation;
note: the difference in letters indicates significant differences between treatments at different times (p < 0.05);
indicates significant differences between different treatments at the same time (p < 0.05).
In FIGS. 17 to 20, A1Test group for short for cold fresh donkey meat added with compound natural preservative；A2The chilled donkey meat without preservative is called as a control group for short.
The invention is further described with reference to the accompanying drawings and the description thereof.
Detailed Description
The invention is further explained below with reference to the figures and the specific embodiments:
the raw materials of example 1 and example 2 were all three-powder black donkey meat.
Example 1: chilled donkey meat added with natural compound natural preservative
1. Extracting ginger alcohol extract, clove alcohol extract and rosemary alcohol extract: weighing 20.00g of 80-mesh spice, placing the spice into a 500mL round-bottom flask, adding 95% ethanol solution according to the material-liquid ratio of 1:10(g/mL), refluxing and extracting for 3h in a water bath kettle at 85 ℃, filtering, repeatedly extracting for 3 times, combining filtrates, performing vacuum concentration (50 ℃ and-0.1 MPa) by using a rotary evaporator, evaporating a solvent, dissolving a crude extract by using sterile distilled water, performing freeze preservation at 20 ℃ for 12h, and performing vacuum freeze drying for 48h to obtain the extract.
2. Preparing a compound natural preservative, and proportioning: 40.00mg/mL of ginger alcohol extract, 5.50mg/mL of clove alcohol extract, 55.00mg/mL of Nisin Z, 30mg/mL of ascorbic acid, 0.65mg/mL of rosemary alcohol extract and 30.00mg/mL of resveratrol.
Firstly, ginger alcohol extract: weighing 1.00g of ginger alcohol extract, dissolving in 5mL of absolute ethyl alcohol, using sterile distilled water to fix and hold in a 25mL volumetric flask, and fully and uniformly mixing.
Alcohol extract of clove: 0.1375g of syringyl alcohol extract is weighed and dissolved in 5mL of absolute ethyl alcohol, and the solution is dissolved in a 25mL volumetric flask with sterile distilled water and mixed well.
③ Nisin Z: weighing 1.375g Nisin Z, dissolving in 0.02mol/L hydrochloric acid dilute solution, and placing in a 25mL volumetric flask, and mixing well.
Extracting ginger with alcohol: alcohol extract of clove: nisin Z is 1:1: 1.
(iv) ascorbic acid: 0.75g ascorbic acid was weighed into sterile distilled water and placed in a 25mL volumetric flask and mixed well.
Fifth, rosemary alcohol extract: 0.01625g of rosemary extract are weighed and dissolved in 2mL of absolute ethyl alcohol, and the solution is dissolved in a 25mL volumetric flask with sterile distilled water and fully mixed.
Sixthly, the resveratrol: weighing 0.75g of resveratrol, dissolving in absolute ethyl alcohol, metering the volume in a 25mL volumetric flask, and fully and uniformly mixing.
Mixing ascorbic acid: rosemary alcohol extract: and (3) preparing the resveratrol in a ratio of 1:1: 1.
3. Vacuum packaging donkey meat slaughtered in a slaughterhouse, transporting the donkey meat to a laboratory through a cold chain, placing the donkey meat in an ultralow-temperature refrigerator at the temperature of-30 ℃ for 1-2 h, and then placing the donkey meat in a refrigerator at the temperature of 4 ℃. Removing fascia and fat in an aseptic operating room, uniformly spraying the composite natural preservative on the surface of donkey meat by using an aseptic spraying kettle, airing for 5min on an aseptic operating platform, sealing, vacuum packaging and storing at 4 ℃.
Example 2: cold fresh donkey meat without added preservative
1. Vacuum packaging donkey meat slaughtered in a slaughterhouse, transporting the donkey meat to a laboratory through a cold chain, placing the donkey meat in an ultralow-temperature refrigerator at the temperature of-30 ℃ for 1-2 h, and then placing the donkey meat in a refrigerator at the temperature of 4 ℃. Fascia and fat were removed in a sterile operating room.
2. Vacuum packaging and storing at 4 deg.C.
TABLE 2 species relationship for sequencing of strains
Table 2 The relationship of species of strain sequence
Analysis of results
The following table compares the results of example 1 with example 2:
TABLE 3 color change on the surface of donkey meat during preservation
Table 3 Color change of the surface of donkey meat during storage
Note: the different letters in each row represent significant differences (p < 0.05)
A1Represents example 1; a. the2Representative of example 2.
TABLE 4 change of fatty acid in fresh donkey meat in storage (%)
Table 4 Changes of fatty acids in cold fresh donkey meat duringstorage
Note: the same row is marked with different letters to indicate the significant difference of fatty acid between different times of treatment (p < 0.05); indicates significant differences between different treatments at the same time (p < 0.05); indicates a very significant difference between the different treatment groups at the same time (p < 0.01); a. the1Represents example 1,; a. the2Representative of example 2; -represents no detection; \\ represents undetermined.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (3)
1. A preparation method of a compound natural preservative for chilled donkey meat is characterized by comprising the following steps:
step 1), identifying dominant spoilage bacteria of donkey meat: vacuum packaging the fresh ass meat sample in sterilized food-grade PE packaging bag, refrigerating at 4 deg.C for 15 daysAfter the meat is putrefactive and deteriorated, the putrefactive bacterial phase of the donkey meat is measured to contain lactobacillus, pseudomonas, enterobacter and thermofuscin, and the lactobacillus, the pseudomonas, the enterobacter and the thermofuscin respectively account for 54.81%, 34.07%, 5.19% and 4.81% of the putrefactive bacterial phase; separating and purifying lactobacillus, Pseudomonas, Enterobacter, and Thermobifida fusca to perform 16SrDNA identification, wherein the putrefactive bacteria of the chilled fresh ass meat is Lactobacillus sake (G)+) And Pseudomonas fluorescens (G)-) The 4 kinds of enterobacteria are Enterobacter halofani (G)-) Serratia liquefaciens (G)-) Hafnia alvei (G)-) Raoultella origani (G)-)；
Extracting spices: weighing 20.00g of 80-mesh spice, placing the 80-mesh spice into a 500mL round-bottom flask, adding a 95% ethanol solution according to the material-liquid ratio of 1:10(g/mL), refluxing the round-bottom flask filled with the spice and the ethanol in a water bath kettle at 85 ℃ for 3 hours, carrying out suction filtration, repeatedly extracting the spice for 3 times, and carrying out suction filtration; mixing filtrates, vacuum concentrating (50 deg.C and-0.1 MPa) filtrate with rotary evaporator, evaporating solvent, dissolving crude extract of spice with sterile distilled water, freezing and storing at-20 deg.C for 12 hr, and vacuum freeze drying for 48 hr to obtain ethanol extract of spice; the extracted ethanol extracts of the spices are respectively as follows: ginger alcohol extract, clove alcohol extract and rosemary alcohol extract; simultaneously, preparing food-grade Nisin Z, ascorbic acid and resveratrol; wherein, the ginger alcohol extract, the clove alcohol extract and Nisin Z are natural bacteriostats, and the ascorbic acid, the rosemary alcohol extract and the resveratrol are natural antioxidants;
step 2), the lactobacillus sake (G) identified by the step 1)+) Pseudomonas fluorescens (G)-) Serratia liquefaciens (G)-) Hafnia alvei (G)-) Raoultella origani (G)-) The strain to be tested; measuring the Minimum Inhibitory Concentration (MIC) of the ginger alcohol extract, the clove alcohol extract and Nisin Z to the tested strain by a 96-well plate micro enzyme-linked immunosorbent assay; treating cold fresh donkey meat with ginger alcohol extract, clove alcohol extract and Nisin Z with different concentrations, vacuum packaging, standing at 4 deg.C for 10 days, and performing sensory evaluation to obtain optimal sensory evaluation concentration; the result obtained by integrating the 96-pore plate micro enzyme-labeling method and sensory evaluation isThe single-factor experiment result is obtained, Design Expert8.0.6 is used for experimental Design on the basis of the single factor, 3-factor 3 horizontal response surface experiments are designed, 41.07mg/mL of ginger alcohol extract, 5.39mg/mL of clove alcohol extract and 56.28mg/mL of Nisin Z are obtained by utilizing response surface optimization, and the reaction is corrected and detected and verified on the basis of the theoretical values to obtain 40.00g/mL of ginger alcohol extract, 5.50mg/mL of clove alcohol extract and 55.00mg/mL of Nisin Z in consideration of feasibility of actual operation; obtaining the best concentration of DPPH free radical clearance of three antioxidants through a single-factor test (DPPH free radical clearance measuring method) for the antioxidant capacity of ascorbic acid, rosemary alcohol extract and resveratrol; treating cold fresh donkey meat with ascorbic acid, rosemary alcohol extract and resveratrol with different concentrations, vacuum packaging, standing at 4 deg.C for 10 days, and performing sensory evaluation to obtain optimal sensory evaluation concentration; the results obtained by the comprehensive antioxidant capacity single-factor test and sensory evaluation are single-factor test results, the design of the test is carried out by applying design expert8.0.6 on the basis of the single factor, a 3-factor 3 horizontal response surface test is designed, the response surface is optimized to obtain 30.64mg/mL of ascorbic acid, 0.68mg/mL of rosemary alcohol extract and 31.83mg/mL of resveratrol, and in consideration of the feasibility of actual operation, the reaction is corrected, detected and verified on the basis of the theoretical values to obtain 30.00mg/mL of ascorbic acid, 0.65mg/mL of rosemary alcohol extract and 30.00mg/mL of resveratrol;
the optimal formula of the compound natural preservative finally obtained comprises 40.00mg/mL of ginger alcohol extract, 5.50mg/mL of clove alcohol extract, 55.00mg/mL of Nisin, 30.00mg/mL of ascorbic acid, 0.65mg/mL of rosemary alcohol extract and 30.00mg/mL of resveratrol.
2. The preparation method of the compound natural preservative for chilled fresh donkey meat according to claim 1, characterized in that when in use, the compound natural preservative prepared in the step 2) is sprayed on the chilled fresh donkey meat, the food-grade PE preservative bag is packaged in vacuum, and the refrigeration is sealed to finish the refrigeration of the chilled fresh donkey meat.
3. The preparation method of the compound natural preservative for chilled fresh donkey meat according to claim 2, characterized in that the ratio of the substances in the natural compound preservative is ginger alcohol extract: alcohol extract of clove: nisin Z ═ 1:1:1, ascorbic acid: rosemary alcohol extract: preparing resveratrol as 1:1: 1; uniformly spraying the mixture on the surface of donkey meat in a sterile environment, spraying 0.1mL for 1 time on the front side and the back side respectively, air drying for 5min, vacuum packaging, and placing in a refrigerator at 4 ℃.
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