Special biological fresh-keeping preservative method for cattle stomach

Abstract

The invention relates to the technical field of biotechnology, and discloses a special biological fresh-keeping preservative method for cattle stomach, which comprises the following steps: firstly, pretreating the cattle stomach; the method comprises the following steps: putting the cattle stomach into flowing clear water, washing off obvious impurities, dirt and mucous membrane liquid on the surface, soaking the cattle stomach in saline water or a food-grade cleaning agent solution in a certain proportion for 10-30 minutes, kneading the surface of the cattle stomach, washing the kneaded cattle stomach with clear water again, and removing impurities and cleaning agent residues after soaking and kneading, and placing the cleaned cattle stomach in a filter screen or a clean container, and naturally draining water. And further determining the strain by adopting a 16SrDNA (Deoxyribose Nucleic Acid) detection technology, and analyzing experimental data to find out a bacteriostatic agent with the best effect and the lowest effective bacteriostatic concentration through a bacteriostatic activity experiment. And finally, determining quality index data of the cattle stomach through a single factor and an orthogonal test, and optimizing the bacteriostatic agent with the best effect, so that the shelf life of the cattle stomach stored at low temperature is prolonged.

Description

Technical Field

[0001] The invention relates to the technical field of biotechnology, in particular to a biological preservation method specially used for tripe. Background Art

[0002] Tripe comes from the stomach of ruminants such as cows. It is the most developed muscular layer in the omasum. Without tripe, there is no hot pot. As a result, the demand for tripe has increased sharply. Tripe is rich in protein, lipids, and mineral nutrients. It has high nutritional value and provides good conditions for the growth and reproduction of microorganisms.

[0003] Fresh tripe is not only expensive but also difficult to preserve. In order to extend its shelf life, the low-temperature preservation method is currently mainly used, but it is difficult to achieve the ideal preservation effect. The addition of biological preservatives will increase the process and cost of tripe production. It needs to be carefully washed before eating, and some biological preservatives will cause slight changes in the color and smell of the tripe. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a biological preservation method specifically for tripe, which solves the problems of unsatisfactory preservation effect and slight changes in the color and smell of tripe caused by some biological preservatives.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: a biological preservation method specifically for tripe, comprising the following steps:

[0006] Step 1: Tripe pretreatment

[0007] Put the tripe under running water to rinse off the obvious impurities, dirt and mucous membrane fluid on the surface, soak the tripe in a certain proportion of salt water or food-grade detergent solution for 10 to 30 minutes, rub the surface of the tripe, rinse the rubbed tripe again with clean water to remove the impurities and detergent residues left by soaking and rubbing, and place the cleaned tripe in a strainer or clean container to drain the water naturally;

[0008] Step 2: Isolation and identification of dominant bacteria

[0009] Take samples from the tripe for culture, select culture dishes with good and dispersed strain growth, observe and record the morphological characteristics of the strains, such as transparency, diameter size 0.5-1mm, picking difficulty, color, appearance, edge state, water content, and adhesion between the colony and the culture medium, perform Gram staining on the separated and purified dominant bacteria colonies, observe and record using an electron microscope, conduct VP experiments, gelatin hydrolysis experiments, biogenic amine experiments, indole physiological and biochemical experiments, and perform polymerase chain reaction, i.e., 16SrDNA detection;

[0010] Step 3: Screening of antibacterial agents

[0011] The Oxford cup method was used to determine the antibacterial activity of different antibacterial agents on the dominant bacteria in tripe, and it was determined that lysozyme, protamine, and ε-polylysine had antibacterial effects on the dominant bacteria, and Nisin was excluded as an inappropriate antibacterial agent;

[0012] Step 4: Single factor test

[0013] Conduct multiple single-factor experiments to explore the effects of pH values ​​between 9.00 and 13.00 on tripe, the effects of lysozyme concentrations between 0.1 and 0.5 g / kg on tripe, the effects of ε-polylysine concentrations between 0.1 and 0.3 g / kg on tripe, and the effects of protamine concentrations between 0.1 and 0.5 g / kg on tripe;

[0014] Step 5: Orthogonal test

[0015] Orthogonal test was conducted to determine the levels of factors that affect the preservation of tripe, lysozyme concentration 0.2-0.4 g / kg, pH value 11.50-12.50, ε-polylysine concentration 0.2-0.3 g / kg, protamine concentration 0.3-0.5 g / kg;

[0016] Step 6: Fresh-keeping

[0017] A fresh-keeping solution containing lysozyme, protamine and ε-polylysine was prepared, with the concentration of each component being 0.3 g / kg, the pH value of the fresh-keeping environment was adjusted to 12.0, the pretreated tripe was soaked in the fresh-keeping solution, and the soaked tripe was refrigerated at 1 to 4°C.

[0018] Preferably, in step six, the stirring speed during the preparation of the fresh-keeping liquid in the fresh-keeping treatment is 50 rpm to 100 rpm, the stirring time is 10 minutes to 20 minutes, and the soaking time of the tripe in the fresh-keeping liquid is 1 hour to 3 hours.

[0019] Preferably, the humidity of the refrigeration is controlled at 70% to 90%.

[0020] Preferably, in the step 1, the tripe is disinfected during pretreatment by irradiating it with ultraviolet light for 5 to 10 minutes.

[0021] Preferably, in step six, the freshness-keeping state of the tripe is checked every 3 days during the freshness-keeping treatment.

[0022] Preferably, the preparation temperature of the fresh-keeping liquid is controlled at 10°C to 25°C.

[0023] Preferably, in step six, the tripe is placed flat in layers during refrigeration.

[0024] Preferably, ascorbic acid is added as an auxiliary preservative ingredient to the preservative liquid in an amount of 0.1 g / kg to 0.3 g / kg.

[0025] Preferably, in the step 2, the step of isolating and identifying the dominant bacteria, the culture temperature is 25° C. to 37° C., and the culture time is 24 hours to 72 hours.

[0026] Preferably, in step 5, the orthogonal test also needs to consider the osmotic pressure of the fresh-keeping solution, which is at a level of 200mOsm / kg to 500mOsm / kg.

[0027] The present invention provides a biological preservation method specifically for tripe, which has the following beneficial effects:

[0028] 1. The present invention separates and purifies through preliminary microbial experiments, and then makes a preliminary judgment on the dominant strain through physiological and biochemical tests and Gram staining microscopy. Then, the strain is further determined through 16SrDNA detection technology. After antibacterial experiments and analysis of experimental data, the best antibacterial agent and the lowest effective antibacterial concentration are found. The best antibacterial agent is found by measuring the quality index data of tripe, thereby extending the shelf life of tripe stored at low temperature.

[0029] 2. The present invention selects lysozyme, protamine and ε-polylysine as the main biological preservatives, and determines the optimal usage concentration through experiments, thereby reducing the usage of the preservatives while ensuring the preservation effect, and at the same time controlling the preparation temperature of the preservative solution, adding ascorbic acid as an auxiliary preservative ingredient, thereby improving the preservation effect and alleviating the color and odor change problems caused by a single preservative.

[0030] 3. The present invention separates the dominant bacteria that cause tripe spoilage from tripe, identifies the characteristics of the dominant bacteria based on biotechnology, and applies biological preservatives to achieve the set antibacterial and preservation effects, providing a certain theoretical and application basis for the healthy development of the tripe industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the technical route of a biological preservation method specifically used for tripe of the present invention;

[0032] Figure 2 It is a schematic diagram of the colony morphology of two dominant bacteria in a biological preservation method specifically used for tripe of the present invention;

[0033] Figure 3 It is a schematic diagram of the Gram staining microscopic examination results of two dominant bacteria in a biological preservation method specifically used for tripe of the present invention;

[0034] Figure 4A schematic diagram of a phylogenetic tree of dominant bacteria in tripe in a biological preservation method specifically used for tripe of the present invention;

[0035] Figure 5 This is a schematic diagram of the antibacterial performance of different antibacterial agents on dominant bacteria in tripe in a biological preservation method specifically used for tripe of the present invention;

[0036] Figure 6 This is a schematic diagram of the effect of different pH values ​​on the total number of dominant bacteria in the tripe according to a biological preservation method specifically used for tripe of the present invention;

[0037] Figure 7 It is a schematic diagram showing the effect of different concentrations of lysozyme on the total number of dominant bacteria in the tripe in a biological preservation method specifically used for the tripe of the present invention;

[0038] Figure 8 It is a schematic diagram showing the effect of different concentrations of ε-polylysine on the total number of dominant bacteria in the tripe according to a biological preservation method specifically used for tripe of the present invention;

[0039] Fig. 9 This is a schematic diagram of the effect of different concentrations of protamine on the total number of dominant bacteria in the tripe in a biological preservation method specifically used for tripe of the present invention. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example:

[0042] Please refer to the attached Figure 1 -Attached Fig. 9 The embodiment of the present invention provides a biological preservation method specifically for tripe, comprising the following steps:

[0043] Step 1: Tripe pretreatment

[0044] Put the tripe under running water to rinse off the obvious impurities, dirt and mucous membrane fluid on the surface, soak the tripe in a certain proportion of salt water or food-grade detergent solution for 10 to 30 minutes, rub the surface of the tripe, rinse the rubbed tripe again with clean water to remove the impurities and detergent residues left by soaking and rubbing, and place the cleaned tripe in a strainer or clean container to drain the water naturally;

[0045] Step 2: Isolation and identification of dominant bacteria

[0046] Take samples from the tripe for culture, select culture dishes with good and dispersed strain growth, observe and record the morphological characteristics of the strains, such as transparency, diameter size 0.5-1mm, picking difficulty, color, appearance, edge state, water content, and adhesion between the colony and the culture medium, perform Gram staining on the separated and purified dominant bacteria colonies, observe and record using an electron microscope, conduct VP experiments, gelatin hydrolysis experiments, biogenic amine experiments, indole physiological and biochemical experiments, and perform polymerase chain reaction, i.e., 16SrDNA detection;

[0047] Step 3: Screening of antibacterial agents

[0048] The Oxford cup method was used to determine the antibacterial activity of different antibacterial agents on the dominant bacteria in tripe, and it was determined that lysozyme, protamine, and ε-polylysine had antibacterial effects on the dominant bacteria, and Nisin was excluded as an inappropriate antibacterial agent;

[0049] Step 4: Single factor test

[0050] Conduct multiple single-factor experiments to explore the effects of pH values ​​between 9.00 and 13.00 on tripe, the effects of lysozyme concentrations between 0.1 and 0.5 g / kg on tripe, the effects of ε-polylysine concentrations between 0.1 and 0.3 g / kg on tripe, and the effects of protamine concentrations between 0.1 and 0.5 g / kg on tripe;

[0051] Step 5: Orthogonal test

[0052] Orthogonal test was conducted to determine the levels of factors that affect the preservation of tripe, lysozyme concentration 0.2-0.4 g / kg, pH value 11.50-12.50, ε-polylysine concentration 0.2-0.3 g / kg, protamine concentration 0.3-0.5 g / kg;

[0053] Step 6: Fresh-keeping

[0054] A fresh-keeping solution containing lysozyme, protamine and ε-polylysine was prepared, with the concentration of each component being 0.3 g / kg, the pH value of the fresh-keeping environment was adjusted to 12.0, the pretreated tripe was soaked in the fresh-keeping solution, and the soaked tripe was refrigerated at 1 to 4°C.

[0055] In step six, the stirring speed during the preparation of the fresh-keeping solution in the fresh-keeping treatment is 50 rpm to 100 rpm, the stirring time is 10 minutes to 20 minutes, and the soaking time of the tripe in the fresh-keeping solution is 1 hour to 3 hours.

[0056] The humidity of refrigeration is controlled at 70% to 90%.

[0057] In step 1, the tripe is disinfected during pretreatment by irradiating it with ultraviolet light for 5 to 10 minutes.

[0058] In step six, the freshness of the tripe is checked every three days.

[0059] The preparation temperature of the preservative solution is controlled at 10℃~25℃.

[0060] In step six, the tripe should be placed flat in layers when refrigerated.

[0061] The auxiliary preservative ingredient ascorbic acid is added to the preservative liquid, and the added amount is 0.1g / kg to 0.3g / kg.

[0062] In step 2, the step of isolating and identifying the dominant bacteria, the culture temperature is 25° C. to 37° C., and the culture time is 24 hours to 72 hours.

[0063] In step five, the orthogonal test also needs to consider the osmotic pressure of the preservation solution, which is at a level of 200mOsm / kg to 500mOsm / kg.

[0064] Take the cultured strains from the incubator, select the culture dishes where the strains grow well and are relatively dispersed, observe and record the morphological characteristics of the strains, including whether the strains are transparent, diameter size, whether they are easy to pick, color, appearance, edge state, water content, and the degree of adhesion between the colony and the culture medium. The colony pictures are as shown in the instructions. Figure 2 As shown, the morphological characteristics of the colonies are shown in Table 1. The base color of the J-1 plate is milky white, the colonies are milky white, the colonies are smooth, round, not convex in the center, round, opaque, with neat edges, moist, with a diameter of about 0.5 mm and easy to pick up. The base color of the J-2 plate is transparent, the colonies are goose yellow, the colonies are smooth, plump, round, convex in the center, translucent, with neat edges, dry, with a diameter of about 1 mm, and not easy to pick up.

[0065] Table 1 Colony morphology characteristics:

[0066]

[0067] Gram staining microscopy of dominant bacteria in tripe

[0068] After Gram staining, the single colonies of the two dominant bacteria that had been separated and purified were observed and recorded using an electron microscope. Figure 3As shown, the two dominant bacteria are numbered J-1 and J-2. It is found that J-1 is red in color, which can be preliminarily determined to be a Gram-negative strain; while J-2 is blue-purple in color, which can be preliminarily determined to be a Gram-positive strain. The cells of strain J-1 are small bean-shaped and plump, without spores, arranged in pairs or chains, and the cells of the strain are densely distributed; the cells of strain J-2 are rod-shaped and plump, without spores, and the cells are often arranged in pairs or chains, and the cells of the strain are dispersed.

[0069] The identification results of the VP test, gelatin hydrolysis test, biogenic amine test, and indole test are shown in Table 2 ;

[0070] Table 2 Physiological and biochemical experimental identification results:

[0071] Strain name VP Experiment Gelatin liquefaction experiment Biogenic amine experiment Indole experiment J-1 Negative Negative Negative Negative J-2 Negative Negative Negative Negative

[0072] From the analysis of the results of physiological and biochemical experiments, it can be concluded that: the result of the VP test is negative, which means that the dominant bacterial strain does not have the ability to decompose glucose and produce the product pyruvic acid; the result of the gelatin liquefaction test is negative, which means that the strain does not have the ability to decompose gelatin and produce amino acids, that is, this strain does not produce the extracellular enzyme-gelatinase; the result of the biogenic amine test is negative, which means that the strain does not have the activity of amino acid decarboxylase, so it will not produce small molecular nitrogen-containing compounds that are harmful to the human body; the result of the indole test is also negative, which indicates that the dominant bacteria cannot use tryptophan to produce indole;

[0073] Results of 16SrDNA Identification of Dominant Bacteria in Tripe

[0074] After polymerase chain reaction, i.e., 16S rDNA detection, it was determined that the two dominant strains were Acinetobacter pituitarius and Xanthomonas candidum of the genus Acinetobacter. Part of the phylogenetic tree of the two strains is shown in the figure and in the description. Figure 4 As shown;

[0075] Preliminary screening of the antibacterial activity of different antibacterial agents on dominant bacteria in tripe

[0076] The Oxford cup method was used to determine the antibacterial activity of different antibacterial agents on the dominant bacteria in tripe. The results obtained after repeated verification were as shown in the description. Figure 5As shown in the figure, by observing the growth of the colonies, compared with the blank control, the blank control had no transparent circle, while the Oxford cups with lysozyme, protamine, ε-polylysine and Nisin had antibacterial circles, while chitosan and kojic acid had no transparent circles, natamycin had poor water solubility and no obvious transparent circle, kojic acid had certain antibacterial properties but produced pigments, and thus it was preliminarily determined that Nisin, ε-polylysine, protamine and lysozyme had antibacterial effects on the two dominant bacteria, and the inhibitory effects were more obvious, but since the optimal pH of Nisin was acidic, its solubility was very low under alkaline conditions, its activity would be greatly reduced or even completely inactivated, and the storage environment of tripe was alkaline, so the effect of Nisin would be very small, and its application in the preservation of tripe would no longer be considered in the future;

[0077] Y and 1 are sterile water blank controls, R is lysozyme, T is protamine, N is Nisin, 2 is natamycin, 3 is lysozyme, 4 is ε-polylysine, PT is Acinetobacter pitei, and TH is Xanthomonas luteus;

[0078] Effect of lysozyme on the quality of tripe

[0079] like Figure 7 As shown, lysozyme has a certain promoting effect on the preservation of tripe, and the five groups of lysozyme solutions with different concentrations have a certain inhibitory effect on the growth of bacterial colonies in the tripe. The strength of the inhibitory effect does not always change with the change of lysozyme concentration. When the concentration is in the range of 0.1-0.40g / kg, the inhibitory effect increases with the increase of lysozyme concentration. When the lysozyme concentration reaches 0.5g / kg, there will be a certain inhibitory effect. Therefore, for the five lysozyme addition concentrations, 0.2g / kg, 0.3g / kg and 0.4g / kg are the most appropriate.

[0080] Effect of ε-polylysine on the quality of tripe

[0081] like Figure 8 As shown in Figure 2, when the ε-polylysine concentration was 0.3 g / kg, the total colony count of tripe did not reach 1.0 × 10 within 60 days. 6 CFU / g, indicating that ε-polylysine has a very good promoting effect on the preservation of tripe. When the concentration of ε-polylysine is 0.15-0.25g / kg, the total colony count exceeds 1.0×10 at 55 days. 6 CFU / g, compared with the blank group, the total number of colonies exceeded 5.0×10 4CFU / g, and other tripes with added ε-polylysine were extended by 5 to 10 days in the fresh tripe stage. The data show that the concentration of ε-polylysine has a correlation effect on the total colony count of tripe. When the concentration of ε-polylysine is between 0.1g / kg and 0.3g / kg, the total colony count of tripe decreases with the increase of concentration. Therefore, for the five added concentrations of ε-polylysine, 0.20g / kg, 0.25g / kg and 0.30g / kg are the most appropriate.

[0082] Effect of protamine on the quality of tripe

[0083] like Fig. 9 As shown in Figure 2, when the protamine concentration was 0.5 g / kg, the total number of colonies in tripe reached 1.1×10 within 60 days. 6 CFU / g, indicating that protamine has a very good effect on the preservation of tripe. When the concentration of protamine is 0.1-0.4 g / kg, the total number of colonies exceeds 1.1×10 at 55 days. 6 CFU / g, compared with the blank group, the total number of colonies exceeded 0.2×10 5 CFU / g, and other tripes with added protamine were extended for 5-10 days in the fresh tripe stage. The data showed that the concentration of protamine had a relevant effect on the total colony count of tripe. When the concentration of protamine was between 0.1g / kg and 0.5g / kg, the total colony count of tripe decreased with the increase of concentration. Therefore, for the five protamine addition concentrations, 0.3g / kg, 0.4g / kg and 0.5g / kg were the most appropriate.

[0084] Table 3 Orthogonal test results of biological preservation method for tripe

[0085]

[0086]

[0087] As shown in Table 3, the order of the influence of the three factors on the total number of colonies in tripe at 1-4°C is pH> protamine> lysozyme> ε-polylysine, that is, the pH has the greatest influence on the total number of colonies in tripe, followed by protamine, lysozyme has a weaker influence, and ε-polylysine has the weakest influence. According to the significance of the four factors on tripe, combined with the stage changes in the total number of colonies and the changes in the quality of tripe, the best level ratio of the three factors is pH 12.0, protamine 0.3g / kg, lysozyme concentration 0.3g / kg, and ε-polylysine concentration 0.3g / kg, which has the best inhibitory effect on the total number of colonies in tripe and plays the best role in promoting the preservation of tripe.

[0088] The following is an introduction in conjunction with specific embodiments:

[0089] Embodiment 1:

[0090] Step 1: Tripe pretreatment

[0091] Rinse the tripe under running water to remove obvious impurities, dirt and mucous membrane fluid on the surface, soak the tripe in a certain proportion of saline solution for 10 minutes, gently rub the surface of the tripe, rinse the tripe again with clean water to remove impurities and residues, place the tripe in a filter to drain the water naturally, and disinfect it with ultraviolet light for 5 minutes;

[0092] Step 2: Isolation and identification of dominant bacteria

[0093] Samples were taken from the tripe for culture at 25°C for 24 hours. The morphological characteristics of the strains were observed, including transparency, diameter of about 0.5 mm, color, and appearance. Gram staining was performed on the dominant bacterial colonies. Electron microscope observation and recording were performed. VP experiments, gelatin hydrolysis experiments, biogenic amine experiments, and indole physiological and biochemical experiments were performed. The dominant bacterial species were determined by polymerase chain reaction, i.e., 16SrDNA detection.

[0094] Step 3: Screening of antibacterial agents

[0095] The Oxford cup method was used to determine the antibacterial activity of different antibacterial agents on the dominant bacteria in tripe, and it was determined that lysozyme, protamine, and ε-polylysine had antibacterial effects on the dominant bacteria, excluding Nisin.

[0096] Step 4: Single factor test

[0097] To explore the effect of pH value of 9.00 on tripe, to explore the effect of lysozyme concentration of 0.1g / kg on tripe, to explore the effect of ε-polylysine concentration of 0.1g / kg on tripe, and to explore the effect of protamine concentration of 0.1g / kg on tripe;

[0098] Step 5: Orthogonal experiment

[0099] The levels of factors affecting the preservation of tripe were determined, with lysozyme concentration of 0.2 g / kg, pH value of 11.50, ε-polylysine concentration of 0.2 g / kg, protamine concentration of 0.3 g / kg, and the osmotic pressure of the preservation solution of 200 mOsm / kg.

[0100] Step 6: Fresh-keeping

[0101] A fresh-keeping solution comprising lysozyme, protamine and ε-polylysine was prepared, wherein the concentration of each component was 0.3 g / kg, the preparation temperature was 10° C., the stirring speed was 50 rpm, the stirring time was 10 minutes, the pH value of the fresh-keeping environment was adjusted to 12.0, the pretreated tripe was soaked in the fresh-keeping solution for 1 hour, the soaked tripe was refrigerated at 1° C., the refrigerated humidity was controlled at 70%, the tripe was laid flat in layers, the fresh-keeping status was checked every 3 days, and ascorbic acid, an auxiliary fresh-keeping component, was added to the fresh-keeping solution in an amount of 0.1 g / kg.

[0102] It can extend the shelf life of tripe to a certain extent and can better maintain the quality of tripe, but the preservation effect may be relatively limited.

[0103] Embodiment 2:

[0104] Step 1: Tripe pretreatment

[0105] Rinse the tripe under running water to remove obvious impurities, dirt and mucous membrane fluid on the surface, soak the tripe in a food-grade detergent solution for 20 minutes, rub the surface of the tripe appropriately, rinse the tripe again with clean water to remove impurities and residues, place the tripe in a clean container to drain naturally, and disinfect it with ultraviolet light for 8 minutes;

[0106] Step 2: Isolation and identification of dominant bacteria

[0107] Samples were taken from the tripe for culture at 31°C for 48 hours. The morphological characteristics of the strains were observed, including transparency, diameter of about 0.75 mm, color, and appearance. Gram staining was performed on the dominant bacterial colonies. Electron microscope observation and recording were performed. VP experiments, gelatin hydrolysis experiments, biogenic amine experiments, and indole physiological and biochemical experiments were performed. The dominant bacterial species were determined by polymerase chain reaction, i.e., 16SrDNA detection.

[0108] Step 3: Screening of antibacterial agents

[0109] The Oxford cup method was used to determine the antibacterial activity of different antibacterial agents on the dominant bacteria in tripe, and it was determined that lysozyme, protamine, and ε-polylysine had antibacterial effects on the dominant bacteria, excluding Nisin.

[0110] Step 4: Single factor test

[0111] To explore the effect of pH value of 11.00 on tripe, to explore the effect of lysozyme concentration of 0.3g / kg on tripe, to explore the effect of ε-polylysine concentration of 0.2g / kg on tripe, and to explore the effect of protamine concentration of 0.4g / kg on tripe;

[0112] Step 5: Orthogonal test

[0113] The levels of factors affecting the preservation of tripe were determined, with lysozyme concentration of 0.3 g / kg, pH value of 12.00, ε-polylysine concentration of 0.25 g / kg, protamine concentration of 0.4 g / kg, and the osmotic pressure of the preservation solution of 350 mOsm / kg.

[0114] Step 6: Fresh-keeping

[0115] A fresh-keeping solution comprising lysozyme, protamine and ε-polylysine is prepared, wherein the concentration of each component is 0.3 g / kg, the preparation temperature is 18° C., the stirring speed is 75 rpm, the stirring time is 15 minutes, the pH value of the fresh-keeping environment is adjusted to 12.0, the pretreated tripe is soaked in the fresh-keeping solution for 2 hours, the soaked tripe is refrigerated at 2.5° C., the refrigerated humidity is controlled at 80%, the tripe is laid flat in layers, the fresh-keeping status is checked every 3 days, and ascorbic acid, an auxiliary fresh-keeping component, is added to the fresh-keeping solution in an amount of 0.2 g / kg.

[0116] It can significantly extend the shelf life of tripe, better maintain the quality of tripe, and keep various indicators relatively stable.

[0117] Embodiment three:

[0118] Step 1: Tripe pretreatment

[0119] Rinse the tripe under running water to remove obvious impurities, dirt and mucous membrane fluid on the surface, soak the tripe in a certain proportion of saline solution for 30 minutes, rub the surface of the tripe vigorously, rinse the tripe again with clean water to remove impurities and residues, place the tripe in a filter to drain the water naturally, and disinfect it with ultraviolet light for 10 minutes;

[0120] Step 2: Isolation and identification of dominant bacteria

[0121] Samples were taken from the tripe for culture at 37°C for 72 hours. The morphological characteristics of the strains were observed, including transparency, diameter of about 1 mm, color, and appearance. Gram staining was performed on the dominant bacterial colonies. Electron microscope observation and recording were performed. VP experiments, gelatin hydrolysis experiments, biogenic amine experiments, and indole physiological and biochemical experiments were performed. The dominant bacterial species were determined by polymerase chain reaction, i.e., 16SrDNA detection.

[0122] Step 3: Screening of antibacterial agents

[0123] The Oxford cup method was used to determine the antibacterial activity of different antibacterial agents on the dominant bacteria in tripe, and it was determined that lysozyme, protamine, and ε-polylysine had antibacterial effects on the dominant bacteria, excluding Nisin.

[0124] Step 4: Single factor test

[0125] To explore the effect of pH value of 13.00 on tripe, the effect of lysozyme concentration of 0.5g / kg on tripe, the effect of ε-polylysine concentration of 0.3g / kg on tripe, and the effect of protamine concentration of 0.5g / kg on tripe;

[0126] Step 5: Orthogonal test

[0127] The levels of factors affecting the preservation of tripe were determined, with lysozyme concentration of 0.4 g / kg, pH value of 12.50, ε-polylysine concentration of 0.3 g / kg, protamine concentration of 0.5 g / kg, and the osmotic pressure of the preservation solution of 500 mOsm / kg.

[0128] Step 6: Fresh-keeping

[0129] A fresh-keeping solution comprising lysozyme, protamine and ε-polylysine is prepared, wherein the concentration of each component is 0.3 g / kg, the preparation temperature is 25° C., the stirring speed is 100 rpm, the stirring time is 20 minutes, the pH value of the fresh-keeping environment is adjusted to 12.0, the pretreated tripe is soaked in the fresh-keeping solution for 3 hours, the soaked tripe is refrigerated at 4° C., the refrigerated humidity is controlled at 90%, the tripe is laid flat in layers, the fresh-keeping status is checked every 3 days, and ascorbic acid, an auxiliary fresh-keeping component, is added to the fresh-keeping solution in an amount of 0.3 g / kg.

[0130] It can extend the shelf life of tripe to the greatest extent, effectively maintain the quality of tripe, and keep it in good condition for a longer period of time.

[0131] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biological preservation method specifically for tripe, characterized in that: The following steps are involved: Step 1: Tripe pretreatment Put the tripe under running water to rinse off the obvious impurities, dirt and mucous membrane fluid on the surface, soak the tripe in a certain proportion of salt water or food-grade detergent solution for 10 to 30 minutes, rub the surface of the tripe, rinse the rubbed tripe again with clean water to remove the impurities and detergent residues left by soaking and rubbing, and place the cleaned tripe in a strainer or clean container to drain the water naturally; Step 2: Isolation and identification of dominant bacteria Take samples from the tripe for culture, select culture dishes with good and dispersed strain growth, observe and record the morphological characteristics of the strains, such as transparency, diameter size 0.5-1mm, picking difficulty, color, appearance, edge state, water content, and adhesion between the colony and the culture medium, perform Gram staining on the separated and purified dominant bacteria colonies, observe and record using an electron microscope, conduct VP experiments, gelatin hydrolysis experiments, biogenic amine experiments, indole physiological and biochemical experiments, and perform polymerase chain reaction, i.e., 16SrDNA detection; Step 3: Screening of antibacterial agents The Oxford cup method was used to determine the antibacterial activity of different antibacterial agents on the dominant bacteria in tripe, and it was determined that lysozyme, protamine, and ε-polylysine had antibacterial effects on the dominant bacteria, and Nisin was excluded as an inappropriate antibacterial agent; Step 4: Single factor experiment Conduct multiple single-factor experiments to explore the effects of pH values ​​between 9.00 and 13.00 on tripe, the effects of lysozyme concentrations between 0.1 and 0.5 g / kg on tripe, the effects of ε-polylysine concentrations between 0.1 and 0.3 g / kg on tripe, and the effects of protamine concentrations between 0.1 and 0.5 g / kg on tripe; Step 5: Orthogonal test Orthogonal test was conducted to determine the levels of factors that affect the preservation of tripe, lysozyme concentration 0.2-0.4 g / kg, pH value 11.50-12.50, ε-polylysine concentration 0.2-0.3 g / kg, protamine concentration 0.3-0.5 g / kg; Step 6: Fresh-keeping A fresh-keeping solution containing lysozyme, protamine and ε-polylysine was prepared, with the concentration of each component being 0.3 g / kg, the pH value of the fresh-keeping environment was adjusted to 12.0, the pretreated tripe was soaked in the fresh-keeping solution, and the soaked tripe was refrigerated at 1 to 4°C.

2. A biological preservation method specifically for tripe according to claim 1, characterized in that: In the step six, the stirring speed during the preparation of the fresh-keeping liquid in the fresh-keeping treatment is 50 rpm to 100 rpm, the stirring time is 10 minutes to 20 minutes, and the soaking time of the tripe in the fresh-keeping liquid is 1 hour to 3 hours.

3. The biological preservation method for tripe according to claim 1, characterized in that: The humidity of the refrigeration is controlled at 70% to 90%.

4. The biological preservation method for tripe according to claim 1, characterized in that: In the step 1, the tripe is disinfected during pretreatment by irradiating it with ultraviolet light for 5 to 10 minutes.

5. The biological preservation method for tripe according to claim 1, characterized in that: In the step six, the freshness-keeping treatment is to check the freshness-keeping state of the tripe every 3 days.

6. The biological preservation method for tripe according to claim 1, characterized in that: The preparation temperature of the fresh-keeping liquid is controlled at 10°C to 25°C.

7. The biological preservation method for tripe according to claim 1, characterized in that: In the step six, the tripe is placed flat in layers during refrigeration.

8. The biological preservation method for tripe according to claim 1, characterized in that: The auxiliary fresh-keeping ingredient ascorbic acid is added to the fresh-keeping liquid in an amount of 0.1 g / kg to 0.3 g / kg.

9. The biological preservation method for tripe according to claim 1, characterized in that: In the step 2, the step of isolating and identifying the dominant bacteria, the culture temperature is 25° C. to 37° C., and the culture time is 24 hours to 72 hours.

10. The biological preservation method for tripe according to claim 1, characterized in that: In step 5, the orthogonal test also needs to consider the osmotic pressure of the fresh-keeping solution, which is at a level of 200mOsm / kg to 500mOsm / kg.

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