Cooked meat product normal-temperature preservation method based on natural components

Abstract

The invention relates to the technical field of normal-temperature preservation of cooked meat products, in particular to a normal-temperature preservation method of cooked meat products based on natural components. The invention provides a method for improving the fresh-keeping degree of cooked meat products during normal-temperature storage, which comprises the following steps: applying a preset amount of compound preservative to the cooked meat products according to the expected fresh-keeping degree requirement, the compound preservative is prepared from epsilon-polylysine salt, nisin and sodium lactate. According to the method provided by the invention, the fresh-keeping degree of the cooked meat product can be comprehensively improved, so that the existing storage limitation is broken through while multiple requirements of consumers on safety, nutrition and flavor are met.

Description

Technical Field

[0001] The present invention relates to the technical field of ambient temperature preservation of cooked meat products, and particularly relates to an ambient temperature preservation method for cooked meat products based on natural ingredients. Background Art

[0002] Approximately 23% of meat products are affected by microbial spoilage every year, resulting in significant losses. Due to the ideal nutritional factors and high water activity of meat products, ambient temperature storage provides a favorable environment for the growth of various microorganisms. These microorganisms and their metabolites may pose food safety hazards and threaten the health of consumers.

[0003] Modern preservation technologies mainly achieve effective inhibition of microbial activities by regulating physical, chemical, and biological factors (such as low-temperature storage, vacuum packaging, and application of preservatives, etc.), thereby ensuring food safety and extending the shelf life. Currently, the mainstream preservation methods can be divided into three categories: physical preservation, chemical preservation, and biological preservation. Physical preservation technologies mainly inhibit microbial growth by changing environmental conditions or applying physical energy. Common means include low-temperature storage (such as refrigeration, freezing), high-pressure processing, microwave-assisted thermal sterilization, modified atmosphere packaging, irradiation treatment, cold plasma, etc. Among them, low-temperature storage delays spoilage by reducing enzyme activity and microbial metabolic rate; high-pressure processing at 400 MPa - 600 MPa can effectively inactivate spoilage bacteria at ambient temperature while reducing the damage to the color and texture of meat products; microwave-assisted thermal sterilization technology achieves efficient sterilization through rapid and uniform heating. Modified atmosphere packaging is a technology that extends the shelf life of food by replacing the gas environment inside the package, creating the most suitable storage gas environment for food. This method is widely used in the fields of fresh agricultural products, meat, etc. Modified atmosphere packaging technology has high requirements for packaging materials, and the gas composition needs to be precisely controlled during packaging. During storage and transportation, affected by various factors such as packaging damage and temperature fluctuations, the gas environment inside the package may change, making it difficult to achieve the expected effect of inhibiting microorganisms.

[0004] Chemical preservation technology focuses on adding safe bacteriostatic agents and achieves anti-corrosion by interfering with microbial metabolism or destroying cell structure. As a traditional chemical preservative, nitrite has both antioxidant and bacteriostatic functions, but also has potential carcinogenic risks. Natural preservatives have attracted much attention due to their safety. For example, they play a broad-spectrum bacteriostatic role by destroying the integrity of cell membranes or inhibiting enzyme activity. However, the control ability of biological preservatives such as Nisin for complex microbial communities is limited. Biological preservation technology relies on microorganisms or their metabolites to achieve bacteriostasis and has the characteristics of green sustainability. For example, technologies such as using phages to target and kill specific pathogenic bacteria and enzyme preparations to decompose biogenic amines have gradually become research hotspots.

[0005] Modern preservation technologies usually combine the above methods for preservation. For example, Chinese Patent Application CN110604167A discloses an anti-corrosion and preservation method for smoked and roasted meat products. An antibacterial agent is added during the cooking process of the meat raw materials. The cooked meat raw materials are subjected to spore induction, and after smoking and roasting, medium-temperature sterilization and vacuum packaging are carried out to extend the shelf life of the smoked and roasted meat products and inhibit Bacillus. Chinese Patent Application CN117796500A discloses a production and processing method for preventing and controlling Bacillus in sausage meat products. It not only adds an antibacterial agent to the meat stuffing, but also inhibits Bacillus by means of vacuum enema and staged variable-temperature sterilization.

[0006] With the development of social economy and the acceleration of the pace of life, cooked meat products are more and more popular among consumers due to their convenience. At present, the market demand for convenient-to-eat cooked meat products is increasing, but there are double challenges of microbial spoilage and oxidative deterioration during storage at room temperature. Under the condition of room temperature storage, the quality of cooked meat products is prone to change, and in severe cases, it will lead to food spoilage. Cooked meat products face severe challenges of microbial contamination during storage at room temperature. Microorganisms decompose nutrients such as proteins and fats, resulting in color deterioration such as yellowing and greening of the product, generation of peculiar smells and formation of surface mucus, significantly shortening the shelf life. Healthy meat products will further accelerate the reproduction of spoilage bacteria under room temperature conditions. In addition, the accumulation of microbial metabolites (such as biogenic amines) not only accelerates spoilage, but may also cause food safety problems.

[0007] Therefore, the control of microbial spoilage of cooked meat products is one of the key challenges faced by the food industry. On the one hand, the cost of cold-chain preservation of cooked meat products in the market is high and the temperature control in the distribution link is prone to failure; on the other hand, existing preservation methods (such as low-temperature storage, vacuum packaging) will affect the immediate edible taste of cooked meat products (such as bulk cooked meat products). Therefore, most of the existing technologies do not match the actual sales situation of non-standardized produced bulk cooked meat products, and their technical solutions are also difficult to apply to their short-term room temperature preservation. There is still little research on the preservation of different types of cooked meat products during storage at room temperature at home and abroad. Therefore, there is an urgent need to develop a room temperature preservation method for cooked meat products that balances the preservation effect and sensory quality to meet the multiple needs of consumers for safety, nutrition and flavor, and thus break through the existing storage limitations of cooked meat products. Summary of the Invention

[0008] In a first aspect, the present invention provides a method for improving the freshness preservation degree of cooked meat products during storage at room temperature, the method comprising: applying a predetermined application amount of a composite preservative to the cooked meat products according to the expected freshness preservation degree requirements.

[0009] As used herein, "cooked meat products" refer to meat products that have been processed by heat treatment (such as at least one of steaming, boiling, roasting, frying, and stir-frying). In some embodiments, the cooked meat products are mainly made from livestock and poultry meats, such as pork, chicken, beef, mutton, duck, and goose. In some embodiments, the cooked meat products may include braised and marinated cooked meat products (such as marinated beef, marinated chicken legs), smoked and cooked meat products (such as smoked chicken), pickled cooked meat products (such as pickled goose, pickled chicken), and boiled / clear-boiled cooked meat products (such as sliced cold chicken, boiled meat). It should be noted that although cooked meat products have been processed by heat treatment (even seasoned), due to reasons such as high moisture and protein content, they are still prone to spoilage at room temperature, and the shelf life is usually about 24 hours.

[0010] As used herein, "freshness preservation degree" refers to the ability of food to maintain its original sensory characteristics (such as color, flavor, appearance, texture, and taste), as well as nutritional components and microbial safety, under certain storage (or storage, preservation) conditions. In some embodiments, the freshness preservation degree reflects the overall quality level of food to maintain an "approximate initial state" under specific storage conditions. In some embodiments, the freshness preservation degree can be evaluated by one or more of physical and chemical indexes (such as volatile basic nitrogen value, thiobarbituric acid value), microbial indexes (such as total number of colonies), and sensory indexes.

[0011] As used herein, "room temperature" refers to the natural temperature range under normal environmental conditions, generally between 15°C and 25°C. In some embodiments, room temperature can also be understood as the temperature for storage, transportation, or use without special temperature control conditions.

[0012] In some embodiments, the composite preservative includes ε-polylysine salt, nisin, and sodium lactate.

[0013] In some embodiments, the ε-polylysine salt is ε-polylysine hydrochloride.

[0014] In some embodiments, when the expected freshness preservation requirement is to comprehensively improve the freshness preservation degree of the cooked meat products during storage at room temperature, the predetermined application amount of nisin is 0.099 - 0.109 g / kg of cooked meat products, the predetermined application amount of ε-polylysine salt is 0.205 - 0.216 g / kg of cooked meat products, and the predetermined application amount of sodium lactate is 3.174 - 3.605 g / kg of cooked meat products.

[0015] In some embodiments, the predetermined application amount of nisin is 0.101 g / kg of cooked meat products, the predetermined application amount of ε-polylysine salt is 0.213 g / kg of cooked meat products, and the predetermined application amount of sodium lactate is 3.379 g / kg of cooked meat products.

[0016] In some embodiments, comprehensively improving the freshness preservation degree of the cooked meat products during storage at room temperature includes comprehensively improving the microbial index, total volatile basic nitrogen index, thiobarbituric acid index, and sensory index of the cooked meat products during storage at room temperature.

[0017] In some embodiments, the method further includes: obtaining the specific application amount of the composite preservative according to the freshness preservation index that is preferentially considered to be improved.

[0018] In some embodiments, when the freshness preservation index that is preferentially considered to be improved is the microbial index, the specific application amount of the composite preservative is obtained according to the first model, and the first model includes: Y1(lg(CFU / g)) = 5.16 - 0.13×A - 0.10×B - 0.048×C - 0.055×A×B - 0.005×A×C + 0.015×B×C + 0.12×A 2 + 0.17×B 2 + 0.26×C 2 , where Y1 is the total colony value, A is the application amount of ε-polylysine salt, B is the application amount of nisin, and C is the application amount of sodium lactate.

[0019] In some embodiments, when the freshness preservation index that is preferentially considered to be improved is the total volatile basic nitrogen index, the specific application amount of the composite preservative is obtained according to the second model, and the second model includes: Y2(TVB-N) = 14.5 - 0.36×A - 0.28×B - 0.14×C - 0.17×A×B - 0.014×A×C + 0.045×B×C + 0.32×A 2 + 0.47×B 2 + 0.74×C 2 , where Y2 is the TVB-N value, A is the application amount of ε-polylysine salt, B is the application amount of nisin, and C is the application amount of sodium lactate.

[0020] In some embodiments, when the freshness preservation index that is preferentially considered to be improved is the thiobarbituric acid index, the specific application amount of the composite preservative is obtained according to the third model, and the third model includes: Y3(TBA) = 24.0 - 0.60×A - 0.48×B - 0.22×C - 0.26×A×B - 0.023×A×C + 0.07×B×C + 0.55×A 2 + 0.78×B 2 + 1.2×C 2 , where Y3 is the TBA value, A is the application amount of ε-polylysine salt, B is the application amount of nisin, and C is the application amount of sodium lactate.

[0021] In some embodiments, when the freshness index to be prioritized is a sensory index, the specific application amount of the composite preservative is obtained according to the fourth model, and the fourth model includes: Y4 (sensory score) = 9.1 + 0.13×A + 0.18×B + 0.43×C + 0.048×A×B + 0.19×A×C + 0.058×B×C - 0.28×A 2 - 0.60×B 2 - 0.63×C 2 , where Y4 is the sensory score value, A is the application amount of ε-polylysine salt, B is the application amount of nisin, and C is the application amount of sodium lactate.

[0022] It should be noted that the method provided by the present invention can not only comprehensively improve the freshness of cooked meat products during storage at room temperature, but also, on the basis of meeting the multiple requirements of safety, nutrition and flavor, customize the specific application amount of the composite preservative according to the above model to improve the freshness index to be prioritized, and thus can realize the customization of the shelf life.

[0023] In some embodiments, the composite preservative is applied in a liquid form. In some embodiments, the composite preservative is applied in a liquid form to the surface of the cooked meat product.

[0024] In some embodiments, the application method of the composite preservative may be: uniformly applying the composite preservative to the surface of the cooked meat product. Specifically, it may include: when the moisture on the surface of the cooked meat product is sufficient to enable the composite preservative to be applied in a liquid manner, the composite preservative (such as in solid form) can be directly and uniformly applied (such as smeared) to the surface of the cooked meat product; when the moisture on the surface of the cooked meat product is less or the moisture is insufficient to enable the composite preservative to be applied in a liquid manner, the composite preservative can be applied by first uniformly applying an appropriate amount of solvent to the surface of the cooked meat product and then uniformly applying the composite preservative to the surface of the cooked meat product, or by first mixing the composite preservative with an appropriate amount of solvent to form a composite preservative solution and then uniformly applying (such as smearing, spraying) the composite preservative solution to the surface of the cooked meat product.

[0025] It should be understood that the solvent only needs to ensure that the composite preservative can be dissolved and does not affect the taste of the cooked meat product, and the present invention does not limit the amount of the solvent.

[0026] In some embodiments, the application method of the composite preservative may also be: soaking the cooked meat product in a composite preservative solution.

[0027] In some embodiments, the application method of the composite preservative may also be: packaging the cooked meat product with a carrier containing the composite preservative.

[0028] In a second aspect, the present invention also provides a compound preservative, which comprises a preservative composed of the following parts by weight: 1 part of nisin, 1.9 - 2.2 parts of ε-polylysine salt, and 29.1 - 36.4 parts of sodium lactate.

[0029] In some embodiments, the compound preservative comprises a preservative composed of the following parts by weight: 1 part of nisin, 2.1 parts of ε-polylysine salt, and 33.6 parts of sodium lactate.

[0030] In some embodiments, the ε-polylysine salt is ε-polylysine hydrochloride.

[0031] By setting the above parts by weight of ε-polylysine salt, nisin and sodium lactate in the compound preservative, the present invention enables these three components to play a synergistic effect, which can not only effectively control the reproduction of microorganisms (including Gram-negative bacteria, Gram-positive bacteria and fungi) in cooked meat products, but also slow down the processes of protein decomposition and fat oxidation, and at the same time maintain good sensory quality to extend the shelf life. However, the bacteriostatic agents disclosed in the prior art (such as CN110604167A, CN117796500A) can only target a single strain (such as Bacillus), and it is difficult to apply them to the short-term preservation of cooked meat products.

[0032] In a third aspect, the present invention also provides the use of the above compound preservative in the preparation of a kit for improving the freshness preservation degree of cooked meat products during storage at room temperature.

[0033] In some embodiments, the compound preservative is configured for the short-term preservation of cooked meat products.

[0034] In some embodiments, the compound preservative comprises nisin in an amount of 0.099 - 0.109 g / kg of cooked meat products, ε-polylysine salt in an amount of 0.205 - 0.216 g / kg of cooked meat products, and sodium lactate in an amount of 3.174 - 3.605 g / kg of cooked meat products.

[0035] In some embodiments, the compound preservative comprises nisin in an amount of 0.101 g / kg of cooked meat products, ε-polylysine salt in an amount of 0.213 g / kg of cooked meat products, and sodium lactate in an amount of 3.379 g / kg of cooked meat products.

[0036] In some embodiments, the compound preservative comprises nisin in an amount of 0.109 g / kg of cooked meat products, ε-polylysine salt in an amount of 0.216 g / kg of cooked meat products, and sodium lactate in an amount of 3.19 g / kg of cooked meat products.

[0037] In some embodiments, the composite preservative comprises nisin at a level of 0.104 g / kg of cooked meat products, ε-polylysine salt at a level of 0.214 g / kg of cooked meat products, and sodium lactate at a level of 3.17 g / kg of cooked meat products.

[0038] In some embodiments, the composite preservative comprises nisin at a level of 0.103 g / kg of cooked meat products, ε-polylysine salt at a level of 0.213 g / kg of cooked meat products, and sodium lactate at a level of 3.51 g / kg of cooked meat products.

[0039] In some embodiments, the composite preservative is applied in liquid form to enhance the freshness preservation of cooked meat products during storage at room temperature. In some embodiments, the composite preservative is applied in liquid form to the surface of the cooked meat products.

[0040] In some embodiments, the application method of the composite preservative may be: applying the composite preservative uniformly to the surface of the cooked meat products. Specifically, it may include: when the moisture on the surface of the cooked meat products is sufficient to enable the composite preservative to be applied in liquid form, the composite preservative (such as in solid form) can be directly and uniformly applied (such as smeared) to the surface of the cooked meat products; when the moisture on the surface of the cooked meat products is less or the moisture is insufficient to enable the composite preservative to be applied in liquid form, it can be applied by first uniformly applying an appropriate amount of solvent to the surface of the cooked meat products and then uniformly applying the composite preservative to the surface of the cooked meat products, or by first mixing the composite preservative with an appropriate amount of solvent to form a composite preservative solution and then uniformly applying (such as smearing, spraying) the composite preservative solution to the surface of the cooked meat products.

[0041] In some embodiments, the application method of the composite preservative may also be: soaking the cooked meat products in a composite preservative solution.

[0042] In some embodiments, the application method of the composite preservative may also be: packaging the cooked meat products with a carrier containing the composite preservative.

[0043] In some embodiments, the use includes extending the shelf life of the cooked meat products by at least 1 day.

[0044] Compared with the prior art, the beneficial effects of the present invention at least include the following aspects: Most of the prior art focuses on combining preservatives with physical freshness preservation methods to extend the shelf life of meat products under low-temperature storage and / or vacuum packaging. However, since the current physical freshness preservation methods (such as low-temperature storage, vacuum packaging) require specific equipment and processes and also affect the ready-to-eat texture of cooked meat products, it is difficult to apply these prior arts to the short-term room-temperature freshness preservation of cooked meat products.

[0045] With the increasing awareness of the risks of chemically synthesized preservatives among people, natural preservatives derived from plants, animals, and microorganisms are generally considered relatively safer. Therefore, consumers have a higher acceptance of natural preservatives. Although natural preservatives are reported to be able to reduce microorganisms in food, the experimental results of the present invention show that the typical representatives of spoilage bacteria in cooked meat products during storage at room temperature include Staphylococcus aureus, Escherichia coli, and Aspergillus flavus, etc. Most commonly used natural preservatives have very limited short-term freshness preservation ability for cooked meat products during storage at room temperature. Substances such as natamycin, chitosan, ascorbic acid, and thyme extract are difficult to inhibit the growth of microorganisms in cooked meat products during storage at room temperature, and a single natural preservative (such as nisin, ε-polylysine salt, sodium lactate) is also difficult to completely cover the complex spoilage bacteria flora (i.e., Gram-negative bacteria, Gram-positive bacteria, and fungi), and thus it is difficult to meet the short-term freshness preservation requirements for cooked meat products at room temperature.

[0046] Based on the dual considerations of the minimum effective dose and regulatory compliance, the present invention provides a method for improving the freshness preservation degree of cooked meat products during storage at room temperature. On the one hand, the experimental results of the present invention show that when the predetermined application amount of nisin in the composite preservative provided by the present invention is 0.099 - 0.109 g / kg of cooked meat products, the predetermined application amount of ε-polylysine salt is 0.205 - 0.216 g / kg of cooked meat products, and the predetermined application amount of sodium lactate is 3.174 - 3.605 g / kg of cooked meat products, the ε-polylysine salt, nisin, and sodium lactate in the composite preservative provided by the present invention can play a synergistic effect, which can not only effectively control the microbial reproduction in cooked meat products, but also slow down the processes of protein decomposition and fat oxidation, and at the same time can maintain good sensory quality to extend the shelf life. The experimental results of the present invention also show that there is almost no residue of the composite preservative in the cooked meat products preserved by the method provided by the present invention, further ensuring food safety. That is to say, the method provided by the present invention can comprehensively improve the freshness preservation degree of cooked meat products, meet the multiple needs of consumers for safety, nutrition, and flavor, and at the same time break through the existing storage limitations.

[0047] In summary, the present invention can not only effectively solve the problem of the limitation of the shelf life of food by current refrigeration or freezing technologies, but also has important practical significance and far-reaching influence in aspects such as ensuring food safety, extending the food shelf life, and reducing food waste. Brief Description of the Drawings

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual scale. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0049] Figure 1 It is a result diagram of the antibacterial zones of 5 kinds of preservatives; Figure 2 It is a result diagram of 10 different spoilage parts on the boiled meat; Figure 3 It is a result diagram of the relative abundances of bacterial communities (A) based on 16S rDNA amplicon sequencing and fungal communities at the species level (B) in the boiled meat samples; Figure 4 It is a result diagram of the prediction of the comprehensive optimal preservative ratio; Figure 5 It is a result diagram of the effects of the compound preservative on the total number of colonies of sliced cooked chicken (A), marinated beef (B), and marinated goose (C) over storage time; Figure 6 It is a result diagram of the effects of the compound preservative on the TVB-N values of sliced cooked chicken (A), marinated beef (B), and marinated goose (C) over storage time; Figure 7 It is a result diagram of the effects of the compound preservative on the TBA values of sliced cooked chicken (A), marinated beef (B), and marinated goose (C) over storage time; Figure 8 It is a result diagram of the effects of the compound preservative on the sensory scores of sliced cooked chicken (A), marinated beef (B), and marinated goose (C) over storage time; Figure 9 It is a result diagram of the effects of the compound preservative on the quality indexes of marinated beef over storage time under refrigeration conditions. Detailed implementation manners

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0051] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0052] As used herein, "a plurality of" means two or more, i.e., it includes two, three, four, five, etc.

[0053] It should be noted that, as used herein, the term "comprising", "including" or any other variation thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or apparatus. Without further limitation, an element defined by the statement "comprising a..." does not exclude the presence of additional identical elements in the process, method, article or apparatus comprising such element.

[0054] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0055] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values within that range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0056] The materials and instruments used in the following examples include: 1. Cooked meat products: Fresh streaky pork of the same batch purchased from a certain supermarket in Beijing was cooked with pure water.

[0057] 2. Preservatives: Nisin (analytical pure), Ascorbic acid (analytical pure), ε-Polylysine hydrochloride (analytical pure), Tea polyphenols (analytical pure), Rosemary extract (98%), Potassium sorbate (analytical pure), Natamycin (analytical pure), Sodium ascorbate (analytical pure), Thymol (analytical pure), Chitosan (98%), Sodium lactate (analytical pure), Macklin company.

[0058] 3. Staphylococcus aureus BNCC186335, Escherichia coli BNCC133264, Aspergillus flavus BNCC142803, BNCC Corporation.

[0059] 4. Nutrient broth agar medium (NB), Comprehensive potato dextrose agar (CPDA) medium, Solarbio Company.

[0060] 5. Antibiotic susceptibility test discs (diameter 6 mm), BKMAM Company; Oxford cups (inner diameter 6 mm ± 0.1 mm), Shanghai Jingchong Electronic Technology Co., Ltd.

[0061] 6. HD-850 tabletop horizontal purification workbench, Shanghai Jingmai Instrument and Equipment Co., Ltd.; SQ810C vertical steam sterilizer, Yamato Company; SHZ-82A air bath constant temperature oscillator, Jintan Kexi Instrument Co., Ltd.; N5000 ultraviolet-visible spectrophotometer, Shanghai Youke Instrument Co., Ltd.; DW-86L388A ultra-low temperature storage box, Hair Company.

[0062] Example 1 1.1 Simulation experiment for screening preservatives Weigh 11 kinds of preservatives according to the maximum dosage of each preservative specified in the national standard GB2760-2024, and dissolve them in 1 mL of pure water respectively. Then evenly apply them on the surface of the cooked meat slices (slice specifications: 5×2×0.5 cm, mass 10 g, with skin, fat and lean meat). Marinate and let stand in a disposable petri dish for 2 h until the additives are absorbed into the meat slices. Place the meat slices in a glass cylinder (100×55×56 cm) that is relatively isolated from the external environment. Use a thermometer and hygrometer to record that the humidity in the cylinder is stable at about 75%, and control the room temperature at 25 °C. Evaluate and record the appearance state of each meat slice every 24 h.

[0063] The experimental results observed with the naked eye show that compared with other experimental groups where plaque hyphae appeared after 48 h, the preservative effects of ε-PL (ε-polylysine hydrochloride), Nisin, SL (sodium lactate), natamycin and chitosan are more prominent, and they can delay the time for plaque hyphae to grow on the meat slices by 24 h.

[0064] 1.2 Inhibition zone test The inhibition zone test was carried out in two forms: antibiotic susceptibility test discs (dry method) and Oxford cups (wet method) to measure the inhibition zones of the above 11 kinds of preservative solutions.

[0065] The determination method of drug - sensitive paper (dry method) is as follows: Take 5 μL of the preservative solution (50 mg / mL) and spot it on a blank drug - sensitive paper, then dry it. Drop two drops of bacterial solution on the NB plate and spread it evenly. Gently place the drug - sensitive paper with the preservative on the plate inoculated with the bacterial solution. After all the plates are incubated at 30 °C for 16 h, take them out, measure and record the diameter of the inhibition zone formed by each preservative on the plate. Each experiment in each group is repeated three times and the average value is taken.

[0066] According to the same method above, inject 200 μL of the preservative solution (50 mg / mL) into the Oxford cup to replace the drug - sensitive paper, and measure the diameter of the inhibition zone of the above 11 kinds of preservative solutions.

[0067] The preparation method of the bacterial solution is as follows: Fresh streaky pork from the same batch as the cooked meat products is boiled in pure water, and then placed in the indoor natural environment (controlled temperature 25 °C, humidity 65%) until it appears spoiled. In the ultra - clean workbench, use a sterile scalpel to pick 10 spoiled parts with different colors and shapes on the boiled meat, named from No. 1 to No. 10 respectively, and transfer them to the NB liquid medium respectively. Then place them in a shaker at 200 r / min and 30 °C for 24 h for enrichment culture. After the OD600 value of each bacterial solution detected by the ultraviolet spectrophotometer meets 0.30 - 0.36, it is reserved for use.

[0068] Due to the lack of the influence of seasonings such as salt and flavoring agents, in this example, the spoilage bacteria community after the spoilage of the boiled meat is selected as the indicator object for the antibacterial experiment, which can directly and effectively reflect the inhibitory effect of each preservative on the spoilage bacteria causing cooked meat spoilage. The inhibition zone method can simultaneously evaluate the broad - spectrum antibacterial activity of multiple preservatives against multiple spoilage bacteria communities in a single experiment, which is efficient and convenient. The larger the diameter of the inhibition zone, the better the antibacterial effect. Specifically, when the inhibition zone < 10 mm, there is no antibacterial effect; when 10 mm < inhibition zone < 15 mm, it is moderately antibacterial; when the inhibition zone > 15 mm, it is highly antibacterial.

[0069] As Figure 1As shown, compared with the simulated application test of the actual pork samples at 1.1, the inhibitory effects of the 11 preservatives on the bacterial solution extracted from pork are somewhat different. Natamycin and chitosan, which have preservative effects in the actual pork samples, do not produce inhibition zones, while tea polyphenols and rosemary extract, which originally have no outstanding preservative effects on pork, can produce relatively large inhibition zones. The inhibitory effects of the 11 preservatives on the ten spoilage bacterial communities are not consistent. Only 5 preservatives, namely ɛ-PL, Nisin, SL, tea polyphenols, and rosemary extract, show inhibition zones. ɛ-PL, tea polyphenols, and rosemary extract generally have good inhibitory effects, but tea polyphenols and rosemary extract have strong dyeing ability and are not suitable for actual application scenarios that require controlling the color of meat products. Although the inhibitory effects of Nisin and SL are weak, the diameters of their inhibition zones basically meet the requirements of the inhibitory effect.

[0070] From the inhibitory effects reflected by the two carriers of Oxford cups and drug sensitivity test paper discs, it can be seen that the inhibitory effect of Oxford cups is more ideal, and the diameter of the inhibition zone is generally above 10 mm. This may be related to the high diffusion rate of the preservative in the solution and the rapid contact with the bacteria. In contrast, as a dry carrier, the drug sensitivity test paper disc gradually releases the preservative through a slow-release effect, and the formation of the inhibition zone is more dependent on the local concentration accumulation of the preservative.

[0071] The inhibitory effects of the preservatives measured under the wet and dry methods provide a preliminary reference for the actual application forms of the subsequent preservatives (such as directly adding them to meat products or doping them into packaging materials). By comparing the two carriers, the diameter of the inhibition zone of ε-PL for most strains in both carriers exceeds 14 mm, indicating its strong application adaptability to wet and dry environments. The high inhibitory effect of Oxford cups suggests that liquid preservatives (such as directly adding them to meat products) can quickly inhibit the growth of microorganisms and are suitable for short-term preservation needs. The slow-release characteristics of the drug sensitivity test paper disc are more suitable for the application scenarios of packaging materials or surface coatings, and the antibacterial effect can be extended by continuously releasing the preservative. SL performs excellently in some cases in Oxford cups, and the diameter of the inhibition zone can reach up to 20 mm, but its effect is weak in the drug sensitivity test paper disc, indicating that it may be more suitable for direct liquid addition. Nisin is highly effective against Staphylococcus aureus in both carriers, and the diameter of the inhibition zone of the drug sensitivity test paper disc is 12 mm - 13 mm, indicating its potential for both immediate and slow release.

[0072] Example Two Determination of the microbial composition of cooked meat products Place the spoiled meat pieces in a laminar flow hood and cut 10 different spoiled parts with a sterile scalpel ( Figure 2 ), named S_1 to S_10, and perform high-throughput sequencing analysis using the 16S rDNA sequencing method and the 16S rRNA sequencing method. Analyze the microbial community composition of cooked meat products stored at room temperature according to the test results and screen the main spoilage bacteria.

[0073] The relative abundances of the microbial communities in the boiled pork spoilage samples at the species level Figure 3 showed that there were abundant microbial species in the samples. The results of the community composition analysis indicated that the top five dominant species in the bacterial communities in S_1 to S_10 accounted for a relative proportion of more than 60% of the total sequences Figure 3 A). Among them, the dominant species were Staphylococcus aureus (32.76 %), Staphylococcus epidermidis (23.70 %), and Escherichia coli (8.63 %), all of which belong to known spoilage and pathogenic bacteria and may play a leading role in the spoilage of boiled pork. Other spoilage bacteria that may play a certain role in the spoilage process of boiled pork, such as Listeria monocytogenes, Bacillus subtilis, and Salmonella enteritidis, were also detected, but their abundances were relatively low and they belonged to secondary spoilage bacteria. Similarly, among the samples numbered 1 to 4 with fungi in S_1 to S_10, the main spoilage bacteria were Aspergillus versicolor (26.19 %), Aspergillus flavus (13.23 %), and Rhodotorula glutinis (10.50 %) with the top three abundance ratios Figure 3 B).

[0074] Based on the results of the microbial community analysis of boiled pork, in this example, Staphylococcus aureus, Escherichia coli, and Aspergillus flavus were selected as representative indicator strains to conduct subsequent antibacterial experiments on preservatives. Among them, both Staphylococcus aureus and Escherichia coli were among the top three bacteria in abundance in the bacterial samples. In addition to their dual attributes of "pathogenicity + spoilage" worthy of key prevention and control, they also represent the typical cell wall structure differences between Gram-negative bacteria and Gram-positive bacteria, and can systematically evaluate the broad-spectrum antibacterial effect of preservatives on bacteria. Aspergillus flavus has a stronger food safety risk compared to Aspergillus versicolor with the same high abundance. The aflatoxin B1 secreted by it belongs to class I carcinogens and is a fungus that must be preferentially blocked in food microbial control. In addition, the three strains respectively correspond to three spoilage pathways: protein spoilage (staphylococcal protease), carbohydrate metabolism (Escherichia coli organic acid), and lipid oxidation (Aspergillus flavus lipase), and can comprehensively evaluate the dual blocking ability of preservatives against bacterial spoilage and fungal spoilage.

[0075] Example 3 Determination of the addition amount of a single preservative The optimal addition amount of a single preservative was determined by a single-factor antibacterial rate test of the preservative dosage.

[0076] The single-factor bacteriostatic rate test method for the dosage of the preservative is as follows: Using Staphylococcus aureus, Escherichia coli, and Aspergillus flavus, which need to be controlled and cause spoilage in cooked pork, as representative indicator strains, the inhibitory effects of the dosages of three preservatives, ε-PL, Nisin, and SL, on the three strains were studied, and the concentration at which each preservative achieves the best bacteriostatic effect when used alone was investigated. The investigation range of the dosage of ε-PL is 0.144, 0.192, 0.24, 0.28, 0.336, the investigation range of the dosage of Nisin is 0.06, 0.08, 0.1, 0.12, 0.14, and the investigation range of the dosage of SL is 10.0, 15.0, 20.0, 25.0, 30.0, with the unit of g / kg for all 熟肉制品 。

[0077] On the plate with the preservative added, divide the zones of each gradient concentration of the bacterial solution. The plate colony counting is carried out by the ten-microliter spotting method. Three parallels and two replicates are made for each bacterial solution concentration, and a blank control is set. Staphylococcus aureus and Escherichia coli are examined for bacteria after culturing at 37 °C for 10 h, and Aspergillus flavus is examined for bacteria after culturing at 37 °C for 36 h. The colony counting is carried out using the image processing software Jingdian Camera, and the bacteriostatic rate of each preservative at each dosage is calculated by comparing with the total number of colonies in the control group (the calculation formula is shown below).

[0078] The experimental results are shown in Table 1. The upper limit value of ε-PL stipulated in the national standard is 0.3 g·kg -1 , and in this example, the influence of ε-PL on the bacteriostatic rate of the three strains was investigated within the dosage range of 0.144 - 0.300 g·kg- 1 . The results show that ε-PL shows significant bacteriostatic effects on the three strains, and as its dosage increases, the bacteriostatic rate gradually increases. When the dosage reaches 0.280 g·kg -1 , the bacteriostatic rates of ε-PL against Staphylococcus aureus and Escherichia coli are both 100%, and the bacteriostatic rate against Aspergillus flavus is 90.0%.

[0079] Table 1. Influence of the dosage of ε-PL on the bacteriostatic rate As shown in Table 2, the bacteriostatic effect of Nisin shows significant strain differences. Within the safe dosage range of 0.06 - 0.50 g·kg -1 , its bacteriostatic rate against Staphylococcus aureus reaches a strong bacteriostatic effect of 98.4% at the lowest dosage of 0.06 g·kg -1 , but it has no inhibition on Escherichia coli. The bacteriostatic rate of Nisin against Aspergillus flavus increases with the increase of the dosage. When the dosage of Nisin reaches the national standard upper limit of 0.50 g·kg -1When it reaches, the antibacterial rate reaches 90.3%.

[0080] Table 2. Effect of Nisin dosage on antibacterial rate As shown in Table 3, SL is required to be added in an appropriate amount in the national standard. However, it is reported that when the addition amount of SL reaches 30 g·kg -1 it will cause the taste of meat products to be salty, with an obvious bitter taste, and even the texture to be loose. It can be seen from Table 3 that the antibacterial effect of SL is similar to that of ε-PL, showing a dose-dependence. When its dosage is 25.0 g·kg -1 the antibacterial rate against 3 strains can reach a strong antibacterial effect (>90%). Even at a lower dose of 15.0 g·kg -1 the antibacterial rates of SL against Staphylococcus aureus and Escherichia coli can reach more than 90%, but the antibacterial efficacy of SL against Aspergillus flavus is relatively weak compared to bacteria.

[0081] Table 3. Effect of SL dosage on antibacterial rate To sum up, under the dual considerations of the minimum effective dose and regulatory compliance, the optimal dosages of ε-PL, Nisin, and SL when used alone are 0.28 g·kg -1 , 0.5 g·kg -1 and 25.0 g·kg -1 . However, it should be noted that there are certain deficiencies when the above-mentioned preservatives are used alone. It is difficult for a single preservative to completely cover the problem of complex spoilage bacteria flora, and thus it is difficult to meet the preservation requirements for typical cooked meat products such as boiled meat at room temperature.

[0082] Example 4 Optimization of the compound preservative ratio The quality evaluation of meat products includes subjective evaluation and objective evaluation. In this example, the two quality evaluation methods are combined to simultaneously measure sensory indicators, physical and chemical indicators, and microbial indicators, which can more comprehensively reflect the preservation effect of the compound preservative on cooked meat products. Among them, the sensory indicators mainly include color, appearance, taste, color, flavor, and tissue state; the physical and chemical indicators include thiobarbituric acid value (TBA) and volatile basic nitrogen value (TVB-N); the microbial indicator is represented by the total number of colonies.

[0083] The experiment of this example was based on single-factor experiments, with boiled meat as the research object. The environmental temperature was controlled at 20 °C ± 2 °C, and the humidity was 45 % ± 5 %. According to the experimental design principle of Box-Behnken response surface method, taking ε-PL, Nisin, and SL as three independent variables, and the total number of colonies, TVB-N, TBA, and sensory evaluation as response indicators, the dosage ratio of preservatives in the composite preservative was optimized (Table 4). ε-PL and Nisin are both preservatives in terms of the functions of food additives. The Food Additive Use Standard GB2760-2014 stipulates that when food additives with the same function are used in combination, the sum of the proportions of their respective dosages to their maximum usage amounts should not exceed 1. Therefore, in the development of the composite preservative, the dosages of the two were appropriately reduced according to the single-factor experimental results and national standard requirements, that is: the highest dosage (level 1) of ε-PL was set at 0.23 g·kg -1 , and the highest dosage (level 1) of Nisin was set at 0.115 g·kg -1 . Since SL can be added in an appropriate amount in the national standard, in this example, SL was set to mainly play a role in maintaining the color of the appearance of meat products. Therefore, the effect of its dosage below 5.0 g·kg -1 in the composite preservative was investigated. The specific experimental factor levels and coding are shown in Table 5, and the index results were measured on the 3rd day.

[0084] Table 4. Response surface experiment factors and levels Table 5. Response surface test design and experimental coding The total number of colonies was determined according to the national standard method. Weigh 10 g of cooked meat samples and place them in a sterile homogenization cup containing 90 mL of sterile normal saline, homogenize at 8000 r / min for 2 min to make a 1:10 sample homogenate. Use a 1 mL sterile pipette to suck 1 mL of the 1:10 sample homogenate, slowly inject it along the tube wall into a sterile test tube containing 9 mL of sterile normal saline, and mix well on a shaker to make a 1:100 sample homogenate. Prepare 10-fold serial dilution sample homogenates in this way until 1:10 6 . According to the estimation of the sample contamination situation, select at least 3 appropriate dilution degrees of sample homogenates, suck 1 mL of sample homogenate into a sterile petri dish, and make two petri dishes for each dilution degree. At the same time, suck 1 mL of sterile normal saline as a blank control respectively. Immediately pour 15 mL of plate count agar medium cooled to 50 °C into each petri dish and rotate the petri dish to mix evenly. Place it horizontally and wait for the agar to solidify, then turn the plate over and culture it at 36 °C ± 1 °C for 48 h ± 2 h. After the culture is completed, perform colony counting and record the dilution multiple and the corresponding colony number.

[0085] Total Volatile Base Nitrogen (TVB-N) can be used to represent the degradation results of microorganisms on protein and non-protein nitrogen-containing compounds, such as ammonia, monoethylamine, dimethylamine, and trimethylamine, etc., and is widely used to evaluate the freshness and degree of spoilage of meat. The metabolic activities of microorganisms will cause more nitrogen-containing substances to form in meat products, especially in the lean part. Among them, TVB-N refers to the total nitrogen content that can be distilled out together with water vapor under alkaline conditions in the water extract of meat products. In the experiment, the semi-micro Kjeldahl method in GB 5009.228-2016 was used and slightly modified to determine the TVB-N content. The lean part of the cooked meat was ground into minced meat. Weighed 10 g of minced meat, accurate to 0.001 g, placed it in a stoppered conical flask, accurately added 100.0 mL of water, used an oscillator to disperse the minced meat evenly in the water, filtered it after impregnating for 30 min, and reserved the filtrate for later use. Accurately pipetted 10.0 mL of the filtrate into the reaction chamber of the Kjeldahl apparatus and immediately tightened it. Then pumped 5 mL of 10 g / L magnesium oxide suspension into the reaction chamber. Added 10 mL of 20 g / L boric acid solution and 5 drops of mixed indicator solution (i.e., 1 part of 1 g / L methyl red ethanol solution and 5 parts of 1 g / L bromocresol green ethanol solution, mixed before use) into the receiving flask, and inserted the lower end of the condenser into the liquid level of the receiving flask. Turned on the circulating cooling water and started distillation. Distilled for 180 s or the distillation volume in the receiving flask reached 180 mL, whichever came first. Then moved the distillate receiving flask to make the lower end of the condenser higher than the liquid level and continued distillation for 10 s. Then rinsed the outside of the lower end of the condenser with a small amount of water and removed the distillate receiving flask. Titrated with 0.0100 mol / L hydrochloric acid standard solution until the end point color was purplish red. Conducted the experiment in parallel three times and did the reagent blank at the same time. The content of TVB-N in the sample was calculated according to the following formula: In the formula, X——The content of volatile base nitrogen in the sample (mg / 100mL); V1——The volume of the hydrochloric acid or sulfuric acid standard titration solution consumed by the test solution (mL); V2——The volume of the hydrochloric acid or sulfuric acid standard titration solution consumed by the reagent blank (mL); c——The concentration of the hydrochloric acid or sulfuric acid standard titration solution (mol / L); 14——The mass of nitrogen equivalent to titrating 1.0 mL of hydrochloric acid [c (HCl ) = 1.000 mol / L] standard solution (g / mol); m——The mass of the sample (g); V——The accurately pipetted volume of the filtrate (mL); V0——The total volume of the sample solution (mL); 100——The conversion factor for converting the calculation result to milligrams per 100 grams (mg / 100g).

[0086] The TBA value (Thiobarbituric acid value, TBA) increases with the accumulation of fatty hydroperoxides and secondary peroxide products and is an important indicator for evaluating lipid oxidation. A major cause of meat product spoilage is the oxidation of fats and oils. When fats and oils are exposed to light, heat, and oxygen in the air, they undergo rancidity reactions and decompose into compounds such as aldehydes and acids. These can react with 2-thiobarbituric acid to produce a red compound with a maximum absorption peak at 538 nm. This property is used to determine the aldehyde content and thus the degree of lipid rancidity in meat products. In the experiment, the TBA value is determined according to the method of GB / T 35252-2017. The fat part of the cooked meat is mixed with anhydrous sodium sulfate in a mass ratio of 10:1, vacuum dried at 50 °C for 1 h, and solidified in a -18 °C refrigerator. Take 50.0 mg of the solidified fat sample and place it in a 25 mL conical flask. Dissolve it with 1-butanol and dilute to the mark. Pipette 5 mL of the sample solution into a stoppered test tube. Using another pipette, pipette 5 mL of the TBA reagent into the stoppered test tube, cover the stopper, and shake well. Place the stoppered test tube in a 95 °C water bath and heat for 2 h. Then remove the stoppered test tube from the water bath and quickly cool it to room temperature. Transfer the solution in the stoppered test tube to a clean and dry 10 mm cuvette, and measure the absorbance (A) of the solution at 530 nm using distilled water as the reference. At the same time, use 1-butanol as the blank sample and measure the absorbance of the blank reagent (B, the B value should not be less than 0.05) according to the same method. The TBA value is calculated according to the following formula: In the formula, A —— The absorbance of the solution to be measured; B —— The absorbance of the blank reagent; m —— The mass of the sample (mg); 50 —— The effective factor for diluting 1-butanol to 25 mL and measuring the absorbance with a 10 mm cuvette.

[0087] The sensory evaluation indicators mainly include color, appearance, taste, color, flavor, and texture. Six experienced food professionals use Williams' 9-point scoring method to score respectively: 9 is excellent, 8 is very good, 7 is good, 6 is fairly good, 5 is average, 4 is below average, 3 is poor, 2 is very poor, and 1 is extremely poor. A score of ≥5 is acceptable (Table 6). That is, take an appropriate amount of the test sample and place it on a clean white porcelain plate. Observe the color and state under natural light, smell its odor, taste its flavor, and give a score, etc.

[0088] Table 6. Standards for sensory evaluation Using Design-Expert 13 software, data processing was carried out through a multi-objective response surface model to obtain the optimization equations for each index and the comprehensive index, and then the optimal ratios of the three composite preservatives were optimized and determined. The core of the Box-Behnken response surface design lies in comprehensively evaluating the multi-factor interaction and non-linear response relationship through a limited number of experimental combinations, which is particularly suitable for the collaborative optimization of multi-index interactions.

[0089] First, based on the determination of each quality evaluation index on the 3rd day of the storage of the boiled meat, the analysis results are shown in Table 7. Then, a quadratic polynomial regression equation was established, and the significance of the model and the lack-of-fit term were verified through analysis of variance (ANOVA). Finally, response surfaces were plotted to explain the interaction between the preservatives. This analytical ability of multi-factor interactions provides a key theoretical basis for subsequent balancing the contradiction between antibacterial and antioxidant effects, and also points out the direction for the optimization of sensory quality.

[0090] Table 7. Results of response surface analysis Effect of the composite preservative on the total number of colonies in cooked meat products As can be seen from Table 8, the regression model of the composite preservative selected in this example is highly significant overall (P < 0.0001). The F value of the model is 50.87, and the lack-of-fit term is 1.97, indicating that the model has reached an extremely significant level and has a good fitting degree. Its corrected determination coefficient is 0.9849, indicating that this model can explain 98.49% of the variation in the response value. From the P value and the F value, it can be seen that the three factors of ε-PL, Nisin, and SL have extremely significant effects on the total number of colonies (P < 0.01). The main effect of ε-PL is the most significant, followed by Nisin, and finally SL. The interaction between ε-PL and Nisin is significant (P < 0.05), indicating that the two have a synergistic effect.

[0091] Table 8. Analysis of variance results of the total number of colonies regression model R 2 = 0.9849, R 2 Adj = 0.9656 The optimization equation for the preservation effect of the boiled meat with different ratios of different preservatives was obtained by the stepwise regression method: Y1(lg(CFU / g)) = 5.16 - 0.13×A - 0.10×B - 0.048×C - 0.055×A×B - 0.005×A×C + 0.015×B×C + 0.12×A 2 + 0.17×B2 +0.26×C 2 (where Y1 is the total number of colony values, A is the content of ε-PL, B is the content of Nisin, and C is the content of SL).

[0092] According to this optimization equation, the influence of each preservative as a linear term on the total number of colonies Y1: the coefficient of the ε-PL term is -0.13, indicating that for every 1 unit increase in ε-PL, the total number of colonies decreases by 0.13; the coefficient of Nisin is -0.10, indicating that for every 1 unit increase in Nisin, the total number of colonies decreases by 0.10; the coefficient of SL is -0.048, indicating that for every 1 unit increase in SL, the total number of colonies decreases by 0.048; the coefficient of the interaction term (AB) of ε-PL and Nisin is -0.055, further proving that the two have a synergistic effect.

[0093] Using the regression model, the optimal ratio of the compound preservative is predicted to be: 0.213 g·kg -1 ε-PL, 0.103 g·kg - 1 Nisin and 3.51 g·kg -1 SL. At this time, the predicted theoretical value of the total number of colonies is lg(CFU / g)=5.105.

[0094] Effect of compound preservative on TVB-N value of cooked meat products The analysis of variance of the regression model of TVB-N value is shown in Table 9. The F value of this model is 60.66, indicating that the model is highly significant (P<0.0001), and the F value of the lack-of-fit term is 1.41, which means that the lack-of-fit term is not significant relative to the pure error (P=0.3628). This model has a good correlation, and the corrected determination coefficient R 2 is 0.9873, and the predicted value is R 2 Adj =0.9711. Within the selected factor range, the influence of ε-PL on TVB-N is very significant (P<0.0001), and the influences of Nisin and SL both reach a highly significant level (P<0.01). The interaction analysis shows that AB (ε-PL and Nisin) is significant (P<0.05). The synergistic inhibitory effect of ε-PL and Nisin has a significant concentration dependence. When the concentrations of the two are increased to 0.216 g·kg -1 and 0.109 g·kg -1 respectively, the synergistic antibacterial effect reaches the peak value, and at this time the TVB-N value is 14.395 mg / 100 g.

[0095] The fitting regression equation is: Y2(TVB-N) = 14.5 - 0.36×A - 0.28×B - 0.14×C - 0.17×A×B - 0.014×A×C + 0.045×B×C + 0.32×A 2 + 0.47×B 2 + 0.74×C 2 (where Y2 is the TVB-N value, A is the ε-PL content, B is the Nisin content, and C is the SL content).

[0096] Table 9. ANOVA Results of the TVB-N Regression Model R 2 = 0.9873, R 2 Adj = 0.9711 The results show that the inhibitory effects of ε-PL and Nisin first increase and then decay with the increase in concentration, that is, a non-linear effect, which is related to the positive coefficients of A² and B² in the regression model. Considering the comprehensive preservation effect and economy, a combination of high-concentration ε-PL and Nisin is preferred, and the dosage of SL is appropriately reduced to achieve optimal control. The optimized ratio of the preservative obtained by the response surface method is: 0.216 g·kg -1 ε-PL, 0.109 g·kg -1 Nisin, 3.19 g·kg -1 SL. At this time, the theoretical value of TVB-N reaches 14.395 mg / 100 g.

[0097] Effect of the Composite Preservative on the TBA Value of Cooked Meat Products By analyzing the variance of the TBA value of the poached meat with the composite preservative (Table 10), the results show that the overall model is highly significant (P < 0.0001) and there is no lack-of-fit problem. The optimized equation for controlling the TBA value of the poached meat with different ratios of the preservative: Y3(TBA) = 24.0 - 0.60×A - 0.48×B - 0.22×C - 0.26×A×B - 0.023×A×C + 0.07×B×C + 0.55×A 2 + 0.78×B 2 + 1.2×C 2 (where Y3 is the TBA value, A is the ε-PL content, B is the Nisin content, and C is the SL content). All three of ε-PL, Nisin, and SL have a highly significant effect on the TBA value (P < 0.01), and the pairwise interactions of ε-PL, Nisin, and SL also show a similar situation to the effects on the total number of colonies and the TVB-N value, that is, the AB effect is significant (P < 0.05).

[0098] Table 10. ANOVA Results of TBA Regression Model R 2 = 0.9833, R 2 Adj = 0.9618 When the concentrations of ε-PL and Nisin increase simultaneously, the TBA value decreases significantly, indicating that their synergistic effect has a greater impact on inhibiting the TBA value, and the synergistic inhibitory effect is the best at high concentrations. The optimal ratio of the three preservatives optimized by the response surface method is: 0.214 g·kg -1 ε-PL, 0.104 g·kg -1 Nisin, 3.17 g·kg -1 SL. The predicted value of the TBA value at this time is 2.378 mg / 100 g.

[0099] Effect of Compound Preservative on Sensory Quality of Cooked Meat Products According to the ANOVA results of the sensory score response surface model (Table 11), the regression model as a whole has a high significance (F = 41.71, P < 0.0001), indicating that the model can effectively explain the changes in sensory scores. The coefficient of determination for model calibration R² = 0.9817, R 2 Adj = 0.9582, further verifying the good fitting ability of the model to the data. From the main effect analysis, the contents of ε-PL (A), Nisin (B), and SL (C) all have significant effects on the sensory score (P < 0.05). However, different from the first three evaluation indicators, the F value of the SL content is as high as 93.63 (P < 0.0001), indicating that its contribution to the score is the most prominent. Among the interaction effects, the AC interaction term is significant (P = 0.0213), indicating that the synergistic effect between ε-PL and SL plays a key role in optimizing the sensory score.

[0100] Table 11. ANOVA Results of Sensory Evaluation Regression Model R 2 = 0.9817, R 2 Adj = 0.9582 The optimization equation of the sensory score of poached meat with different ratios of different preservatives was obtained by the stepwise regression method: Y4 (sensory score) = 9.1 + 0.13×A + 0.18×B + 0.43×C + 0.048×A×B + 0.19×A×C + 0.058×B×C - 0.28×A 2 - 0.60×B 2 - 0.63×C 2(where Y4 is the sensory score value, A is the ε-PL content, B is the Nisin content, and C is the SL content). The quadratic coefficients of the model are all negative values, indicating that there is a peak effect on the sensory score for each factor, that is, excessive addition will cause the sensory score to decline.

[0101] The sensory score increases with the increase of the variable within a certain range, but when the variable exceeds a certain critical value (ε-PL 0.205 g·kg -1 , Nisin 0.099 g·kg -1 , SL 3.61 g·kg -1 ), the sensory score begins to decline. The positive coefficient of AC (0.19) indicates that the synergistic effect of ε-PL and SL has a positive improvement on the sensory score, while the positive coefficient of BC (0.058) shows that the synergistic effect of Nisin and SL also has a certain promoting effect on the sensory score. The optimal ratio of the composite preservative obtained by optimizing through Box-Behnken design is: 0.205 g·kg -1 ε-PL, 0.099 g·kg -1 Nisin, 3.61 g·kg -1 SL. At this time, the predicted value of the sensory score is 9.203.

[0102] Example 5 Optimizing the ratio of the composite preservative with multiple indicators According to the multi-index optimization of three factors, three levels, and four responses processed by Design-Expert 13, the optimal ratio of the composite preservative was obtained, that is, when the total number of colonies, TVB-N value, and TBA value meet the minimum expected value, and the sensory score meets the maximum expectation, the optimal ratio group content of ε-PL, Nisin, and SL predicted. That is, ε-PL is 0.213 g·kg -1 , Nisin is 0.101 g·kg -1 , SL is 3.379 g·kg -1 . At this time, the total number of colonies lg(CFU / g), TVB-N value (mg / 100g), and TBA value (mg / 100g) are 5.097, 14.530, and 0.238 respectively, and the sensory score is 9.142 ( Figure 4 ).

[0103] The best prediction groups optimized for each index obtained according to the above examples and the comprehensive optimal prediction group obtained after index optimization in this example were respectively subjected to verification tests, and the test results are shown in Table 12.

[0104] Table 12. Comparison between the response optimization value and the true value R 2 =97.84 The degree of coincidence between the single-index predicted value and the measured value is extremely high, and the difference ≤ 0.055. The difference degree of 0.452 between the multi-index predicted value and the measured value is greater than the difference between the single-index predicted value and the measured value, but there is still a high correlation. Under the optimized conditions, the correlation between the predicted value and the measured value is 97.84. Thus, it can be seen that the optimized model can be used to predict and control the preservation of actual cooked meat products, and the model has reliability and practicability.

[0105] According to the experimental results, after optimizing and verifying each preservative, it can be concluded that when the addition amount of ε-PL is in the range of 0.205 g·kg -1 -0.216 g·kg -1 , the addition amount of Nisin is in the range of 0.099 g·kg -1 -0.109 g·kg -1 , and the addition amount of SL is in the range of 3.174 g·kg -1 -3.605 g·kg -1 , the synergistic effect of the three can not only effectively control the microbial reproduction in cooked meat products, but also slow down the processes of protein decomposition and fat oxidation, and at the same time can maintain good sensory quality to extend the shelf life.

[0106] Example Six Through judgment criteria such as total bacterial count, TVB-N value, TBA value, and sensory evaluation, this example investigated the influence of the addition of the compound preservative of the optimal ratio group obtained in Example Five on the shelf life of cooked meat products such as white-cut chicken, marinated beef, and marinated goose. Among them, white-cut chicken is a typical cooked meat product of the white-boiled type, without added seasonings, with high water content, and microorganisms are easy to breed, and it is extremely perishable at room temperature; marinated beef, as a common marinated and braised cooked meat product, is rich in protein and water. Although the sauce may cover up the signs of early spoilage, it is still easy to deteriorate at room temperature; for the marinated cooked meat product, marinated goose is selected. Marinated goose uses fermented grains to give a unique flavor. Although the fermented grains itself have a certain antibacterial effect, its preservation at room temperature is still limited. Marinated goose represents traditional process products, and white-cut chicken and marinated beef have a large market demand and an urgent need for preservation. These actual samples help to verify the effect of the compound preservative provided by the present invention in the complex matrix of cooked meat products with high protein and high water content, and further verify the expansion effect of the compound preservative provided by the present invention on its room-temperature circulation scenario.

[0107] In this embodiment, the application method of the composite preservative includes: calculating the specific application amounts of ε-PL, Nisin, and SL in the composite preservative according to the masses of the samples of sliced cold chicken, marinated beef, and marinated goose, and uniformly mixing ε-PL, Nisin, and SL. Since there is a certain amount of moisture on the surfaces of sliced cold chicken, marinated beef, and marinated goose themselves, in this embodiment, the composite preservative is uniformly applied to the surfaces of sliced cold chicken, marinated beef, and marinated goose, so that the composite preservative is applied in a liquid state. It should be understood that if the surface moisture of the cooked meat products is less or the moisture is insufficient to enable the composite preservative to be applied in a liquid state, the composite preservative can be applied by first uniformly applying an appropriate amount of solvent to the surface of the cooked meat products and then uniformly applying the composite preservative to the surface of the cooked meat products, or by first mixing the composite preservative with an appropriate amount of solvent to form a composite preservative solution and then uniformly applying the composite preservative solution to the surface of the cooked meat products. In addition, it should be understood that as long as the solvent can ensure that the composite preservative can be dissolved and does not affect the taste of the cooked meat products, the present invention places no limitation on the amount of the solvent.

[0108] Total number of colonies By measuring the total number of colonies of sliced cold chicken, marinated beef, and marinated goose, the preservation effect of the composite preservative at room temperature was evaluated. The results showed that the total number of colonies in each treatment group showed an upward trend with the extension of the storage time ( Figure 5 ). The microbial growth rates in the composite preservative groups were all significantly lower than those in the blank control group (P < 0.05), indicating that the composite preservative has a significant inhibitory effect on microbial growth.

[0109] After the sliced cold chicken was stored at room temperature for 2 days, the logarithm of the total number of colonies in the blank control group reached 5.43, exceeding the standard limit of the total number of colonies of 5.00 lg(CFU / g), while the logarithm of the total number of colonies in the preservative treatment group after 2 days was 4.57, indicating that the composite preservative can extend the shelf life of sliced cold chicken from 1.5 days to 2.5 days; similarly, the shelf life of marinated goose was also extended from 1 day to 2.5 days. The preservation effect of marinated beef was more significant. The preservative group could extend the shelf life by 1.5 days compared with the blank group, and the shelf life reached 4.5 days.

[0110] Figure 6Data of group A showed that the TVB-Nz value of sliced boiled chicken presented a significant storage temperature-time dependence (P<0.05). After 2 days of storage, the TVB-N value in the blank control group increased from the initial 8.805 mg / 100 g to 15.228 mg / 100 g, with an increase of 72.9%, exceeding the limit of first-class fresh meat of 15.000 mg / 100 g. After 2 days of storage in the preservative-treated group, the TVB-N value only increased to 14.778 mg / 100 g, with an increase of about 67.8%. This might be due to the fact that the composite preservative reduced the activation energy of chemical reactions and effectively delayed the spoilage reaction process such as protein decomposition. Currently, there is no clear standard for the spoilage limit of TVB-N value. According to industry experience, it is acceptable when not exceeding 20 mg / 100 g. The experimental results showed that the use of the composite preservative with an optimized formula could extend the shelf life of sliced boiled chicken to about 3.5 days, providing a longer time guarantee for the quality control of boiled meat products.

[0111] The difference between groups in the braised beef group was very small in the first 1 day ( Figure 6 B). After 1 day, the increase in the TVB-N value in the blank group compared with the initial stage of storage was as high as 66.6%, while the TVB-N value in the preservative group was only 9.393 mg / 100 mg, with an increase of 22.6%. This indicated that the composite preservative formula had a significant control effect on microorganisms in the early stage of ambient temperature storage and could extend the shelf life of braised beef from 3 days to 5 days.

[0112] The change in the TVB-N value of the marinated goose group was between that of the sliced boiled chicken and the braised beef ( Figure 6 C). The TVB-N value in the blank group exceeded the standard limit after 2.5 days of storage, while the preservative-treated group remained within the acceptable range within 3.5 days. This showed that the antibacterial effect of the distiller's grains was limited, and the addition of the composite preservative significantly enhanced the antibacterial effect. The composite preservative effectively inhibited the growth of microorganisms and protein decomposition, extending the ambient temperature shelf life of sliced boiled chicken, braised beef, and marinated goose by 1 day, 2 days, and 1.5 days, respectively.

[0113] By measuring the TBA value of sliced boiled chicken, its degree of fat oxidation was evaluated. Regarding the limit of the TBA value, there is no clear regulation at home and abroad yet. The index value can refer to the limit of malondialdehyde (MDA) included in the TBA value, that is, the EU standard stipulates that foods with a safe shelf life do not exceed 0.2 mg / 100 g, and fresh foods do not exceed 0.1 mg / 100 g. Figure 7The results showed that the TBA values of both the preservative group and the blank control group gradually increased with the storage time. The TBA value of the preservative group exceeded 0.2 mg / 100 g at 3 d of storage, while that of the blank control group exceeded the standard as early as 2 d. This indicated that the composite preservative treatment had a significant inhibitory effect on the lipid oxidation of sliced cooked chicken, and there were certain differences between the preservative treatment group and the blank control group. Under normal temperature storage conditions, this composite preservative could extend the shelf life of sliced cooked chicken from 2 d to 3 d.

[0114] The sensory qualities of sliced cooked chicken treated with the composite preservative are as Figure 8 shown. Different additions of preservatives led to different degrees and rates of sensory quality decline. After 1 d of storage, the sensory quality of the sliced cooked chicken without any treatment declined the fastest, followed by marinated goose, and finally marinated beef. Before 3 d of storage, the samples of sliced cooked chicken and marinated goose had become spoiled meat and were judged inedible from aspects such as smell and color. However, each group of cooked meat samples in the preservative treatment group maintained good sensory quality (score ≥ 5) within 3 d, and their color, texture, and flavor were better than those of the control group. This indicated that from the perspective of sensory quality, the composite preservative could extend the shelf life of poached meat represented by sliced cooked chicken by at least 1 d, and extend the shelf life of marinated meat products such as marinated beef and marinated meat products such as marinated goose by at least 1.5 d.

[0115] In summary, using the optimized composite preservative could effectively extend the shelf lives of sliced cooked chicken, marinated beef, and marinated goose by 1 d, 2 d, and 1.5 d, respectively.

[0116] To more intuitively reflect the effectiveness of the preservative, the test results of the control group (n = 3) stored at 4℃ are shown in Figure 9 . The results showed that the shelf life of the preservative group was much longer than that of the blank control group without adding preservatives, and it could extend the shelf life by up to 6 d, and was longer than the shelf life under normal temperature storage conditions, with the maximum extension of 10 d.

[0117] Example 7 In this example, an HPLC detection method for ε-PL and Nisin with easy operation, high recovery rate, high precision, and low detection limit was established. This not only improved the safety control of Nisin and ε-PL in food, but also was used for the determination of the residual amounts of ε-PL and Nisin in poached meat preserved with the composite preservative.

[0118] Sample pretreatment: Accurately weigh a certain amount of the poached meat minced after preservation, mix it evenly with an appropriate amount of Wahaha brand pure water, transfer it into a 50 mL centrifuge tube, centrifuge at 8000 r / min for 20 min, and take the supernatant for filtration through a 0.45 μm filter membrane for standby.

[0119] Preparation of ε-PL and Nisin standard solutions: Accurately weigh 264.7 mg of Nisin standard product, dilute it with pure water to prepare Nisin standard series solutions with concentrations of 0.50 mg / mL - 2.00 mg / mL. After filtering through a 0.45 μm filter membrane, perform detections in ascending order of concentration to obtain the standard curve of the chromatographic peak area versus concentration. According to the same method, weigh 500.0 mg of ε-PL, prepare ε-PL standard series solutions with concentrations of 0.05 mg / mL - 2.00 mg / mL, and perform determinations using HPLC in sequence to establish a linear standard curve.

[0120] Chromatographic conditions: Use a C18 chromatographic column (4.6 mm × 250 mm, 5 μm) as the separation column, with an injection volume of 10 μL. Set the flow rate to 0.5 mL / min for the determination of Nisin and 1.0 mL / min for the determination of ε-PL. The detection wavelengths for the two are set to 220 nm and 210 nm respectively. For Nisin, use acetonitrile (99.9%) + trifluoroacetic acid (0.1%) as the mobile phase. For ε-PL, use phosphate buffer solution (98%) + acetonitrile (2%) as the mobile phase.

[0121] Sample determination: Inject the pretreated sample into a high-performance liquid chromatograph, measure the chromatographic peak area of the sample analyte, and calculate the contents of ε-PL and Nisin in the sample according to the linear equation of the standard curve.

[0122] Linear equation and detection limit: Prepare a series of standard solutions of ε-PL and Nisin (2.00, 1.50, 1.25, 1.0, 0.75, 0.50 mg / mL). After filtering through a 0.45 μm filter membrane, under suitable chromatographic separation conditions, perform separation and detection in an HPLC system. Use the peak areas ( y ) and the mass concentrations of the corresponding standard solutions ( x , mg / mL) of the two to plot the standard curve, fit the linear equation, and calculate the linear correlation coefficient (R 2 ); and calculate the instrument detection limit (LOD) and quantification limit (LOQ) respectively with 3 times and 10 times the signal-to-noise ratio. As shown in Table 13, within the concentration range of 0.50 - 2.00 mg / mL for the target analytes, there is a good linear relationship between the peak area and the mass concentration, and the linear correlation coefficients are all greater than 0.99. The LODs are 0.1495 mg / mL and 0.1313 mg / mL respectively.

[0123] Table 13. Linear ranges, linear equations, LODs, and RSDs of migration time and peak area for the detection of ε-PL and Nisin Precision: Respectively take the ε-PL and Nisin standard solutions of 0.50 mg / mL and measure them 5 times under the above chromatographic conditions. Calculate the RSD of the retention time and peak area. As can be seen from Table 13, the RSD of the retention time and peak area of the two do not exceed 2.98% and 3.13% respectively.

[0124] Accuracy: Select an appropriate amount of the water extract of boiled meat and conduct a standard addition recovery experiment at different standard addition concentrations (0.50, 1.00, 1.50 mg / mL). The results of 3 repeated experiments show that the average standard addition recovery rates of ε-PL and Nisin are both in the range of 96.62 - 104.05%, and the RSD value is lower than 3.24%. Detection of actual samples found that ε-PL and Nisin were not detected in the boiled meat. The results show that the method established in this example is accurate and reliable and can be used for the simultaneous separation and detection of ε-PL and Nisin in cooked meat products preserved with this composite preservative.

[0125] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims. All of these fall within the protection scope of the present invention.

Claims

1. A method for improving the freshness preservation of cooked meat products during storage at room temperature, characterized in that, The method includes: applying a predetermined dosage of a composite preservative to the cooked meat products according to the expected freshness preservation requirement; the composite preservative includes ε-polylysine salt, nisin and sodium lactate; Wherein, when the expected freshness preservation requirement is to comprehensively improve the freshness preservation of the cooked meat products during storage at room temperature, the predetermined dosage of nisin is 0.099 - 0.109 g / kg of cooked meat products, the predetermined dosage of ε-polylysine salt is 0.205 - 0.216 g / kg of cooked meat products, and the predetermined dosage of sodium lactate is 3.174 - 3.605 g / kg of cooked meat products.

2. The method according to claim 1, wherein The method further includes: obtaining the specific dosage of the composite preservative according to the freshness preservation index to be preferentially improved; wherein When the freshness preservation index to be given priority to improvement is the microbial index, the specific application amount of the composite freshness preservation agent is obtained according to the first model, and the first model includes: Y1 (lg (CFU / g)) = 5.16 - 0.13×A - 0.10×B - 0.048×C - 0.055×A×B - 0.005×A×C + 0.015×B×C + 0.12×A 2 + 0.17×B 2 + 0.26×C 2 , where Y1 is the total colony value, A is the application amount of ε-polylysine salt, B is the application amount of nisin, and C is the application amount of sodium lactate; When the freshness preservation index to be prioritized for improvement is the total volatile basic nitrogen index, the specific application amount of the composite freshness preservative is obtained according to the second model, and the second model includes: Y2(TVB-N)=14.5 - 0.36×A - 0.28×B - 0.14×C - 0.17×A×B - 0.014×A×C + 0.045×B×C + 0.32×A 2 + 0.47×B 2 + 0.74×C 2 , where Y2 is the TVB-N value, A is the application amount of ε-polylysine salt, B is the application amount of nisin, and C is the application amount of sodium lactate; When the freshness preservation index to be prioritized for improvement is the thiobarbituric acid index, the specific application amount of the composite freshness preservation agent is obtained according to the third model, and the third model includes: Y3(TBA)=24.0 - 0.60×A - 0.48×B - 0.22×C - 0.26×A×B - 0.023×A×C + 0.07×B×C + 0.55×A 2 + 0.78×B 2 + 1.2×C 2 , where Y3 is the TBA value, A is the application amount of ε-polylysine salt, B is the application amount of nisin, and C is the application amount of sodium lactate; When the freshness preservation index to be prioritized for improvement is the sensory index, the specific application amount of the composite freshness preservative is obtained according to the fourth model, and the fourth model includes: Y4 (sensory score) = 9.1 + 0.13×A + 0.18×B + 0.43×C + 0.048×A×B + 0.19×A×C + 0.058×B×C - 0.28×A 2 - 0.60×B 2 - 0.63×C 2 , where Y4 is the sensory score value, A is the application amount of ε - polylysine salt, B is the application amount of nisin, and C is the application amount of sodium lactate.

3. The method according to claim 1, wherein The application methods of the composite preservative include: uniformly applying the composite preservative to the surface of the cooked meat products, soaking the cooked meat products in a solution formed by the composite preservative, and packaging the cooked meat products with a carrier containing the composite preservative, one or more of them.

4. The method according to claim 1, wherein The predetermined dosage of nisin is 0.101 g / kg of cooked meat products, the predetermined dosage of ε-polylysine salt is 0.213 g / kg of cooked meat products, and the predetermined dosage of sodium lactate is 3.379 g / kg of cooked meat products.

5. A composite preservative, characterized in that, The composite preservative includes a preservative composed of the following weight parts: 1 part of nisin, 1.9 - 2.2 parts of ε-polylysine salt, and 29.1 - 36.4 parts of sodium lactate.

6. The composite preservative according to claim 5, characterized in that, A preservative composed of the following weight parts: 1 part of nisin, 2.1 parts of ε-polylysine salt, and 33.6 parts of sodium lactate.

7. Use of the composite preservative according to claim 5 or 6 in the preparation of a kit for improving the freshness preservation of cooked meat products during storage at room temperature.

8. The use according to claim 7, characterized in that, The composite preservative is configured for short-term freshness preservation of cooked meat products, wherein the composite preservative includes nisin in an amount of 0.099 - 0.109 g / kg of cooked meat products, ε-polylysine salt in an amount of 0.205 - 0.216 g / kg of cooked meat products, and sodium lactate in an amount of 3.174 - 3.605 g / kg of cooked meat products.

9. The use according to claim 8, characterized in that, The composite preservative includes nisin in an amount of 0.103 g / kg of cooked meat products, ε-polylysine salt in an amount of 0.213 g / kg of cooked meat products, and sodium lactate in an amount of 3.510 g / kg of cooked meat products.

10. The use according to claim 7, characterized in that, The use includes extending the shelf life of the cooked meat products by at least 1 day.

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