Low-salt pickling agent for inhibiting growth of microorganisms generating putrefying peculiar smell in ham as well as method and application of low-salt pickling agent

Abstract

The invention relates to the technical field of food processing, in particular to a low-salt pickling agent for inhibiting growth of microorganisms generating putrefying peculiar smell in ham and a method and application of the low-salt pickling agent, and the low-salt pickling agent comprises table salt, Nisin, epsilon-PL and chitosan. The compound preservative is prepared from Nisin, epsilon-PL and chitosan, and the compound preservative is added into a dry-cured meat block system for fermentation, so that the formula can completely inhibit the growth of putrefying bacteria, and meets the national regulations on the compound preservative. Due to the natural source and low toxicity of the additive, the additive is safer when being used in food, the requirements of consumers on natural and safe food are met, and the synergistic effect of the three can also play an effective antibacterial role at low concentration. The low-dosage use is beneficial to reducing the influence on the sensory quality (such as taste and color) of food, the natural flavor and taste of the food are kept, and the effect of prolonging the shelf life of the food is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and particularly relates to a low-salt curing agent for inhibiting the growth of microorganisms that produce putrid odors in ham, and a method and application thereof. Background Art

[0002] With the continuous improvement of people's requirements for food safety and quality, how to extend the shelf life of traditional foods has become a research hotspot. As a traditional Chinese cured meat product with a unique flavor, Xuanwei ham has a high salt and low water content, which although inhibits the growth of microorganisms to a certain extent, still has a risk of being contaminated by spoilage microorganisms. The growth and reproduction of spoilage microorganisms not only affect the quality of ham, but may also pose a threat to the health of consumers. Therefore, it is particularly important to find effective preservatives to inhibit the growth of these microorganisms.

[0003] However, there are mainly the following problems in the current ham processing:

[0004] (1) The traditional ham curing process usually relies on high salt to inhibit the growth of microorganisms, but excessive salt intake will increase the risk of diseases such as hypertension, cardiovascular diseases and kidneys. Therefore, the current market demand for ham products with low salt content or moderate salt content has increased, but the traditional process is difficult to meet these demands.

[0005] (2) Currently, most of the applications of preservatives are for fish and shrimp with a water content as high as 60%-80%, which allows the preservatives to easily diffuse and penetrate into the tissues, enabling the preservatives to quickly penetrate into the fish and shrimp tissues, evenly distribute and play an antibacterial role. However, the water content of ham is less than 50%, resulting in limited diffusion and penetration ability of the preservatives in ham, and thus the antiseptic effect is weakened. Compared with fish and shrimp, ham has a higher fat content, lower water activity, and a relatively dense protein structure. This greatly reduces the penetration ability of the preservatives in ham, resulting in a weakened efficacy of the preservatives. Therefore, directly applying the preservative ratio of fish and shrimp to ham may not achieve the desired antiseptic effect.

[0006] (3) In terms of microorganisms, ham contains a relatively high salt content. Although this environment inhibits the growth of some microorganisms, it also provides suitable growth conditions for certain salt-tolerant bacteria such as lactic acid bacteria, Staphylococcus aureus, and some molds. These microorganisms can cause the spoilage of ham, leading to a decline in quality. And the long-term use of the same preservative may cause microorganisms to develop drug resistance, reduce the effectiveness of the preservative, and increase the risks of food spoilage and safety. Most of the preservatives for fish and shrimp mainly inhibit Gram-negative bacteria and some molds. These microorganisms grow rapidly in a high-moisture environment. Fish meat preservatives may not be able to effectively inhibit the growth of these salt-tolerant and acid-tolerant microorganisms because these microorganisms usually have stronger tolerance to common preservatives. Therefore, it is necessary to develop a compound preservative that can improve the inhibitory ability against salt-tolerant microorganisms and reduce the risk of microbial drug resistance through the synergistic effect of multiple preservatives, thereby enhancing the safety and stability of ham products.

[0007] Some microorganisms and their metabolites have shown the characteristics of inhibiting the growth and reproduction of spoilage microorganisms in nature, and possess the advantages of being safe, non-toxic, natural, and highly efficient, and can be used as preservatives for meat products. ε-PL has a wide range of antibacterial properties and has a certain astringent taste at high concentrations, which may bring an adverse sensory experience. Nisin has a strong inhibitory effect on Gram-positive bacteria, especially Bacillus, but its antibacterial range is relatively narrow. Chitosan and its derivatives exhibit extremely high antibacterial activity and can effectively inhibit the growth and reproduction of bacteria, viruses, and fungi. Therefore, the use of compound preservatives is of great significance for reducing the spoilage of Xuanwei ham. Summary of the Invention

[0008] The purpose of the present invention is to provide a low-salt pickling agent and its method and application for inhibiting the growth of microorganisms that produce spoilage odors in ham, effectively inhibiting the growth and reproduction of spoilage microorganisms in Xuanwei ham, and laying a foundation for the future application of compound preservatives in actual Xuanwei ham.

[0009] To achieve the above technical purposes and reach the above technical effects, the present invention is realized through the following technical solutions:

[0010] A low-salt pickling agent for inhibiting the growth of microorganisms that produce spoilage odors in ham, including but not limited to table salt, Nisin, ε-PL, and chitosan.

[0011] Furthermore, the table salt is 70 g / kg, Nisin is 0.2 mg / kg, ε-PL is 0.1 mg / kg, and chitosan is 1.8 mg / kg.

[0012] On the other hand, the present invention proposes a pickling method using the above low-salt pickling agent to prevent spoilage odors in fermented meat products, including the following steps:

[0013] S1: Selection and trimming of raw meat: Select fresh pork hind legs with a mass of about 500 g and consistent size.

[0014] S2: Marinating: Apply and rub the above-mentioned marinade on the hind leg meat blocks, drain the blood, and stack the meat blocks in a marinating chamber at a temperature of 4 °C and a relative humidity of 80% - 90% for 3 days after salting.

[0015] S3: Air-drying: Hang the meat blocks at a temperature of 12 °C and a relative humidity of 50% - 60% to dehydrate and air-dry for 3 days.

[0016] S4: Fermentation: Place the air-dried meat blocks at a temperature of 20 °C and a relative humidity of 60% - 70% for 15 days, then ferment at a temperature of 28 °C and a relative humidity of 75% - 85% until mold grows, and finally ferment at a temperature of 20 °C and a relative humidity of 75% - 85% for 5 days to complete the fermentation.

[0017] On the other hand, the present invention proposes the application of the above-mentioned low-salt marinade in preventing the spoilage odor of fermented meat products.

[0018] Advantages of the present invention:

[0019] Currently, there are few patents on ham preservatives. Most of them are preservatives for fish and shrimp preservation. The water content of fish and shrimp is about 60% - 80%, and the water activity is relatively high, which is conducive to the diffusion and action of some preservatives such as phosphoric acid complex salts. However, for ham, the water content of ham is very low, and traditional fish and shrimp preservatives are difficult to achieve the same effect in ham. But after using the compound preservative, its total bacterial count is significantly reduced, indicating that it can achieve an effective antibacterial effect. And in the sensory evaluation, it also has good acceptability and meat flavor. Moreover, the results of biogenic amines show that it effectively inhibits the growth of harmful microorganisms with decarboxylase activity (able to produce biogenic amines), resulting in a decrease in the content of biogenic amines, clarifying the application effect of the compound preservative in simulated dry-cured meat blocks, and laying a foundation for the future application of the compound preservative in actual Xuanwei ham.

[0020] Based on the molecular interaction characteristics of ε-PL, Nisin and chitosan, the present invention forms a triple action mechanism of "membrane potential disruption - cell wall lysis - membrane structure damage". As a cationic polypeptide, ε-PL adsorbs on the negatively charged salt-tolerant bacterial cell membrane through electrostatic interaction, destroys its transmembrane proton gradient, and blocks ATP synthesis; Nisin, as a lantibiotic, specifically binds to the lipid II precursor of Gram-positive bacteria and inhibits the synthesis of cell wall peptidoglycan; chitosan forms a positively charged polymer through amino protonation and binds to the lipopolysaccharide on the microbial surface, causing a change in membrane permeability. The steric hindrance effect and charge complementarity characteristics of the three make the composite system produce a synergistic antibacterial effect at low concentrations, overcoming the defect of a single preservative having a single action target in a low-moisture system.

[0021] In view of the dense tissue structure of ham with a moisture content <50% and a fat content >30%, the present invention innovatively adopts an infiltration enhancement strategy of "physical property modification - gradient drive - phase change diffusion". During the air-drying stage, myofibrils contract to form surface micropore channels, improving the adsorption efficiency of preservatives; during the early fermentation stage, a moisture gradient is established inside the meat blocks to drive the small molecule ε-PL to penetrate deep along the myofibril gaps; during the middle and late fermentation stages, the phase change interface generated by the softening of adipose tissue is utilized to promote the directional distribution of chitosan at the lipid-water interface.

[0022] Through the coupling effect of composite preservatives and process parameters, the present invention constructs a three-in-one regulation system of "enzyme inhibition - antioxidant - flavor guidance". Nisin inhibits the proliferation of lipase-producing bacteria and reduces the activity of lipolytic enzymes; chitosan chelates pro-oxidant metal ions such as Fe 2+ / Cu 2 + to block the lipid peroxidation chain reaction; ε-PL regulates the microbial community structure and promotes the metabolism of beneficial bacteria to produce aldehyde flavor substances. Especially in terms of flavor formation, the gradient fermentation process of 20°C → 28°C → 20°C activates the temperature-sensitive flora in sequence to guide the synthesis path of volatile substances: the low temperature in the early stage promotes the hydrolysis of esterase to produce free fatty acids, the high temperature in the middle stage accelerates the Strecker degradation to form methyl aldehydes, and the low temperature in the later stage stabilizes aromatic compounds.

[0023] Of course, it is not necessary for any product implementing the present invention to simultaneously achieve all the above-mentioned advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts.

[0025] Figure 1 It is a schematic diagram of the sensory evaluation of dry-cured meat blocks;

[0026] Figure 2 It is a schematic diagram of biogenic amines in dry-cured meat blocks;

[0027] Figure 3 It is a schematic diagram of volatile flavor compounds in dry-cured meat blocks. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0029] Process 1:

[0030] In the embodiment, the concentration of ε-PL is 0.1 mg / kg, the concentration of Nisin is 0.2 mg / kg, and the concentration of chitosan is 1.8 mg / kg. The three solutions are mixed into the MRS medium to prepare a compound preservative.

[0031] In the embodiment, bacteria are inoculated into 5 mL of MRS broth medium. After incubating at 37 °C for 24 hours, 1 mL of the bacterial suspension is taken, centrifuged at 10,000 g for 10 minutes, then the precipitate is washed with sterile physiological saline, and subsequently the precipitate is resuspended again with 0.5 mL of sterilized physiological saline. Finally, the concentration of the bacterial suspension obtained is 106 CFU / mL. Bacterial suspensions of different types are mixed to obtain a mixed microorganism.

[0032] In the embodiment, fresh pig hind legs with a mass of about 500 g and the same size are selected, and the salt content is 12% of the meat weight. First, it is marinated at 4 °C and a relative humidity of 80% - 90% for 3 days, then dehydrated and air-dried at 12 °C and a relative humidity of 50% - 60% for 3 days. Then it is fermented at 20 °C and a relative humidity of 60% - 70% for 15 days. Then it is fermented at 28 °C and a relative humidity of 75% - 85% until mold grows, and finally it is fermented at 20 °C and a relative humidity of 75% - 85% for 5 days, and the fermentation is completed.

[0033] Process 2:

[0034] In the embodiment, the salt content is changed to 7% of the meat weight, and the remaining steps remain unchanged.

[0035] Process 3:

[0036] In the embodiment, fresh pig hind legs with a mass of about 500 g and the same size are selected, and the salt content is 7% of the meat weight. After the air-drying period ends, the compound preservative is applied, and after the initial fermentation ends, the mixed spoilage bacteria are applied, and the remaining steps remain unchanged.

[0037] Process 4:

[0038] In the embodiment, fresh pig hind legs with a mass of about 500 g and the same size are selected, and the salt content is 7% of the meat weight. After the air-drying period ends, the mixed spoilage bacteria are applied, and after the initial fermentation ends, the compound preservative is applied, and the remaining steps remain unchanged.

[0039] Process Five:

[0040] In the example, fresh pork hind legs with a mass of about 500 g and consistent size were selected, and the amount of salt added was 7% of the meat weight. After the end of the air-drying period, only the mixed spoilage bacteria were applied, and the remaining steps remained unchanged.

[0041] Example 1

[0042] The dry-cured meat blocks were cut into small pieces, a certain amount (such as 25 g) was weighed, 225 ml of sterile diluent was added, and the sample was homogenized to fully release the microorganisms from the meat blocks. Then, the homogenate was serially diluted 10-fold (for example, from 10 -1 diluted to 10 -6 ). An appropriate diluent (usually 10 -3 or 10 -4 ) was selected, and 1 ml of the diluent was inoculated into a pre-prepared sterile culture medium plate, and inoculation was carried out using the pour plate method or the spread plate method. The inoculated plate was placed in an incubator at 37 °C and cultured for 24 - 48 hours. After the culture was completed, the plates with colony counts between 30 and 300 were selected for counting, and the total number of colonies in the original dry-cured meat blocks was calculated according to the dilution factor, and the results were expressed as CFU / g (colony forming units / gram).

[0043] Table 1 Total number of colonies in dry-cured meat blocks

[0044]

[0045] In the experiment of this example, a total of 5 batches of dry-cured meat blocks were prepared and analyzed, and the total number of colonies is shown in Table 1. The experimental data showed that the experimental groups with the compound preservative added were significantly reduced in terms of the total number of colonies. Among them, Process Three was 2805.00 CFU / g, and Process Four was 3324.86 CFU / g, both of which were significantly lower than Process Five (p < 0.05). This result indicates that the compound preservative used has a significant antibacterial effect under the experimental conditions. In addition, the total number of colonies of the pickled meat blocks in Process One was lower than that in Process Two. It is speculated that the reason may be related to the inhibition of microbial growth in a high-salt environment. Under the premise of low salt, the total number of colonies in Process Three and Process Four was not only significantly lower than that in Process Five and Process Two, but also significantly lower than that in the high-salt group Process One (p < 0.05), verifying the "low-salt without reduced efficacy" effect achieved by the compound preservative through a synergistic mechanism in a low-salt system.

[0046] Example 2

[0047] Determination of moisture content: The direct drying method in the national standard was used for determination. Place the aluminum box in an oven at 105 °C and dry it to a constant mass (the mass difference between two consecutive weighings does not exceed 2 mg, which is considered a constant mass); separately crush the meat pieces from five pickling processes, then weigh 3 g of the sample, take it out after drying for 2 h, put it in a desiccator to cool for 0.5 h and then weigh; repeat the above operations until the sample reaches a constant mass, and conduct 3 parallel tests for each sample.

[0048] Determination of water activity: Select meat pieces from different processes, crush them, weigh 3 g, put them into a water activity meter, and use the water activity meter for determination. Repeat the determination 3 times for each sample.

[0049] Determination of pH: Take 5 g of dry-cured meat pieces, crush them and place them in 50 ml, add 5 times the volume of distilled water, mix well, filter with gauze, and take the supernatant for pH measurement using a pH meter.

[0050] Determination of TBARS: Accurately weigh 2 g of the homogenized dry-cured meat piece sample, place it in a 50 mL centrifuge tube, add 17 mL of 2.5% trichloroacetic acid solution, homogenize at 8000 rpm / min for 30 s, add 3 mL of 1% thiobarbituric acid, and mix well. Heat in a boiling water bath for 30 minutes and cool to room temperature. Accurately transfer 4 mL of the supernatant to a 10 mL centrifuge tube, add an equal volume of chloroform, mix well, centrifuge at 3000 r / min for 10 min, suck out the supernatant, measure the absorbance at 532 nm (perform a blank test simultaneously), record the absorbance, and calculate the TBARS value (mg / kg) using the following formula:

[0051] TBARS (mg / kg) = (A 532 / Ws) × 9.48

[0052] where A532 is the absorbance of the measured solution (532 nm), Ws is the muscle weight (g), and "9.48" is a constant obtained from the dilution factor and the molar extinction coefficient of the TBA reaction product [152,000 (mol / L) -1 cm -1 .

[0053] Table 2 Moisture content, water activity, pH, TBARS of dry-cured meat pieces

[0054]

[0055] As shown in Table 2, in the results of moisture content, it was found that the normal moisture content of dry-cured meat chunks was between 35% and 38%. The moisture content of Process 1 was significantly lower than that of other processes. This is because when a large amount of salt covers the surface of the meat chunks, the salt will form a high osmotic pressure environment on the surface of the meat chunks. Since the osmotic pressure of the moisture inside the meat chunks is lower than that outside, the moisture will diffuse outward through the cell membrane, resulting in the moisture in the meat chunks reaching the surface and finally being evaporated or discharged. There was no significant difference between Process 3 and Process 4 with compound preservatives added and Process 2 with 7% salt added. In terms of water activity (aw), it was found that there was a significant difference between Process 5 and other groups of samples (p < 0.05). The water activity of Process 3 was 0.73 (p < 0.05), which was significantly different from other processes, indicating that the addition of compound preservatives reduced the water activity compared with Process 5, and there was also no significant difference compared with Process 1 and Process 2. In terms of pH, it was also found that there was a significant difference between Process 5 and other groups of samples (p < 0.05). There was no significant difference between Process 3 and Process 4, but there was a significant difference between Process 1 and Process 2 (p < 0.05). The possible reason is that the lactic acid bacteria in the compound preservative treatment group had stronger activity and enhanced acid production ability, resulting in a decrease in the pH of the system. The low pH had certain antibacterial ability and also enhanced the protonation effect of ε-polylysine (ε-PL), thereby improving its penetration ability into the microbial cell membrane; Nisin also showed better stability and antibacterial performance under acidic conditions. Therefore, the decrease in pH not only reflected the regulation effect of the compound preservative but also provided a suitable environment for inhibiting spoilage bacteria for the synergistic antibacterial effect of the compound preservative under low-salt conditions.

[0056] The values of TBARS were all within 1, indicating that their lipid oxidation was within an acceptable range. Among them, the TBARS value of Process 5 without preservatives added was the highest (0.82 ± 0.02), showing the most serious degree of lipid oxidation. The TBARS values of Process 3 (0.48 ± 0.00) and Process 4 (0.54 ± 0.01) were significantly lower than that of Process 5 (p < 0.05), indicating that under low-salt (7%) conditions, the compound preservative effectively slowed down the lipid oxidation reaction and had good antioxidant stability. The TBARS value of Process 1 was the lowest (0.38 ± 0.00), indicating the natural inhibitory effect of the high-salt environment on fat oxidation. However, Processes 3 and 4 still maintained relatively low TBARS values under salt reduction treatment, indicating that through the synergistic effect of ε-polylysine, Nisin, and chitosan, lipid stability similar to that of the high-salt environment could also be achieved. From the mechanism of the compound preservative, ε-polylysine can reduce the pressure of fat degradation by inhibiting microbial metabolic activities; chitosan chelates Fe 2+ / Cu 2+Transition metal ions such as inhibit the free radical-induced lipid peroxidation chain reaction; Nisin indirectly controls the oxidation process by weakening the activity of oxidation-promoting bacteria. At the same time, it promotes the acid production of lactic acid bacteria at 28°C and superimposes the effect with preservatives to control the proliferation of miscellaneous bacteria. When the temperature is reduced to 20°C, it slows down the activity of lipase and the oxidation reaction rate, which is beneficial to Nisin maintaining stable antibacterial performance. This antioxidant performance achieved through "multi-mechanism coordination + low-salt environment" is one of the key innovations in the anti-corrosion strategy of the present invention.

[0057] Example 3

[0058] Measurement of color difference: Cut the dry-cured meat blocks into meat slices with a length, width, and thickness of 3 cm, 3 cm, and 1 cm respectively. Take 3 different cut surfaces for each curing process, and then measure 6 times on the cut surface. Calibrate the color difference meter before measurement and express it with L* value (brightness), a* value (redness), and b* value (yellowness).

[0059] Measurement of texture: Cut the cured dry-cured meat block samples into cube small pieces with a size of 10 mm × 10 mm × 10 mm. The diameter of the P50 probe is 50 mm, its initial measurement speed is 1.0 mm / s, and the subsequent measurement speed is also 1.0 mm / s. The trigger force reaches 30.0 g, and the compression rate reaches 70%. Measure the samples in an environment with a temperature of 25°C.

[0060] Table 3 Parameters such as color difference, hardness, and elasticity of dry-cured meat blocks

[0061]

[0062]

[0063] The color difference analysis of the dry-cured meat blocks is shown in Table 2. Among the five fermentation processes, the L* value of Process Five is significantly higher than that of other experimental groups (p < 0.05), while the brightness of Process Three with added preservatives is the lowest at 33.02 (p < 0.05), and there is no significant difference between their a* and b*. The texture analysis of the dry-cured meat blocks is shown in Table 3. The hardness values of Process Four and Process Five are significantly lower than those of other process groups (p < 0.05), and there is no significant difference between Process Three and Processes One and Two. The adhesiveness has the same trend as the hardness, and there is no obvious difference in elasticity, chewiness, and resilience in the dry-cured meat blocks, probably because the fermentation time of the dry-cured meat blocks is relatively short.

[0064] Example 4

[0065] The color, brightness, hardness, texture, off-odor, and overall acceptability of Xuanwei ham were evaluated. Before the sensory evaluation, 20 panelists (10 males and 10 females, aged 21 - 25 years) were selected from teachers, staff, and postgraduate students who had participated in a professional sensory training course. The sensory attributes were evaluated using a 10-point intensity line scale: red color (1 = light red, 10 = dark red); brightness (1 = dull, 10 = bright); hardness (1 = hard or loose, 10 = moderate toughness); meat aroma (1 = hardly detectable, 10 = strong); off-odor (1 = hardly detectable, 10 = strong); overall acceptability (1 = unacceptable, 10 = very like). On a white plastic board, the dry-cured meat pieces after fermentation were cut into slices with a thickness of 1 mm and then randomly placed. Then, random three-digit labels were pasted on them, and the samples were brought into a sensory evaluation room at a temperature of 20 - 22 °C for tasting. Between the samples of dry-cured meat pieces with different processes, about 50 mL of pure water was provided for the judges. The members of the sensory evaluation panel recorded according to their perceived intensity of each attribute.

[0066] As Figure 1 shown, the pickled meat pieces of Process 1 and Process 2 had higher meat aroma, acceptability, and hardness. Process 3 and Process 4 were significantly superior to Process 5 without added preservatives in terms of redness, brightness, meat aroma, and acceptability, indicating that the addition of the compound preservative effectively inhibited the accumulation of metabolites of spoilage bacteria. Through the multi-target synergistic mechanism of ε-polylysine, nisin, and chitosan, it not only delayed the spoilage reaction but also stabilized the color and flavor basis of the product. In particular, ε-PL and Nisin can reduce the formation of off-odor substances such as off-odor amines and aldehydes and ketones, and chitosan inhibits color deterioration through a film protection effect, thus maintaining better redness and brightness. At the same time, the temperature gradient fermentation process of 20 °C → 28 °C → 20 °C also played an important role in flavor formation and flora regulation. In the early 20 °C stage, the reproduction of miscellaneous bacteria was moderately inhibited, providing a stable environment for the dominant fermenting bacteria; in the middle stage, the temperature was raised to 28 °C to activate the metabolism of lactic acid bacteria and yeast, enhancing the acid production and aroma production ability, and promoting the formation of flavor precursors such as esters and aldehydes; in the later stage, the temperature was lowered to 20 °C again to inhibit the restorative proliferation of spoilage bacteria, while slowing down the enzymatic inactivation of Nisin and stabilizing the final flavor structure, improving the overall coordination and consumer acceptability. Process 5 only added mixed spoilage bacteria without adding the compound preservative, so it had a higher off-odor score. In contrast, the off-odor scores of Process 3 and Process 4 were within an acceptable range. Compared with Process 4, Process 3 had higher acceptability and meat aroma. This may be because spoilage microorganisms were added first in Process 4. During the pickling process, the spoilage microorganisms grew on the pickled meat pieces for a period of time and may have entered the interior of the meat pieces, so the compound preservative added subsequently only played a role in inhibiting the continued growth of spoilage microorganisms. This is consistent with the total colony count results.

[0067] Example 5

[0068] Prepare standard solutions of biogenic amines with different concentrations (0.5, 1, 2, 5, 20, 50, and 100 μg / mL) using 0.4 mol / L perchloric acid, and determine biogenic amines by high performance liquid chromatography (HPLC). Take 5 g of the sample, add it to 20 mL of 0.4 mol / L perchloric acid, mix evenly, centrifuge at 5000 g for 10 min at 4 °C; repeat the extraction twice under the same conditions. After combining the supernatants, make up the volume to a total of 50 mL with 0.4 mol / L perchloric acid. Then, add 200 μL of 2 mol / L sodium hydroxide, 300 μL of saturated sodium bicarbonate, and 2 mL of 10 mg / mL dansyl chloride to 1 mL of the solution and mix. Incubate the mixture in the dark in a water bath at 40 °C for 30 min, and then add 100 μL of ammonia water to terminate the reaction. Finally, make up the volume of the mixture to 5 mL with acetonitrile and filter it through a 0.22 μm membrane filter. [[ID=—3]]

[0069] Perform on an Agilent ZORBAX XDB-C18 chromatographic column (5 μm, 4.6 mm × 250 mm) at 254 nm. The mobile phase consists of eluent A (ultrapure water) and B (acetonitrile), with 35% and 65% respectively, the injection volume is 20 μL, the flow rate of the two phases is 1 mL / min, and the temperature is 40 °C. Calculate the biogenic amine content according to the standard curve.

[0070] The total biogenic amine content of dry-cured meat chunks is as Figure 2 shown. The research results show that the biogenic amine content in Process Five is significantly higher than that in other groups of dry-cured meat chunks (p < 0.05). The reason for the higher biogenic amine content in Process Five may be the excessive hydrolysis of proteins and the rapid growth of spoilage bacteria and the occurrence of amino acid decarboxylation reactions in the dry-cured meat chunks of Process Five, resulting in the large generation of amine metabolites. The biogenic amine content in Process Three is significantly higher than that in Process One (p < 0.05). ε-PL disrupts the cell membrane potential and interferes with energy metabolism; Nisin inhibits the construction of the cell wall of Gram-positive bacteria and reduces the activity of enzyme-producing bacteria; chitosan weakens the decarboxylation activity of microorganisms by changing the membrane permeability and chelating metal ions. It shows an inhibitory effect on spoilage flora under low-salt conditions, significantly reducing the accumulation risk of potentially toxic amines such as histamine and putrescine, and ensuring product safety. In the regulation of fermentation temperature, at 20 °C in the first stage, it inhibits the early growth rate of miscellaneous bacteria and spoilage bacteria represented by Pseudomonas and Morganella, avoiding the rapid accumulation of spoilage metabolites such as biogenic amines and odor compounds in the early stage of fermentation; at 28 °C in the second stage, the fermentation temperature promotes lactic acid bacteria to produce acid and reduces the pH, significantly inhibiting the expression and activity of amine decarboxylase; in the third stage, the fermentation temperature is reduced to 20 °C to stabilize the activity of Nisin, delay the recovery of spoilage bacteria and inhibit the re-accumulation of amines in the later stage, so as to achieve a better inhibitory effect.

[0071] Example 6

[0072] The volatile compounds in Xuanwei ham were determined by headspace-solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry. The minced ham (3.0 g) was placed in a sealed headspace vial, and 3 μL of 1,2-dichlorobenzene was added as an internal standard (concentration 100 mg / mL). After equilibration at 40 °C for 10 min in a heating device, a 50 / 30 μm thick DVB / CAR / PDMS fiber was inserted into the headspace vial for extraction for 30 min. Finally, the adsorbed volatile compounds were desorbed in the gas chromatography injector at 240 °C for 5 min, and identification and quantification were performed using a gas chromatography-mass spectrometry system with a DB-WAX (30 m × 0.25 mm × 0.25 μm) column. The gas chromatography temperature program was as follows: hold at 40 °C for 3 min, increase to 90 °C at a rate of 3 °C / min and hold for 5 min, then increase to 200 °C at a rate of 3 °C / min; finally, increase to 230 °C at a rate of 15 °C / min and hold for 5 min; the split ratio was 5:1. The carrier gas was high-purity helium with a flow rate of 2.0 mL / min. The mass spectrometry parameters were 70 eV, electron ionization mode, and the scanning range was m / z 35 - 500. The mass spectrometry ion source and quadrupole temperatures were 200 °C and 230 °C, respectively.

[0073] The volatile compounds were identified by comparing the mass spectra with the reference mass spectra in the NIST21 library data, and the similarity between the two was 90%. For further confirmation, the retention index (RI) was calculated using a mixture of n-alkanes (C5 - C25). Finally, the odor activity value (OAV) of the volatile compounds was calculated by dividing the concentration of the compound in the sample by the threshold value of the compound:

[0074] The area chart of the contents of different categories of volatile compounds in dry-cured meat chunks is as Figure 3As shown. It can be seen from the figure that Process 1 has more aldehyde volatile compounds, Process 2 has more ketone substances, Process 4 has more alcohols and esters, Process 3 has more aldehyde substances and ester substances, and Process 5 has more ester substances. Both Process 1 and Process 3 contain more aldehyde substances in the volatile compounds. Aldehyde compounds have antioxidant effects, can slow down fat oxidation, delay the process of fatty acid rancidity in ham, and thus extend the shelf life of the product. This is of great significance for the storage and sale of traditional fermented and air-dried hams. While the main substances in Process 4 and Process 5 are ester substances. Ester substances are usually formed by the reaction of fatty acids and alcohols catalyzed by esterase, and are the key components presenting fruity and fatty flavors in dry-cured meat products; aldehyde substances mainly come from the Strecker degradation reaction of amino acids and are important carriers endowing cooked and caramel flavors. On the contrary, the accumulation of alcohols and ketones is often related to the abnormal fermentation or oxidative decomposition process of spoilage bacteria, and excessive alcohols and ketones will cause the generation of off-flavors. The anti-corrosion technologies used in traditional high-water-activity fish and shrimp products are difficult to apply to the low-moisture and high-fat dense system of hams. In the present invention, ε-polylysine weakens microbial metabolism through membrane potential interference, Nisin targets and blocks cell wall synthesis, and chitosan regulates by changing membrane permeability and enzymes. The synergy of the three in molecular structure, action targets and physicochemical properties makes up for the problem of limited action of a single preservative in a low-moisture system, promotes the metabolic advantages of flavor-beneficial bacteria such as lactic acid bacteria and some yeasts, restricts the generation of spoilage bacteria, and thus the product obtains good flavor. In addition, it is related to the "20°C → 28°C → 20°C" gradient temperature control fermentation process for the distribution of flavor substances. In the first stage (20°C), the low temperature effectively restricts mesophilic spoilage bacteria, helps to weaken miscellaneous bacteria and delay the degradation process of protein and fat substrates, and provides a stable matrix environment for the subsequent preferential colonization of beneficial microorganisms and the activation of target metabolic pathways. In the second stage (28°C), the metabolism of lactic acid bacteria and yeasts is activated, the activities of functional enzymes such as esterase and decarboxylase are increased, and the synthesis of flavor core substances such as esters and aldehydes is rapidly promoted; in the third stage (20°C), the growth and reproduction of spoilage bacteria are reduced, and at the same time the stability of Nisin and ε-PL is maintained to avoid late flavor deterioration or abnormal accumulation of alcohols and ketones. The synergistic effect of temperature regulation in the three stages and the antibacterial mechanism of the compound preservative realizes the technology of low salt without reducing efficacy.

[0075] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor limit the invention to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments to better explain the principle and practical application of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A low-salt curing agent for inhibiting the growth of microorganisms that produce putrid odors in ham, characterized in that, Including but not limited to table salt, Nisin, ε-PL and chitosan.

2. The low-salt curing agent for inhibiting the growth of microorganisms that cause putrid and off-flavor in ham, characterized in that: The table salt is 70 g / kg, Nisin is 0.2 mg / kg, ε-PL is 0.1 mg / kg, and chitosan is 1.8 mg / kg.

3. The pickling method for preventing spoilage and off-odor of fermented meat products with the low-salt pickling agent according to claim 1 or 2, characterized in that: It includes the following steps: S1: Select fresh pig hind legs with a mass of 500 g and the same size. S2: Apply and rub the low-salt curing agent described in Claim 1 or 2 on the hind leg meat blocks, drain the blood, stack the meat blocks in a curing chamber at a temperature of 4°C and a relative humidity of 80% - 90% for 3 days after salting. S3: Hang the meat blocks at a temperature of 12°C and a relative humidity of 50% - 60% for 3 days for dehydration and air drying. S4: Place the air-dried meat blocks at a temperature of 20°C and a relative humidity of 60% - 70% for 15 days of fermentation; then ferment at a temperature of 28°C and a relative humidity of 75% - 85% until mold grows, and finally ferment at a temperature of 20°C and a relative humidity of 75% - 85% for 5 days to complete fermentation.

4. The application of the low-salt curing agent according to Claim 1 or 2 in preventing the spoilage odor of fermented meat products.

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