Preparation method of ready-to-eat fermented and pickled horse meat with low nitrite and nitrosamine content

Abstract

The invention discloses a preparation method of ready-to-eat fermented and pickled horse meat with low nitrite and nitrosamine content, and belongs to the field of processing of pickled meat products and food safety control. The preparation method comprises the following steps: slicing horse meat, adding ingredients and compound bacteria, performing room-temperature fermentation and pickling, after fermentation is completed, performing baking, performing drying, performing sterilization, and performing packaging so as to obtain the ready-to-eat fermented and pickled horse meat, wherein based on the weight of 100 kg of horse meat, 2%-6% of table salt, 2%-6% of cane sugar, 0.2%-0.4% of Chinese prickly ash and 0.05%-0.15% of nitrite are added; the ratio of the inoculum size of lactobacillus plantarum to the inoculum size of lactobacillus reuteri in the compound bacteria is 1: 1-3: 1, and the total inoculum size is 108 CFU / mL. By optimizing process conditions and parameters, the ready-to-eat fermented and pickled horse meat can be obtained by fermenting and pickling at room temperature for 24 hours, the nitrite content in the horse meat is less than 20% of the national standard, no nitrosamine is detected, and the invention provides a safer preparation technology of the ready-to-eat fermented and pickled horse meat.

Description

Technical Field

[0001] The invention relates to the field of processing and food safety control of pickled meat products, in particular to a method for preparing instant fermented pickled horse meat with low nitrite and nitrosamine. Background Art

[0002] Cured meat products refer to meat products that have a long shelf life and undergo a series of biochemical and physical changes in raw meat under natural or artificial controlled conditions, through the action of curing liquid, microbial fermentation or enzymes, producing special flavor, color and texture. Horse meat has the characteristics of high protein (20% to 24%), high fatty acid (more than 55%), low cholesterol (2.48%), and high glycogen content (1%). It contains more iron and zinc than other meats; horse meat has low fat content and good lipid distribution. The concentrations of unsaturated fatty acids arachidonic acid and eicosapentaenoic acid can reach 2.97% and 0.43%, and the ratio of polyunsaturated fatty acids to saturated fatty acids (PUFA / SFA) is 0.97. As consumer attitudes change, horse meat consumption is currently slowly rising worldwide, with horse meat production increasing by 7.57% between 2010 and 2020 (from 718,433 tons in 2010 to 772,829 tons in 2020), of which 55.58% came from Asia.

[0003] Horse meat processing products in my country are at a relatively low level of smoking and boiling, lacking fine and deep processing of horse meat, and lacking representative horse meat deep-processing products; currently, the main horse meat products are dry cured meat, horse intestines and other horse meat products. Cured meat began in the Shang and Zhou dynasties. With the development of the times, in order to better extend the quality and shelf life of cured meat, nitrite has become an important preservative and colorant, which can effectively reduce the harm of botulism and bring a bright rose color and good pickled flavor to cured meat; GB 2760 currently has clear requirements for the maximum amount of nitrite used in cured meat products, which is 0.15g / kg; at the same time, it also requires that its residual amount is ≤30mg / kg (calculated as sodium nitrite); however, some manufacturers will add excessive amounts to achieve better color and flavor, and cannot effectively control the residual nitrite during processing, resulting in excessive nitrite in the product. At the same time, the residual nitrite will react with free amino acids and amines during storage to form nitrosamines. When nitrite and nitrosamine are excessive, they can cause poisoning and even cancer. Therefore, it is necessary to control the nitrite and nitrosamine in pickled horse meat to ensure food safety. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing ready-to-eat fermented pickled horse meat with low nitrite and nitrosamine, so as to solve the problems existing in the above-mentioned prior art. By improving and optimizing the fermentation and pickling bacteria, process conditions, etc., the nitrite content in the ready-to-eat fermented pickled horse meat can be significantly reduced, and nitrosamines are not detected.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for preparing instant fermented pickled horse meat with low nitrite and nitrosamine, comprising the following steps:

[0007] After the horse meat is cut into pieces and weighed, ingredients and purified water are added at the same time, and composite bacteria are inoculated, and fermented and marinated at room temperature. After the fermentation is completed, it is baked and dried, packaged and sterilized to obtain ready-to-eat fermented marinated horse meat; the ingredients include salt, sucrose, pepper and nitrite, and the composite bacteria are Lactobacillus plantarum and Lactobacillus reuteri;

[0008] Among them, based on the weight of 100kg of horse meat, 2% to 6% of salt, 2% to 6% of sucrose, 0.2% to 0.4% of Sichuan pepper and 0.05% to 0.15% of nitrite are added.

[0009] Optionally, the ingredients are dissolved in water when used, and the material-to-water ratio of the ingredients is 1:2 to 1:4.

[0010] Optionally, the inoculum ratio of Lactobacillus plantarum to Lactobacillus reuteri in the composite bacteria is 1:1 to 3:1, and the total inoculum amount is 10 8 CFU / mL~10 9 CFU / mL; and / or the Lactobacillus plantarum and the Lactobacillus reuteri are both inoculated into the horse meat during the logarithmic growth phase of culturing for 9 to 12 hours.

[0011] Optionally, the fermentation and pickling conditions are: fermentation and pickling at 20°C to 30°C for 20 to 24 hours, and an initial pH value of 4.0 to 4.5.

[0012] Preferably, the optimal fermentation and pickling time is 24 hours.

[0013] Optionally, the baking and drying conditions are: baking temperature 100-180° C., and baking time 60-180 min.

[0014] Preferably, the optimal baking temperature is 140° C. and the baking time is 120 min.

[0015] Optionally, the sterilization conditions are: sterilization at 121° C. for 15 to 25 minutes.

[0016] Preferably, based on 100 kg of horse meat, 3.3% salt, 4.3% sucrose, 0.4% prickly ash and 0.15% nitrite are added.

[0017] Optionally, the nitrite content of the ready-to-eat fermented and cured horse meat is 5.23±0.09 mg / kg, and the ready-to-eat fermented and cured horse meat does not contain nitrosamines.

[0018] The present invention discloses the following technical effects:

[0019] The present invention can significantly reduce the content of nitrite in the fermented horse meat pickled product by inoculating specific Lactobacillus plantarum and Lactobacillus reuteri composite bacteria in pickled horse meat. And by optimizing conditions, the present invention can obtain the product by fermenting and pickling for 24 hours at room temperature (25°C). Compared with the traditional pickling method, which strictly controls the pickling and fermentation temperatures and requires long pickling and fermentation to obtain the product, the present invention significantly shortens the fermentation and pickling time, and the pickling and fermentation temperatures are easy to control and achieve, and can more efficiently prepare the fermented horse meat pickled product.

[0020] The present invention improves and optimizes the process flow and parameters of fermented pickled horse meat. In the production of pickled horse meat, after adding nitrite according to the national standard, the residual nitrite (calculated as sodium nitrite) in the finished pickled horse meat product can be effectively controlled to be less than 20% of the national standard, and nitrosamines are not detected, under the premise that the quality is not reduced. It is explained that the instant fermented pickled horse meat obtained by the preparation method provided by the present invention is a safe, high-quality instant pickled horse meat product, which provides a new method for the preparation of refined and deep-processed horse meat products, and also enriches the types of pickled horse meat products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The effect of pepper addition on nitrite residue, sensory evaluation and comprehensive score in pickled horse meat; different letters indicate significant differences P<0.05;

[0023] Figure 2 The effect of salt addition on nitrite residue, sensory evaluation and comprehensive score in pickled horse meat; different letters indicate significant differences P<0.05;

[0024] Figure 3The effect of sucrose addition on nitrite residue, sensory evaluation and comprehensive score in pickled horse meat; different letters indicate significant differences P<0.05;

[0025] Figure 4 The effect of baking temperature on nitrite residue, sensory evaluation and comprehensive score in pickled horse meat; different letters indicate significant differences P<0.05;

[0026] Figure 5 The effect of baking time on nitrite residue, sensory evaluation and comprehensive score in pickled horse meat; different letters indicate significant differences P<0.05;

[0027] Figure 6 The response surface diagram of the interaction between salt addition and sugar addition on the comprehensive score of pickled horse meat;

[0028] Figure 7 The response surface diagram of the interaction between salt addition and baking temperature on the comprehensive score of cured horse meat;

[0029] Figure 8 The response surface diagram of the interaction between salt addition and baking time on the comprehensive score of cured horse meat;

[0030] Fig. 9 The response surface diagram of the interaction between sugar addition and baking temperature on the comprehensive score of pickled horse meat;

[0031] Fig.10 The response surface diagram of the interaction between sugar addition and baking time on the comprehensive score of pickled horse meat;

[0032] Fig.11 The response surface diagram of the interaction between baking temperature and baking time on the comprehensive score of pickled horse meat;

[0033] Fig.12 Flow chart for fermenting cured horse meat with strains;

[0034] Fig.13 Effects of different strains on nitrite residues in pickled horse meat Note: Different letters indicate significant differences P<0.05;

[0035] Fig.14 The nitrate resistance characteristics of different strains;

[0036] Fig.15 The effect of the composite strain on the residual nitrite in cured horse meat;

[0037] Fig.16 These are the growth and acid production curves of the strains; A: growth curve, B: acid production curve;

[0038] Fig.17 The effect of salt concentration on the growth and acid production of the strain; A: growth curve, B: acid production curve;

[0039] Fig.18 The effect of NaNO2 on the growth and acid production of the strain; A: growth curve, B: acid production curve;

[0040] Fig.19 The effect of pH on the growth and acid production of the strain; A: growth curve, B: acid production curve;

[0041] Fig. 20 is the nitrite degradation rate of the strain; different letters indicate significant differences P < 0.05;

[0042] Fig.21 The effect of bacterial strain ratio on nitrite residue; different letters indicate significant differences (P<0.05);

[0043] Fig. 22 The effect of inoculum size on residual nitrite; different letters indicate significant differences (P<0.05);

[0044] Fig.23 The effect of pickling time on residual nitrite; different letters indicate significant differences (P<0.05);

[0045] Fig.24 Electronic nose radar charts for the control group and the optimized group;

[0046] Fig.25 Radar chart of sensory scores for the control group and the optimized group. DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0049] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0050] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0051] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0052] The detection method involved in the embodiment of the present invention is as follows:

[0053] 1. Determination of indicators of pickled horse meat

[0054] 1.1 Determination of nitrite

[0055] The residual nitrite in cured horse meat was determined according to "GB 5009.33-2016 National Food Safety Standard Determination of Nitrite and Nitrate in Food".

[0056] 1.2 Determination of nitrosamines

[0057] The nitrosamine content in pickled horse meat was determined according to the second method of "GB 5009.26-2023 National Food Safety Standard Determination of N-Nitrosamine Compounds in Foods".

[0058] 1.3 Sensory evaluation of pickled horse meat

[0059] Ten sensory comprehensive evaluators (half male and half female) were selected to conduct sensory evaluation according to the standards in Table 1. The 10 sensory comprehensive evaluators evaluated the taste, smell, color, and acceptability of the pickled horse meat before and after optimization compared with the control group. In order to avoid the possible influence of the presentation order, the samples were presented to the evaluation members in a random order and provided together with purified water.

[0060] Table 1 Sensory scoring table for pickled horse meat

[0061]

[0062]

[0063] 1.4 Overall score of pickled horse meat

[0064] The comprehensive scoring formula for horse meat is as follows: Mmin represents the minimum value of nitrite residue, Mi represents the nitrite residue; Nmax represents the maximum value of sensory score, Ni represents the sensory score.

[0065] Example 1

[0066] 1. Determination of basic formula and process

[0067] The preliminary formula of the present invention is: based on 100kg of horse meat, add 4% salt, 4% sucrose, 0.4% pepper, 0.15% nitrite, a material-water ratio of 1:3, and add appropriate amounts of phosphate and soy sauce. The preliminary process conditions are: pickling time 20h, pickling temperature 4°C, baking temperature 120°C, baking time 120min. Nitrite is added at the maximum allowable amount of 150mg / kg according to national standards. Nitrite inhibits bacterial reproduction during the pickling process, and can also promote moisture retention of meat products, improve color, and increase flavor. It is mainly used as a colorant and preservative in pickled meat products. During the processing, nitrite decomposes to form NO + It combines with secondary amines to generate nitrosamines. Therefore, the present invention first conducts preliminary experiments on nitrites and nitrosamines to explore the effects of different factors on them.

[0068] In the early stage of the experiment, phosphates, soy sauce and other ingredients were added to the pickled horse meat formula, and the nitrosamine content of the pickled horse meat was measured to be 0.53±0.21μg / kg. After inoculation with Lactobacillus plantarum, the nitrosamine content of the pickled horse meat was 0.47±0.02μg / kg, indicating that the inoculated microorganisms can regulate the formation of nitrosamines. After further optimization of the formula and process, it was found that nitrosamines were not detected, which may be due to the optimization of the processing technology and formula to avoid the formation of nitrosamines.

[0069] 1.1 Determination of the amount of pepper added

[0070] The preliminary experiment screened the single factor conditions and adjusted the addition amount of pepper, salt, sucrose and other ingredients in the formula. It was found that different addition amounts of salt and sucrose had significant effects on the residual nitrite, but the addition amount of pepper had no significant effect on the residual nitrite. Figure 1 As shown, the results showed that when the amount of pepper added exceeded 0.8%, the pepper flavor was too strong and the sensory score was low, so the amount of pepper added was fixed at 0.4%.

[0071] 1.2 Determination of the scope of salt addition

[0072] The effects of adding 2%, 3%, 4%, 5% and 6% salt on nitrite residue, sensory evaluation, comprehensive score and nitrosamine were investigated. Figure 2 As shown in the results, the nitrite residue was the highest when the salt addition was 4%. As the salt addition increased, the nitrite residue showed a trend of first increasing and then slowly decreasing. This may be related to the osmotic pressure. When the salt content was low, the osmotic pressure gradually increased, the horse meat absorbed more marinating liquid, and the nitrite content increased; when the salt addition exceeded 4%, the osmotic pressure was too high, the meat lost water, the intracellular fluid was pressed into the extracellular space, and the space between cells decreased, which led to a decrease in capillary force, and the marinating liquid was more easily squeezed out of the muscle, and the water holding capacity decreased, resulting in a decrease in nitrite content. At relatively low salt concentrations, salt anions bind to muscle filaments, thereby increasing water holding capacity; at higher salt concentrations, proteins denature and unfold, and the exposure of hydrophobic areas in proteins increases, resulting in water accumulation and loss in the muscle. Cross-linking between proteins and muscle contraction may lead to a reduction in water absorption space, reducing the absorption rate of marinating liquid in meat. The amount of salt added will also affect the activity of nitrite reductase in microorganisms and thus affect the nitrite content. As the amount of salt added increased, the sensory evaluation and comprehensive evaluation showed a trend of first increasing and then decreasing, and both reached the highest score when the salt addition was 3%. As shown in Table 2, no nitrosamines were detected in pickled horse meat with salt addition of 2% to 6%.

[0073] Table 2 Effect of salt addition on nitrosamines in pickled horse meat

[0074]

[0075] Note: “+” indicates detection; “-” indicates no detection; NDMA: N-nitrosodimethylamine, NDEA: N-nitrosodiethylamine, NDPA: N-nitrosodi-n-propylamine, NDELA: N-nitrosodiethanolamine, NDBA: N-nitrosodibutylamine, NPYR: N-nitrosopyrrolidine, NPIP: N-nitrosopiperidine, NMOR: N-nitrosomorpholine.

[0076] 1.3 Determination of the scope of sucrose addition

[0077] The addition of salt was fixed at 3%, pepper at 0.4%, nitrite at 0.15%, baking temperature at 120℃, and baking time at 120min. The effects of sucrose addition at 2%, 3%, 4%, 5%, and 6% on nitrite residue, sensory evaluation, comprehensive score, and nitrosamines were investigated. The results are as follows Figure 3As shown in the table, the highest nitrite residual amount was 27.16 mg / kg when the sugar addition amount was 2%, and the nitrite residual amount gradually decreased with the increase of sugar addition amount. This may be because the higher sugar concentration produced lower water activity and higher osmotic pressure, which led to a decrease in the absorption rate of pickled horse meat to the pickling liquid and reduced the nitrite residual amount. On the other hand, sugar can serve as a carbon source for microorganisms, promoting the growth and reproduction of nitrite reductase microorganisms in the pickling liquid, thereby reducing the nitrite residual amount. At the same time, as the sugar addition amount increases, the pH value may gradually decrease, thereby affecting the nitrite residual amount. The sensory score and the comprehensive score both reached the highest score at a sugar addition amount of 4%. When the sugar addition amount exceeded 4%, the pickled horse meat tasted sweet and greasy, and a small amount of sugar crystal particles precipitated on the surface after drying, which was not sensory. As shown in Table 3, no nitrosamines were detected when the sugar addition amount was 2% to 6%.

[0078] Table 3 Effect of sucrose addition on nitrosamines in pickled horse meat

[0079]

[0080] Note: + indicates detected; - indicates not detected

[0081] 1.4 Determination of baking temperature range

[0082] The salt content was fixed at 3%, sucrose at 4%, pepper at 0.4%, nitrite at 0.15%, and baking time at 120 min. The effects of baking temperatures at 100°C, 120°C, 140°C, 160°C, and 180°C on nitrite residue, sensory evaluation, comprehensive score, and nitrosamines were investigated. Figure 4 As shown in Table 4, when the baking temperature increases, the residual nitrite decreases accordingly, and the highest residual nitrite is 25.03 mg / kg at 100°C. Baking temperature can significantly reduce the nitrite level in meat. At higher temperatures, the percentage of activated molecules increases, thereby increasing the rate of nitrite decomposition reaction. At the same time, in the marinade, nitrite penetrates into the meat through water. The reduction in water reduces its migration rate, thereby reducing the residual nitrite. The sensory score and the comprehensive score both reach the highest value at 140°C. When the baking temperature is 100°C, the finished horse meat has more water analysis and the taste is too soft; when the baking temperature is 180°C, the marinated horse meat is brown and the meat is dry and woody. As shown in Table 4, no nitrosamines were detected in marinated horse meat at drying temperatures of 100-180°C.

[0083] Table 4 Effect of baking temperature on nitrosamines in pickled horse meat

[0084]

[0085] Note: + indicates detected; - indicates not detected

[0086] 1.5 Determination of baking time range

[0087] The salt content was fixed at 3%, sucrose at 4%, pepper at 0.4%, nitrite at 0.15%, and the drying temperature at 140°C. The effects of baking time at 60 min, 90 min, 120 min, 150 min, and 180 min on nitrite residue, sensory evaluation, comprehensive score, and nitrosamine were investigated. Figure 5 As shown in Table 5, the residual nitrite content decreased with the extension of drying time, and the residual nitrite content was the highest at 60 minutes, and the comprehensive score was the lowest. After baking for 60 minutes, the surface of the dried horse meat was not dried, and there was still pickling liquid on the surface of the horse meat after sterilization, which was not sensory. When the horse meat was dried for 180 minutes, it changed from reddish brown to dark brown, and the horse meat was dry, hard, and had a poor color. As shown in Table 5, no nitrosamines were detected in the pickled horse meat when the drying time was 60 to 180 minutes.

[0088] Table 5 Effect of baking time on nitrosamines in pickled horse meat

[0089]

[0090] Note: + indicates detected; - indicates not detected

[0091] 1.6 Determination of sterilization conditions

[0092] Using a high pressure autoclave to sterilize at 121°C for 15 to 30 minutes can meet the sterilization requirements, but preliminary experiments have found that when the sterilization time exceeds 20 minutes, the finished meat is relatively loose and does not achieve the ideal taste, so the sterilization conditions are fixed at 121°C for 15 minutes.

[0093] According to the above single factor experimental results, the present invention selects salt addition amount of 2%, 3%, 4%; sucrose addition amount of 3%, 4%, 5%, drying temperature of 90°C, 120°C, 150°C, and drying time of 120min, 140min, 160min for response surface optimization.

[0094] 1.7 Response surface optimization experiment

[0095] The response surface experimental factors and levels are shown in Table 6.

[0096] Table 6 Response surface test factors and levels

[0097]

[0098] The response surface software was used to analyze and process the results in Tables 7 and 8, and the experimental data were fitted with a quadratic multinomial regression. The multivariate regression equation for the comprehensive evaluation of the amount of salt added (A), the amount of sugar added (B), the baking temperature (C), and the baking time (D) in the pickled horse meat was obtained as follows: R = 92.18 + 1.60A + 4.36B-4.89C + 3.07D-1.07AB + 1.30AC + 3.95AD + 5.20BC-1.35BD-3.06CD-5.82A 2 -3.08B 2 -12.42C 2 -5.63D 2 .

[0099] Table 7 Nitrite residue and sensory scoring results

[0100]

[0101]

[0102] Table 8 Response surface experimental design and results

[0103]

[0104]

[0105] As can be seen from Table 9, the P value of the model is less than 0.01, indicating that the equation has a good fit. 2 =0.9765, indicating that the actual value and the predicted value have a high correlation. Therefore, the model can better reflect the relationship between each factor and the response value in the preparation process of pickled horse meat and predict the optimal process conditions. From the P value, it can be seen that the effects of salt addition (A), sucrose addition (B), drying temperature (C), drying time (D), interaction terms AD, BC, CD, and A2, B2, C2, and D2 on the comprehensive score of pickled horse meat are extremely significant (P<0.05). From the F value, it can be seen that the order of the four factors on the comprehensive score of pickled horse meat is: C>B>D>A, that is, baking temperature>sugar addition>baking time>salt addition.

[0106] Table 9 Analysis of variance of regression model

[0107]

[0108]

[0109] Note: “*” means P<0.05, there is a statistical difference; “**” means P<0.01, there is a significant statistical difference; “***” means P<0.001, there is an extremely significant difference.

[0110] The contour lines and response surfaces of the interaction between various factors on the residual nitrite in pickled horse meat are shown in Figure 6-Figure 11 The horizontal projection of the response surface is the contour line. If the contour line is elliptical, it means that the two factors have a very significant interaction, and if it is circular, it means that it is not significant. And the steepness of the slope of the response surface of each factor reflects the influence of the factor on the residual nitrite in pickled horse meat. The steeper the slope, the greater the influence.

[0111] The experimental data were optimized and predicted by Design-Expert 13 software, and the optimal process formula was obtained as follows: the amount of salt added was 3.315%, the amount of sugar added was 4.269%, the baking temperature was 138.734°C, and the baking time was 121.886min. The comprehensive score of pickled horse meat under this process condition was 93.35 points. According to the actual operation needs, the present invention selects the amount of salt added to be 3.3%, the amount of sugar added to be 4.3%, the baking temperature to be 139°C, and the baking time to be 122min. The experiment was repeated 3 times, and the average value was taken to measure the comprehensive score value to be 93.50±0.44 points, which was close to the predicted value. Under this condition, the residual nitrite was 19.74±0.30mg / kg, the sensory score was 32.50±0.41 points, and nitrosamines were not detected.

[0112] Table 10 Determination of nitrosamines

[0113]

[0114] Nitrite reacts with H under acidic conditions. + The reaction generates nitrous acid, which is unstable and decomposes to produce N2O3, which further decomposes to produce NO + It reacts with amines to generate nitrosamines, and finally generates NAs, among which only secondary amines can form stable nitrosamines. Factors affecting the production of nitrosamines mainly include processing technology and methods (heating temperature, heating method), pH, microbial action, NAs precursors, additives and fats. It can be seen from the table that in the pickling process of the present invention, under the conditions of salt addition of 2% to 6%, sugar addition of 2% to 6%, baking temperature of 100℃ to 180℃min, and baking time of 60 to 180min, nitrosamines were not detected.

[0115] The temperature for nitrosamine formation is met during the production of pickled horse meat. A comprehensive analysis shows that the lack of nitrosamine formation may be related to pH, water activity aw, and fat content. Secondary amines, as precursors of nitrosamines, require an acidic environment (pH < 5) for nitrosation, and the optimal pH for nitrosation is about pH 3.0. The pH of the pickling solution for pickled horse meat is generally around 4.5-5.5, and aw is relatively low, which can easily lead to the inhibition of nitrosamine synthesis. The raw material this time is horse hind leg meat. In the previous determination of basic indicators of horse meat, the fat content of horse hind leg meat was relatively low, which reduced the formation of nitrosamines caused by oxidation products formed by oil oxidation.

[0116] 2. Screening of nitrite-degrading strains and fermentation characteristics

[0117] By measuring the effects of process and formula on nitrite, it was found that the formula and process had a significant impact on the residual nitrite, and the control effect on nitrite was small after taking into account the sensory organs. According to the literature, lactic acid bacteria have a good nitrite degradation effect and can add flavor to meat products. Therefore, this experiment uses the following strains to further reduce nitrite and nitrosamines, with an inoculation size of 1×10 8 Since the strains had different degradation abilities in meat products and culture medium, we chose to directly put the edible fungi into pickled horse meat as the initial screening condition for the strains (see the flowchart for details). Fig.12 ). During the experiment, it was found that when the strain was added, the fermentation temperature exceeded 30°C, and the pickled horse meat produced had an unpleasant flavor, while the nitrite degradation effect was not obvious when the temperature was below 20°C, so 25°C was selected as the fixed temperature for pickling horse meat to carry out the experiment.

[0118] Process steps:

[0119] (1) Thaw, cut and sort the quick-frozen or fresh horse meat that meets the quarantine and processing requirements for later use;

[0120] (2) Wash and cut the horse meat into pieces (4 x 8 cm), drain and weigh them for later use;

[0121] (3) adding ingredients (3.3% salt, 4.3% sucrose, 0.4% pepper and 0.15% nitrite), purified water and composite bacteria according to the screened optimal formula, stirring and mixing, and then fermenting and pickling;

[0122] (4) After the fermentation is completed, the product is baked and dried, packaged and sterilized, and the obtained product is ready-to-eat fermented pickled horse meat.

[0123] Wa: Wickerhamomyces anomalus, purchased from Linyi Xiaolu Biotechnology Co., Ltd.

[0124] Kt: Kluyveromyces thermotolerans, purchased from Angel Technology Co., Ltd.

[0125] Mt: Angel Saccharomyces cerevisiae, purchased from Angel Technology Co., Ltd.;

[0126] Kl: Kluyveromyces lactis, with the deposit number of CICC 32428, purchased from China Industrial Microbiological Culture Collection Center;

[0127] Pg: Pichia guilliermondii, purchased from Beina Biotechnology Co., Ltd.

[0128] Mp: Metschnikowia pulcherrima, purchased from Linyi Xiaolu Biotechnology Co., Ltd.

[0129] Td: Torulaspora delbrueckii, purchased from Linyi Xiaolu Biotechnology Co., Ltd.

[0130] LP: Lactobacillus plantarum, with the deposit number of CICC 24936, purchased from China Industrial Microbiological Culture Collection Center;

[0131] GL: Lactobacillus casei, with the accession number of CICC 6112, purchased from China Industrial Microbiological Culture Collection Center;

[0132] DS: Lactobacillus delbrueckiisubsp.lactis, purchased from Linyi Xiaolu Biotechnology Co., Ltd.

[0133] LYS: Lactobacillus reuteri, with the collection number of CICC 6123, purchased from China Industrial Microbiological Culture Collection Center;

[0134] LA: Lactobacillus acidophilus, purchased from Linyi Xiaolu Biotechnology Co., Ltd.;

[0135] BV: Bacillus coagulans, purchased from Linyi Xiaolu Biotechnology Co., Ltd.;

[0136] SLT: Lactobacillus rhamnosus, with the accession number CICC 6137, purchased from China Industrial Microbiological Culture Collection Center;

[0137] 2.1 Preliminary screening of strains

[0138] Depend on Fig.13 It can be seen that among the 14 strains screened, four strains, Mt, LP, LYS, and SLT, showed good nitrite reduction and control effects. Compared with lactic acid bacteria, the addition of yeasts such as Wa, Kt, Kl, Pg, MP, and Td increased the residual nitrite, and the pickled horse meat with the addition of the above yeasts had a poor flavor. Based on the initial screening results of the strains, four strains, Mt, LP, LYS, and SLT, were selected for compounding.

[0139] like Fig.14 As shown in the figure, the four strains screened were tested for nitrate resistance to verify whether they are suitable for high-concentration nitrite environments. Different strains have their own unique biological and growth characteristics, and their absorbances will be different even if they are cultured under the same conditions. The results show that all four strains have good nitrite resistance and can be used as subsequent experimental strains.

[0140] like Fig.15 As shown in the figure, the four strains were compounded in a 1:1 ratio. The experimental results showed that the 1:1 ratio of SLY and LYS strains had the best effect on reducing nitrite in pickled horse meat. The pickled horse meat with the addition of Mt compound strains showed a rancid smell and poor flavor. The residual nitrite was 15.92 mg / kg when SLT and LYS strains were compounded in a 1:1 ratio, the residual nitrite was 15.85 mg / kg when LP and SLT strains were compounded in a 1:1 ratio, and the residual nitrite was 13.17 mg / kg when LP and LYS strains were compounded in a 1:1 ratio.

[0141] The three strains of lactic acid bacteria had a good effect on reducing nitrite. After the three strains were mixed in a ratio of 1:1:1, the residual nitrite in pickled horse meat was 13.01 mg / kg. Considering the effect and the production cost of the enterprise, LP and LYS strains were selected for subsequent experiments.

[0142] 2.2 Fermentation characteristics of strains

[0143] Determining the growth curve of the strain is helpful to study its growth characteristics and determine the optimal inoculation period. The growth and acid production curves of LP and SLT strains are as follows: Fig.16As shown. Lactic acid bacteria produce organic acids, mainly lactic acid, by consuming nutrients, which affects the pH value of the fermentation environment. As can be seen from the figure, both strains rapidly produce acid within 0-9h, and LP reaches a stable acid production rate after 18h, which is consistent with the growth curve of the strain. The low pH environment inhibits the growth of the strain, and the activity of the strain gradually weakens. The growth curves and acid production curves of the two strains enter the stable period after 9h. The three periods of growth and reproduction of the two strains are basically the same. 0-3h is the strain retardation period, which is a short-term adaptation process for the strain to adjust its own metabolic system to the new environment. 3-9h is the logarithmic period, and the OD value of the strain increases rapidly in a stable geometric series; after 9h, it enters the stable period. Due to the consumption of nutrients in the culture medium and the change of environmental pH caused by lactic acid bacteria acid production, the growth rate of the strain slows down; after 30h of culture, the two strains did not reach the decline period, and the optimal inoculation period of the two strains was 9-12h.

[0144] Depend on Fig.17 It can be seen that the two strains grow well when the NaCl concentration in the culture medium is 6%, and they have good activity when the NaCl concentration is lower than 6%. When the NaCl concentration is higher than 6%, the bacterial density of LP and LYS decreases rapidly, and the activity of the strains decreases significantly. The bacterial density of LP is slightly higher than that of LYS, indicating that the strain performance of LP is slightly higher than that of LYS, which is also demonstrated by the growth curve and acid production curve of the strain. Both strains decrease rapidly when the NaCl concentration is higher than 6%, and almost do not grow at NaCl concentrations of 10% and 12%. The acid production ability is affected when the NaCl concentration is higher than 6%. In a high-salt environment, osmotic pressure changes the intracellular ion balance, causing cell structure and physiological damage to microorganisms, and even causing cells to stop growing or die. The salt concentration required for this experiment is 3.3%. Both strains can be used for fermentation of pickled horse meat in terms of salt tolerance.

[0145] The results of nitrate resistance of the two strains are as follows Fig.18 As shown, the results show that both LP and LYS strains have good nitrate resistance and can survive well in the medium with 0mg / kg to 250mg / kg nitrite addition. As the nitrite concentration increases, the OD values ​​of the two strains change slightly, and the acid production curve trend is gentle. In this experiment, the amount of nitrite added to pickled horse meat is 150mg / kg, and both strains are suitable for the growth of pickled horse meat.

[0146] During the pickling process, lactic acid bacteria will continuously consume nutrients to generate organic acids, causing the pH of the fermentation environment to drop. Therefore, it is necessary to select strains with good acid resistance so that they can maintain good growth in an acidic environment. Fig.19It can be seen that the two lactic acid bacteria have poor vitality in the environment of pH=3, among which LYS grows well in the environment of pH4-8, and the OD value reaches the maximum at pH=6; LP grows well in the environment of pH5-8, and the OD value reaches the maximum at pH=7; at pH5-8, LP has higher vitality than LYS. The growth situation corresponds to the pH value. At pH=3, the strain does not grow, so the pH value remains unchanged. When the pH is suitable for the growth of the strain, the strain grows and metabolizes to accumulate organic acids to reduce the pH value of the culture medium, making the pH value of the culture medium lower than the initial pH value.

[0147] Depend on Fig. 20 It can be seen that both LP and LYS strains have good nitrite degradation capabilities, with degradation capabilities of more than 90%, and the residual nitrite gradually decreases with the extension of strain culture time. The nitrite degradation rate of the two strains showed a rapid growth trend within 4 to 20 hours. After 20 hours of fermentation, the nutrients and nitrite residual content of the culture medium decreased, and the nitrite degradation rate decreased. Among them, the nitrite degradation rate of the LP strain in 24 hours was 98.54% ± 0.39, and the nitrite degradation rate of the LYS strain in 24 hours was 91.73 ± 1.09. LP showed a strong degradation advantage.

[0148] Biogenic amines are indirect precursors of nitrosamines, which are formed by decarboxylation of amino acids under the action of amino acid decarboxylase, so decarboxylase-negative bacteria need to be selected as fermentation agents. The results of the amino acid decarboxylase test of the two strains are shown in Table 11. As can be seen from Table 11, both Lactobacillus plantarum and Lactobacillus reuteri are negative, which meets the standards for meat product fermentation agents. Nitrate reductase promotes nitrite production. As can be seen from the table, both Lactobacillus plantarum and Lactobacillus reuteri are negative. The free amino acids generated by protein hydrolysis can give fermented meat products a unique flavor. As can be seen from the table, both Lactobacillus plantarum and Lactobacillus reuteri have protease activity.

[0149] Table 11 Fermentation characteristics of strains

[0150]

[0151] Note: + indicates detected; - indicates not detected.

[0152] 2.3 Reduction and control of nitrite and nitrosamine in pickled horse meat by room temperature mixed bacteria fermentation

[0153] Effect of bacterial strain ratio on residual nitrite in pickled horse meat Fig.21As shown, inoculation of lactic acid bacteria can effectively reduce the residual nitrite. When the compound ratio is 1:1, the residual nitrite is the highest at 13.31±0.40mg / kg, and when the compound ratio is 2:1, the residual nitrite is the lowest at 8.64±0.23mg / kg, and the nitrite reduction effect is the best. When the proportion of LP strains is larger, the degradation effect is better, which is consistent with the growth characteristics and acid resistance characteristics measured previously. LP strains have better activity and stronger acid production ability. The degradation of nitrite by lactic acid bacteria is divided into two stages: nitrite reductase degradation and acid degradation. When pH>4.5, lactic acid bacteria mainly degrade by nitrite reductase; when pH<4.0, the degradation of nitrite is mainly acid degradation. The pickling environment of this experiment was pH4.0-4.5, which may be due to the presence of nitrite reductase in LP and LYS strains, resulting in a decrease in nitrite residues.

[0154] Effect of inoculum size on residual nitrite in cured horse meat Fig. 22 As shown in the figure, as the amount of strain added increased, the residual nitrite in pickled horse meat decreased. 8 When the pH of pickled horse meat was lower than 4.0, the horse meat tasted sour and the meat was too soft and rotten, so the inoculation amount was selected to be 10 8 CFU / mL, at this time the residual nitrite is 9.39±0.13mg / kg. As the inoculation amount increases, metabolites continue to accumulate and the pH gradually decreases. The acid produced during the fermentation process can inhibit the growth of nitrite-reducing bacteria and gradually reduce the nitrite content by combining with hydrogen ions to form nitrous acid. Nitrous acid then undergoes a disproportionation reaction to produce nitrogen dioxide, ultimately reducing the nitrite content.

[0155] Effect of curing time on residual nitrite in cured horse meat Fig.23 As shown in the figure, the residual nitrite gradually increased with the extension of time during the 12-20h process, and a nitrite peak appeared at 20h of pickling, after which the nitrite content decreased. It may be because the pickling liquid did not completely penetrate into the meat at the beginning, and the osmotic equilibrium was reached at 20h, resulting in an increase in the nitrite content. In addition, the nitrite-reducing bacteria carried in the meat reduced and accumulated nitrite, and then the nitrite content decreased as LP and LYS lactobacilli multiplied. After 24 hours of inoculation, there was no significant difference in the residual nitrite, so the pickling time of 24h was selected as the optimal condition, at which time the residual nitrite was 5.82±0.05mg / kg.

[0156] 2.4 Changes in physical and chemical indicators before and after optimization of fermentation conditions

[0157] The moisture content and pH value of pickled horse meat are shown in Table 12. After adding Lactobacillus plantarum and Lactobacillus reuteri, the moisture content and pH decreased slightly. During the pickling process, lactic acid bacteria produced acid during the fermentation process, which led to protein denaturation, reduced water holding capacity, and decreased pH. After fermentation with Lactobacillus plantarum and Lactobacillus reuteri, its brightness and redness values ​​increased. The texture is affected by many factors. It can be seen from Table 12 that the hardness decreased after adding Lactobacillus plantarum and Lactobacillus reuteri, which may be related to the change in pickling time. The TBARS value can reflect the secondary oxidation of fat, that is, the final oxidation degree, indicating that the addition of Lactobacillus plantarum and Lactobacillus reuteri can help improve the antioxidant properties of pickled horse meat, thereby delaying rancidity.

[0158] Table 12 Changes in physical and chemical indicators of pickled horse meat before and after optimization

[0159]

[0160] Note: Control group: pickled horse meat without adding bacteria according to the optimal response surface result; Optimized group: pickled horse meat with added bacteria after optimization.

[0161] The electronic nose was used to detect subtle changes in volatile flavor compounds between the control group and the experimental group. Figure 24-25 It can be seen that the response values ​​of pickled horse meat samples to sensors such as WIC, W3S, W2W, W2S, and W5S are relatively high, and the flavor profiles of the two samples are slightly different, indicating that the addition of microorganisms causes a slight change in the types of volatile flavor substances. After adding Lactobacillus plantarum and Lactobacillus reuteri, the response values ​​of the three sensors W2W, WIW, and W5S changed significantly, while the changes of other sensors were not obvious. This shows that the volatile substance content of the optimized pickled horse meat has increased significantly in aromatic components, organic sulfide sensitivity, and nitrogen oxides. The volatile components reflect that Lactobacillus plantarum and Lactobacillus reuteri have a certain contribution to the improvement of the flavor of pickled horse meat.

[0162] According to the formula in the previous invention patent authorized by this team (CN 101099575 B): 6% salt, 20% sugar, 0.4% phosphate, 0.003% nitrite, 0.5% sodium ascorbate, 0.6% white pepper, 0.15% sage, 0.1% nutmeg, 0.1% cinnamon, 0.3% coriander, 0.15% basil and 3% bacteria, wherein the bacteria consisted of 2% lactic acid bacteria and 1% mold; fermented horse meat was prepared, and the residual nitrite content was measured to be 21.14±0.21 (mg / kg), and the nitrosamine content was 2.74±0.03 (μg / kg), both of which were lower than the national standard; but much higher than the content in the optimized horse meat product of the present invention.

[0163] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing ready-to-eat fermented pickled horse meat low in nitrite and nitrosamine, characterized in that: The following steps are involved: After the horse meat is cut into pieces, ingredients and composite bacteria are added to perform fermentation and pickling at room temperature, and after the fermentation and pickling are completed, the meat is baked and dried, packaged and sterilized to obtain ready-to-eat fermented and pickled horse meat; the ingredients include salt, sucrose, pepper and nitrite, and the composite bacteria are Lactobacillus plantarum and Lactobacillus reuteri; Among them, based on the weight of 100kg of horse meat, 2% to 6% of salt, 2% to 6% of sucrose, 0.2% to 0.4% of Sichuan pepper and 0.05% to 0.15% of nitrite are added.

2. The preparation method according to claim 1, characterized in that The ingredients are dissolved in water when used, and the material-water ratio of the ingredients to water is 1:2 to 1:

4.

3. The preparation method according to claim 1, characterized in that: The inoculation ratio of Lactobacillus plantarum to Lactobacillus reuteri in the composite bacteria is 1:1 to 3:1, and the total inoculation amount is 10 8 CFU / mL~10 9 CFU / mL; and / or the Lactobacillus plantarum and the Lactobacillus reuteri are both inoculated into the horse meat during the logarithmic growth phase of culturing for 9 to 12 hours.

4. The preparation method according to claim 1, characterized in that: The fermentation and pickling conditions are: fermentation and pickling at 20° C. to 30° C. for 20 to 24 hours, and an initial pH value of 4.0 to 4.

5.

5. The preparation method according to claim 4, characterized in that: The best fermentation and pickling time is 24 hours.

6. The preparation method according to claim 1, characterized in that: The baking and drying conditions are: baking temperature 60-180° C., and baking time 100-180 min.

7. The preparation method according to claim 6, characterized in that: The optimal baking temperature is 140℃ and the baking time is 120min.

8. The preparation method according to claim 1, characterized in that: The sterilization conditions are: sterilization at 121° C. for 15 to 25 minutes.

9. The preparation method according to claim 1, characterized in that: Based on 100 kg of horse meat, add 3.3% salt, 4.3% sucrose, 0.4% prickly ash and 0.15% nitrite.

10. The preparation method according to claim 1, characterized in that: The nitrite content of the instant fermented pickled horse meat is 5.23±0.09 mg / kg, and the instant fermented pickled horse meat does not contain nitrosamines.

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