Low-salt soy sauce meat based on probiotic fermentation and preparation method thereof

Abstract

The invention relates to the technical field of food processing, in particular to low-salt soy sauce meat based on probiotic fermentation and a preparation method thereof.The low-salt soy sauce meat is prepared from pork as a raw material through pretreatment, inoculation of a compound probiotic leavening agent (lactobacillus plantarum CGMCC No. 1.555 and staphylococcus carnosus CGMCC No. 1.274), vacuum rolling, kneading and pickling, stepped temperature control fermentation and low-temperature air drying and curing; the sodium chloride content is 2.0%-4.5%, the water activity is 0.80-0.84, the probiotics comprise 0.4%-0.7% of lactic acid and 0.08%-0.15% of acetic acid, and the total viable count of the two probiotics is not less than 1.0 * 10 CFU / g. According to the preparation method, lactobacillus plantarum CGMCC No.1.555 and staphylococcus carnosus CGMCC No.1.274 are taken as compound probiotic leavening agents, gradient temperature control fermentation is combined, sodium chloride of the soy sauce meat is reduced to 2.0%-4.5%, lactic acid and acetic acid are generated to construct a natural antibacterial system, and chemical preservatives are not needed; through processes of micropore puncture, vacuum intermittent rolling and kneading and the like, consistent indexes are ensured, two-stage fermentation and low-temperature air drying are easy to control, and the quality and flavor are improved.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a low-salt soy sauce meat based on probiotic fermentation and its preparation method. Background Technology

[0002] Soy sauce-braised meat, as a traditional cured product, holds an important position in the food consumption sector due to its unique flavor and long shelf life. However, existing soy sauce-braised meat production technology has significant shortcomings, making it difficult to meet modern consumer demands and industrialized production requirements. The main problems are concentrated in the following two aspects:

[0003] On the one hand, traditional processes require the addition of high-concentration sodium chloride (5% or more) to achieve preservation and flavor development. Long-term consumption of high-salt products can pose health risks, which is severely inconsistent with the current trend of low-salt diets. Furthermore, most products rely on chemical preservatives to extend shelf life, raising not only safety concerns but also potentially masking the product's natural flavor, resulting in a monotonous flavor profile. Some attempts to reduce salt content, however, lack effective natural antibacterial methods, generally facing the technical contradiction of easy spoilage and significantly shortened shelf life, failing to achieve a balance between health benefits and shelf life.

[0004] On the other hand, the existing production process has a low degree of standardization. The raw material marinating stage mostly adopts static marinating or simple tumbling, which easily leads to uneven flavoring and large fluctuations in the absorption efficiency of the inoculum. This results in significant differences in key indicators such as salt content and viable count of functional bacteria between different batches of products, and poor quality stability. The fermentation process is mostly a crude control under single temperature and humidity conditions, without clear criteria for determining the fermentation endpoint. It relies on the experience of operators, which leads to high risk of process fluctuations and high operational difficulty. In the air-drying and maturation stage, improper control of temperature, humidity and wind speed often leads to hardening of meat and loss of nutrients, which further affects the taste and quality of the product. Moreover, the overall process is difficult to adapt to large-scale industrial production, limiting its industrial application. Summary of the Invention

[0005] Technical problems to be solved

[0006] To address the aforementioned shortcomings of existing technologies, this invention uses *Lactobacillus plantarum* CGMCC No. 1.555 and *Staphylococcus aureus* CGMCC No. 1.274 as a compound probiotic starter, combined with stepped temperature-controlled fermentation, to reduce the sodium chloride content of soy sauce meat to 2.0%–4.5%, generating lactic acid and acetic acid to construct a natural antibacterial system, eliminating the need for chemical preservatives. Through processes such as microporous puncture and vacuum intermittent tumbling, consistent indicators are ensured, and the two-stage fermentation and low-temperature air drying are easily controlled, improving quality and flavor.

[0007] Technical solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] On the one hand, there is a low-salt soy sauce meat based on probiotic fermentation, which is prepared by pre-treating pork raw materials, inoculating with compound probiotic fermentation agents, vacuum tumbling and marinating, step-controlled temperature fermentation, and low-temperature air drying and maturation.

[0010] The compound probiotic starter consists of Lactobacillus plantarum CGMCC No. 1.555 and Staphylococcus aureus CGMCC No. 1.274;

[0011] The composition of the soy sauce meat includes, by weight percentage: 2.0%–4.5% sodium chloride, a water activity (Aw) value of 0.80–0.84, 0.4%–0.7% lactic acid, and 0.08%–0.15% acetic acid, and the total viable count of *Lactobacillus plantarum* and *Staphylococcus carotidum* is not less than 1.0 × 10⁻⁶. 6 CFU / g.

[0012] Furthermore, the ratio of viable Lactobacillus plantarum to Staphylococcus aureus is (2:1) to (3:1).

[0013] Furthermore, the soy sauce-braised meat has a pH value of 5.3 to 5.6 and contains no chemical preservatives.

[0014] On the other hand, a method for preparing low-salt soy sauce meat based on probiotic fermentation includes the following steps:

[0015] Step 1: Raw material pretreatment: Select qualified pork hind leg or pork belly, trim and cut into uniform strips, and pierce micro-holes on the surface of the strips.

[0016] Step 2: Preparation of low-salt marinade: Add brewed soy sauce, white sugar, spice extract and salt to drinking water at 2℃~5℃ in sequence, stir to dissolve, and prepare low-salt marinade; the amount of salt added is 2.0%~4.0% of the weight of the meat raw material.

[0017] Step 3, Inoculation and Vacuum Dynamic Marinating: Lactobacillus plantarum and Staphylococcus aureus are mixed in a specific ratio to form a compound probiotic starter. After activation, the compound probiotic starter is inoculated into the low-salt marinating solution prepared in Step 2 and mixed thoroughly to obtain an inoculation solution. The inoculation amount of the compound probiotic starter is 1.0% to 2.5% of the meat raw material mass. The meat strips treated in Step 1 and the inoculation solution are placed in a vacuum tumbler and intermittently tumbled for 2.5 to 4.0 hours under vacuum conditions of -0.07 MPa to -0.09 MPa and a temperature of 2℃ to 5℃, until the meat strip absorption rate reaches 18% to 22%.

[0018] Step 4, Step-by-Step Temperature-Controlled Fermentation: Hang the tumbled meat strips and proceed with two stages of fermentation as follows:

[0019] First stage: Ferment for 20-28 hours at a temperature of 30℃~32℃ and a relative humidity of 88%~92%;

[0020] Second stage: Ferment for 60-72 hours at a temperature of 20℃~22℃ and a relative humidity of 78%~82%;

[0021] Step 5: Low-temperature air drying and maturation: Transfer the fermented meat strips into an air drying room with an ambient temperature of 12℃~14℃, relative humidity of 65%~68%, and wind speed of 2.0m / s~2.5m / s. Air dry for 6~8 days until the water activity drops to 0.80~0.84, and the finished product is obtained.

[0022] Furthermore, in step two, the composition of the low-salt marinade, based on the percentage of the meat raw material's mass, includes: 3.0% to 5.0% brewed soy sauce, 0.8% to 1.5% white sugar, 0.2% to 0.4% spice extract, and 2.5% to 3.5% salt.

[0023] Furthermore, in step two, the spice extract is a mixture of star anise essential oil and Sichuan pepper essential oil obtained by supercritical CO2 extraction in a mass ratio of (3:1) to (5:1).

[0024] Furthermore, in step three, the intermittent tumbling process involves rotating clockwise for 15 minutes, pausing for 8 minutes, then rotating counterclockwise for 15 minutes, and repeating this cycle.

[0025] Furthermore, in step four, the endpoint of the first stage of fermentation is determined by the following criteria: the pH value of the fermentation system drops to 5.4-5.5, and there is uniform mucus exudation on the surface of the meat strips, emitting a rich aroma of wine and fruit esters.

[0026] Beneficial effects

[0027] Compared with known public technologies, the technical solution provided by this invention has the following beneficial effects:

[0028] I. This invention combines a compound probiotic fermentation agent (Lactobacillus plantarum CGMCC No. 1.555 and Staphylococcus aureus CGMCC No. 1.274) with a stepped temperature-controlled fermentation process to reduce the sodium chloride content of the finished product to 2.0%–4.5%, significantly meeting the needs of modern low-salt diets. Simultaneously, the probiotic fermentation process directionally generates 0.4%–0.7% lactic acid and 0.08%–0.15% acetic acid. Combined with low-temperature air-drying and ripening to control the water activity to 0.80–0.84, a natural antibacterial system is constructed, ensuring stable product preservation without the need for any chemical preservatives. Furthermore, by precisely controlling the fermentation endpoint (pH 5.3–5.6, uniform mucus precipitating on the surface of the meat strips, and the release of wine and fruit ester aromas), the probiotic metabolites synergistically enhance the flavor substances of brewed soy sauce and spice extracts (a blend of star anise oil and Sichuan pepper oil in a 3:1–5:1 ratio), enriching the product's flavor profile while reducing salt content.

[0029] Second, this invention employs a microporous piercing design on the surface of meat strips during the raw material pretreatment stage, combined with an intermittent tumbling process at a vacuum of -0.07MPa to -0.09MPa and a temperature of 2℃ to 5℃ during the marinating stage. This allows for precise control of the inoculum absorption rate to 18% to 22%, ensuring that the salt content and probiotic live bacteria count (total amount not less than 1.0 × 10⁻⁶) of each batch of product are within acceptable limits. 6 The consistency of key indicators such as CFU / g significantly improves product quality stability. Furthermore, this invention breaks down the fermentation process into a two-stage controllable process (stage 1: fermentation at 30℃~32℃ and 88%~92% relative humidity for 20~28h; stage 2: fermentation at 20℃~22℃ and 78%~82% relative humidity for 60~72h). The invention also clearly defines the criteria for determining the end point of the first stage fermentation (pH 5.4~5.5 + surface mucus + characteristic aroma), reducing the difficulty of process operation. Simultaneously, the low-temperature air-drying and ripening parameters (12℃~14℃ temperature, 65%~68% relative humidity, and 2.0~2.5m / s wind speed) can be precisely controlled using conventional industrial air-drying equipment, avoiding meat hardening and nutrient loss caused by high-temperature processing. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments thereof. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] The present invention will be further described below with reference to embodiments.

[0032] I. Preparation of Raw Materials and Equipment

[0033] (I) Raw material selection and pretreatment specifications

[0034] The pork used is selected from qualified pork hind leg or pork belly, meeting the requirements of the National Food Safety Standard for Fresh (Frozen) Livestock and Poultry Products GB2707-2016. The meat must be fresh, free of tendons, odor, visible blood clots, and fat spots. Specifically, the fat content of the pork hind leg is controlled at 15%–20% to ensure firmness and prevent the finished product from being too greasy; the fat content of the pork belly is controlled at 25%–30% to balance texture and flavor release. During trimming, stainless steel knives are used to remove subcutaneous connective tissue, tendons, and visible blood vessels, cutting the meat into uniform strips with dimensions of 20cm ± 1cm in length, 5cm ± 0.5cm in width, and 3cm ± 0.3cm in thickness. This ensures even heating and consistent water loss during subsequent fermentation and air-drying. Using a stainless steel piercing needle with an aperture of 1mm ± 0.1mm, micropores are pierced into the surface of the meat strips in a crisscross pattern along the length and width directions, with a spacing of 2cm ± 0.2cm between the micropores. Each meat strip is pierced with at least 30 micropores, and the piercing depth is 1 / 2 to 2 / 3 of the meat strip's thickness. This avoids piercing the meat strip, which could lead to loss of the marinade or seepage of juices during fermentation. The purpose is to disrupt the dense structure of the meat tissue, providing a channel for the marinade to penetrate and for beneficial bacteria to colonize the meat. The processed meat strips are placed on a sterile stainless steel tray and refrigerated at 4℃ ± 1℃ for no more than 2 hours to prevent the slow growth of microorganisms at low temperatures from affecting the freshness of the raw material.

[0035] The compound probiotic starter culture is composed of *Lactobacillus plantarum* (strain number CGMCC No. 1.555) and *Staphylococcus aureus* (strain number CGMCC No. 1.274). Both strains are sourced from the China General Microbiological Culture Collection Center (CGMCC), ensuring clear traceability, stable activity, and no pathogenicity risk. The activation medium is MRS liquid medium, composed of 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 5 g / L sodium acetate, 2 g / L diammonium citrate, 1 mL / L Tween-80, 0.58 g / L magnesium sulfate, and 0.25 g / L manganese sulfate. The pH of the medium is adjusted to 6.2 ± 0.2. Both strains were inoculated separately into MRS liquid medium and activated by static incubation at 37℃±1℃ for 24h±1h. After activation, the viable count was determined using the plate count method according to GB4789.2-2022, "National Food Safety Standard for Microbiological Examination of Food: Determination of Total Colony Count". The viable count of *Lactobacillus plantarum* must be no less than 1.0 × 10⁻⁶. 9 CFU / mL, Staphylococcus aureus viable count not less than 5.0 × 10⁻⁶ 8CFU / mL. Mix the two activated bacterial solutions at a live bacteria ratio of 2:1 to 3:1. The mixing process should be carried out in a Class 100 aseptic operating table. Use a sterile pipette to accurately measure the bacterial solution to avoid contamination by external bacteria. The mixed compound probiotic fermentation agent should be used for inoculation immediately. If it needs to be temporarily stored due to process connection, it should be placed in a refrigerated environment at 4℃±1℃ and the storage time should not exceed 2 hours to prevent the activity of the strain from decreasing due to prolonged low temperature.

[0036] Low-salt brine raw materials

[0037] Brewed soy sauce: High-salt, dilute-state fermented soy sauce is selected, which meets the requirements of Grade I of "Brewed Soy Sauce" GB / T18186-2000. The amino acid nitrogen content is not less than 1.2g / 100mL to ensure the basic umami flavor of the finished product. Artificial umami agents are avoided, and no caramel coloring or preservatives are added to prevent exogenous additives from affecting the fermentation efficiency of probiotics or changing the flavor of the finished product.

[0038] White sugar: Selected premium white sugar conforming to GB / T317-2018 "White Sugar", with a purity of not less than 99.5% and a sucrose content of not less than 99.6%, providing a carbon source for probiotic fermentation and promoting the reproduction of strains and the generation of metabolites.

[0039] Salt: Iodized refined salt conforming to the "Edible Salt" standard GB / T5461-2020 is selected, with a sodium chloride content of not less than 99.1% and a moisture content of not more than 0.5%. The low salt range is precisely controlled to avoid the high salt environment inhibiting the activity of probiotics.

[0040] Spice Extract: Prepared using supercritical CO2 extraction, the raw materials are freshly harvested star anise and Sichuan peppercorns free from mold. The star anise is pulverized to 40 mesh ± 5 mesh, and the Sichuan peppercorns are seeded and pulverized to 40 mesh ± 5 mesh to ensure sufficient contact between the raw materials and the extractant, thereby improving the extraction efficiency of the active ingredients. The extraction process parameters are set as follows: extraction pressure 30 MPa ± 2 MPa, extraction temperature 45℃ ± 2℃, CO2 flow rate 20 L / h ± 2 L / h, and extraction time 2 h ± 0.2 h. The star anise essential oil and Sichuan peppercorn essential oil are collected separately and mixed evenly at a mass ratio of 3:1 to 5:1 to obtain the spice extract, which enhances the flavor complexity of the finished product and helps extend the shelf life by utilizing the natural antibacterial properties of the spice components.

[0041] The spice extract should be stored in a brown, light-proof, airtight bottle at 4℃±1℃. The shelf life is no more than 6 months. Before use, it should be brought to room temperature and shaken well to ensure that the components in the extract are evenly dispersed and to avoid local flavors that are too strong or too weak.

[0042] Other auxiliary materials and drinking water must comply with the "Standards for Drinking Water Quality" GB5749-2022. Before use, they should be filtered through a 0.22μm microporous membrane to remove microorganisms from the water and prevent contamination of the pickling liquid or impact on the probiotic fermentation process. The purity of lactic acid and acetic acid standards should not be less than 99.0% and should be used for the physicochemical index testing of the finished product to ensure the accuracy of the test results.

[0043] (II) Equipment Selection and Parameter Calibration

[0044] The vacuum tumbler should be selected with a vacuum range of -0.10MPa to 0MPa, a temperature control range of 0℃ to 15℃, and an adjustable drum speed of 5r / min to 15r / min. Before use, the inner wall of the drum should be wiped and disinfected with a 75% ethanol solution, followed by a vacuum system leak test: the vacuum level should be reduced to -0.07MPa and left to stand for 30 minutes, ensuring that the vacuum level drop does not exceed 0.005MPa. This ensures a stable vacuum environment during tumbling and prevents residual air inside the meat strips from affecting the penetration of the marinade. The temperature control system must be calibrated with a standard thermometer to ensure that the error between the set temperature and the actual temperature does not exceed ±0.5℃, preventing temperature fluctuations from causing meat spoilage or premature inactivation of probiotics.

[0045] The fermentation chamber is equipped with a temperature control range of 5℃ to 60℃, a relative humidity control range of 40% to 95%, a temperature control accuracy of ±0.5℃, and a humidity control accuracy of ±2%. It also features a ventilation and stirring system to ensure uniform temperature and humidity distribution within the chamber, preventing inconsistent fermentation results due to localized environmental differences. Before use, it must be sterilized with 121℃ high-pressure steam for 30 minutes. After cooling, sterile air is introduced to room temperature. Fermentation parameters are then set according to process requirements to prevent residual bacteria in the fermentation chamber from contaminating the meat strips and affecting the fermentation process.

[0046] The drying chamber is custom-made from stainless steel, offering excellent corrosion resistance and easy cleaning. It is equipped with a temperature sensor with an accuracy of ±0.1℃, a humidity sensor with an accuracy of ±1%, a fan speed regulator adjustable from 0.5m / s to 3.0m / s, and a three-stage air filtration system (pre-filter + medium-efficiency filter + high-efficiency filter, with a high-efficiency filter membrane pore size of 0.3μm) to remove microorganisms and impurities from the air. The airflow organization is designed with top supply and bottom exhaust to ensure that the wind speed uniformity error does not exceed ±0.2m / s, guaranteeing even evaporation of moisture from the meat strips and preventing localized shrinkage or mold growth. Before use, the meat strips must be disinfected by irradiation with a 30W ultraviolet lamp at a height of 2m above the ground for 60 minutes, followed by 30 minutes of ventilation to prevent residual ultraviolet light from affecting the activity of probiotics.

[0047] Testing equipment

[0048] pH meter: accuracy ±0.01pH. It needs to be calibrated with pH 4.00 and pH 6.86 standard buffer solutions before use to ensure accurate pH measurement results.

[0049] Water activity meter: Measurement range 0.03 Aw to 1.00 Aw, accuracy ±0.003 Aw. Before use, it needs to be calibrated with saturated NaCl solution Aw=0.753 and saturated KCl solution Aw=0.842 to ensure the accuracy of water activity detection.

[0050] Microbial incubator: Temperature control range 5℃ to 60℃, accuracy ±0.5℃, used for strain culture in the process of determining the number of live probiotics.

[0051] High-performance liquid chromatograph: Equipped with a UV detector, the chromatographic column is a reversed-phase column suitable for the detection of organic acids (such as 4.6mm×250mm, 5μm specification), used for the determination of lactic acid and acetic acid content. Before detection, a system suitability test must be performed to ensure that the resolution of the target component is not less than 1.5, and to ensure the reliability of the organic acid content detection results. II. Specific Implementation Methods

[0053] Example 1

[0054] For raw material pretreatment, 10.0 kg of inspected and qualified pork hind leg meat was weighed and trimmed using a stainless steel knife to remove subcutaneous fat and connective tissue. The meat was then cut into strips 20 cm long, 5 cm wide, and 3 cm thick. Using a 1 mm diameter stainless steel piercing needle, micro-holes were pierced into the surface of each strip along both the length and width directions, with a spacing of 2 cm between the micro-holes. Each strip was pierced with 30 micro-holes. The processed meat strips were placed on a sterile stainless steel tray and refrigerated at 4℃ for 1.5 hours.

[0055] To prepare the low-salt marinade, take 2.0L of drinking water filtered through a 0.22μm microporous membrane and place it in a sterile stainless steel container. Lower the temperature of the drinking water to 2℃, and then add 300.0g of brewed soy sauce (3.0% of the meat raw material mass), 80.0g of white sugar (0.8% of the meat raw material mass), 20.0g of spice extract (star anise oil and Sichuan pepper oil in a 3:1 mass ratio, accounting for 0.2% of the meat raw material mass), and 200.0g of salt (2.0% of the meat raw material mass). Stir the mixture at 300r / min for 15min using a sterile stirrer until all ingredients are completely dissolved and there are no visible particles. The low-salt marinade is then prepared. The marinade temperature is maintained at 2℃, and the preparation time is 40min.

[0056] For inoculation and vacuum dynamic marinating, 100.0g of compound probiotic starter (Lactobacillus plantarum and Staphylococcus aureus live bacteria ratio of 2:1) was weighed out as 1.0% of the meat raw material weight and added to the above low-salt marinating solution. The mixture was stirred for 5 minutes with a sterile glass rod to ensure thorough mixing of the bacterial solution and the marinating solution, thus obtaining the inoculation solution. The pre-treated meat strips and the inoculation solution were placed together into the drum of a vacuum tumbler. The drum door was closed, and the vacuum system was activated to achieve a vacuum level of -0.07MPa. The tumbling temperature was set to 2℃, and an intermittent tumbling program was used (15 minutes forward rotation, 8 minutes pause, 15 minutes reverse rotation), repeated for 2.5 hours. During the tumbling process, the vacuum level and temperature were recorded every 30 minutes to ensure that the vacuum level remained stable at -0.07MPa±0.005MPa and the temperature remained stable at 2℃±0.5℃. After tumbling, three meat strips were randomly selected, and the absorption rate of the meat strips was determined by weighing (the absorption rate calculation formula is: absorption rate = mass of meat strip after tumbling - mass of meat strip before tumbling / mass of meat strip before tumbling × 100%). The result was 18%, which meets the process requirement of 18% to 22%.

[0057] For the stepped temperature-controlled fermentation, the tumbled meat strips are suspended in the fermentation chamber using sterile hooks, with a 5cm gap between them to prevent uneven fermentation due to contact. The fermentation chamber is started, with the first stage set at 30℃ and 88% relative humidity for 20 hours. During this period, the pH value of the fermentation system is measured every 4 hours (using a calibrated pH meter to detect the juice seeping from the surface of the meat strips). The first stage of fermentation is considered complete when the pH value drops to 5.4, and a uniform, transparent mucus appears on the surface of the meat strips (approximately 0.1mm thick), emitting a rich aroma of wine and fruit esters (without rancid, fishy, ​​or other off-odors). The second stage of fermentation then begins, with the chamber parameters adjusted to 20℃ and 78% relative humidity for 60 hours. During this period, samples are taken daily (three samples from different parts of each meat strip are mixed and tested). The MRS plate count method is used to determine the viable probiotic count, ensuring that the total viable probiotic count in each test result is not less than 1.0 × 10⁻⁶. 6 CFU / g.

[0058] After fermentation, the meat strips were transferred to a drying chamber and hung using sterile hooks, with an 8cm spacing between them to ensure adequate airflow contact. The drying chamber parameters were set at 12℃, 65% relative humidity, and 2.0m / s air velocity for 6 days. The water activity of the meat strips was measured daily (a sample from the center of each strip was crushed and tested using a calibrated water activity meter). Drying was stopped when the water activity dropped to 0.80, and the meat strips were removed and cooled to 25℃ to obtain the finished low-salt soy sauce meat.

[0059] Finished product indicator test results:

[0060] Sodium chloride content: determined according to the national food safety standard GB5009.44-2016 "Determination of Chloride in Food", the result was 2.0%, which meets the requirement of 2.0% to 4.5%.

[0061] Water activity: Determined according to the national food safety standard GB5009.238-2016 "Determination of water activity in food", the result was 0.80, which meets the requirements of 0.80 to 0.84.

[0062] Lactic acid content: The lactic acid content was determined by high performance liquid chromatography according to the national food safety standard GB5009.157-2016 "Determination of organic acids in food". The result was 0.4%, which meets the requirement of 0.4% to 0.7%.

[0063] Acetic acid content: The test method is the same as that for lactic acid content, and the result is 0.08%, which meets the requirement of 0.08% to 0.15%.

[0064] pH value: The pH value was determined according to the national food safety standard GB5009.237-2016 "Determination of pH value of food". The result was 5.3, which meets the requirements of 5.3 to 5.6.

[0065] Probiotic viable count: The total viable count of Lactobacillus plantarum and Staphylococcus aureus was determined using the MRS plate count method, and was 1.2 × 10⁻⁶. 6 CFU / g, not less than 1.0×10 6 Requirements for CFU / g.

[0066] Chemical preservatives: Common chemical preservatives were tested according to the standards such as "Determination of Benzoic Acid, Sorbic Acid and Sodium Saccharin in Food" GB5009.28-2016 and "Determination of Sodium Dehydroacetate in Food" GB5009.29-2016. No chemical preservatives were detected, which meets the requirement of no chemical preservatives.

[0067] Example 2

[0068] For raw material pretreatment, 15.0 kg of qualified pork hind leg meat was weighed, trimmed and cut to the same specifications as in Example 1, and the number of micro-holes pierced on the surface of the meat strip was 32 per strip. The processed meat strip was placed on a sterile stainless steel tray and refrigerated at 4°C for 1.8 hours.

[0069] To prepare the low-salt marinade, take 3.0L of drinking water filtered through a 0.22μm microporous membrane and place it in a sterile stainless steel container. Lower the temperature of the drinking water to 3℃, then add 525.0g of brewed soy sauce (3.5% of the meat raw material's mass), 187.5g of white sugar (1.25% of the meat raw material's mass), 45.0g of spice extract (star anise oil and Sichuan pepper oil in a mass ratio of 4:1, accounting for 0.3% of the meat raw material's mass), and 450.0g of salt (3.0% of the meat raw material's mass). Stir the mixture for 18 minutes at 350r / min using a sterile stirrer until all ingredients are completely dissolved to obtain the low-salt marinade. Maintain the marinade temperature at 3℃ and allow it to stand for 45 minutes.

[0070] For inoculation and vacuum dynamic marinating, 262.5g of compound probiotic starter (Lactobacillus plantarum to Staphylococcus aureus live bacteria ratio of 2.5:1) was weighed out at 1.75% of the meat raw material weight and added to the low-salt marinating solution. The solution was stirred with a sterile glass rod for 6 minutes to obtain the inoculation solution. The meat strips and inoculation solution were placed in the drum of a vacuum tumbler. The vacuum system was activated to -0.08MPa, and the tumbling temperature was set to 3.5℃. The same intermittent tumbling program as in Example 1 was used, and the cycle was repeated for 3.2 hours. After tumbling, the absorption rate of the meat strips was measured, and the result was 20%, which met the process requirements.

[0071] For the stepped temperature-controlled fermentation, meat strips were suspended in the fermentation chamber using sterile hooks. The first stage was set at 31℃ and 90% relative humidity for 24 hours. Fermentation ended when the pH dropped to 5.45, the surface mucus of the meat strips was uniform, and the aroma was satisfactory. The second stage involved adjusting the fermentation chamber parameters to 21℃ and 80% relative humidity for 66 hours. During this period, the viable probiotic count was measured daily, and the results were consistently no less than 1.5 × 10⁻⁶. 6 CFU / g.

[0072] The parameters of the low-temperature air-drying and maturation drying chamber were set as follows: temperature 13℃, relative humidity 66.5%, and wind speed 2.25m / s, for 7 days. When the water activity of the meat strips dropped to 0.82, the air-drying was stopped, and the finished product, low-salt soy sauce meat, was obtained after cooling.

[0073] Finished product test results: Sodium chloride content 3.2%, water activity 0.82, lactic acid content 0.55%, acetic acid content 0.11%, pH value 5.45, total live probiotic count 2.5 × 10⁻⁶. 6 The CFU / g value showed no detectable chemical preservatives, and all specifications met the technical requirements of the claims of this invention.

[0074] Example 3

[0075] For raw material pretreatment, weigh 20.0 kg of qualified pork belly, remove excess fat during trimming (retaining a fat layer thickness of 0.5 cm), cut into strips 20 cm long, 5 cm wide, and 3 cm thick, and pierce the surface of the strips with 35 micro-holes per strip. Place the processed strips on a sterile stainless steel tray and refrigerate at 4℃ for 2 hours.

[0076] To prepare the low-salt marinade, take 4.0L of drinking water filtered through a 0.22μm microporous membrane and place it in a sterile stainless steel container. Lower the temperature of the drinking water to 5℃, and then add 1000.0g of brewed soy sauce (5.0% of the meat raw material mass), 300.0g of white sugar (1.5% of the meat raw material mass), 80.0g of spice extract (star anise oil and Sichuan pepper oil in a mass ratio of 5:1, accounting for 0.4% of the meat raw material mass), and 800.0g of salt (4.0% of the meat raw material mass). Stir the mixture at 400r / min for 20min using a sterile stirrer until all ingredients are completely dissolved to obtain the low-salt marinade. Maintain the temperature of the marinade at 5℃ and allow it to stand for 50min.

[0077] For inoculation and vacuum dynamic marinating, 500.0g of compound probiotic starter (Lactobacillus plantarum and Staphylococcus aureus live bacteria ratio of 3:1) was weighed out as 2.5% of the meat raw material weight and added to the low-salt marinating solution. The solution was stirred with a sterile glass rod for 8 minutes to obtain the inoculation solution. The meat strips and inoculation solution were placed in the drum of a vacuum tumbler. The vacuum system was activated to -0.09MPa, and the tumbling temperature was set to 5℃. An intermittent tumbling program was used for 4.0 hours. After tumbling, the absorption rate of the meat strips was measured, and the result was 22%, which met the process requirements.

[0078] The stepped temperature-controlled fermentation process involves suspending meat strips in the fermentation chamber using sterile hooks. The first stage is set at 32℃ and 92% relative humidity for 28 hours. Fermentation ends when the pH drops to 5.5, the surface mucus of the meat strips is uniform, and the aroma is satisfactory. The second stage involves adjusting the fermentation chamber parameters to 22℃ and 82% relative humidity for 72 hours, during which the viable probiotic count remains at least 2.0 × 10⁻⁶. 6 CFU / g.

[0079] The parameters of the low-temperature air-drying and maturation drying chamber were set as follows: temperature 14℃, relative humidity 68%, and wind speed 2.5m / s, for 8 days. When the water activity of the meat strips dropped to 0.84, the air-drying was stopped, and the finished product, low-salt soy sauce meat, was obtained after cooling.

[0080] Finished product test results: Sodium chloride content 4.5%, water activity 0.84, lactic acid content 0.7%, acetic acid content 0.15%, pH value 5.6, total live probiotic count 3.8 × 10⁻⁶. 6 The CFU / g concentration showed no detectable chemical preservatives, fully satisfying all the technical features of the claims of this invention.

[0081] III. Process Validation and Effect Analysis

[0082] (I) Verification of key process parameters

[0083] The validation of the ratio of compound probiotics was conducted to verify the effect of the ratio of viable Lactobacillus plantarum to Staphylococcus aureus on the quality of the finished product. Ratio gradients of 1:1, 2:1, 2.5:1, 3:1, and 4:1 were set. Other process parameters were as described in Example 2. The viable probiotic count, pH value, and flavor score of the finished product were measured. The results showed:

[0084] Ratio 1:1: Total live probiotic count is only 0.8 × 10⁻⁶ 6 CFU / g, not reaching at least 1.0×10 6 The required CFU / g and pH value are 5.8. If the pH value exceeds the range of 5.3 to 5.6, the finished product will have a bland flavor and no obvious fermentation aroma.

[0085] A 4:1 ratio resulted in meat strips with a surface mucus thickness exceeding 0.2mm, a slightly sour taste in the finished product, and an abnormal flavor. Furthermore, although the number of live probiotics reached 1.3 × 10⁻⁶, the quality was poor. 6 The CFU / g value did not meet quality requirements due to flavor defects.

[0086] Ratio 2:1 to 3:1: The total number of live probiotics is not less than 1.2 × 10⁻⁶. 6 The CFU / g concentration and pH value remained stable between 5.3 and 5.6. The finished wine exhibited a rich aroma of fruit esters and a harmonious flavor, verifying the rationality and necessity of the probiotic ratio range specified in this invention.

[0087] The parameters for the stepped temperature-controlled fermentation were validated by setting temperature and humidity gradients for the two stages of the fermentation process: Stage 1: temperatures of 28℃, 30℃, 32℃, and 34℃, and relative humidity of 85%, 88%, 92%, and 95%; Stage 2: temperatures of 18℃, 20℃, 22℃, and 24℃, and relative humidity of 75%, 78%, 82%, and 85%. Other parameters were as described in Example 2. The final product indicators were then measured. Results showed:

[0088] The first stage, at a temperature of 28℃ or a relative humidity of 85%, resulted in a pH value of only 5.9 after 20 hours of fermentation. This did not meet the requirement of a pH value of 5.4-5.5 at the end of the first stage, and the probiotic reproduction rate was slow, which prevented the subsequent second stage of fermentation from proceeding normally.

[0089] In the first stage, at a temperature of 34℃ or a relative humidity of 95%, the total number of bacterial colonies on the surface of the meat strips exceeded 5000 CFU / g, indicating contamination by other microorganisms. Some meat strips showed mold spots, and the finished product failed to meet the microbial safety standards.

[0090] The second stage, at a temperature of 18℃ or a relative humidity of 75%, results in a lower content of flavor compounds (such as esters and alcohols) in the finished product, a mild aroma, and a sensory score below 8 out of 10.

[0091] In the second stage, at a temperature of 24℃ or a relative humidity of 85%, the number of live probiotics decreased to 0.9 × 10⁻⁶. 6 The CFU / g level was below the standard, and the water activity of the finished product was likely to exceed the upper limit of 0.84, increasing the risk of mold growth.

[0092] Only when the temperature in the first stage is 30℃~32℃ and the relative humidity is 88%~92%, and the temperature in the second stage is 20℃~22℃ and the relative humidity is 78%~82%, the number of live probiotics, pH value, flavor and microbial safety of the finished product all meet the requirements, proving the scientificity and feasibility of the step-controlled temperature fermentation parameters of this invention.

[0093] The salt addition level was validated by setting salt addition gradients of 1.5%, 2.0%, 3.0%, 4.0%, and 5.0%, with other parameters following those in Example 2. The sodium chloride content, sensory quality, and microbiological indicators of the finished product were measured. Results showed:

[0094] Salt addition of 1.5%: During the curing process, the meat strips developed a putrid odor, the volatile basic nitrogen content exceeded 20mg / 100g, which did not meet the requirements for the freshness of the raw materials, and the total number of colonies in the finished product exceeded the standard, indicating insufficient microbial safety.

[0095] Salt addition of 5.0%: The sodium chloride content of the finished product reaches 5.8%, which exceeds the low salt range of 2.0% to 4.5%. The taste is too salty, and the sensory score is less than 7 points, which does not meet the positioning of low salt food.

[0096] Salt addition of 2.0% to 4.0%: The sodium chloride content of the finished product is stable between 2.0% and 4.5%, with a moderate taste. Moreover, the meat strips do not show signs of spoilage during the marinating and fermentation process, and the microbial indicators are qualified, which verifies the rationality of the salt addition range specified in this invention.

[0097] II) Product Performance Analysis

[0098] For sensory quality comparison, 10 professional sensory evaluators (5 males and 5 females, aged 25-45 years) who had passed the training in "General Guidelines for the Selection, Training and Management of Sensory Analysis Evaluators" GB / T16291.1-2012 were invited to conduct sensory evaluations on the finished products of Examples 1-3 and traditional soy sauce meat (sodium chloride content 7%, potassium sorbate added 0.05%). Evaluation indicators included color (uniformity of meat color, gloss), aroma (fermentation aroma, spice aroma, off-flavors), taste (saltiness, umami, aftertaste), and texture (hardness, elasticity, chewiness). Each indicator was scored out of 10, and the average value was taken. The results are as follows:

[0099]

[0100] Microbiological safety analysis was conducted on the finished products of Examples 1-3 according to the series of standards of "National Food Safety Standard for Microbiological Examination of Food".

[0101] Total bacterial count: determined according to GB4789.2-2022, all results were ≤3000 CFU / g, which meets the requirements of the National Food Safety Standard for Cooked Meat Products GB2726-2016.

[0102] Coliform bacteria: According to GB4789.3-2022, none were detected (<30 CFU / 100g).

[0103] Pathogenic bacteria: According to GB4789.10-2022 (Staphylococcus aureus) and GB4789.4-2022 (Salmonella), no pathogens were detected (Staphylococcus aureus <10 CFU / g, Salmonella not detected in 25g sample).

[0104] This proves that the product of this invention has high microbial safety and no risk of pathogenic bacteria contamination.

[0105] The shelf life test involved vacuum-packing the finished products from Examples 1-3 and traditional soy sauce meat using polyethylene film (0.08 mm thick) and placing them in an environment of 25℃±2℃ and 60%±5% relative humidity for a room temperature shelf life test. Sensory quality, physicochemical indicators, and microbiological indicators were measured periodically (0 days, 15 days, 30 days, 45 days, 60 days, and 75 days). The results showed:

[0106] Example 1 Finished product: No significant changes in sensory properties within 60 days, stable physicochemical indicators (sodium chloride, water activity, pH value), and qualified microbiological indicators.

[0107] Example 2 Finished product: Maintains good quality for 75 days, with all indicators meeting the requirements (longest shelf life).

[0108] Example 3 Finished product: The product meets the required standards within 65 days and shows no signs of spoilage or deterioration.

[0109] Traditional soy sauce meat: After 45 days, it developed a rancid odor and the total bacterial count exceeded 10,000 CFU / g, which is excessive and can no longer be consumed.

[0110] This invention demonstrates that by utilizing the antibacterial substances such as lactic acid and acetic acid produced during the fermentation process of probiotics, the shelf life of the product can be significantly extended without the addition of chemical preservatives, thus solving the technical pain point of traditional soy sauce meat relying on preservatives.

[0111] IV. Precautions and Quality Control

[0112] Raw material quality control

[0113] Pork raw materials: Each batch of raw materials must be provided with a quarantine certificate. Sampling tests must be conducted on the moisture content (≤77%) and volatile basic nitrogen (≤15mg / 100g). Only raw materials that meet the requirements of GB2707-2016 can be used. Unqualified raw materials are prohibited from being put into production.

[0114] Compound probiotic starter: The number of live bacteria needs to be tested for each batch. If the number of live Lactobacillus plantarum is less than 1.0 × 10⁻⁶, the result will be negative. 9 CFU / mL or Staphylococcus aureus viable count less than 5.0 × 10⁻⁶ 8 If the concentration is CFU / mL, the inoculum needs to be reactivated to ensure that its activity meets the standard.

[0115] Spices extracts: The content of volatile components is tested in each batch (anethole content ≥80% in star anise oil, limonene content ≥15% in Sichuan pepper oil). Products with substandard component content are prohibited from use to avoid affecting the flavor of the finished product.

[0116] Aseptic operation control

[0117] Personnel operation: Operators must wear sterile work clothes, gloves, and masks. Before entering the sterile operation area, they must pass through an air shower for 10 seconds and disinfect their hands with 75% ethanol to avoid contamination of raw materials or equipment by microorganisms carried by personnel.

[0118] Equipment cleaning and disinfection: Stainless steel trays, agitators, hooks and other utensils that come into contact with raw materials must be sterilized by high-pressure steam at 121℃ for 30 minutes before use; after each use, the inner wall of the vacuum tumbler should be cleaned with 2% sodium hydroxide solution, then rinsed with sterile water, and finally wiped with 75% ethanol for disinfection to prevent residual contaminants from affecting the next batch of production.

[0119] Environmental control: The cleanliness of the aseptic operating table is tested regularly (it must meet the Class 100 standard). The fermentation chamber and drying room are disinfected according to the prescribed procedures after each use to ensure that the production environment meets the aseptic requirements.

[0120] Process parameter monitoring

[0121] Vacuum tumbling stage: Record the vacuum degree and temperature every 30 minutes to ensure that the vacuum degree is stable between -0.07MPa and -0.09MPa and the temperature is stable between 2℃ and 5℃. If the parameters fluctuate, the equipment should be adjusted in time to avoid affecting the marinating effect.

[0122] The first stage of fermentation requires that the final stage of fermentation simultaneously meet three conditions: pH value of 5.4-5.5, uniform mucus on the surface of the meat strips, and qualified aroma. None of these conditions can be missing. In the second stage, temperature, humidity, and the number of live probiotics are recorded daily to ensure that the parameters meet the process requirements and to prevent over- or under-fermentation.

[0123] Low-temperature air-drying stage: Record temperature, humidity and wind speed every 8 hours, and measure the water activity of meat strips daily to avoid over-drying, which would cause the meat strips to become hard (water activity < 0.80) or under-drying, which would cause the meat strips to become moldy (water activity > 0.84).

[0124] Finished Product Inspection Control

[0125] Sampling rules: Each batch of finished products must be randomly sampled, with a sampling size of 0.5% of the total production of each batch, and no less than 5 samples, covering products from different production periods to ensure that the samples are representative.

[0126] Testing items: Full-item testing of sensory quality, physicochemical indicators (sodium chloride, water activity, lactic acid, acetic acid, pH value), microbiological indicators (total bacterial count, coliform bacteria, pathogenic bacteria, live probiotic count), and chemical preservatives. Only products that pass all tests can leave the factory. Unqualified products must be handled according to the prescribed procedures and are prohibited from entering the market.

[0127] Storage requirements: The finished product should be refrigerated at 4℃±2℃. The shelf life is 6 months under vacuum packaging. It should be consumed within 24 hours after opening to avoid microbial contamination and spoilage.

[0128] In summary, this invention, by selecting specific strains (Lactobacillus plantarum CGMCC No. 1.555 and Staphylococcus aureus CGMCC No. 1.274) to form a compound probiotic starter, and optimizing low-salt pickling, vacuum dynamic tumbling, step-controlled temperature fermentation, and low-temperature air-drying processes, successfully prepared a probiotic starter with a sodium chloride content of 2.0%–4.5%, no chemical preservatives, and a probiotic viable count of not less than 1.0 × 10⁻⁶. 6 This product is a low-sodium soy sauce meat product with a CFU / g concentration. This technology not only solves the technical shortcomings of traditional soy sauce meat, such as high salt content and reliance on preservatives, but also enhances the flavor profile and extends shelf life through probiotic metabolism.

[0129] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A low-salt fermented soy sauce-based meat product based on probiotic fermentation, characterized in that, The low-salt soy sauce meat is prepared from pork raw material through pretreatment, inoculation of compound probiotic starter, vacuum tumbling and pickling, stepwise temperature control fermentation and low-temperature air-drying curing. The compound probiotic starter is composed of Lactobacillus plantarum CGMCC No.1.555 and Staphylococcus carnosus CGMCC No.1.

274. The composition of the soy sauce meat comprises: sodium chloride 2.0%-4.5% by weight, water activity (Aw) value 0.80-0.84, lactic acid 0.4%-0.7%, acetic acid 0.08%-0.15%, and the total amount of viable bacteria of the Lactobacillus plantarum and Staphylococcus carnosus is not less than 1.0x10 6 CFU / g.

2. The low-salt fermented soy sauce-based meat of claim 1, wherein the low-salt fermented soy sauce-based meat is prepared by adding 0.1 to 1 wt% of the low-salt fermented soy sauce to 100 wt% of the meat. The ratio of the viable cell count of the Lactobacillus plantarum to that of the Staphylococcus carnosus is (2:1) to (3:1).

3. The low-salt fermented soy sauce meat based on probiotics according to claim 1, characterized in that, The pH value of the soy sauce meat is 5.3-5.6, and the soy sauce meat does not contain any chemical preservatives.

4. A method for preparing a low-salt fermented soy sauce meat based on probiotic fermentation, suitable for a low-salt fermented soy sauce meat based on probiotic fermentation according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: Step one, raw material pretreatment: select quarantine-approved pork ham or pork belly, trim and cut into uniform-sized strips, and puncture micro-holes on the surface of the meat strips; Step two, preparation of low-salt pickling solution: add brewing soy sauce, white granulated sugar, spice extract and salt in sequence into drinking water at 2-5 DEG C, stir and dissolve to prepare a low-salt pickling solution; the amount of salt added is 2.0-4.0% based on the mass of the meat raw material; Step three, inoculation and vacuum dynamic pickling: mix Lactobacillus plantarum and Staphylococcus carnosus in a certain ratio to prepare a compound probiotic starter, inoculate the compound probiotic starter into the low-salt pickling solution prepared in step two after activation, mix well to prepare inoculum, and the inoculation amount of the compound probiotic starter is 1.0-2.5% based on the mass of the meat raw material; place the meat strips treated in step one and the inoculum into a vacuum tumbling machine, and perform intermittent tumbling at a vacuum degree of-0.07 to-0.09 MPa and a temperature of 2-5 DEG C for 2.5-4.0 hours until the meat strip absorption rate reaches 18-22%; Step four, stepwise temperature control fermentation: hang the tumbled meat strips, and perform two-stage fermentation according to the following program: First stage: fermentation at a temperature of 30-32 DEG C and a relative humidity of 88-92% for 20-28 hours; Second stage: fermentation at a temperature of 20-22 DEG C and a relative humidity of 78-82% for 60-72 hours; Step five, low-temperature air-drying curing: move the fermented meat strips into an air-drying room at an ambient temperature of 12-14 DEG C, a relative humidity of 65-68% and an air speed of 2.0-2.5 m / s, and air-dry for 6-8 days until the water activity is reduced to 0.80-0.84 to obtain the finished product.

5. The method of claim 4, wherein the low-salt fermented soy sauce-based meat is prepared by adding 0.1 to 1 wt% of the low-salt fermented soy sauce to 100 wt% of the meat. In step two, the low-salt pickling solution comprises brewing soy sauce 3.0-5.0%, white granulated sugar 0.8-1.5%, spice extract 0.2-0.4% and salt 2.5-3.5% based on the mass percentage of the meat raw material.

6. The method of claim 4, wherein the low-salt fermented soy sauce-based meat is prepared by adding 0.1 to 1 wt% of the low-salt fermented soy sauce to 100 wt% of the meat. In step two, the spice extract is star anise essential oil and pepper essential oil obtained by supercritical CO2 extraction and compounded in a mass ratio of (3:1) to (5:1).

7. The method of claim 4, wherein the low-salt fermented soy sauce-based meat is prepared by adding 0.1 to 1 wt% of the low-salt fermented soy sauce to 100 wt% of the meat. In step three, the intermittent tumbling program is forward rotation for 15 minutes, pause for 8 minutes, and reverse rotation for 15 minutes, and the cycle is repeated.

8. The method of claim 4, wherein the low-salt fermented soy sauce-based meat is prepared by adding 0.1 to 1 wt% of the low-salt fermented soy sauce to 100 wt% of the meat. In step four, the end point determination standard of the first stage fermentation is that the pH value of the fermentation system is reduced to 5.4-5.5, and uniform mucus is excreted on the surface of the meat strips, and the meat strips emit rich wine aroma and ester aroma.

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