Method for fermenting smelly mandarin fish by inoculating with potassium chloride instead of salt

Abstract

The invention discloses a method for fermenting smelly mandarin fish by using potassium chloride to replace salt and inoculate bacteria, and belongs to the technical field of food fermentation processing. Potassium chloride is used for replacing part of sodium chloride, and the content of sodium chloride in the smelly mandarin fish can be remarkably reduced. Potassium chloride is added to replace sodium chloride, the metabolic activity of lactic acid bacteria in the inoculation fermentation process is effectively adjusted, the growth activity of microorganisms such as putrefying bacteria is inhibited, and the number of enterococcus, pseudomonas and hydrogen sulfide producing bacteria in each gram of fish meat in the fermentation process is obviously reduced. Potassium chloride is added to replace sodium chloride, accumulation of volatile basic nitrogen and biogenic amine in the fermentation process is inhibited, the volatile basic nitrogen is as low as 28.87 mg / 100g, putrescine is 137.45 mg / Kg, and tyramine is 18.34 mg / Kg.

Description

Technical Field

[0001] This invention relates to a method for fermenting stinky mandarin fish using potassium chloride as a salt substitute, specifically a method for preparing stinky mandarin fish by partially replacing sodium chloride with potassium chloride and combining it with inoculation of a compound fermentation agent, belonging to the field of food fermentation and processing technology. Background Technology

[0002] Mandarin fish (Siniperca chuatsi) has tender flesh and is rich in nutrients, making it widely used in high-end aquatic products and processed products. Stinky mandarin fish, a traditional fermented food made from mandarin fish, is a representative dish of Anhui cuisine. Its production process mainly involves short-term salting and fermentation. Through the synergistic action of microorganisms and endogenous enzymes, proteins are degraded into small peptides, amino acids, and various volatile flavor compounds, ultimately creating a unique sensory characteristic of "smelling pungent but tasting delicious," while also enhancing the umami flavor and nutritional value of the fish.

[0003] To improve fermentation efficiency and product quality, existing research has explored the application of starter cultures. For example, Chinese patent CN109402010A discloses a mixed starter culture using *Lactococcus lactis* M10 and *Weissella cibaria* M3. This technology can accelerate the fermentation process, promote the effective accumulation of flavor compounds, and inhibit the production of harmful substances such as biogenic amines. Another example is CN117511770A, which, by inoculating *Bacillus halophilus* SJ1-6 and *Staphylococcus aureus* F2, not only increased the content of the key flavor compound indole but also effectively inhibited the growth of spoilage bacteria.

[0004] However, current methods for preparing stinky mandarin fish generally rely on high-concentration salting processes, with salt content typically ranging from 6% to 12% (China Food Journal, 2019, 19(5):253-262). According to the World Health Organization (WHO), the daily salt intake for adults should be less than 5 grams. Taking stinky mandarin fish produced using this process as an example, consuming just 100 grams of fish could result in a salt intake exceeding 6 grams, easily surpassing the recommended daily limit. Long-term consumption of this could increase the risk of chronic diseases such as hypertension, cardiovascular disease, and stomach cancer. Therefore, developing low-salt or reduced-salt fermentation processes has become an inevitable trend in the development of the health food industry.

[0005] While reducing salt intake is imperative, salt plays a crucial role not only as a seasoning in the processing of stinky mandarin fish, but also in inhibiting spoilage microorganisms, ensuring product safety, and shaping the traditional flavor. Simply reducing the amount of salt can easily lead to fermentation failure, spoilage, and flavor deterioration, thereby posing serious food safety risks.

[0006] To address this challenge, some studies have attempted to partially replace sodium chloride with potassium salts (such as potassium chloride) in fermented meat products, achieving some salt reduction effects. However, research on the application of this strategy in fermented fish products is still limited, and the impact of potassium salt substitution on the unique flavor, safety, and microbial characteristics of fish products remains unclear. Especially in environments with reduced salt content, effectively controlling the accumulation of volatile basic nitrogen and biogenic amines to prevent product quality deterioration remains an unresolved challenge for this technological approach. Summary of the Invention

[0007] This invention aims to address the health risks posed by the excessively high salt content in existing stinky mandarin fish products, as well as the technical bottleneck of traditional salt reduction processes that struggle to balance product safety and flavor quality. To this end, this invention provides a novel method for fermenting and producing stinky mandarin fish by synergistically applying a compound fermenting agent and potassium chloride to partially replace sodium chloride.

[0008] The core technical idea of ​​this invention lies in utilizing a compound fermentation agent with specific functions, such as Lactococcus lactis M10 and Weissella asiatica M3, which have been previously proven to inhibit putrefactive bacteria. Through their biological activity during fermentation, these agents actively inhibit the growth of harmful microorganisms, thereby creating a safe microbial environment for reducing the concentration of pickling salt. Based on this, potassium chloride is introduced as a partial substitute for sodium chloride, which can significantly reduce the sodium content of the final product while maintaining the necessary osmotic pressure and saltiness.

[0009] The first technical solution provided by the present invention is a mixed fermentation agent containing L. lactis M10 and W. cibaria M3, which are deposited at the Guangdong Provincial Center for Microbial Culture Collection with accession numbers CGMCC 16612 and CGMCC 16611, respectively.

[0010] In some implementations, L. lactis M10 and W. cibaria M3 are picked from storage conditions at -80°C and revived using MRS solid medium at 30–37°C for 16–24 hours. Single colonies after revival are picked and inoculated into MRS broth liquid medium and incubated at 100–200 rpm, 37°C for 16–20 hours to obtain the desired bacterial cells.

[0011] The second technical solution provided by the present invention is a method for fermenting stinky mandarin fish. The method involves introducing the mixed fermentation agent described in the first technical solution into a fermentation system in which potassium chloride partially replaces sodium chloride, thereby fermenting the mandarin fish.

[0012] In some implementations, the following steps are included:

[0013] S1. Pre-treatment: Remove the scales, gills, and internal organs from the mandarin fish;

[0014] S2. Preparation of fermentation liquid: Weigh water equal to the weight of the mandarin fish obtained in step S1, add seasonings and the mixed fermentation agent described in the second technical solution to obtain fermentation liquid;

[0015] S3. Fermentation: The mandarin fish obtained in step S1 is soaked in the fermentation liquid obtained in step S2 to ferment, resulting in stinky mandarin fish.

[0016] In S2, the seasoning contains potassium chloride and sodium chloride, and the total amount of potassium chloride and sodium chloride added is 6 wt%.

[0017] In some embodiments, in step S3, the fermentation temperature is 12±1℃ and the fermentation time is 6 days.

[0018] In some embodiments, the total amount of the L. lactis M10 and W. cibaria M3 mixed bacteria added to the fermentation system is 1 × 10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g.

[0019] In some implementations, the ratio of viable bacteria counts of L. lactis M10 to W. cibaria M3 is 1:1.

[0020] In some embodiments, the seasoning specifically comprises, by weight of drinking water added, 5 wt% sodium chloride, 1 wt% potassium chloride, 0.4 wt% ginger, 0.2 wt% star anise, 0.06 wt% fennel, 0.06 wt% cumin, 0.03 wt% chili pepper, and 0.03 wt% Sichuan peppercorn.

[0021] The third technical solution provided by the present invention is a method for reducing the salt content in fermented mandarin fish. The method is to introduce a mixed fermentation agent into a fermentation system in which potassium chloride partially replaces sodium chloride, and then ferment the mandarin fish.

[0022] In some implementations, the following steps are included:

[0023] S1. Pre-treatment: Remove the scales, gills, and internal organs from the mandarin fish;

[0024] S2. Preparation of fermentation liquid: Weigh water equal to the weight of the mandarin fish obtained in step S1, add seasonings and the mixed fermentation agent described in the second technical solution to obtain fermentation liquid;

[0025] S3. Fermentation: The mandarin fish obtained in step S1 is soaked in the fermentation liquid obtained in step S2 to ferment, resulting in stinky mandarin fish.

[0026] In S2, the seasoning contains potassium chloride and sodium chloride, and the total amount of potassium chloride and sodium chloride added is 6 wt%.

[0027] In some embodiments, in step S3, the fermentation temperature is 12±1℃ and the fermentation time is 6 days.

[0028] In some embodiments, the total amount of the L. lactis M10 and W. cibaria M3 mixed bacteria added to the fermentation system is 1 × 10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g.

[0029] In some implementations, the ratio of viable bacteria counts of L. lactis M10 to W. cibaria M3 is 1:1.

[0030] In some embodiments, the seasoning specifically comprises, by weight of drinking water added, 5 wt% sodium chloride, 1 wt% potassium chloride, 0.4 wt% ginger, 0.2 wt% star anise, 0.06 wt% fennel, 0.06 wt% cumin, 0.03 wt% chili pepper, and 0.03 wt% Sichuan peppercorn.

[0031] Among them, the amount of sodium chloride used decreased by 16.67%.

[0032] The fourth technical solution provided by the present invention is the application of potassium chloride in improving the fermentation quality of stinky mandarin fish. The application is to use a mixed bacterial culture of L. lactis M10 and W. cibaria M3 as a fermentation agent and to replace 1 wt% sodium chloride with potassium chloride to ferment the mandarin fish.

[0033] In some embodiments, the improvement of the fermentation quality of stinky mandarin fish includes at least one of the following effects:

[0034] (1) Regulate the metabolic activity of microorganisms in fermented mandarin fish and inhibit the growth of putrefactive bacteria;

[0035] (2) Inhibit the accumulation of volatile basic nitrogen and biogenic amines during the bacterial fermentation of mandarin fish.

[0036] In some embodiments, the putrefactive bacteria include Enterococcus, Pseudomonas, and hydrogen sulfide-producing bacteria.

[0037] In some implementations, biogenic amines include putrescine and tyramine.

[0038] The present invention also provides a method for regulating the metabolic activity of microorganisms and inhibiting the growth of putrefactive bacteria in fermented mandarin fish. The method utilizes the fermentation method described in the third technical solution to inhibit the growth of putrefactive bacteria during the fermentation process of the mandarin fish and improve food safety.

[0039] The fifth technical solution provided by the present invention is a method for inhibiting the accumulation of volatile basic nitrogen and biogenic amines during the inoculation fermentation of stinky mandarin fish. The method uses the fermentation method described in the second technical solution to inhibit the accumulation of volatile basic nitrogen and biogenic amines and improve the safety of fermented food.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] (1) Replacing part of the sodium chloride with potassium chloride can significantly reduce the sodium chloride content in stinky mandarin fish.

[0042] (2) By adding potassium chloride to replace sodium chloride, the metabolic activity of lactic acid bacteria during the inoculation fermentation process is effectively regulated, and the growth activity of putrefactive bacteria and other microorganisms is inhibited. Among them, the number of enterococci, pseudomonas and hydrogen sulfide-producing bacteria per gram of fish meat is significantly reduced during the fermentation process.

[0043] (3) By adding potassium chloride to replace sodium chloride, the accumulation of volatile basic nitrogen and biogenic amines during fermentation was inhibited. The volatile basic nitrogen was as low as 28.87 mg / 100g, putrescine was 137.45 mg / Kg, and tyramine was 18.34 mg / Kg. Detailed Implementation

[0044] The following embodiments are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. The present invention mainly describes a method for fermenting stinky mandarin fish using potassium chloride as a salt substitute. The substitution of simple parameters in the embodiments cannot be described one by one in the embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions. Any person skilled in the art should be covered within the scope of protection of the present invention within the technical scope disclosed in the present invention.

[0045] Test method:

[0046] 1. The Na+ content in fish meat was determined using an atomic absorption spectrophotometer, following Method 1 of the national standard GB5009.91-2017 "Determination of Potassium and Sodium in Food", namely, flame atomic absorption spectrometry. The results are expressed as mg / 100g.

[0047] 2. Microorganisms in the stinky mandarin fish samples were cultured using the plate count method. Specific culture conditions were as follows: Fish heads were chopped in a clean bench. 5g of sample was placed in a sterile homogenizing bag, and 45mL of sterile physiological saline was added. The mixture was homogenized for 5 minutes. A gradient dilution was prepared using sterile physiological saline. 1mL of each appropriate dilution was dispersed on agar plates. Lactic acid bacteria were cultured on MRS agar at 37°C for two days; sulfur-producing bacteria were cultured on iron agar at 37°C for two days; Pseudomonas bacteria were cultured on CFC selective medium at 37°C for two days; and Enterococci were cultured on VRBA solid plates at 37°C for two days. Results are expressed as 1g CFU / g.

[0048] 3. The Kjeldahl method was used to determine the volatile basic nitrogen content in fish meat, following Method 3, namely the micro-diffusion method, in the national standard GB 5009.228-2016 "Determination of Volatile Basic Nitrogen in Food". The results are expressed as mg / 100g.

[0049] 4. The content of biogenic amines in fish meat was determined by high performance liquid chromatography (HPLC), following method one of the national standard GB 5009.208-2016 "Determination of Biogenic Amines in Food", i.e., liquid chromatography. The results are expressed as mg / Kg.

[0050] Raw materials used in the examples:

[0051] 1. Mandarin fish, seasonings, etc. were purchased from Changxing Market, Dalian City, Liaoning Province. After fermentation, the samples were stored at -20℃ for later use.

[0052] 2. MRS liquid culture medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH 5.7±0.2.

[0053] 3. MRS solid medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5 ± 0.2.

[0054] 4. Potassium chloride was purchased from Jiahe Xuri flagship store on Taobao.

[0055] 5. The Lactococcus lactis M10 and Weissella multocida M3 have been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession numbers CGMCC 16612 and CGMCC 16611 respectively, and have been disclosed in patent CN109402010A.

[0056] 6. Lactobacillus sakei CICC 22728 and Staphylococcus saprophyticus CICC 24371 were purchased from the China Industrial Microbial Culture Collection Center.

[0057] Example 1

[0058] L. lactis M10 and W. cibaria M3 were inoculated in a 1:1 ratio, with a total bacterial count of 10. 7 CFU / g 肉 The application of adding 5 wt% sodium chloride and 1 wt% potassium chloride in fermented stinky mandarin fish. The specific method is as follows:

[0059] S1. Preparation of the starter culture: L. lactis M10 and W. cibaria M3 were picked from storage conditions at -80℃ and revived using MRS solid medium at 30–37℃ for 16–24 hours. Single colonies after revival were inoculated into MRS broth liquid medium and incubated at 100–200 rpm, 37℃ for 16–20 hours to obtain the desired bacterial cells. The bacterial cells were washed twice with sterile physiological saline, centrifuged again, and the precipitate was collected. The cells were then resuspended in an appropriate amount of sterile water and prepared with 10... 7 CFU / mL was used to obtain L. lactis M10 and W. cibaria M3 bacterial cultures for later use.

[0060] S2. Pre-treatment: Remove the scales, gills, and internal organs from the mandarin fish;

[0061] S3. Preparation of fermentation liquid: Weigh water equal to the weight of the mandarin fish obtained in step S2, add seasonings and the mixed fermentation agent obtained in S1 to obtain fermentation liquid;

[0062] The seasonings are specifically: based on 100% of the weight of the added drinking water, 5 wt% sodium chloride, 1 wt% potassium chloride, 0.4 wt% ginger, 0.2 wt% star anise, 0.06 wt% fennel, 0.06 wt% cumin, 0.03 wt% chili pepper, and 0.03 wt% Sichuan peppercorn.

[0063] S4. Fermentation: The mandarin fish obtained in step S1 is immersed in the fermentation liquid obtained in step S2 for fermentation. The fish is then pressed down with stones to obtain stinky mandarin fish.

[0064] The total amount of L. lactis M10 and W. cibaria M3 added was 10. 7 CFU / g 肉And the ratio of the two is 1:1.

[0065] The fermentation conditions are as follows: temperature set at 12±1℃, fermentation time 6 days.

[0066] The above product is named Example 1.

[0067] Comparative Example 1:

[0068] L. lactis M10 and W. cibaria M3 were inoculated in a 1:1 ratio, with a total bacterial count of 10. 7 CFU / g 肉 Application of adding 5.5wt% sodium chloride and 0.5wt% potassium chloride in fermented stinky mandarin fish.

[0069] The amount of sodium chloride added in Example 1 was changed to 5.5 wt%, and the amount of potassium chloride added was changed to 0.5 wt%, while other conditions or parameters remained the same as in Example 1.

[0070] The above products are named Comparative Example 1.

[0071] Comparative Example 2: Natural Fermentation

[0072] The specific method is as follows:

[0073] S1. Remove the scales, gills, and internal organs from the fresh mandarin fish;

[0074] S2. Preparation of fermentation liquid: Weigh water equal to the weight of the mandarin fish obtained in step S1, add seasonings and the mixed fermentation agent obtained in S1 to obtain fermentation liquid;

[0075] The seasonings are specifically: based on the mass of drinking water, 6 wt% sodium chloride, 0.4 wt% ginger, 0.2 wt% star anise, 0.06 wt% fennel, 0.06 wt% cumin, 0.03 wt% chili pepper, and 0.03 wt% Sichuan peppercorns are added.

[0076] S3. Fermentation: Immerse the mandarin fish obtained in step S1 into the fermentation liquid obtained in S2, and press the fish body with stones to obtain stinky mandarin fish.

[0077] The fermentation conditions are as follows: temperature set at 12±1℃, fermentation time 6 days.

[0078] The above products are named Comparative Example 2.

[0079] Comparative Example 3:

[0080] L. lactis M10 and W. cibaria M3 were inoculated in a 1:1 ratio, with a total bacterial count of 10. 7 CFU / g 肉 Application of adding 6wt% sodium chloride in fermented stinky mandarin fish.

[0081] S1. Preparation of the starter culture: L. lactis M10 and W. cibaria M3 were picked from storage conditions at -80℃ and revived using MRS solid medium at 30–37℃ for 16–24 hours. Single colonies after revival were inoculated into MRS broth liquid medium and incubated at 100–200 rpm, 37℃ for 16–20 hours to obtain the desired bacterial cells. The bacterial cells were washed twice with sterile physiological saline, centrifuged again, and the precipitate was collected. The cells were then resuspended in an appropriate amount of sterile water and prepared with 10... 7 CFU / mL was used to obtain L. lactis M10 and W. cibaria M3 bacterial cultures for later use.

[0082] S2. Pre-treatment: Remove the scales, gills, and internal organs from the mandarin fish;

[0083] S3. Preparation of fermentation liquid: Weigh water equal to the weight of the mandarin fish obtained in step S2, add seasonings and the mixed fermentation agent obtained in S1 to obtain fermentation liquid;

[0084] The seasonings mentioned are specifically: 6 wt% sodium chloride, 0.4 wt% ginger, 0.2 wt% star anise, 0.06 wt% fennel, 0.06 wt% cumin, 0.03 wt% chili pepper, and 0.03 wt% Sichuan pepper, based on 100% of the weight of the added drinking water.

[0085] S4. Fermentation: The mandarin fish obtained in step S1 is immersed in the fermentation liquid obtained in step S2 for fermentation. The fish is then pressed down with stones to obtain stinky mandarin fish.

[0086] The total amount of L. lactis M10 and W. cibaria M3 added was 10. 7 CFU / g 肉 And the ratio of the two is 1:1.

[0087] The fermentation conditions are as follows: temperature set at 12±1℃, fermentation time 6 days.

[0088] The above products are named Comparative Example 3.

[0089] Comparative Example 4:

[0090] L. lactis M10 and W. cibaria M3 were inoculated at a ratio of 1:2, with a total bacterial count of 10. 7 CFU / g 肉 Application of adding 6wt% sodium chloride in fermented stinky mandarin fish.

[0091] L. lactis M10 and W. cibaria M3 from Comparative Example 3 were mixed and inoculated at a ratio of 1:2, with other conditions or parameters being the same as those in Comparative Example 3.

[0092] The above products are named Comparative Example 4.

[0093] Comparative Example 5:

[0094] L. lactis M10 and W. cibaria M3 were inoculated at a ratio of 2:1, with a total bacterial count of 10. 7 CFU / g 肉 Application of adding 6wt% sodium chloride in fermented stinky mandarin fish.

[0095] L. lactis M10 and W. cibaria M3 from Comparative Example 3 were mixed and inoculated at a ratio of 2:1, with other conditions or parameters being the same as those in Comparative Example 3.

[0096] The above products are named Comparative Example 5.

[0097] Comparative Example 6:

[0098] L. lactis M10 and W. cibaria M3 were mixed in a 1:1 ratio and the total inoculation amount was 10. 7 CFU / g 肉 Application of adding 4wt% sodium chloride and 2wt% potassium chloride in fermented stinky mandarin fish.

[0099] The amount of sodium chloride added in Example 1 was changed to 4 wt%, and the amount of potassium chloride added was changed to 2 wt%, while other conditions or parameters remained the same as in Example 1.

[0100] The above products are named Comparative Example 6.

[0101] Comparative Example 7:

[0102] L. lactis M10 and W. cibaria M3 were inoculated in a 1:1 ratio, with a total bacterial count of 10. 7 CFU / g 肉 Application of adding 3wt% sodium chloride and 3wt% potassium chloride in fermented stinky mandarin fish.

[0103] The amount of sodium chloride added in Example 1 was changed to 3wt%, the amount of potassium chloride added was changed to 3wt%, and other conditions or parameters were the same as in Example 1.

[0104] The above products are named Comparative Example 7.

[0105] Comparative Example 8:

[0106] L. lactis M10 and W. cibaria M3 were mixed in a 1:1 ratio and the total inoculation amount was 10. 7 CFU / g 肉Application of adding 5wt% sodium chloride and 1wt% calcium chloride in fermented stinky mandarin fish.

[0107] The amount of sodium chloride added in Example 1 was changed to 5 wt%, and calcium chloride was added to 1 wt%, while other conditions or parameters remained the same as in Example 1.

[0108] The above products are named Comparative Example 8.

[0109] Comparative Example 9:

[0110] L. lactis M10 and W. cibaria M3 were inoculated in a 1:1 ratio, with a total bacterial count of 10. 7 CFU / g 肉 Application of adding 5wt% sodium chloride and 1wt% magnesium chloride in fermented stinky mandarin fish.

[0111] The amount of sodium chloride added in Example 1 was changed to 5 wt%, and magnesium chloride was added to 1 wt%, while other conditions or parameters remained the same as in Example 1.

[0112] The above products are named Comparative Example 9.

[0113] Comparative Example 10

[0114] Lactobacillus sakei CICC 22728 and Staphylococcus saprophyticus CICC 24371 were inoculated at a 1:1 ratio, with a total bacterial count of 10. 7 CFU / g 肉 Application of adding 5wt% sodium chloride and 1wt% potassium chloride in fermented stinky mandarin fish.

[0115] The microorganisms inoculated in Example 1 were replaced with Lactobacillus sakei CICC 22728 and Staphylococcus saprophyticus CICC24371, while other conditions or parameters remained the same as in Example 1.

[0116] The above products are named Comparative Example 10.

[0117] Test case

[0118] The stinky mandarin fish obtained in Example 1 and Comparative Examples 1-10 were subjected to index testing.

[0119] I. Sensory Evaluation

[0120] The present invention uses sensory evaluation to determine the inoculation ratio of fermenting agent in stinky mandarin fish. The sensory evaluation results are shown in Table 1.

[0121] Comparisons of Comparative Examples 2-5 show that when *Lactococcus lactis* M10 and *Westernella* M3 were mixed and inoculated at a 1:1 ratio, the sensory evaluation was superior to that of the naturally fermented group. Comparisons of Comparative Example 3, Example 1, and Comparative Example 1 show that when 6% sodium chloride (Comparative Example 3) was gradually replaced with 0.5% potassium chloride (Comparative Example 1) and 1% potassium chloride (Example 1), the sensory properties were not significantly affected, and even improved. However, when the concentration of potassium chloride continued to increase (Comparative Examples 6 and 7) to 2% or even 3%, the sensory results deteriorated, with the degree of deterioration increasing with the higher the proportion of potassium chloride replacement. This indicates that a 0.5%-1% potassium chloride replacement does not affect the sensory properties of the product. However, using other salts, such as 1% calcium chloride (Comparative Example 8) or magnesium chloride (Comparative Example 9), can also produce unpleasant odors and tastes, which are detrimental to the sensory quality of the product. Finally, comparing Comparative Example 10 with Example 1, it can be seen that using Lactococcus lactis M10 and Weissella M3 (1:1) yields better sensory quality, while fermentation using other strains results in even worse results than the natural fermentation group without added bacteria (Comparative Example 2). Therefore, using a 1:1 ratio of Lactococcus lactis M10 and Weissella M3 as a starter culture, and replacing it with KCl at a concentration of approximately 0.5%-1% NaCl, is a process that does not affect and may even slightly improve the sensory quality of stinky mandarin fish.

[0122] Table 1 Sensory evaluation results of fermented mandarin fish samples

[0123]

[0124]

[0125] Table 2 Sensory Evaluation Criteria for Fermented Stinky Mandarin Fish

[0126]

[0127] II. Na in each group + Content determination

[0128] Na+ in the fish obtained from each group was analyzed using an atomic absorption spectrophotometer. + K + Content (mg / g) 肉 The results of the measurements are shown in Table 3. As can be seen from Table 3, compared to the fermentation groups without salt substitution (Comparative Examples 2-3), the Na+ content in the experimental groups with salt substitution (Example 1, Comparative Example 1, Comparative Examples 6-10) was significantly higher. + The content of sodium chloride in the fish was significantly reduced. This indicates that fermenting fish with alternative metal salts can effectively reduce the sodium chloride content in the fish meat to some extent.

[0129] Table 3. Na content in fermented mandarin fish samples + content

[0130]

[0131]

[0132] III. Counting of Various Microorganisms

[0133] The number of lactic acid bacteria, hydrogen sulfide-producing bacteria, enterococci, and pseudomonococci in each group was determined using plate counting, as shown in Table 4. Comparing Example 1, Comparative Example 1, and Comparative Example 3, we found that when L. lactis M10 and W. cibaria M3 were inoculated, gradually replacing 6% sodium chloride (Comparative Example 3) with 0.5% potassium chloride (Comparative Example 1) and 1% potassium chloride (Example 1) significantly promoted the growth activity of lactic acid bacteria and inhibited the growth of putrefactive bacteria (hydrogen sulfide-producing bacteria, pseudomonococci, and enterococci). However, as the potassium chloride replacement concentration increased to 2%-3% (Comparative Examples 6-7), although there was a further inhibitory effect on hydrogen sulfide-producing bacteria, the promoting effect on lactic acid bacteria gradually weakened, the inhibitory effect on Pseudomonas growth was not significant, and it even had a promoting effect on enterococci. Replacing lactic acid bacteria with other salts, such as 1% calcium chloride (Comparative Example 8) or magnesium chloride (Comparative Example 9), did not promote the growth of lactic acid bacteria. Furthermore, the inhibitory effect on putrefactive bacteria decreased, and it even promoted their proliferation. Finally, comparing Example 1 and Comparative Example 10, it was found that when using other microbial starter cultures, the activity of lactic acid bacteria in the fermentation system was low, and putrefactive bacteria grew rapidly, resulting in poor fermentation. Therefore, using a 1:1 ratio of Lactococcus lactis M10 and Weissella M3 as a mixed starter culture, and replacing 0.5%-1% of NaCl with KCl for the fermentation of stinky mandarin fish, effectively enhanced the activity of lactic acid bacteria and effectively inhibited the growth of putrefactive bacteria.

[0134] Table 4. Number of various microorganisms in fermented stinky mandarin fish

[0135]

[0136] IV. Determination of Volatile Basic Nitrogen Content in Each Group

[0137] The changes in volatile basic nitrogen content during fermentation are closely related to microbial activity. The volatile basic nitrogen content (mg / 100g meat) in each group of samples was determined by the Kjeldahl method, and the results are shown in Table 5. Comparison with Comparative Examples 2 and 3 shows that the volatile basic nitrogen content increases on the sixth day after inoculation with *Lactococcus lactis* M10 and *Weisseria gonorrhoeae* M3 for fermenting stinky mandarin fish. This may be due to the inoculation promoting fermentation. However, previous experiments have demonstrated that inoculation fermentation can improve the fermentation quality of stinky mandarin fish, such as the formation of volatile flavors, and contributes to rapid fermentation. Comparing Example 1, Comparative Example 1, and Comparative Example 3, it can be seen that when 6% sodium chloride (Comparative Example 3) is gradually replaced with 0.5% potassium chloride (Comparative Example 1) and 1% potassium chloride (Example 1), the accumulation of volatile basic nitrogen content can be significantly inhibited. However, when 6% sodium chloride (Comparative Example 3) is gradually replaced with 2% potassium chloride (Comparative Example 6) and 3% potassium chloride (Example 7), this inhibitory effect gradually weakens. It is evident that the inhibitory effect is best when 1% sodium chloride is replaced with potassium chloride (Example 1), with its volatile basic nitrogen content significantly lower than the national standard requirement of 30 mg / 100g. Using other salts, such as 1% calcium chloride (Comparative Example 8) or magnesium chloride (Comparative Example 9), does not significantly inhibit the accumulation of volatile basic nitrogen content. Finally, comparing Example 1 and Comparative Example 10, it was found that the addition of potassium chloride significantly inhibited the increase in volatile basic nitrogen content only when fermenting stinky mandarin fish with *Lactococcus lactis* M10 and *Westernella esculenta* M3, while it had no effect on other microbial fermentation groups. Therefore, only by using a 1:1 ratio of *Lactococcus lactis* M10 and *Westernella esculenta* M3 as a mixed starter culture, and replacing approximately 1% NaCl with KCl, can the accumulation of volatile basic nitrogen content be limited to the greatest extent, thereby improving food safety.

[0138] Table 5. Volatile basic nitrogen content in fermented mandarin fish

[0139]

[0140] V. Determination of biogenic amine content in each group

[0141] The changes in biogenic amine content during fermentation are closely related to microbial activity. The contents of putrescine and tyramine in each group of samples were determined by high-performance liquid chromatography (HPLC), and the results are shown in Table 5. Comparison with Comparative Examples 2 and 3 shows that inoculating fermented mandarin fish with *Lactococcus lactis* M10 and *Westernella* M3 may promote the accumulation of putrescine and tyramine. This is consistent with the previous findings. Comparison with Examples 1, 1, and 3 shows that when 6% sodium chloride (Comparative Example 3) was gradually replaced with 0.5% potassium chloride (Comparative Example 1) and 1% potassium chloride (Example 1), the accumulation of putrescine and tyramine was significantly inhibited. However, when 6% sodium chloride (Comparative Example 3) was gradually replaced with 2% potassium chloride (Comparative Example 6) and 3% potassium chloride (Example 7), the accumulation of putrescine and tyramine was promoted. Therefore, replacing 1% potassium chloride showed a good fermentation effect. Using other salts, such as 1% calcium chloride (Comparative Example 8) or magnesium chloride (Comparative Example 9), did not inhibit the accumulation of putrescine and tyramine. Finally, comparing Example 1 and Comparative Example 10, it was found that only when inoculated with *Lactococcus lactis* M10 and *Westernella* M3 for fermenting stinky mandarin fish did the addition of potassium chloride significantly inhibit the increase in putrescine and tyramine content; this was ineffective for other microbial fermentation groups. Therefore, only by using a 1:1 ratio of *Lactococcus lactis* M10 and *Westernella* M3 as a mixed starter culture, and replacing approximately 1% NaCl with KCl, can the accumulation of putrescine and tyramine be limited to the greatest extent, thus improving food safety.

[0142] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for fermenting stinky mandarin fish, characterized in that, The method involves introducing a mixed fermentation agent into a fermentation system in which potassium chloride partially replaces sodium chloride, and then fermenting mandarin fish. The mixed fermentation agent contains Lactococcus lactis M10 and Weissellacibaria M3, which have been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession numbers CGMCC 16612 and CGMCC 16611, respectively. The total amount of potassium chloride and sodium chloride added to the fermentation system is 6 wt%.

2. The method according to claim 1, characterized in that, Includes the following steps: S1. Pre-treatment: Remove the scales, gills, and internal organs from the mandarin fish; S2. Preparation of fermentation liquid: Weigh water equal to the weight of the mandarin fish obtained in step S1, add seasoning containing potassium chloride and sodium chloride, and the mixed fermentation agent to obtain fermentation liquid; S3. Fermentation: The mandarin fish obtained in step S1 is soaked in the fermentation liquid obtained in step S2 to ferment and obtain stinky mandarin fish.

3. The method according to claim 1 or 2, characterized in that, The total amount of the mixed Lactococcus lactis M10 and Weissella M3 bacteria added to the fermentation system was 1×10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g; the ratio of viable Lactococcus lactis M10 to Weissella M3 was 1:

1.

4. The method according to claim 2, characterized in that, Based on 100% of the weight of drinking water added, the seasoning contains 5 wt% sodium chloride and 1 wt% potassium chloride.

5. The method according to claim 4, characterized in that, The seasoning also contains 0.4 wt% ginger, 0.2 wt% star anise, 0.06 wt% fennel, 0.06 wt% cumin, 0.03 wt% chili pepper, and 0.03 wt% Sichuan peppercorn.

6. The method according to claim 2, characterized in that, In step S3, the fermentation temperature is 12±1℃, and the fermentation time is at least 6 days.

7. A method for reducing the salt content in fermented mandarin fish, characterized in that, Includes the following steps: S1. Pre-treatment: Remove the scales, gills, and internal organs from the mandarin fish; S2. Preparation of fermentation liquid: Weigh water equal to the weight of the mandarin fish obtained in step S1, add seasonings containing potassium chloride and sodium chloride, and mixed fermentation agent to obtain fermentation liquid; S3. Fermentation: The mandarin fish obtained in step S1 is soaked in the fermentation liquid obtained in step S2 to ferment, resulting in stinky mandarin fish; In S2, the mixed fermentation agent contains *Lactococcus lactis* M10 and *Weissella cibaria* M3, which are deposited at the Guangdong Provincial Microbial Culture Collection Center with accession numbers CGMCC 16612 and CGMCC 16611, respectively. The total amount of the mixed *Lactococcus lactis* M10 and *Weissella cibaria* M3 added to the fermentation system is 1 × 10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g; the ratio of viable Lactococcus lactis M10 to Weissella M3 was 1:1; the amount of sodium chloride used was reduced by 16.67%.

8. The application of potassium chloride in improving the fermentation quality of stinky mandarin fish, characterized in that, The application involves using a mixed culture of Lactococcus lactis M10 and Weissella esculenta M3 as a starter culture, and replacing 1 wt% of sodium chloride with potassium chloride to ferment mandarin fish; the amount of sodium chloride used in the entire fermentation system is reduced by 16.67%.

9. The application according to claim 8, characterized in that, The improvement of fermented mandarin fish quality includes at least one of the following effects: (1) Regulate the metabolic activity of microorganisms in fermented mandarin fish and inhibit the growth of putrefactive bacteria, wherein the putrefactive bacteria include enterococci, pseudomonas, and hydrogen sulfide-producing bacteria; (2) Inhibit the accumulation of volatile basic nitrogen and biogenic amines during the bacterial fermentation of mandarin fish, wherein the biogenic amines include putrescine and tyramine.

10. A method for inhibiting the accumulation of volatile basic nitrogen and biogenic amines during the bacterial fermentation of mandarin fish, characterized in that, The method involves fermenting mandarin fish in a fermentation system where potassium chloride partially replaces sodium chloride, using a mixed fermentation agent to inhibit the accumulation of volatile basic nitrogen and biogenic amines in the fish.

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