A cold water fish fermentation product obtained by synergistic fermentation with a microbial composition and a method of making the same

Abstract

The application discloses a cold water fish fermentation product obtained through synergistic fermentation of a microbial composition and a preparation method thereof, and belongs to the field of microbial fermentation food. The application comprises the following steps: compounding Lactobacillus plantarum, Lactobacillus sakei and Saccharomyces cerevisiae into a microbial composition, preparing a pickling liquid from the complex microbial liquid of the three kinds of microorganisms, taking the dorsal muscle of a cold water fish as raw material, and performing pickling and drying fermentation treatment to obtain the cold water fish fermentation product. Experimental results show that inoculation of the microbial composition provided by the application can quickly produce acid to form a low pH environment, efficiently regulate protein hydrolysis, significantly shorten the fermentation period, inhibit lipid oxidation and reduce the TBARS value. The application provides a new microbial composition and fermentation method for low-salt, efficient and safe fermentation of cold water fish, and lays a foundation for improving the fermentation flavor of the cold water fish fermentation product, stabilizing the product quality and improving the product edible safety.

Description

Technical Field

[0001] This invention relates to the field of microbial fermented food, and in particular to a cold-water fish fermentation product obtained by co-fermentation of a microbial composition and its preparation method. Background Technology

[0002] Cold-water fish (such as rainbow trout, golden trout, and sturgeon) have advantages such as firm flesh and high content of unsaturated fatty acids and high-quality protein. Due to their slow growth, cold-water fish have firmer, more flavorful flesh and accumulate more nutrients. To adapt to the low-temperature environment, cold-water fish typically contain a higher proportion of unsaturated fatty acids, which produce a unique aroma during cooking.

[0003] However, traditional processing of cold-water fish primarily focuses on frozen sales, with limited deep-processed products and low added value. Existing fermented fish products largely rely on natural fermentation, which presents several problems: high salt dependence (salt content in pickling is generally as high as 8%-15%, easily leading to excessive sodium intake); long fermentation cycle (natural fermentation requires 3-6 months and is easily affected by fluctuations in environmental temperature and humidity, resulting in poor quality stability); limited flavor (relying on a single bacterial strain (such as *Lactobacillus plantarum*) for fermentation, leading to insufficient production of esters, aldehydes, and ketones, and a noticeable earthy taste); and safety risks (contamination by other microorganisms poses a high risk of exceeding limits for harmful substances such as biogenic amines and nitrites). Furthermore, the unique low-temperature growth characteristics of cold-water fish result in high muscle fiber density, making it difficult for traditional fermentation to fully degrade proteins and fats, resulting in a coarse texture. While existing technologies have attempted to improve flavor by artificially inoculating bacterial strains (such as *Staphylococcus xylose*) or adding sugars, these methods suffer from insufficient bacterial metabolic activity and unclear mechanisms of synergistic action between additives and fish proteins.

[0004] Therefore, there is an urgent need to develop a low-salt, high-efficiency, and flavor-controllable fermentation method for cold-water fish. Summary of the Invention

[0005] The purpose of this invention is to provide a fermented cold-water fish product obtained by co-fermentation of a microbial composition and its preparation method, so as to solve the problems existing in the prior art. This invention provides a new microbial composition and fermentation method for low-salt, high-efficiency and safe fermentation of cold-water fish, and lays the foundation for improving the fermentation flavor of cold-water fish fermented products, stabilizing product quality and improving product safety for consumption.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a microbial composition for fermenting cold-water fish, the microbial composition comprising Lactobacillus plantarum, Lactobacillus sakei, and Saccharomyces cerevisiae.

[0007] The present invention also provides an application of the above-mentioned microbial composition in the preparation of fermented cold-water fish products.

[0008] Furthermore, the cold-water fish is a rainbow trout.

[0009] This invention also provides a method for preparing fermented cold-water fish products, comprising the following steps: Take the back muscle of a cold-water fish and cut it into pieces; The above-mentioned microbial composition was prepared into a bacterial-fungal mixed bacterial solution, which was then mixed with salt solution, sucrose and glucose to prepare a pickling solution. Mix the fish pieces with the marinade, marinate, remove and drain to obtain marinated fish; The pickled fish is dried and fermented to obtain the fermented cold-water fish product.

[0010] Furthermore, the cold-water fish is a rainbow trout.

[0011] Furthermore, in the bacterial-fungal mixed bacterial solution, the ratio of the number of viable Lactobacillus plantarum, Lactobacillus sakei, and Saccharomyces cerevisiae is 10:10:1.

[0012] Furthermore, the salt solution has a mass fraction of 3%; The volume ratio of the saline solution to the bacterial-fungal mixed solution is 9:1.

[0013] Furthermore, the marinating temperature is 20°C and the time is 18 hours.

[0014] Furthermore, the drying fermentation temperature is 18-22℃, the humidity is 50%-60%, and the time is 24 hours.

[0015] The present invention also provides a fermented cold-water fish product obtained according to the above preparation method.

[0016] The present invention discloses the following technical effects: This invention combines *Lactobacillus plantarum*, *Lactobacillus sakei*, and *Saccharomyces cerevisiae* into a microbial composition. The compound bacterial solution of these three microorganisms is used to prepare a pickling solution. Using the dorsal muscle of cold-water fish as raw material, the product undergoes pickling, drying, and fermentation to obtain a fermented cold-water fish product. Experimental results show that inoculating with the microbial composition provided by this invention can rapidly produce acid to create a low-pH environment, efficiently regulate protein hydrolysis, significantly shorten the fermentation cycle, inhibit lipid oxidation, and reduce TBARS values. This invention provides a new microbial composition and fermentation method for low-salt, efficient, and safe fermentation of cold-water fish, laying the foundation for improving the fermented flavor, stabilizing product quality, and enhancing the food safety of fermented cold-water fish products. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A statistical graph showing the change in pH value of fermented cold-water fish products over fermentation time; Figure 2 The TVB-N content of fermented cold-water fish products after 23 hours; Figure 3 The thiobarbituric acid reactant value of fermented cold-water fish products after 23 hours; Figure 4 Electronic nose radar image of fermented cold-water fish products after 23 hours; Figure 5 Linear discriminant analysis was performed on fermented cold-water fish products over 23 hours. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] For cold-water fish of varying freshness, pretreatment with microorganisms or enzymes ensures stable and consistent quality while developing unique flavors. During fermentation, the metabolic activities of microorganisms break down proteins, fats, and other components in the fish meat, generating flavor compounds such as amino acids, organic acids, alcohols, and esters. Different microorganisms produce different metabolic products, resulting in varying flavors after fermentation. Therefore, the key technologies for fermented cold-water fish products lie in efficient deodorization and flavor control.

[0025] This invention has the following advantages: (1) Lactobacillus plantarum, Lactobacillus sakei, and Saccharomyces cerevisiae form a micro- and controllable ecosystem. The acidic environment created by lactic acid bacteria provides selective pressure for yeast growth, while the metabolic activities of yeast further enrich the flavor. After autolysis, the yeast cells can also provide nutrients for the lactic acid bacteria, forming a benign interaction. The acidity of lactic acid bacteria and the aroma of yeast can effectively neutralize and mask the inherent fishy smell of fish products, transforming it into a "fermented flavor" that is more acceptable to a wider range of consumers.

[0026] (2) The low pH environment formed by rapid acid production, combined with the bacteriocins produced by lactic acid bacteria, has a strong inhibitory effect on common foodborne pathogens such as Staphylococcus aureus and Listeria, and its safety is significantly higher than that of naturally fermented products.

[0027] (3) Using a starter culture can significantly shorten the fermentation cycle and ensure batch-to-batch stability and consistency, overcoming the pain point of unstable product quality in traditional natural fermentation.

[0028] (4) Biogenic amines are common harmful substances in fermented fish products, produced by the decarboxylation of amino acids by putrefactive bacteria. Inoculation with dominant Lactobacillus plantarum and Lactobacillus sakei can quickly occupy the site and inhibit the growth of amine-producing bacteria, thereby significantly reducing the accumulation of biogenic amines from the source and improving the food safety of the product.

[0029] The fish used in this embodiment of the invention is a cold-water rainbow trout, purchased from Xinjiang Zungui Fresh Food Technology Co., Ltd.

[0030] The plant lactobacillus used in this invention ( Lactiplantibacillus plantarum Lactobacillus sakei () Lactobacillus sakei ) and brewer's yeast ( Saccharomyces cerevisiae All were purchased from Beina Biotechnology - Henan Provincial Industrial Microbial Strains Engineering Technology Research Center.

[0031] Among them, the strain number of Lactobacillus plantarum is BNCC 337069. When using it, the dilution plating method is used to culture it on MRS medium. The strain of Lactobacillus sakei is numbered BNCC 192620. When using it, the dilution plating method should be used to culture it on MRS medium. The strain number of Saccharomyces cerevisiae is BNCC 336054. It is used by dilution plating method and cultured on YM medium.

[0032] Example 1 1. Raw material pretreatment Remove the skin, scales, and entrails from the fresh cold-water fish and clean it thoroughly. After rinsing, take the back muscle and cut it into 3 cm × 2 cm × 2 cm pieces. Wash the pieces with sterile distilled water and drain.

[0033] 2. Strain activation Lactobacillus plantarum was activated and cultured in MRS liquid medium at 37°C for 48 h; Lactobacillus sakei was activated and cultured in MRS liquid medium at 37°C for 48 h; and Saccharomyces cerevisiae was activated and cultured in YM liquid medium at 30°C for 24 h.

[0034] 3. Preparation of the marinating solution Collect the culture solutions of *Lactobacillus plantarum*, *Lactobacillus sakei*, and *Saccharomyces cerevisiae* after expansion culture, centrifuge and resuspend, and adjust the concentration of each culture solution to 10. 8 A mixed bacterial-fungal culture was prepared by mixing *Lactobacillus plantarum* culture, *Lactobacillus sakei* culture, and *Saccharomyces cerevisiae* culture at a volume ratio of 10:10:1 (CFU / mL).

[0035] Prepare a 3% (w / w) salt solution. Mix the salt solution with the above-mentioned bacterial-fungal mixed bacterial solution at a volume ratio of 9:1 to achieve a total viable bacteria concentration of 10 in the pickling solution. 7 CFU / mL, with the addition of sucrose (final concentration 30 g / L) and glucose (final concentration 25 g / L).

[0036] 4. Marinating The pre-treated fish pieces are placed in the marinade and marinated at 20°C for 18 hours. They are then removed, drained, and the marinated fish is obtained.

[0037] 5. Fermentation The marinated fish was placed in a fermentation box and fermented in a constant temperature and humidity incubator at a temperature of 20±2℃ and a humidity of 55±5%. After 24 hours of drying and fermentation, fermented fish was obtained. Samples were taken at 0 h, 4 h, 10 h, 18 h, and 23 h for quality determination.

[0038] Comparative Example 1 The fermentation method is the same as in Example 1, except that the mixed bacterial strains used for fermenting the fish are different when preparing the marinade in "3. Preparation of the marinade" and no exogenous bacterial strains are added.

[0039] Comparative Example 2 The fermentation method is the same as in Example 1, except that the mixed bacterial strains used for fermenting the fish are different in "3. Preparation of the pickling solution", and only 2% of Lactobacillus plantarum is added.

[0040] Comparative Example 3 The fermentation method is the same as in Example 1, except that the mixed bacterial strains for fermenting fish are different when preparing the marinade in "3. Preparation of marinade", only 2% Lactobacillus sakei is added.

[0041] Comparative Example 4 The fermentation method is the same as in Example 1, except that the mixed bacterial strains used for fermenting the fish are different in "3. Preparation of the pickling solution". Only 2% Lactobacillus plantarum and 2% Saccharomyces cerevisiae are added.

[0042] Comparative Example 5 The fermentation method is the same as in Example 1, except that the mixed bacterial strains used for fermenting the fish are different in "3. Preparation of the marinade". Only 2% Lactobacillus sakei and 2% Saccharomyces cerevisiae are added.

[0043] Example 1 of effect verification The fermented fish prepared in Example 1 was named PLS group, and the fermented fish prepared in Comparative Examples 1-5 were named CK, P, L, PS, and LS groups, respectively. The pH, amino acid nitrogen (ANN) content, and other indicators of the fermented products were detected at different times after the start of fermentation.

[0044] I. Experimental Methods 1. pH measurement Accurately weigh 10 g of fish meat, add 100 mL of distilled water and homogenize using a high-speed disperser for 1 min. Immediately after homogenization, measure the pH value of the homogenate with a pH meter and repeat the measurement 3 times.

[0045] 2. Determination of volatile basic nitrogen (TVB-N value) Refer to the automatic Kjeldahl nitrogen analyzer method in GB 5009.228-2016 "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food".

[0046] 3. Determination of Thiobarbituric Acid Reactive Substances (TBARS) Value 10.0 g of fish meat was placed in a stoppered conical flask, and 50 mL of trichloroacetic acid mixture (7.5% trichloroacetic acid + 0.1% EDTA) was added. The mixture was shaken in a 50℃ constant temperature shaker for 30 min. After cooling to room temperature, it was filtered twice with double-layered filter paper. 5 mL of the filtrate was transferred, and 5 mL of 0.02 mol / L 2-thiobarbituric acid solution was added. A blank control was also prepared. The experimental group and the control group were reacted simultaneously in a 90℃ water bath for 40 min. After 40 min, they were removed and cooled to room temperature for about 20 min. Then, they were transferred to centrifuge tubes and centrifuged at 4000 r / min for 10 min. The supernatant was collected, and the absorbance was measured at 532 nm and 600 nm, respectively. Each sample was measured in triplicate. The TBARS value was calculated using the following formula: ; In the formula, A 532 nm The absorbance at 532 nm; A 600 nm The absorbance at 600 nm; 4. Electronic nose measurement The volatile flavor compounds in the samples were investigated using an E-nose assay (ALPHA MOS, Heracles, France) combined with an interaction-sensitive sensor array and data analysis software. The detection conditions were set as follows: headspace inlet temperature 50 °C; headspace time 120 s; stirring speed 500 r / min; injection volume 2.5 mL; sample acquisition time 120 s; and delay time 300 s. Three parallel samples were prepared for E-nose analysis.

[0047] 5. Determination of volatile compound content 2.0 g of sample was weighed and transferred to a 20 mL headspace vial. Volatile compounds were extracted by SPME. The volatile compounds were analyzed by GC-MS using a 30 mm × 0.25 mm × 0.25 μm DB-WAX spectrometer with high-purity helium (99.999%) as the carrier gas. The target substances were separated at a flow rate of 1 mL / min. The temperature program was set to 40 °C for 3 min, followed by heating to 240 °C at 5 °C / min and holding for 15 min. Volatile flavor compounds were identified according to the NIST 2005 and Willey 7 standard libraries, and semi-quantitative analysis was performed using the internal standard 2,4,6-trimethylpyridine. Results are expressed in μg·kg⁻¹. -1 express.

[0048] II. Experimental Results 1. pH measurement The pH value of the fish meat system during fermentation is one of the important indicators reflecting the degree of fermentation.

[0049] like Figure 1 As shown, the pH value of fresh rainbow trout decreased after fermentation. Compared with the comparative group, in Example 1, Lactobacillus plantarum and Lactobacillus sakei can rapidly metabolize sugars to produce a large amount of lactic acid and other organic acids during fermentation, which are the main forces for lowering pH. Meanwhile, Saccharomyces cerevisiae creates an anaerobic environment by rapidly consuming oxygen, which promotes the efficient production of acid by lactic acid bacteria. At the same time, it releases nutrients to help lactic acid bacteria proliferate. The three work together to greatly increase the acid production rate and achieve a rapid decrease in pH.

[0050] 2. Determination of volatile basic nitrogen (TVB-N value) The TVB-N value in fish reflects the extent to which enzymes and microorganisms break down proteins, producing ammonia and alkaline nitrogenous substances. It is one of the important indicators for evaluating the freshness of meat products. like Figure 2 As shown, although the TVB-N value of the samples after 23 h of fermentation did not exceed the upper limit (30 mg / 100 g) specified in GB 10136-2015, the TVB-N content in the CK group was higher than that in other groups. The growth rate of TVB-N value in the inoculated fermentation groups was significantly inhibited, and the PLS group had the lowest content. This is because *Lactobacillus plantarum* and *Lactobacillus sakei* rapidly produce acid to lower the pH, inhibiting the growth of amine-producing putrefactive bacteria and secreting bacteriocins to directly inhibit bacteria; *Saccharomyces cerevisiae* consumes oxygen and competes with other bacteria for nutrients, further reducing protein decomposition. The three work synergistically to block amino acid deamination and decarboxylation, thereby significantly delaying the formation and accumulation of TVB-N, further enhancing its sensory properties.

[0051] 3. Determination of the reactant value of thiobarbituric acid The TBARS (Thiobarbituric Acid Reactive Matter) value is an important indicator for assessing the degree of lipid peroxidation. It mainly reflects the level of oxidative stress in biological samples or food by detecting the amount of oxidation products such as malondialdehyde (MDA). An excessively high TBARS value often indicates spoilage of fermented meat products due to fat oxidation, causing the fermented meat products to lose their good color and texture, and even produce an unpleasant pungent odor.

[0052] like Figure 3 As shown, it can be seen that after 23 hours of fermentation, the TBARS value of the inoculated fermentation group was significantly lower than that of the CK group, and the TBARS value of the PLS group was the lowest. This indicates that the synergistic fermentation of the three bacteria can reduce the degree of fat oxidation in fermented fish meat and reduce the production of fat oxidation products such as aldehydes and ketones.

[0053] 4. Changes in volatile flavor compounds The electronic nose is equipped with a gas-sensitive sensor that produces a unique response pattern to volatile flavor compounds in a sample. This technology enables rapid and comprehensive detection of volatile odor characteristics, providing detailed information about the aroma profile of a sample. The flavor characteristics of rainbow trout during the smoking process are shown in the radar chart. Figure 4 The relatively high response values ​​of sensors W5S, W1S, W1W, and W2W indicate that hydrocarbons, alcohols, aldehydes, and ketones are the main aromatic compounds in the rainbow trout samples. Sensors W6 (alkane-sensitive) and W7 (sulfur oxide-sensitive) showed the most significant changes throughout the processing, indicating that the smoking process altered the flavor profile of the rainbow trout samples. Linear discriminant analysis (LDA) showed ( Figure 5 These five samples are clearly distinguishable. LDA-1 and LDA-2 contributed 90.96% and 5.02% respectively, with a cumulative contribution of 95.98%, representing the main flavor information of rainbow trout. Further qualitative and quantitative determination of flavor compounds is needed to identify which volatile components show significant differences.

[0054] A total of 27 VOCs were detected by GC-MS, including 4 aldehydes, 4 ketones, 5 alcohols, 10 hydrocarbons, 2 acids, and 2 other types. Table 1 shows the volatile flavor compounds in each group.

[0055] Table 1. Content of volatile flavor compounds As shown in Table 2, the volatile flavor compounds in the CK group were significantly lower than those in the inoculated group, while the volatile flavor compounds in the Example 1 group were the highest. This is because after inoculation, the microorganisms grow and multiply. *Lactobacillus plantarum* and *Lactobacillus sakei* metabolize sugars to produce lactic acid, acetic acid, and ester precursors, while simultaneously breaking down proteins to produce amino acid flavor precursors. *Saccharomyces cerevisiae* converts sugars into ethanol and higher alcohols, and undergoes esterification reactions with organic acids to generate aromatic substances such as esters and aldehydes. The synergistic metabolism of these three processes significantly increases the variety and content of volatile flavor compounds, resulting in a richer and more harmonious flavor.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial composition for fermenting cold-water fish, characterized in that, The microbial composition includes Lactobacillus plantarum, Lactobacillus sakei, and Saccharomyces cerevisiae.

2. The use of the microbial composition of claim 1 in the preparation of fermented cold-water fish products.

3. The application according to claim 2, characterized in that, The cold-water fish mentioned is a rainbow trout.

4. A method for preparing a fermented cold-water fish product, characterized in that, Includes the following steps: Take the back muscle of a cold-water fish and cut it into pieces; The microbial composition of claim 1 is prepared into a bacterial-fungal mixed bacterial solution, which is then mixed with salt solution, sucrose and glucose to prepare a pickling solution; Mix the fish pieces with the marinade, marinate, remove and drain to obtain marinated fish; The pickled fish is dried and fermented to obtain the fermented cold-water fish product.

5. The preparation method according to claim 4, characterized in that, The cold-water fish mentioned is a rainbow trout.

6. The preparation method according to claim 4, characterized in that, In the bacterial-fungal mixed bacterial solution, the ratio of viable counts of *Lactobacillus plantarum*, *Lactobacillus sakei*, and *Saccharomyces cerevisiae* is 10:10:

1.

7. The preparation method according to claim 4, characterized in that, The salt solution has a mass fraction of 3%; The volume ratio of the saline solution to the bacterial-fungal mixed solution is 9:

1.

8. The preparation method according to claim 4, characterized in that, The pickling temperature is 20°C and the time is 18 hours.

9. The preparation method according to claim 4, characterized in that, The drying fermentation is carried out at a temperature of 18-22℃, a humidity of 50%-60%, and a time of 24 hours.

10. A fermented cold-water fish product obtained by the preparation method according to any one of claims 4-9.

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