Fish sauce, method of making and use thereof

Abstract

The application discloses fish sauce and a preparation method and application thereof. The preparation method of the fish sauce comprises the following steps: frying fish material in edible oil, performing solid-liquid separation, collecting the solid to be crushed to prepare surimi; mixing the surimi, edible salt and water to prepare fish paste, mixing the fish paste and a fermentation agent, performing fermentation treatment, standing for maturation, filtering, sterilizing the collected filtrate to prepare the fish sauce; wherein the fermentation agent comprises Pediococcus pentosaceus and Lactobacillus plantarum. The prepared fish sauce is high in safety, free of fishy smell, bitterness and raw taste, rich in flavor and stable in quality.

Description

Technical Field

[0001] This application belongs to the field of fermented food and condiment technology, and relates to fish sauce, its preparation method and application. Background Technology

[0002] Fish sauce is a liquid umami seasoning made from low-value fish or seafood processing by-products through natural fermentation. It is rich in nutrients such as amino acids and nucleotides and is widely used in cooking. However, current methods of preparing fish sauce result in low safety and inconsistent quality. Summary of the Invention

[0003] Therefore, it is necessary to provide a fish sauce with high safety and stable quality, as well as its preparation method and application.

[0004] In some embodiments, a method for preparing fish sauce is provided, comprising the following steps:

[0005] Fish raw materials are fried in edible oil, solid and liquid are separated, the solid is collected and crushed to prepare fish paste;

[0006] Fish paste is prepared by mixing fish paste, edible salt and water. The fish paste is then mixed with a fermentation agent, fermented, allowed to stand and mature, filtered, and the collected filtrate is sterilized to prepare fish sauce.

[0007] The fermentation agent contains Pediococcus pentosaceus and Lactobacillus plantarum.

[0008] In some embodiments, the frying temperature in the provided method for preparing fish sauce is 160°C to 180°C, and / or the frying time is 30s to 60s.

[0009] Optionally, during the frying process, the core temperature of the fish raw material is controlled to be greater than or equal to 75°C and then fried for 30 to 60 seconds.

[0010] In some embodiments, the provided method for preparing fish sauce satisfies one or both of the following conditions:

[0011] (1) In the fermentation agent, the ratio of viable counts of Pediococcus pentosaceus and Lactobacillus plantarum is 1:(0.7~1.5).

[0012] (2) Based on the amount of feed, the viable cell concentration of the fermentation agent in the fermentation system mixture is 1.0 × 10⁻⁶. 6 CFU / mL ~1.0×10 8 CFU / mL.

[0013] In some embodiments, the provided method for preparing fish sauce satisfies one or both of the following conditions:

[0014] (1) The mass ratio of the fish paste to the water is (1.5~5):1;

[0015] (2) The mass fraction of the edible salt in the fish paste is 10%~12%.

[0016] In some embodiments, the fermentation conditions in the provided method for preparing fish sauce include: a temperature of 35°C to 37°C, and / or a time of 7 to 10 days, and / or fermentation in a closed anaerobic environment, and / or fermentation under light-protected conditions.

[0017] In some embodiments, the conditions for static aging in the provided fish sauce preparation method include: a temperature of 50°C to 70°C and / or a time of 12h to 24h.

[0018] In some embodiments, the provided method for preparing fish sauce satisfies one or both of the following conditions:

[0019] (1) Sterilization is carried out by ultra-high temperature instantaneous sterilization method. Optionally, the sterilization temperature is 135℃~138℃ and the sterilization time is 3s~5s.

[0020] (2) Fish raw materials include one or two of fish bones and fish meat.

[0021] In some embodiments, a fish sauce is provided, which is prepared by the aforementioned preparation method;

[0022] Optionally, the content of amino acid nitrogen in each 100g of the fish sauce is greater than or equal to 0.8g.

[0023] In some embodiments, the fish sauce is provided for use in the preparation of food or condiments.

[0024] In some embodiments, a food or condiment is provided, the raw materials of which include the aforementioned fish sauce.

[0025] The aforementioned method for preparing fish sauce involves frying the fish until it is thoroughly cooked, which kills parasites, parasite eggs, and pathogens (such as Salmonella and Vibrio parahaemolyticus) in the fish, reducing the number of spoilage bacteria and pathogens at the source. The frying pretreatment also volatilizes and removes most of the raw, fishy, ​​and astringent odors from the fish. It causes moderate denaturation of the fish protein, stretching the peptide chains and loosening the meat structure, generating basic roasted aroma compounds and constructing a rich base aroma system. Simultaneously, it promotes moderate precipitation and mild thermal degradation of fish fat. The frying process involves the combined effects of oil oxidation and Maillard reactions, increasing the production of oxygen- and nitrogen-containing heterocyclic compounds such as pyrazines, pyridines, and furans, contributing to the caramel and meaty aromas. A large amount of aldehydes, ketones, and alcohols generate basic roasted aroma compounds, effectively masking unpleasant fishy odors and creating a roasted meat and oily aroma. Because the raw materials have been thoroughly sterilized in the early stages, a pure fermentation environment is created. The inoculated compound probiotics do not need to compete with endogenous bacteria, allowing them to proliferate rapidly and become the dominant bacterial group, achieving targeted and controllable fermentation. This targeted fermentation not only ensures controllable flavor during the fish sauce fermentation process, but also increases the rate at which the strains decompose proteins due to low-salt fermentation, resulting in a short-term increase in ammonia nitrogen. The resulting fish sauce is highly safe, free of fishy, ​​bitter, and raw tastes, with a rich flavor and stable quality. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0027] Figure 1 Growth curves of *Lactobacillus plantarum* and *Pediococcus pentosaceus* cultured for 24 h under 10 wt% NaCl and 12 wt% NaCl conditions.

[0028] Figure 2 The image shows the sensory evaluation results of fish sauce.

[0029] Figure 3 This is a graph showing the results of the analysis of aroma components in fish sauce. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0031] Unless otherwise defined, 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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0033] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B."

[0034] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0035] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0036] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0037] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0038] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0039] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0040] In this invention, the terms "first aspect," "second aspect," "third aspect," and "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," and "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0041] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0042] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0043] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0044] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0045] In this application, "room temperature" or "normal temperature" generally refers to 5℃~30℃, and more preferably 25±5℃.

[0046] Traditional fish sauce preparation methods involve long-term natural fermentation with high salt content, utilizing environmental microorganisms as well as the microorganisms and enzymes carried by the fish itself. Because each batch of raw materials is different, the microorganisms vary, leading to variations in flavor. Furthermore, harmful substances such as histamine and volatile basic nitrogen are prone to exceeding safe limits. Moreover, because it cannot completely kill endogenous harmful microorganisms such as parasites, eggs, and pathogens in raw fish, the fresh fish contains a large amount of volatile fishy-smelling substances, and unsaturated fats are easily oxidized and rancid, producing unpleasant flavors such as fishy, ​​bitter, and sour tastes. In addition, to inhibit spoilage bacteria and other microorganisms in raw fish, 25% to 30% salt is usually added, resulting in excessively high sodium content in the product, which does not meet modern healthy eating requirements. Natural fermentation takes 6 to 12 months, resulting in low production efficiency, a long cycle, and high risks. Although there are currently methods for preparing fish sauce, such as enzymatic hydrolysis and microbial fermentation, these methods rely solely on salt and fermentation bacteria to inhibit the production of fish sauce. However, these methods cannot completely kill endogenous harmful microorganisms during the preparation process. Fish sauce is prone to spoilage under high temperature conditions, and a large number of miscellaneous bacteria, such as Geotrichum candida and Aspergillus flavus, can easily produce biotoxins. These toxins cannot be decomposed after fermentation.

[0047] In some embodiments, a method for preparing fish sauce is provided, comprising the following steps:

[0048] Fish raw materials are fried in edible oil, solid and liquid are separated, the solid is collected and crushed to prepare fish paste;

[0049] Fish paste is prepared by mixing fish paste, salt and water. The fish paste is then mixed with a fermentation agent, fermented, allowed to stand and mature, filtered, and the collected filtrate is sterilized to prepare fish sauce.

[0050] The fermentation agents include Pediococcus pentosaceus and Lactobacillus plantarum.

[0051] The dense, tightly folded protein structure of fresh fish makes it difficult for probiotics to penetrate and decompose, resulting in slow hydrolysis, long cycles, and low amino acid yield in traditional fermentation. The proposed preparation method involves frying the fish raw material. The high-temperature frying thoroughly kills the bacteria, pathogens, and spoilage bacteria carried by the fresh fish, as well as the fish's own endogenous enzymes, preventing natural fermentation. This reduces bacterial contamination, biogenic amine formation, and off-flavors during fermentation from the source. By removing the native microbial flora from the raw material, specific strains can be introduced for targeted fermentation, avoiding inconsistencies in endogenous enzymes and microbial composition due to different origins, times, and species of raw materials, which can lead to fluctuations in fish sauce flavor. Simultaneously, high-temperature frying fully denatures the fish proteins, expanding their spatial structure and loosening their texture. This breaks down the dense protein structure, making it easier for probiotics to decompose it into flavor-enhancing amino acids and small peptides. It also promotes the appropriate release and oxidation of fish fat, weakening the raw fishy smell and laying the foundation for a richer flavor. In addition, the Maillard reaction during frying produces a large number of heterocyclic compounds, aldehydes and ketones, resulting in a rich aroma.

[0052] The provided fish sauce preparation method is standardized, with minimal batch-to-batch quality variation, making it suitable for large-scale industrial production. The fermentation cycle is maintained at 7 to 10 days, with amino acid nitrogen ≥0.8g / 100mL, shortening the process by more than 90% compared to traditional methods, significantly improving production efficiency.

[0053] In some embodiments, the frying temperature in the provided method for preparing fish sauce is 160°C to 180°C, and / or the frying time is 30s to 60s. For example, the frying temperature can be 160°C, 165°C, 170°C, 175°C, 180°C, etc., or any range of two of the aforementioned values; the frying time can be 30s, 35s, 40s, 45s, 50s, 55s, 60s, etc., or any range of two of the aforementioned values.

[0054] In some embodiments, in the provided method for preparing fish sauce, during the frying process, the temperature of the fish raw material is controlled to be greater than or equal to 75°C and then kept at that temperature for 30 to 60 seconds. For example, the time for keeping the fish raw material at a temperature of greater than or equal to 75°C is 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 60 seconds, etc., or it can be a range composed of any two of the aforementioned values.

[0055] For example, during the frying process, the temperature of the edible oil is controlled at 160℃~180℃, and the fish raw material is fried for 30s~60s after the center temperature is greater than or equal to 75℃.

[0056] The frying temperature should be between 160℃ and 180℃, and the frying time should be between 30 and 60 seconds. These conditions ensure that the core temperature of the fish reaches 75℃ or higher and is maintained at this temperature for at least 30 seconds during frying. Below 160℃, the oil temperature is too low, resulting in poor oil flow and the fish remaining submerged in oil for too long. This causes the fish to absorb excessive oil, resulting in a greasy and dry finished product. Above 180℃, the oil temperature is too high, causing the surface to cook extremely quickly. The fish skin will instantly burn and turn black while the inside remains uncooked. The surface proteins of the fish will harden rapidly, locking in internal moisture. High-temperature oil is also prone to smoking and producing harmful substances, and oil consumption will be even greater.

[0057] In some embodiments, in the provided method for preparing fish sauce, the ratio of viable counts of Pediococcus pentosaceus and Lactobacillus plantarum in the fermentation agent is 1:(0.7~1.5), for example, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc., or it can be a range of any two of the aforementioned ratios.

[0058] An excessive amount of Pediococcus pentosaceus will result in a fish sauce with a fishy, ​​greasy, ammonia-heavy, weak umami flavor and insufficient soy sauce aroma. During the preparation process, protein hydrolysis will be slow, amino acid nitrogen levels will be low, and the process will be prolonged. Conversely, an excessive amount of Lactobacillus plantarum will cause the pH to drop rapidly below 4.8 during the fermentation process, inhibiting Pediococcus, reducing the number of viable bacteria, and causing an imbalance in the bacterial flora. This will result in a fish sauce with a strong, bitter, and pungent sour taste, where the sourness overpowers the umami flavor, lacks milky / nutty aroma, and has a thin flavor profile.

[0059] In some embodiments, in the provided method for preparing fish sauce, the concentration of viable bacteria in the fermentation system mixture is 1.0 × 10⁻⁶ based on the amount of feed. 6 CFU / mL ~1.0×10 8 CFU / mL, for example, 1.0 × 10⁻⁶ 6 CFU / mL, 5.0×10 6 CFU / mL, 1.0×10 7 CFU / mL, 5.0×10 7 CFU / mL, 1.0×10 8 CFU / mL, etc., can also be a range consisting of any two of the aforementioned values.

[0060] In some embodiments, the mass ratio of fish paste to water in the provided fish sauce preparation method is (1.5~5):1, for example, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc., or any range of any two of the aforementioned ratios. In some embodiments, the mass fraction of edible salt in the provided fish sauce preparation method is 10%~12%, for example, 10%, 10.5%, 11%, 11.5%, 12%, etc., or any range of any two of the aforementioned values. The edible salt content is reduced to 10%~12%, compared to the high-salt fermentation of over 30% in traditional preparation methods, the salt content is reduced by more than 50%, meeting the requirements of modern healthy diets. The preparation method provided in this application achieves low-salt production, reducing the sodium content of the finished product and health risks. The *Lactobacillus plantarum* and *Pediococcus pentosaceus* used for fermentation can tolerate a salt concentration of up to 12 wt%, maintaining good growth characteristics (OD value > 0.8) at this level, while being significantly inhibited at salt concentrations ≥ 15 wt%. Under conditions of 10-12% edible salt content, they can inhibit the growth of other microorganisms during subsequent fermentation, avoiding the possibility of spoilage due to excessively low salt levels.

[0061] In some embodiments, the fermentation conditions in the provided method for preparing fish sauce include: a temperature of 35°C to 37°C, and / or a time of 7 to 10 days, and / or fermentation in a closed anaerobic environment, and / or fermentation under light-protected conditions.

[0062] In some embodiments, the conditions for static aging in the provided fish sauce preparation method include: a temperature of 50°C to 70°C and / or a time of 12h to 24h.

[0063] The aging process at 50℃~70℃ helps to volatilize and remove some low-boiling-point substances that may cause a fishy or foul odor, making the flavor of the fish sauce purer. On the other hand, it can generate aging aroma. The heating process itself will induce some non-enzymatic reactions (such as Maillard reaction), which can generate new aroma substances, making the fish sauce more savory and mellow.

[0064] In some embodiments, the fish sauce preparation method provides an ultra-high temperature instantaneous sterilization method for sterilization.

[0065] In some embodiments, the sterilization temperature in the provided fish sauce preparation method is 135℃~138℃, and the sterilization time is 3s~5s.

[0066] The process employs ultra-high temperature instantaneous sterilization, which rapidly kills residual yeast, mold, other microorganisms, and spores in the original liquid, completely eliminating problems such as fermentation spoilage, mold growth, and souring. This process features rapid heating and cooling, maximizing the preservation of core flavor compounds such as amino acids and flavor peptides generated during fermentation, and avoiding issues like aroma loss, dull color, and bitter taste caused by prolonged high-temperature cooking.

[0067] In some embodiments, the fish raw materials provided in the fish sauce preparation method include one or both of fish bones and fish meat.

[0068] In some embodiments, a fish sauce is provided, which is prepared by the aforementioned preparation method.

[0069] In some embodiments, the provided fish sauce contains 0.8g or more of amino acid nitrogen per 100g of fish sauce.

[0070] In some embodiments, the aforementioned fish sauce is provided for use in the preparation of food or condiments.

[0071] In some embodiments, a food or condiment is provided, the raw materials of which include the aforementioned fish sauce.

[0072] The following are specific embodiments. They are intended to provide a more detailed description of this application to help those skilled in the art and researchers better understand it. The technical conditions described do not constitute any limitation on this application. Any modifications made within the scope of the claims of this application are protected by the claims.

[0073] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods not specifying particular conditions in the examples were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0074] Example 1: Screening of salt content in fermentation system

[0075] 1. Raw material pretreatment

[0076] Fresh or frozen anchovies are selected as raw materials. Rotten, spoiled, severely mechanically damaged, or odorous fish are removed. Mud, scales, and impurities are removed from the fish surface. The selected anchovies are placed in a clean running water washing tank and rinsed repeatedly 2-3 times to thoroughly clean the surface. The surface water is drained and set aside. The raw material processing temperature is controlled below 25℃ throughout the process to prevent spoilage from prolonged exposure to room temperature. The constant temperature frying equipment is preheated, and the oil temperature is maintained at 160℃-180℃. The drained, whole anchovies are evenly placed into the frying equipment, laid out in a single layer to avoid uneven heating. During frying, the fish are continuously turned to ensure even heating. Strict cooking standards are maintained: the center temperature of the fish is measured using a handheld thermometer, and frying continues for at least 30 seconds after the center temperature reaches ≥75℃. After frying, the fish are removed and placed on a clean, draining cooling table to cool naturally to room temperature, draining excess oil.

[0077] 2. Preparation of fish paste

[0078] After cooling, the cooked anchovies are put into a food-grade stainless steel grinder for fine grinding. The grinding speed is controlled at 2800~3200r / min. The mixture is ground into a fine fish paste without obvious fish bones or fish meat particles to ensure that the fish paste has a uniform texture. This increases the contact area between the subsequent bacteria and the fish substrate, improves the fermentation rate and fermentation uniformity, and avoids problems such as insufficient local fermentation and uneven flavor.

[0079] The fish paste and purified water were precisely mixed in a 2:1 mass ratio, and the pulverized fish paste and room-temperature purified water were sequentially added to a sterile fermentation mixing tank. Food-grade refined edible salt was then added to prepare fish paste. The salt content of the system was precisely adjusted according to the salt percentage in the fish paste as shown in Table 1, with a live bacteria concentration of 1.0 × 10⁻⁶ in the fermentation substrate at each salt content. 6 Lactobacillus plantarum and Pediococcus pentosaceus were added at CFU / mL, respectively, and fermented at a controlled temperature of 35-37℃ for 24 hours in a sealed, static environment. Table 1 below shows the growth of Lactobacillus plantarum and Pediococcus pentosaceus under different salinities over 24 hours. Figure 1 The figures show the growth curves of *Lactobacillus plantarum* and *Pediococcus pentosaceus* after 24 hours of cultivation in 10 wt% NaCl and 12 wt% NaCl.

[0080] Table 1

[0081]

[0082] From Table 1 and Figure 1 The results show that *Lactobacillus plantarum* and *Pediococcus pentosus* grow well in salt concentrations ≤12wt%. Considering the possibility of spoilage if the salt concentration is too low during fermentation, a salt concentration of 10wt%~12wt% was selected.

[0083] Example 2: Preparation of Fish Sauce

[0084] This embodiment provides a fish sauce, the preparation method of which includes the following steps:

[0085] 1. Raw material pretreatment

[0086] Fresh or frozen anchovies are selected as raw materials. Rotten, spoiled, severely mechanically damaged, or odorous fish are removed. Mud, scales, and impurities are removed from the fish surface. The selected anchovies are placed in a clean running water washing tank and rinsed repeatedly 2-3 times to thoroughly clean the surface. The surface water is drained and set aside. The raw material processing temperature is controlled below 25℃ throughout the process to prevent spoilage from prolonged exposure to room temperature. The constant temperature frying equipment is preheated, and the oil temperature is maintained at 160-180℃. The drained, whole anchovies are evenly placed into the frying equipment, laid out in a single layer to avoid uneven heating. During frying, the fish are continuously turned to ensure even heating. Strict cooking standards are maintained: the center temperature of the fish is measured using a handheld thermometer. Once the center temperature reaches ≥75℃, fry for at least 30 seconds. After frying, the fish are removed and placed on a clean, draining cooling table to cool naturally to room temperature and drain excess oil.

[0087] 2. Preparation of fish paste

[0088] After cooling, the cooked anchovies are put into a food-grade stainless steel grinder for fine grinding. The grinding speed is controlled at 2800~3200r / min. The mixture is ground into a fine fish paste without obvious fish bones or fish meat particles to ensure that the fish paste has a uniform texture. This increases the contact area between the subsequent bacteria and the fish substrate, improves the fermentation rate and fermentation uniformity, and avoids problems such as insufficient local fermentation and uneven flavor.

[0089] The fish paste and purified water were precisely mixed in a 2:1 mass ratio, and the crushed fish paste and room-temperature purified water were added sequentially into a sterile fermentation mixing tank. Food-grade refined edible salt was then added to precisely adjust the salt content in the system to 10 wt%.

[0090] After adding the ingredients, turn on the stirring device and set the speed to 60~80 r / min. Stir at a constant temperature of 25℃ for 15~20 minutes to fully integrate the fish paste, water, and salt, forming a low-salt fish paste substrate with a fine texture, uniform concentration, no lumps, and no layering. After stirring, let it stand for 5 minutes to remove any floating foam on the surface, ensuring the fermentation substrate is clean and stable.

[0091] 3. Probiotic-directed fermentation

[0092] A compound probiotic fermentation agent (a 1:1 ratio of viable Pediococcus pentosaceus and Lactobacillus plantarum) was inoculated into low-salt fish paste. Pediococcus pentosaceus and Lactobacillus plantarum were inoculated onto MRS medium and cultured in a shaker at 37°C for 24 hours. The cells were then collected by centrifugation at 4°C and 8000 rpm, resuspended in 0.85% physiological saline, and inoculated into the low-salt fish paste with the compound probiotic fermentation agent at a viable cell concentration of 1.0 × 10⁻⁶ cells / mL. 6 The inoculated fish paste was sealed and placed in a constant temperature fermentation device at a concentration of CFU / mL. The entire fermentation process was carried out at a controlled temperature of 37℃, with the mixture kept in a sealed, static environment. The total fermentation period was controlled within 7 days, and the ammonia nitrogen content was ≥0.8g / 100mL. The entire fermentation process was conducted in a dark, sealed, and anaerobic environment.

[0093] The fermentation system completely removes the fishy, ​​bitter, and astringent taste of raw fish, presenting a pure and rich fish sauce aroma with a mellow taste and no off-flavors. The system is stable and does not spoil.

[0094] 4. Post-processing refining

[0095] After the fermentation cycle reaches its target and fermentation is terminated, the fermented fish mash is not filtered immediately. Instead, it is placed in a sealed, sterile container and left to stand at 70°C for 12 hours to complete the final maturation. This step allows unreacted small-molecule peptides and amino acids in the fermentation system to fully integrate, stabilizing the flavor system and mitigating slight fermentation off-flavors. Simultaneously, it releases trace amounts of carbon dioxide and volatile odor gases produced during fermentation, preventing issues such as bubbles, astringent taste, and impure aroma in the finished fish sauce. During the settling period, the container must be sealed and protected from light to prevent contamination by external microorganisms.

[0096] A three-stage filtration system—coarse filtration, fine filtration, and plate and frame filtration—is employed to thoroughly remove impurities and improve the clarity and stability of the finished product. The first step, coarse filtration, uses an 80-mesh food-grade stainless steel filter to initially filter the fermentation mash, trapping large solid particles such as residual fish bones, coarse fish fibers, and large protein residues, thus initially separating the fish sauce concentrate from solid waste. The second step, fine filtration, involves a second filtration through a 200-mesh nylon precision filter to remove fine suspended particles and small protein flocculents, initially reducing the turbidity of the liquid. The third step, plate and frame filtration clarification, involves sending the finely filtered concentrate into a plate and frame filter press. High-density food-grade pure cotton filter cloth is used, and the filtration pressure is controlled at 0.3~0.4MPa and the filtration temperature at 20~25℃. Uniform pressure filtration thoroughly removes tiny colloidal particles and insoluble impurities, ultimately yielding a high-purity fish sauce concentrate that is clear, free of turbidity, sediment, and suspended impurities, effectively preventing backflow and sedimentation problems after storage.

[0097] To ensure thorough sterilization while preserving flavor and nutrients, the traditional prolonged boiling sterilization method is abandoned in favor of an industrially standardized UHT (Ultra-High Temperature) instantaneous sterilization process. The sterilization temperature is set at 135-138℃ and held at that temperature for 3-5 seconds. The sterilized high-temperature fish sauce concentrate is then rapidly cooled through a closed, aseptic plate cooling system, reaching below 25℃ within 10 seconds to prevent residual heat from further aging and damaging the flavor. The entire cooling process utilizes closed, aseptic pipelines to isolate the product from air and external contaminants, eliminating the possibility of secondary bacterial contamination.

[0098] Examples 3-4

[0099] Examples 3 and 4 are similar to Example 2, except that the frying temperature is different. In Example 3, the frying temperature is 140°C, and in Example 4, the frying temperature is 200°C.

[0100] Examples 5 to 8

[0101] Examples 5-8 are similar to Example 2, except that the ratio of viable cells of Pediococcus pentosaceus and Lactobacillus plantarum is different from that in Example 2.

[0102] In Example 5, the ratio of viable Pediococcus pentosaceus to Lactobacillus plantarum was 2:1.

[0103] In Example 6, the ratio of viable cells of Pediococcus pentosaceus and Lactobacillus plantarum was 3:1.

[0104] In Example 7, the ratio of viable Pediococcus pentosaceus to Lactobacillus plantarum was 1:2.

[0105] In Example 8, the ratio of viable counts of Pediococcus pentosaceus and Lactobacillus plantarum was 1:3.

[0106] Examples 9 to 12

[0107] Examples 9-12 are similar to Example 2, except that the mass ratio of fish paste to purified water is different from that in Example 2.

[0108] In Example 9, the mass ratio of fish paste to purified water was 5:1.

[0109] In Example 10, the mass ratio of fish paste to purified water was 1:0.

[0110] In Example 11, the mass ratio of fish paste to purified water was 1:1.

[0111] In Example 12, the mass ratio of fish paste to purified water was 1:2.

[0112] Comparative Example 1

[0113] This comparative example is similar to Example 2, except that the frying step is omitted in this comparative example.

[0114] Comparative Example 2

[0115] This comparative example is similar to Example 2, except that steaming is used instead of frying in this comparative example. The operation steps are as follows: boil the water completely, and after a large amount of steam is generated, maintain the water temperature at around 100°C, and then put the fish in to steam. Use a handheld thermometer to measure the temperature of the fish's center. When the temperature of the fish's center is ≥75°C, start timing and keep it for 30 seconds.

[0116] Comparative Example 3

[0117] This comparative example is similar to Example 2, except that the fermentation agent in this comparative example is *Lactobacillus plantarum*, and the viable concentration of *Lactobacillus plantarum* in the fermentation system mixture is controlled at 1.0 × 10⁻⁶. 6 CFU / mL.

[0118] Comparative Example 4

[0119] This comparative example is similar to Example 2, except that the fermentation agents in this comparative example are Aspergillus oryzae, Lactobacillus plantarum, and yeast, wherein the ratio of viable cells of Aspergillus oryzae, Lactobacillus plantarum, and yeast is 6:1:2, and the total viable cell concentration in the fermentation system mixture is controlled at 1.0 × 10⁻⁶. 6 CFU / mL.

[0120] Effect evaluation

[0121] (1) Sensory evaluation

[0122] Quantitative descriptive analysis (QDA) was used to evaluate the differences in sensory aroma characteristics among different samples. Ten trained sensory experts (5 men and 5 women, aged 25-35 years) were selected to evaluate the aroma characteristics of the fish sauce samples. Prior to conducting the formal sensory evaluation, the team members underwent multiple rounds of training to ensure consistency in the evaluation results. The training focused on clearly defining and accurately describing the sensory attributes involved in the fish sauce evaluation. Through discussion, the team members determined that aroma, taste, color, form, and clarity would be used to describe the sensory characteristics of the samples. The fish sauce samples were placed in white porcelain dishes and labeled with numerical codes before being submitted to the team members for sensory evaluation and scoring. The scoring criteria are shown in Table 2.

[0123] Table 2

[0124]

[0125] The results are as follows Figure 2As shown, after sensory evaluation, it was found that (1) the fish sauce prepared in Comparative Example 1 had a more obvious fishy and raw aroma. Since it was not sterilized, the fish itself had microorganisms, and the directional fermentation of lactic acid bacteria strains did not dominate. In terms of taste, it lacked umami and had an off-flavor. In terms of color, it was light brown and cloudy. (2) The fish sauce prepared in Example 2 had an obvious oily and fried aroma. The flavor had a mild sour taste from lactic acid bacteria fermentation and a unique milky aroma and soy sauce aroma. The overall aroma was more harmonious and rich. In terms of taste, it was more intense and complex, with a caramel and fat aroma. In terms of color, it was dark reddish-brown and amber. (3) The fish sauce prepared in Comparative Example 2 produced a more intense cooked meat aroma, soy sauce aroma or caramel aroma. After directional fermentation of lactic acid bacteria strains, the fishy smell was reduced, but it still existed slightly. In terms of taste, the umami was more mellow and soft. In terms of color, it was reddish-brown. The fish sauce prepared in Example 2, after 7 days of low-salt fermentation, has a stronger and fuller aroma, no fishy or raw taste, and the highest overall sensory score.

[0126] (2) Evaluation of the flavor and aroma of fish sauce

[0127] Quantitative descriptive analysis (QDA) was used to evaluate the differences in sensory aroma characteristics among different samples. Ten trained sensory experts (5 men and 5 women, aged 25-35 years) were selected to evaluate the flavor and aroma characteristics of the fish sauce samples. Prior to conducting the formal sensory evaluation, the team members underwent multiple rounds of training to ensure the consistency of the evaluation results. The training focused on clearly defining and accurately describing the sensory attributes involved in the fish sauce evaluation. The fish sauce samples were placed in white porcelain dishes and labeled with numerical codes before being submitted to the team members for sensory evaluation scoring. The scoring criteria for the flavor and aroma of fish sauce are shown in Table 3, and the results are shown in Table 4.

[0128] Table 3

[0129]

[0130] Table 4

[0131]

[0132] As shown in Table 4, the fish sauce prepared in Example 2 using a 1:1 ratio of live bacteria *Lactobacillus plantarum* and *Pediococcus pentosaceus* exhibited a superior aroma compared to the fish sauce prepared in Comparative Example 3 using a single *Lactobacillus plantarum* strain. *Lactobacillus plantarum*'s flavor-producing ability was relatively weak, while the umami and lingering aftertaste produced by *Pediococcus pentosaceus* were crucial for enhancing the umami flavor of the fish sauce. The combination of the two strains not only quickly established an acidic protective environment but also laid a more robust foundation for umami. Furthermore, the compound strain showed a better effect on increasing ammonia nitrogen compared to the single strain, resulting in a superior sensory flavor compared to single-strain fermentation.

[0133] The fish sauce prepared by fermentation of Aspergillus oryzae, Lactobacillus plantarum and Saccharomyces hanssonii in Comparative Example 4 with a live bacteria ratio of 6:1:2 had a poorer aroma than the fish sauce prepared by fermentation of Lactobacillus plantarum and Pediococcus pentosaceus in Example 2 with a live bacteria ratio of 1:1. The characteristic aroma of the fish sauce was not obvious, and the fish sauce fermented by Aspergillus oryzae had a distinct soy sauce aroma, which diluted the original fish flavor of the fish sauce.

[0134] (2) Determination of aroma components by gas chromatography-mass spectrometry (GC-MS)

[0135] The aroma components in the prepared fish sauce were determined by GC-MS. The detection method is as follows:

[0136] Take 5ml of fish sauce obtained after fermentation and put it into a headspace vial. Adjust the salt concentration to a suitable level and use an extraction head to complete the aroma extraction.

[0137] GC-MS detection conditions: HP-Innowax fused silica capillary columns (60m × 0.225mm × 0.25μm, Agilent, Santa Clara, CA) and DB-5MS fused silica capillary columns (60m × 0.25mm × 0.25μm, Agilent, Santa Clara, CA) were used. Hydrogen was used as the carrier gas at a flow rate of 0.8 mL / min. Gas chromatography-mass spectrometry (GC-MS) was performed in split-injection mode. The injection volume was 1 μL, the initial temperature was set to 40°C for 4 min, increased to 160°C after 6 min, and then increased to 160-220°C after 10 min, with a split ratio of 10:1. Mass spectrometry conditions: electron energy 70 eV, ion source temperature 230°C, transfer temperature 250°C, mass scan: 35-500 m / z, full scan mode. Results are as follows: Figure 3 As shown in Table 5.

[0138] Table 5

[0139]

[0140] From Table 5 and Figure 3 As can be seen, the fish sauce prepared in Example 2 contains a large number of heterocyclic compounds, aldehydes, and ketones. High-temperature fat oxidation during frying significantly increases aldehyde levels, and frying promotes lipid oxidation to generate ketones. Only frying produces a large amount of roasted pyrazines. Heat treatment significantly degrades trimethylamine and dimethyl sulfide. Frying has the best deodorizing effect and produces the richest variety of flavor compounds, reaching 102 types. Comparative Example 1 shows weak endogenous enzyme decomposition, more spoilage precursors, and a higher level of small-molecule organic acids, with the fewest varieties at 66. Comparative Example 2 shows mild hydrolysis through cooking, resulting in a large amount of alcohol produced by lactic acid bacteria, with 84 varieties. In conclusion, the flavor compounds produced by the frying pretreatment process in Example 2 are the best among the three groups.

[0141] (3) Detection of physicochemical indicators

[0142] Volatile basic nitrogen was tested in accordance with GB 5009.228-2016 "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food".

[0143] Amino acid nitrogen was tested in accordance with GB 5009.235-2016, "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food".

[0144] Biogenic amines (histamine) were tested in accordance with GB 5009.208-2016 "National Food Safety Standard - Determination of Biogenic Amines in Food".

[0145] The test results are shown in Table 6.

[0146] Table 6

[0147]

[0148] As shown in Table 6, the fish sauces of Comparative Examples 2, 1, and 3 were compared in terms of various physicochemical indicators. The fish sauce prepared in Example 2, after frying and subsequent targeted fermentation, showed a significant increase in amino acid nitrogen content due to the absence of interference from other microorganisms. Furthermore, the volatile basic nitrogen content of Comparative Example 1 was significantly higher than that of Example 2 and Comparative Example 2, while there were no significant differences in volatile basic nitrogen and ammonia nitrogen content between Example 2 and Comparative Example 2. The histamine content of Comparative Example 1 was 62.51 mg / kg, exceeding the safety limit of 50 mg / kg for aquatic products, posing a risk to food safety; the biogenic amine content was much higher than in the two heat-treated groups, indicating the most severe spoilage. In Comparative Example 2, steaming only mildly inactivated the biogenic amines, and a small amount of endogenous amino acids in the fish meat had already slightly decomposed in the early stages, resulting in slightly higher biogenic amine content than in Example 2. Example 2 had the lowest content of various spoilage-causing biogenic amines among the three groups, with histamine at only 9.43 mg / kg, indicating the best safety.

[0149] Table 7

[0150]

[0151] As shown in Table 7, if the material-to-water ratio is too concentrated (e.g., the mass ratio of fish paste to water is 1:0), the viscosity is high, the fermentation is slow, and it is easy to spoil. If the material-to-water ratio is too diluted (e.g., the mass ratio of fish paste to water is 1:2 or 1:1), the ammonia nitrogen content is less than 0.8g / 100mL, the flavor is bland, it is easy to spoil, and the yield is low.

[0152] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0153] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing fish sauce, characterized in that, Includes the following steps: Fish raw materials are fried in edible oil, solid and liquid are separated, the solid is collected and crushed to prepare fish paste; Fish paste is prepared by mixing fish paste, edible salt and water. The fish paste is then mixed with a fermentation agent, fermented, allowed to stand and mature, filtered, and the collected filtrate is sterilized to prepare fish sauce. The fermentation agent contains Pediococcus pentosaceus and Lactobacillus plantarum.

2. The method for preparing fish sauce according to claim 1, characterized in that, The frying temperature is 160℃~180℃, and / or the frying time is 30s~60s; Optionally, during the frying process, the core temperature of the fish raw material is controlled to be greater than or equal to 75°C and then fried for 30 to 60 seconds.

3. The method for preparing fish sauce according to claim 1, characterized in that, One or both of the following conditions must be met: (1) In the fermentation agent, the ratio of viable counts of Pediococcus pentosaceus and Lactobacillus plantarum is 1:(0.7~1.5). (2) Based on the amount of feed, the viable cell concentration of the fermentation agent in the fermentation system mixture is 1.0 × 10⁻⁶. 6 CFU / mL ~1.0×10 8 CFU / mL.

4. The method for preparing fish sauce according to any one of claims 1 to 3, characterized in that, One or both of the following conditions must be met: (1) The mass ratio of the fish paste to the water is (1.5~5):1; (2) The mass fraction of the edible salt in the fish paste is 10%~12%.

5. The method for preparing fish sauce according to any one of claims 1 to 3, characterized in that, The fermentation conditions include: a temperature of 35℃~37℃, and / or a time of 7 to 10 days, and / or fermentation in a closed anaerobic environment, and / or fermentation under light-proof conditions.

6. The method for preparing fish sauce according to any one of claims 1 to 3, characterized in that, The conditions for static curing include a temperature of 50℃~70℃ and / or a time of 12h~24h.

7. The method for preparing fish sauce according to any one of claims 1 to 3, characterized in that, One or both of the following conditions must be met: (1) Sterilization is carried out by ultra-high temperature instantaneous sterilization method. Optionally, the sterilization temperature is 135℃~138℃ and the sterilization time is 3s~5s. (2) Fish raw materials include one or two of fish bones and fish meat.

8. A fish sauce, characterized in that, Prepared by the preparation method according to any one of claims 1 to 7; Optionally, the content of amino acid nitrogen in each 100g of the fish sauce is greater than or equal to 0.8g.

9. The use of the fish sauce according to claim 8 in the preparation of food or condiments.

10. A food product or condiment, characterized in that, The raw materials used in its preparation include the fish sauce described in claim 8.

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