A compound probiotic fermented fish sauce and its preparation method

Abstract

This invention discloses a compound probiotic fermented fish sauce and its preparation method. Fish and by-products are cleaned and pulped with distilled water to obtain fish paste. β-D-glucose, glucose oxidase, and catalase are added to the fish paste, and a first enzymatic hydrolysis is performed under stirring. The pH of the first enzymatic hydrolysate is adjusted to neutral, and a compound enzyme is added for a second enzymatic hydrolysis. After enzyme inactivation, a second enzymatic hydrolysate is obtained. Salt is added to obtain the second enzymatic hydrolysate. Soybean meal, green beans, wheat bran, and water are mixed to obtain koji (fermentation starter). The koji is autoclaved and cooled to room temperature, then inoculated with compound probiotics to make koji. The starter is added to the enzymatic hydrolysate for fermentation. The fermentation broth is desalted by electrodialysis and then autoclaved to obtain fish sauce. This invention removes the fishy smell from the by-products by adding β-D-glucose, glucose oxidase, and catalase, and further degrades the fishy smell through fermentation. By adding green beans to the koji, the fermented fish sauce retains its unique aroma, resulting in a fish sauce product with good flavor and low fishy smell.

Description

Technical Field

[0001] This invention relates to the field of aquatic product processing technology, specifically to a compound probiotic fermented fish sauce and its preparation method. Background Technology

[0002] Traditional fish sauce fermentation involves using low-value anchovies as raw material. Salt and fish are thoroughly mixed in a specific ratio, and this mixture is then used for fermentation. Without adding any external enzymes, the various enzymes naturally present in the fish, including cathepsins and other enzymes, work together with natural microorganisms to degrade the fish's proteins, fats, and polysaccharides into easily absorbed small molecules such as amino acids, fatty acids, and monosaccharides. While this process saves costs by eliminating the need for additives, its overall economic efficiency is very low. There are three reasons for this: First, the natural fermentation process is lengthy, typically taking two to three years, resulting in high time costs. Second, the yield from natural fermentation is low, and production capacity cannot meet demand. Third, the nutritional content is insufficient; because no enzymes are added during the fermentation process, the fermentation process is undirected, leading to a final product that lacks comprehensive nutritional value.

[0003] Fermenting fish sauce with probiotics can significantly shorten the fermentation cycle. However, the raw materials for fish sauce fermentation are generally low-value anchovies or other fish and by-products. These by-products typically take a certain amount of time to reach the fish sauce production site, and even with freezing, they will still oxidize during this process, producing more fishy odor. Therefore, how to remove the fishy smell has always been a key issue to be addressed in the production of fish sauce. Currently, the main methods for removing the fishy smell include adding substances such as activated charcoal or using nanofiltration membranes. However, activated charcoal and similar methods are not very effective, and nanofiltration membranes are not only costly but also inefficient. In addition, due to the short fermentation time of probiotics, the unique aroma of fish sauce is relatively weak. Therefore, a method of fermenting fish sauce with compound probiotics is needed that can shorten the fermentation time, maintain the unique aroma of fish sauce, and effectively remove the fishy smell, resulting in a fish sauce product with a better taste. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a compound probiotic fermented fish sauce and its preparation method. This invention removes fishy-smelling substances from fish and by-products by adding β-D-glucose, glucose oxidase, and catalase, and further degrades these substances through fermentation. By adding green soybeans to the koji (fermentation starter), the fermented fish sauce retains its unique aroma, resulting in a fish sauce product with good flavor and low fishy smell.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing a compound probiotic fermented fish sauce, comprising the following steps:

[0007] (1) After cleaning the fish and offal, add distilled water to make fish paste. Add β-D-glucose, glucose oxidase and catalase to the fish paste and perform a first enzymatic hydrolysis under stirring. After inactivating the enzyme, obtain a first enzymatic hydrolysate. Adjust the pH of the first enzymatic hydrolysate to neutral, add a compound enzyme to perform a second enzymatic hydrolysis, and after inactivating the enzyme, obtain a second enzymatic hydrolysate. Add salt to the second enzymatic hydrolysate to obtain an enzymatic hydrolysate.

[0008] (2) Mix soybean meal, green soybean, wheat bran and water to obtain koji material. After high pressure sterilization, cool the koji material to room temperature, inoculate with compound probiotics to make koji seed. Add the koji seed to the enzymatic hydrolysate for fermentation. Desalinate the fermentation liquid by electrodialysis and then sterilize it under high pressure to obtain fish sauce.

[0009] Preferably, in step (1), the amount of β-D-glucose added is 1% of the mass of fish and by-products; the amount of glucose oxidase added is 0.1-0.5% of the mass of fish and by-products; and the amount of catalase added is 1-3% of the mass of fish and by-products.

[0010] Preferably, in step (1), the temperature of the first enzymatic hydrolysis is 25~30℃ and the time is 4~8h; the stirring speed is ≥200rpm.

[0011] Preferably, in step (1), the complex enzyme is selected from at least one of neutral protease, flavor protease, pepsin, bromelain, and papain; the amount of the complex enzyme added accounts for 0.1-1% of the mass of the first enzymatic hydrolysate; the temperature of the second enzymatic hydrolysis is 50°C, and the hydrolysis time is 4-8 h.

[0012] Preferably, in step (1), the amount of salt added accounts for 9% of the mass of the secondary enzymatic hydrolysate.

[0013] Preferably, in step (2), the green soybeans are soybeans obtained 12-16 days after the soybean flowers; the mass ratio of soybean meal, green soybeans, wheat bran and water is 25:5:20:50; the compound probiotics are obtained by mixing Zygosacchariformis, Sacchariformis and Bacillus subtilis in a mass ratio of 1:1:1; the inoculation amount of the compound probiotics accounts for 1-5% of the mass of the koji material; the koji-making temperature is 37℃ and the time is 3-7 days.

[0014] The strain of *Zygosaccharomyces rouxii*, with the culture accession number CICC 33378, was purchased from the China Industrial Microbial Culture Collection Center.

[0015] The strain preservation number of Saccharomyces cerevisiae is CICC 1017, and it was purchased from the China Industrial Microbial Culture Collection Center.

[0016] The Bacillus subtilis strain has the preservation number CGMCC 1.14985 and was purchased from the China General Microbiological Culture Collection Center.

[0017] Preferably, in step (2), the amount of seed culture added accounts for 20% of the mass of the enzymatic hydrolysate; the fermentation temperature is 40℃ and the fermentation time is 30 days.

[0018] Preferably, in step (2), the voltage of the electrodialysis desalination is 30V and the sample loading flow rate is 50L / hour.

[0019] A second aspect of the present invention provides the application of the above-described preparation method in improving the flavor of fish sauce and reducing its fishy odor.

[0020] In a third aspect, the present invention provides a compound probiotic fermented fish sauce obtained by the above preparation method.

[0021] The beneficial effects of this invention are:

[0022] (1) The present invention adopts the traditional probiotic fermentation method for fish sauce. First, the fish and by-products are enzymatically hydrolyzed, then koji is made and fermented with compound probiotics, which shortens the fermentation time of fish sauce. The process is simple and suitable for large-scale production.

[0023] (2) This invention removes fishy substances from fish and by-products by adding β-D-glucose, glucose oxidase and catalase, and further degrades fishy substances through fermentation; by adding green beans to the koji to make koji, the fish sauce obtained by fermentation can retain its unique aroma, and obtain fish sauce products with good flavor and low fishy smell. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, 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 application pertains.

[0025] As introduced in the background section, the main methods for removing fishy odors currently include adding activated charcoal or using nanofiltration membranes. However, activated charcoal and similar methods are not very effective at removing odors, while nanofiltration membranes are not only costly but also inefficient. In addition, due to the short fermentation time of probiotics in fish sauce, the unique aroma of fish sauce is relatively weak.

[0026] Therefore, the purpose of this invention is to provide a compound probiotic fermented fish sauce and its preparation method. Because the raw materials for making fish sauce, such as fish and by-products, not only contain their own fishy odor, but also produce aldehydes, ketones, trimethylamine, and other fishy-smelling substances during transportation due to oxidation. Anchovies, in particular, are a low-value fish, mainly used for feed preparation. This is because anchovies easily spoil after being brought ashore and need to be frozen immediately; even then, their fishy odor is stronger than that of other fish.

[0027] This invention first adds β-D-glucose and catalase to fish and by-products. β-D-glucose, catalyzed by glucose oxidase, reacts with oxygen to produce hydrogen peroxide and gluconic acid. Glucoic acid reacts with amines produced by the fish and by-products, thus removing the fishy odor. Hydrogen peroxide not only kills bacteria in the fish and by-products, preventing further spoilage, but also catalyzes catalase to decompose peroxides produced by oxidation in the fish and by-products, further removing the fishy odor. However, if hydrogen peroxide is not removed, it will not only oxidize the fish meat but also inhibit bacterial growth during subsequent fermentation. Therefore, catalase consumes hydrogen peroxide to produce oxygen, which provides oxygen for the β-D-glucose reaction, forming a positive cycle. An excess of catalase needs to be added to ensure that all hydrogen peroxide is consumed; stirring helps accelerate the consumption of hydrogen peroxide by catalase. The fermentation process using compound probiotics further degrades residual fishy-smelling substances. Although this process significantly shortens the production time of fish sauce, the aroma is relatively weak. Adding green soybeans (obtained 12-16 days after soybean flowering) during the koji-making process helps to enhance the aroma. Compared to soybeans, green soybeans obtained 12-16 days after flowering have a very high content of hexadecanoic acid (C16:1). Although hexadecanoic acid is a monounsaturated fatty acid, it is an omega-7 fatty acid. Adding green soybeans to the koji-making process allows them to be utilized by probiotics during fermentation, resulting in a richer aroma in the fish sauce. This overcomes the problem of weak aroma in fish sauce produced by short-term fermentation and improves the overall quality of the fish sauce.

[0028] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0029] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0030] Example

[0031] (1) After cleaning the anchovies (including internal organs), mix them with distilled water at a ratio of 1:1 to obtain fish paste. Take 1 kg of fish paste, add 5 g of β-D-glucose (Hubei Biaoyue Biotechnology Development Co., Ltd. CAS 492-61-5), 1.5 g of glucose oxidase (10,000 U / g Leshengyuan Biotechnology (Nanjing) Co., Ltd.), and 10 g of catalase (100,000 U / g Shaanxi Chenming Biotechnology Co., Ltd.), stir at 300 rpm, and control the enzymatic hydrolysis temperature at 28℃ for 6 h. After the enzymatic hydrolysis is completed, a primary enzymatic hydrolysate is obtained, which is then inactivated at 121℃ for 20 min. Add 0.3 mol / L NaOH solution to the primary enzymatic hydrolysate to adjust the pH to neutral. Add 0.3% flavor protease (100,000 U / g, Hebei Zhongzhisheng Biotechnology Co., Ltd.) and 0.3% neutral protease (50,000 U / g, Sichuan Huanxu Biotechnology Co., Ltd.) to the primary enzymatic hydrolysate, and hydrolyze at 50℃ for 6 hours. Then, inactivate the enzyme at 121℃ for 20 minutes to obtain the secondary enzymatic hydrolysate. Add 9 wt% sodium chloride to the secondary enzymatic hydrolysate and mix thoroughly to obtain the final enzymatic hydrolysate.

[0032] (2) Mix 250g soybean meal, 50g green soybeans, 200g wheat bran and 500g distilled water, sterilize with high pressure steam at 98℃ and cool to room temperature, add 10g of Zygosaccharomyces rouxii (CICC 33378), 10g of Saccharomyces cerevisiae (CICC 1017) and 10g of Bacillus subtilis (CGMCC 1.14985), and culture at 37℃ for 5 days, turning the starter once a day to obtain the starter culture.

[0033] (3) Take 500g of the enzymatic hydrolysate prepared in step (1), add 100g of the starter culture prepared in step (2), and ferment at 40℃ for 30 days. After fermentation, desalinate the fermentation broth by electrodialysis at an operating voltage of 30V and a loading flow rate of 50L / hour. After desalination, use a plate and frame filter press with 100-mesh filter cloth at 0.4MPa to remove impurities, and then sterilize by high-pressure steam at 98℃ to obtain fish sauce.

[0034] Comparative Example 1

[0035] (1) After cleaning the anchovies (including internal organs), mix them with distilled water at a ratio of 1:1 to obtain fish paste. Take 1 kg of fish paste, add 0.3% flavor protease (100,000 U / g, Hebei Zhongzhisheng Biotechnology Co., Ltd.) and 0.3% neutral protease (50,000 U / g, Sichuan Huanxu Biotechnology Co., Ltd.), and enzymatically hydrolyze at 50℃ for 6 h, then inactivate the enzyme at 121℃ for 20 min to obtain the enzymatic hydrolysate. Add 9 wt% salt (sodium chloride) to the enzymatic hydrolysate and mix well.

[0036] (2) Mix 250g soybean meal, 50g soybeans, 200g wheat bran and 500g distilled water, sterilize with high pressure steam at 98℃ and cool to room temperature, add 10g of Zygosacchariformis (CICC 33378), 10g of Saccharomyces cerevisiae (CICC 1017) and 10g of Bacillus subtilis (CGMCC No. 23784), and culture at 37℃ for 5 days, turning the starter once a day to obtain the starter culture.

[0037] (3) Take 500g of the enzymatic hydrolysate prepared in step (1), add 100g of the starter culture prepared in step (2), and ferment at 40℃ for 30 days. After fermentation, desalinate the fermentation broth by electrodialysis at an operating voltage of 30V and a loading flow rate of 50L / hour. After desalination, use a plate and frame filter press with 100-mesh filter cloth at 0.4MPa to remove impurities, and then sterilize by high-pressure steam at 98℃ to obtain fish sauce.

[0038] Comparative Example 2

[0039] (1) After cleaning the anchovies (including internal organs), mix them with distilled water at a ratio of 1:1 to obtain fish paste. Take 1 kg of fish paste, add 5 g of β-D-glucose (Hubei Biaoyue Biotechnology Development Co., Ltd. CAS 492-61-5), 1.5 g of glucose oxidase (10,000 U / g Leshengyuan Biotechnology (Nanjing) Co., Ltd.), and 10 g of catalase (100,000 U / g Shaanxi Chenming Biotechnology Co., Ltd.), stir at 300 rpm, and control the enzymatic hydrolysis temperature at 28℃ for 6 h. After the enzymatic hydrolysis is completed, a primary enzymatic hydrolysate is obtained, which is then inactivated at 121℃ for 20 min. Add 0.3 mol / L NaOH solution to the primary enzymatic hydrolysate to adjust the pH to neutral. Add 0.3% flavor protease (100,000 U / g, Hebei Zhongzhisheng Biotechnology Co., Ltd.) and 0.3% neutral protease (50,000 U / g, Sichuan Huanxu Biotechnology Co., Ltd.) to the primary enzymatic hydrolysate, and hydrolyze at 50℃ for 6 hours. Then, inactivate the enzyme at 121℃ for 20 minutes to obtain the secondary enzymatic hydrolysate. Add 9 wt% sodium chloride to the secondary enzymatic hydrolysate and mix thoroughly to obtain the final enzymatic hydrolysate.

[0040] (2) Mix 250g soybean meal, 50g soybeans, 200g wheat bran and 500g distilled water, sterilize with high pressure steam at 98℃ and cool to room temperature, add 10g of Zygosacchariformis (CICC 33378), 10g of Saccharomyces cerevisiae (CICC 1017) and 10g of Bacillus subtilis (CGMCC No. 23784), and culture at 37℃ for 5 days, turning the starter once a day to obtain the starter culture.

[0041] (3) Take 500g of the enzymatic hydrolysate prepared in step (1), add 100g of the starter culture prepared in step (2), and ferment at 40℃ for 30 days. After fermentation, desalinate the fermentation broth by electrodialysis at an operating voltage of 30V and a loading flow rate of 50L / hour. After desalination, use a plate and frame filter press with 100-mesh filter cloth at 0.4MPa to remove impurities, and then sterilize by high-pressure steam at 98℃ to obtain fish sauce.

[0042] Comparative Example 3

[0043] (1) After cleaning the anchovies (including internal organs), mix them with distilled water at a ratio of 1:1 to obtain fish paste. Take 1 kg of fish paste, add 0.3% flavor protease (100,000 U / g, Hebei Zhongzhisheng Biotechnology Co., Ltd.) and 0.3% neutral protease (50,000 U / g, Sichuan Huanxu Biotechnology Co., Ltd.), and enzymatically hydrolyze at 50℃ for 6 h, then inactivate the enzyme at 121℃ for 20 min to obtain the enzymatic hydrolysate. Add 9 wt% salt (sodium chloride) to the enzymatic hydrolysate and mix well.

[0044] (2) Mix 250g soybean meal, 50g green soybeans (16 days after soybean flowering), 200g wheat bran and 500g distilled water, sterilize with high pressure steam at 98℃ and cool to room temperature, add 10g of Zygosaccharomyces rouxii (CICC 33378), 10g of Saccharomyces cerevisiae (CICC 1017) and 10g of Bacillus subtilis (CGMCC No. 23784), and culture at 37℃ for 5 days, turning the starter once a day to obtain the starter culture.

[0045] (3) Take 500g of the enzymatic hydrolysate prepared in step (1), add 100g of the starter culture prepared in step (2), and ferment at 40℃ for 30 days. After fermentation, desalinate the fermentation broth by electrodialysis at an operating voltage of 30V and a loading flow rate of 50L / hour. After desalination, use a plate and frame filter press with 100-mesh filter cloth at 0.4MPa to remove impurities, and then sterilize by high-pressure steam at 98℃ to obtain fish sauce.

[0046] Experimental Example 1: Detection of Physicochemical Indicators of Fish Sauce

[0047] The total nitrogen, amino acid nitrogen, trimethylamine content, amino acid content, volatile ester content, and volatile acid content of the fish sauce products prepared in the test examples and comparative examples 1-3 were tested; the results are shown in Table 1.

[0048] The total nitrogen content was determined using the Kjeldahl method (GB 5009.5-2010);

[0049] The trimethylamine content was determined using the headspace gas chromatography-mass spectrometry method according to GB5009.179-2016;

[0050] The amino acid nitrogen content was determined by formaldehyde titration (GB / T 5009.39-2003);

[0051] The amino acid content was determined using a fully automated amino acid analyzer.

[0052] The content of volatile esters and volatile acids was determined by solid phase microextraction gas chromatography-mass spectrometry (HPMS-HS-GC-MS).

[0053] Table 1 Physicochemical Indicators

[0054]

[0055] Total nitrogen and amino acid nitrogen are indicators of fish sauce quality. Although water content is the main component of green soybeans and nitrogen content is higher in soybeans than in green soybeans, fish sauce is a product of fish product fermentation, and its nitrogen content mainly comes from fish and by-products. The better the fermentation, the higher the content of protein and amino acids in the fish sauce. Among amino acids, glutamic acid can increase the umami flavor of fish sauce, glycine can increase the sweetness, and histidine can increase the bitterness. These three representative amino acids were selected, and their content can be used to judge the taste of fish sauce. Trimethylamine, volatile esters, and acids can indicate the flavor of fish sauce. The lower the trimethylamine content, the less fishy the fish sauce, and the higher the volatile esters and acids, the better the aroma of fish sauce. As shown in Table 1, the fish sauce prepared in the examples is superior to that of comparative examples 1-3 in terms of quality, taste, and aroma. Comparing Comparative Examples 1 to 3, it can be seen that adding β-D-glucose, glucose oxidase, and catalase can remove the fishy smell and partially enhance the aroma of fish sauce. While adding green beans to make koji is difficult to reduce the fishy smell of fish sauce, it can enhance the aroma of fish sauce, indicating that adding green beans can increase the degree of fermentation.

[0056] Experiment Example 2: Sensory Test

[0057] Sensory evaluation was conducted to assess the sensory preferences of the fish sauces prepared in Examples 1-3. An evaluation panel of 10 individuals trained in sensory evaluation was formed, and they were randomly assigned to evaluate the fish sauce based on its umami, fishy smell, aftertaste, richness, and seafood flavor. Total scores were calculated, and the sensory evaluation forms are shown in Table 2. The sensory evaluation results are shown in Table 3.

[0058] Table 2 Sensory Evaluation Form

[0059]

[0060] Table 3 Sensory test results

[0061]

[0062] As shown in Table 2, the fish sauce products prepared in the examples have higher sensory evaluations compared to those prepared in Comparative Examples 1 to 3. The score of Comparative Example 3 is lower than that of Comparative Example 2, mainly because although the fish sauce of Comparative Example 3 has a distinct aroma, it also has a distinct fishy smell. The two smells conflict, giving a poor sensory experience, so the sensory evaluation is slightly lower than that of Comparative Example 2.

[0063] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of a method for preparing compound probiotic fermented fish sauce in improving its flavor, texture, and reducing its fishy odor, characterized in that... The preparation method includes the following steps: (1) After cleaning the fish and by-products, distilled water is added to make a fish paste. β-D-glucose, glucose oxidase, and catalase are added to the fish paste, and a first enzymatic hydrolysis is performed under stirring. After enzyme inactivation, a first enzymatic hydrolysate is obtained. The pH of the first enzymatic hydrolysate is adjusted to neutral, and a compound enzyme is added for a second enzymatic hydrolysis. After enzyme inactivation, a second enzymatic hydrolysate is obtained. Salt is added to the second enzymatic hydrolysate to obtain the enzymatic hydrolysate. The amount of β-D-glucose added is 1% of the mass of the fish and by-products; the amount of glucose oxidase added is 0.1~0.5% of the mass of the fish and by-products; The amount of catalase added is 1-3% of the mass of the fish and by-products; the fish and by-products are from anchovies; the temperature of the first enzymatic hydrolysis is 25-30℃, and the time is 4-8 hours; the stirring speed is ≥200 rpm; the compound enzyme is selected from at least one of neutral protease, flavor protease, pepsin, bromelain, and papain; the amount of the compound enzyme added accounts for 0.1-1% of the mass of the first enzymatic hydrolysate; the temperature of the second enzymatic hydrolysis is 50℃, and the hydrolysis time is 4-8 hours; the amount of salt added accounts for 9% of the mass of the second enzymatic hydrolysate. (2) Soybean meal, green beans, wheat bran and water are mixed to obtain koji material. After high-pressure sterilization, the koji material is cooled to room temperature and inoculated with compound probiotics to make koji seed. The koji seed is added to the enzymatic hydrolysate for fermentation. The fermentation liquid is desalted by electrodialysis and then sterilized by high pressure to obtain fish sauce. The green beans are beans obtained 12-16 days after soybean flowering. The mass ratio of soybean meal, green beans, wheat bran and water is 25:5:20:

50. The compound probiotics are obtained by mixing Zygosacchariformis, Sacchariformis and Bacillus subtilis in a mass ratio of 1:1:

1. The inoculation amount of the compound probiotics accounts for 1-5% of the mass of the koji material. The koji making temperature is 37℃ and the time is 3-7 days. The amount of koji seed added accounts for 20% of the mass of the enzymatic hydrolysate. The fermentation temperature is 40℃ and the fermentation time is 30 days. The voltage of electrodialysis desalination is 30V and the sample loading flow rate is 50L / hour.

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