Preparation method of low-salt fish sauce by rapid fermentation of composite microorganisms

Abstract

The invention discloses a method for preparing a low-salt fish sauce by rapid fermentation of composite microorganisms, and belongs to the technical field of food processing. The preparation method of the invention specifically comprises the following steps: cleaning marine fish and removing the internal organs to prepare minced meat; performing composite enzymatic hydrolysis on the minced meat and then killing enzymes at high temperature; preparing seed koji using Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis, and using the seed koji for fermentation of minced meat; after the fermentation is completed, performing impurity removal and sterilization, ultrafiltration purification and nanofiltration desalination and fishy removal to obtain a low-salt fish sauce. The fish sauce prepared by the invention has low salt content, short fermentation cycle, high amino acid nitrogen content, clear color, strong sauce-flavored seafood flavor, and high antioxidant performance and uric acid-lowering activity, meeting the needs of consumers for healthy and delicious food.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing and relates to a method for preparing a low-salt fish sauce by rapid fermentation of composite microorganisms. Background Art

[0002] Fish sauce is a delicious condiment produced by fermenting low-value fish, shrimp, shellfish and their waste. It is rich in nutrition and has a strong flavor. Fish sauce contains a large number of degraded small peptide substances, which can act on related protein targets and have many potential biological activities, including anti-oxidation, blood pressure reduction, blood sugar reduction, uric acid reduction and other effects.

[0003] The traditional fish sauce fermentation process uses a high-salt pickling method, using the fish's own endogenous protease and fermented by multiple microorganisms. However, due to the high salt content and long production cycle, its ability to be directly used as a functional food and the large-scale development of seafood fermented fish sauce are limited. At the same time, the fish sauce produced has a certain ammonia smell and fishy smell. The rapid fermentation process can accelerate the fermentation process of fish sauce through protease hydrolysis, but due to the short fermentation time, the unique aroma of fish sauce is relatively thin, and there are odors such as rancid smell, rust smell, fish smell, and fatty acid rancidity. Summary of the invention

[0004] The invention aims to provide a method for preparing a low-salt fish sauce by rapid fermentation of composite microorganisms. The method adopts composite enzymolysis and multi-strain fermentation technology and combines ultrafiltration and nanofiltration experiments to produce a fish sauce with sufficient flavor, no fishy smell or ammonia odor, low salt and sodium, rapid fermentation, and antioxidant and uric acid-lowering activities.

[0005] In a first aspect, the present invention provides a method for preparing a low-salt fish sauce by rapid fermentation of composite microorganisms, comprising: cleaning marine fish and removing the internal organs to prepare minced meat; performing composite enzymatic hydrolysis on the minced meat and killing enzymes at high temperature to obtain an enzymatic solution; preparing seed koji using Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis, and using the seed koji for fermentation of the enzymatic solution to obtain a fermentation product; removing impurities and sterilizing, ultrafiltration purification and nanofiltration desalting and removing fishy smell from the fermentation product to obtain a low-salt fish sauce;

[0006] The seed koji is a mixture of Lactobacillus plantarum seed koji, Saccharomyces rouxii seed koji, Aspergillus oryzae seed koji and Bacillus subtilis seed koji in a mass ratio of 1:1:1:1;

[0007] The ultrafiltration purification comprises: defatting and removing impurities from the fish sauce using a 10000Da ultrafiltration membrane to obtain a finished fish sauce;

[0008] The nanofiltration desalination and fishy smell removal comprises: the finished fish sauce is mixed with water, passed through a 300Da nanofiltration membrane, concentrated, and mixed with water again, passed through a 300Da nanofiltration membrane, and concentrated.

[0009] Furthermore, the raw material of the fish sauce in the present invention is sea tilapia, and the composite enzymolysis of the present invention comprises: mixing minced meat with deionized water, adjusting the pH to 7 with NaOH and HCl, adding protease and performing enzymolysis at 50° C. for 8 hours by shaking; through proteolysis, different enzymes produce synergistic effects, thereby shortening the fermentation time;

[0010] The protease is a combination of neutral protease and flavor protease, or a combination of neutral protease, flavor protease, pepsin, bromelain and papain;

[0011] The high temperature enzyme inactivation includes: high temperature enzyme inactivation at 121° C. for 20 minutes.

[0012] Furthermore, the present invention provides a method for preparing seed koji, which is as follows: soybean meal, bran and water are mixed evenly and sterilized with high-pressure steam to obtain koji material; Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis are respectively inoculated on the koji material at an inoculation amount of 1% and mixed evenly, and then cultured at 37°C for 3 days to obtain Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis seed koji, and the koji material is added to provide a carbon source for the growth of microorganisms, and at the same time, the sugars, acids, aldehydes and other substances produced by the decomposition of the koji material provide a richer flavor for the fish sauce; the mixed strain fermentation can not only improve the enzyme system in the fish sauce fermentation process, improve the utilization rate of raw materials, and degrade fishy and odorous substances, but also produce different flavor substances, thereby improving and enriching the flavor of the fish sauce.

[0013] Furthermore, the present invention provides a method for preparing a Lactobacillus plantarum liquid, a Saccharomyces rouxii liquid, an Aspergillus oryzae liquid and a Bacillus subtilis liquid, comprising: culturing Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis separately for 2 days.

[0014] Furthermore, the fermentation of the enzymatic hydrolysate of the present invention comprises: mixing the enzymatic hydrolysate, sodium chloride and seed koji, and fermenting at 37° C. and 60% humidity for 30 days.

[0015] Specifically, the mass of sodium chloride is 8% of the mass of the enzymatic hydrolysate;

[0016] The mass of the seed koji is 20% of the mass of the enzymatic hydrolysate.

[0017] Furthermore, the impurity removal and sterilization of the present invention comprises: filtering the fermented product and sterilizing it in high pressure steam at 98°C for 20 minutes;

[0018] The ultrafiltration purification comprises: defatting and removing impurities from the fish sauce using a 10000Da ultrafiltration membrane to obtain a finished fish sauce;

[0019] The nanofiltration desalination and fishy smell removal comprises: the finished fish sauce is mixed with water and then passed through a 300Da nanofiltration membrane for concentration, and then mixed with water again and then passed through a 300Da nanofiltration membrane for concentration. The repeated nanofiltration operation can remove small molecular trimethylamine fishy smell substances, sulfides and sodium chloride along with water, thereby achieving the effect of reducing salt and removing fishy smell. The trimethylamine fishy smell substances, sulfides and sodium chloride in the fish sauce are small molecular substances, less than 300 Daltons, and at the same time, many umami peptides and active functional substances are still retained above 300 Daltons.

[0020] In a second aspect, the present invention provides a low-salt fish sauce, wherein the amino acid nitrogen content of the low-salt fish sauce is 1.051±0.004 g / 100 mL, the total acid content is 1.913±0.022 g / 100 mL, the total nitrogen content is 2.234±0.053 g / 100 mL, and the sodium chloride content is 10.15±0.10%.

[0021] In a third aspect, the present invention provides the use of low-salt fish sauce in the preparation of antioxidant products.

[0022] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0023] (1) The fish sauce prepared by the present invention has a lower salt content of 8% than that of the traditional fermentation, which is less than 20% to 30% of the traditional fish sauce fermentation. In the early stage, neutral protease and flavor protease are used for composite enzymatic hydrolysis, which shortens the fermentation time of the fish sauce. The fish sauce can be matured after 30 days of fermentation, which is less than 1 to 3 years of traditional fermentation.

[0024] (2) The fish sauce prepared by the present invention has a high amino acid nitrogen content, reaching the level of special-grade fish sauce, with the content reaching 1.051 g / 100 mL.

[0025] (3) Use multiple microorganisms (Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae, Bacillus subtilis) for mixed fermentation to produce more flavors and accelerate the fermentation process. Among them, Lactobacillus plantarum, Saccharomyces rouxii, and Aspergillus oryzae can provide rich flavors. Lactobacillus plantarum decomposes koji and raw materials to produce acids, Saccharomyces rouxii decomposes to produce aldehydes, and the reaction of acids and aldehydes can produce esters. Aspergillus oryzae produces rich sauce-flavored substances. At the same time, the mixed strains can degrade fishy and ammonia-odor substances. Among them, Bacillus subtilis and Aspergillus oryzae can produce highly active proteases, which accelerate the decomposition of proteins during the fermentation process and shorten the fermentation time.

[0026] (4) The fish sauce produced is clear in color, has no fishy smell, no peculiar smell, and no ammonia smell. It has a strong seafood flavor, is less salty, and has a high acidity. Compared with commercially available fish sauce, it has a better sensory evaluation.

[0027] (5) The fish sauce prepared by the present invention has high antioxidant activity and uric acid-lowering biological activity. After the fish sauce was diluted 100 times, the DPPH scavenging rate was 43.92±4.56%. The fish sauce was freeze-dried, and it was found that the IC50 half inhibition concentration of the prepared fish sauce for scavenging DPPH was 941.8ug / mL. After the fish sauce was diluted 10 times, the xanthine oxidase inhibition rate was 44.30±6.78%. The fish sauce was freeze-dried, and the xanthine oxidase inhibition rate of the fish sauce at 8000ug / mL was 45.34±5.92%.

[0028] (6) The present invention combines ultrafiltration and nanofiltration operations to purify, desalinate and remove fishy smell from fish sauce. The salt, trimethylamine fishy smell substances and sulfide substances are less than 300 Daltons. By adding water to the fish sauce and performing repeated nanofiltration, these substances are removed along with the water, and then concentrated to the original volume, which can reduce the salinity, fishy smell and ammonia odor. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the amino acid nitrogen content produced by enzymatic hydrolysis with different enzymes and enzyme combinations.

[0030] Figure 2 The effect of fermentation time on the amino acid nitrogen content in the finished fish sauce. A is the change of amino acid nitrogen content in the finished fish sauce with fermentation time; B is the comparison of amino acid nitrogen content in the finished fish sauce, Chutang fish sauce, Fengqiu brand fish sauce and Mintian special fish sauce; ** represents significant difference.

[0031] Figure 3 The effect of fermentation time on the total acid content in the finished fish sauce. A is the change of total acid content in the finished fish sauce with fermentation time; B is the comparison of total acid content in the finished fish sauce, Chutang fish sauce, Fengqiu brand fish sauce and Mintian special fish sauce; ** represents significant difference.

[0032] Figure 4 The effect of fermentation time on the non-enzymatic browning of finished fish sauce. A is the change of non-enzymatic browning of finished fish sauce with fermentation time; B is the comparison of browning values ​​of finished fish sauce, Chutang fish sauce, Fengqiu brand fish sauce and Mintian special fish sauce; ** represents significant difference.

[0033] Figure 5 Comparison of total nitrogen content and soluble solid content in finished fish sauce, first soup fish sauce, Fengqiu brand fish sauce and Mintian premium fish sauce. A is total nitrogen content; B is soluble solid content; ** represents significant difference.

[0034] Figure 6 Comparison of sodium chloride content in finished fish sauce, first soup fish sauce, Fengqiu brand fish sauce and Mintian premium fish sauce. ** indicates significant difference.

[0035] Figure 7This is the PCA diagram of the electronic nose detecting finished fish sauce, Chutang fish sauce, Fengqiu brand fish sauce and Mintian premium fish sauce.

[0036] Figure 8 The loading diagram of the electronic nose for finished fish sauce detection is shown in Figure 1. Among them, W1W is a sulfur compound; W1C is a benzene compound; W1S is a methyl compound; W2W is an organic sulfide; W2S is an aldehyde ketone compound; W3S is a long-chain alkane compound; W3C is an amine compound; W5C is a short-chain alkane compound; W5S is a nitrogen oxide compound; and W6S is a hydrogen compound.

[0037] Fig. 9 This is an electronic tongue radar chart of the finished fish sauce.

[0038] Fig.10 This is a radar chart for the sensory evaluation of finished fish sauce.

[0039] Fig.11 The figure is the result of the antioxidant activity test of the finished fish sauce. Among them, A is the DPPH removal rate of the finished fish sauce diluted to different contents; B is the DPPH removal rate of the finished fish sauce and its components diluted to different concentrations; >1KDa is the component larger than 1KDa of the finished fish sauce after passing through the 1KDa nanofiltration membrane; <1KDa is the component less than 1KDa of the finished fish sauce after passing through the 1KDa nanofiltration membrane.

[0040] Fig.12 The figure is a diagram of the xanthine oxidase inhibition effect of the finished fish sauce. Among them, A is the xanthine oxidase inhibition rate of the finished fish sauce diluted to different contents; B is the xanthine oxidase inhibition rate of the finished fish sauce and its components diluted to different concentrations; >1KDa is the component larger than 1KDa of the finished fish sauce after passing through the 1KDa nanofiltration membrane; <1KDa is the component less than 1KDa of the finished fish sauce after passing through the 1KDa nanofiltration membrane. DETAILED DESCRIPTION

[0041] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0042] Example 1

[0043] This embodiment provides a method for preparing fish sauce.

[0044] 1. Microbial inoculation culture: Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis (Lactobacillus plantarum is Lactobacillus plantarum subsp. Plantarum ATCC 14917(T); Saccharomyces rouxii Zygosaccharomyces rouxii GIM2.54, purchased from Guangdong Provincial Culture Collection Center; Aspergillus oryzae Aspergillus oryzae ATCC 42149, purchased from Guangdong Provincial Culture Collection Center; Bacillus subtilis Bacillus subtilis HL-1, provided by the Marine Biological Products Research Team of the School of Food Science and Technology of Guangdong Ocean University) was dissolved and inoculated in different liquid culture media. Among them, Lactobacillus plantarum was inoculated in MRS medium, Saccharomyces rouxii was inoculated in PDB medium, Aspergillus oryzae was inoculated in PDB medium, and Bacillus subtilis was inoculated in nutrient broth medium. The culture medium inoculated with microorganisms was placed in a constant temperature and humidity environment at 37°C and 60% humidity for 2 days to obtain Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae, and Bacillus subtilis liquids.

[0045] 2. Koji making: put soybean meal, bran and water (mass ratio is 3:2:6) into a bottle to prepare koji material. After stirring and mixing evenly, sterilize with high-pressure steam (121℃, 20min). After cooling naturally to room temperature, inoculate Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis at an inoculation rate of 1% into the koji material, stir evenly, stir and turn the koji once a day, and culture at 37℃ for 3 days. When Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis cover the koji material, the koji making is mature to obtain Lactobacillus plantarum seed koji, Saccharomyces rouxii seed koji, Aspergillus oryzae seed koji and Bacillus subtilis seed koji.

[0046] 3. Pretreatment and pulverization of fish raw materials: Clean the whole tilapia, remove the internal organs, cut into small pieces, and use a meat grinder to grind the small pieces of tilapia to obtain minced meat.

[0047] 4. Enzyme hydrolysis: Add deionized water to the minced meat, the ratio of water to minced meat is the solid-liquid ratio, that is, water: minced meat = 0.6:1, then use 0.5 mol / L NaOH and 0.5 mol / L HCl to adjust the pH of the minced meat to 7. After adding 2000 U / g of neutral protease and 1000 U / g of flavor protease, place it in a constant temperature water bath at 50°C and shake for 8 hours to obtain an enzymolysis solution. In this embodiment, the composite protease hydrolysis allows different enzymes to produce a synergistic effect, which can shorten the fermentation time.

[0048] 5. Inactivate enzyme: Inactivate the enzyme in the hydrolyzate at 121°C for 20 min.

[0049] 6. Mixed strain fermentation: add the enzymatic solution to the fermentation tank, add 8% sodium chloride by mass fraction of the enzymatic solution, and 20% mixed koji (Lactobacillus plantarum: Saccharomyces rouxii: Aspergillus oryzae: Bacillus subtilis = 1:1:1:1), seal the bottle mouth, and place it in a constant temperature and humidity environment at 37°C and 60% humidity for 30 days to obtain a fermented product. In this embodiment, the carbon source for microbial growth is provided by adding koji material, and the sugars, acids, aldehydes and other substances produced by the decomposition of the koji material provide a richer flavor for the fish sauce; mixed strain fermentation can not only improve the enzyme system in the fish sauce fermentation process, improve the utilization rate of raw materials, and degrade fishy and odorous substances, but also produce different flavor substances, improving and enriching the flavor of the fish sauce.

[0050] 7. Removal of impurities and sterilization: After the fermentation is completed, use gauze to filter and remove impurities, and then sterilize with high-pressure steam at 98°C for 20 minutes to obtain fish sauce.

[0051] 8. Ultrafiltration purification: The fish sauce is passed through a 100,000 Da ultrafiltration membrane for degreasing and impurity removal to produce finished fish sauce.

[0052] 9. Nanofiltration desalination for initial fishy smell: Add 6 times the mass of water to the finished fish sauce, mix evenly, pass through a 300Da nanofiltration membrane, discard the part below 300Da, concentrate to the original volume, add 6 times the water and repeat the nanofiltration once, concentrate to about the original volume, achieve the removal of small molecule salt, fishy substances and sulfides, and obtain low-salt fish sauce. Repeated nanofiltration operations can remove small molecule trimethylamine fishy substances, sulfides and sodium chloride along with water, achieving the effect of reducing salt and removing fishy smell. Trimethylamine fishy substances, sulfides and sodium chloride in fish sauce are small molecules, less than 300 Daltons, while more than 300 Daltons still retain many umami peptides and active functional substances.

[0053] Example 2

[0054] This example provides relevant physicochemical properties of fish sauce.

[0055] 1. Selection of enzyme types: Taking the amino acid nitrogen content in the finished fish sauce as an indicator, different enzymes are used for combined enzymolysis to screen out the best enzyme combination for enzymolysis of fish minced meat. The experiment was carried out with a material-liquid ratio of 1:1 (water: minced meat), an enzymolysis time of 8 hours, and an enzymolysis temperature of 50°C as the quantitative, and different types of enzyme combinations as variables. Among them, the types and addition amounts of enzymes are: neutral protease 2000U / g, flavor protease 1000U / g, pepsin 900U / g, bromelain 1000U / g, papain 1000U / g. Figure 1As shown, the amino acid nitrogen content produced by the enzymatic hydrolysis reaction of neutral protease alone is the lowest, which is 0.39±0.003g / 100mL; while the amino acid nitrogen content produced by the combined enzymatic hydrolysis of neutral protease and flavor protease and the combined enzymatic hydrolysis of neutral protease, flavor protease, pepsin, bromelain, and papain is the highest, which is 0.60±0.008g / 100mL and 0.60±0.003g / 100mL respectively. In the case where the enzymatic hydrolysis effects produced by the two enzyme combinations are the same, considering the economic cost, the present invention subsequently uses neutral protease and flavor protease for combined enzymatic hydrolysis.

[0056] 2. The effect of fermentation time on the amino acid nitrogen content of fish sauce: refer to the formaldehyde titration method in the national food standard GB 5009.235-2016 "Determination of amino acid nitrogen in food" for detection. Figure 2 As shown in A, as the fermentation time increases, the amino acid nitrogen content in the finished fish sauce starts to increase from 0.766±0.010g / 100mL on the second day. On the 18th day, the growth trend slows down until it reaches 1.111±0.049g / 100mL on the 30th day. The combined enzymatic hydrolysis of neutral protease and flavor protease gives the fish sauce a higher initial amino acid nitrogen content. The addition of Bacillus subtilis and Aspergillus oryzae during the fermentation process promotes the production of proteases, and the hydrolysis of proteins and polypeptides during the fermentation process can produce free amino acids. Figure 2 As shown in B, the amino acid nitrogen content in the finished fish sauce after ultrafiltration is reduced to 1.051±0.004g / 100mL, which is higher than the amino acid nitrogen content of 0.775±0.006g / 100mL of Chutang Fish Sauce and 0.595±0.023g / 100mL of Fengqiuma Fish Sauce, which are three fish sauce products on the market, and is equivalent to the amino acid nitrogen content of 1.087±0.041g / 100mL of Mintian Special Fish Sauce, which meets the requirements of special fish sauce. In summary, the finished fish sauce produced by the present invention contains a higher amino acid nitrogen content.

[0057] 3. Effect of fermentation time on total acid content of fish sauce: Refer to the pH meter potentiometric titration method in the national food standard GB 12456-2021 "Determination of total acid in food" for detection. Figure 3 As shown in A in Figure 1, as the fermentation time increases, the total acid content in the finished fish sauce decreases, but there is no significant change. Figure 3As shown in Figure B, the total acid content of the finished fish sauce after ultrafiltration is 1.913±0.022 g / 100 mL, which is more than twice the total acid content of three fish sauce products on the market (Chu Tang Fish Sauce, Fengqiu Brand Fish Sauce, Mintian Special Fish Sauce). During the fermentation process, a large amount of lactic acid is produced by adding plant lactobacillus, thereby increasing the total acid content in the finished fish sauce. In summary, the finished fish sauce produced by the present invention contains a higher total acid content.

[0058] 4. Effect of fermentation time on non-enzymatic browning of fish sauce: The non-enzymatic browning in finished fish sauce was detected by using a microplate reader at a wavelength of 420 nm. Figure 4 As shown in A, there is no obvious change in the browning degree of the finished fish sauce during the fermentation period of 0 to 10 days, but during the fermentation period of 10 to 30 days, the browning degree begins to deepen as the fermentation time increases, i.e., OD 420nm The absorbance at the 2nd day increased from 2.726±0.25 to 3.600±0.292 on the 30th day. Figure 4 As shown in B, after ultrafiltration, the finished fish sauce is sterilized by high temperature and high pressure steam. 420nm The value increased to 3.986±0.105, which was much higher than 0.760±0.013 (OD 420nm ), the first soup fish sauce 0.966 ± 0.006 (OD 420nm ), and Fengqiu Brand fish sauce 0.592±0.011 (OD 420nm ). Therefore, the finished fish sauce prepared by the present invention contains a higher browning value and has a darker color.

[0059] 5. Determination of total nitrogen and soluble solids content in finished fish sauce: Figure 5 From A in the figure, we can see that the total nitrogen content in the finished fish sauce is 2.234±0.053g / 100mL, which is much higher than the total nitrogen content of 1.216±0.046g / 100mL in Mintian Special Grade Fish Sauce, 1.044±0.011 g / 100mL in Chutang Fish Sauce, and 0.597±0.0255g / 100mL in Fengqiu Brand Fish Sauce. Figure 5 From B in the figure, we can see that the soluble solids in the finished fish sauce are lower than other fish sauces on the market. The reason is that ultrafiltration is carried out at the end of fermentation to remove macromolecular substances, making the soluble solids content in the finished fish sauce lower.

[0060] 6. Determination of sodium chloride content in finished fish sauce: refer to the silver volume method (direct titration method) in GB 5009.44-2016 "National Food Safety Standard for the Detection of Chloride in Food" for determination, and then convert the mass fraction of chloride into the mass fraction of sodium chloride. Figure 6It can be seen that the sodium chloride content in the finished fish sauce is 10.15±0.10%, which is much lower than the 25.29±0.72% of the sodium chloride content in Mintian Special Grade Fish Sauce. The sodium chloride content in Chutang Fish Sauce is 25.13±0.30%, and the sodium chloride content in Fengqiuma Fish Sauce is 20.31±0.81%. During the fermentation process, since the fermentation time is much shorter than that of other fish sauce products, the amount of sodium chloride required to inhibit the growth of miscellaneous bacteria is less, that is, the amount of sodium chloride added is 8%. However, due to the loss of water during high-temperature sterilization, the sodium chloride content in the finished fish sauce increases to about 10.15%.

[0061] 7. Detection of fish sauce odor: This embodiment uses an electronic nose to detect the odor of fish sauce. Figure 7 From A in , we can see that the first principal component is the horizontal coordinate (accounting for 95.64%), and the second principal component is the vertical coordinate (accounting for 3.30%), and the sum of the two accounts for 98.84%. Figure 8 It can be seen that the sensors with the greatest impact of the first principal component are W5S, W1W, and W2S, and the corresponding odors are nitrogen oxides, sulfur compounds, and aldehydes and ketones. The flavors of the finished fish sauce and the other three fish sauces on the market (Chu Tang Fish Sauce, Fengqiu Brand Fish Sauce, and Mintian Special Fish Sauce) can be clearly distinguished on the PCA graph. At the same time, the value of the first principal component is significantly higher than that of the other three fish sauces, and the smell is better than the other three fish sauces, proving that multi-bacteria fermentation has a good effect on the odor of fish sauce.

[0062] 8. Fish sauce taste detection: This embodiment uses an electronic tongue to detect the taste of fish sauce. The fish sauce is diluted 30 times with distilled water and then tested with the electronic tongue. Fig. 9 As shown in the figure, in terms of taste, the finished fish sauce can be clearly distinguished from the other three fish sauces (Chu Tang Fish Sauce, Fengqiu Brand Fish Sauce, and Mintian Special Fish Sauce). The finished fish sauce scored less in saltiness and astringency, which was better than the three commercially available fish sauces. The finished fish sauce scored higher in bitterness, which may be because the degree of fermentation and hydrolysis of the fish sauce was not as high as that of the three commercially available fish sauces, resulting in the accumulation of bitter peptides. In terms of umami, the finished fish sauce scored lower in umami.

[0063] 9. Sensory evaluation: The sensory evaluation was conducted by a panel of 10 people who had received sensory evaluation training, and the evaluation was conducted using random numbers. The fish sauce was evaluated from nine dimensions, including freshness, saltiness, fishy smell, sauce flavor, mellow taste, aftertaste, color, clarity, and seafood flavor. The sensory evaluation table is shown in Table 1.

[0064] Table 1 Sensory evaluation table

[0065]

[0066]

[0067] Depend on Fig.10It can be seen that compared with the other three commercially available fish sauces (Chu Tang Fish Sauce, Fengqiu Brand Fish Sauce, and Mintian Special Fish Sauce), the finished fish sauce has a bright and uniform color, a strong sauce flavor, a mellow taste, a light fishy smell, appropriate freshness, a strong seafood flavor, and a lasting aftertaste, and has obtained a high sensory evaluation, but the sensory evaluation of the freshness and clarity of the finished fish sauce is not as good as the three commercially available fish sauces, which is consistent with the test results of the electronic tongue. The overall score of the finished fish sauce is 83.3±2.65, and the total scores of the other three commercially available fish sauces (Mintian Special Fish Sauce, Chu Tang Fish Sauce, and Fengqiu Brand Fish Sauce) are 52.4±2.80, 49.6±4.32, and 46.6±3.53, respectively. Therefore, the finished fish sauce has a higher score in the comprehensive evaluation of sensory evaluation, which is better than the other three commercial fish sauces.

[0068] 10. Antioxidant activity test of fish sauce: The antioxidant activity of fish sauce was evaluated by DPPH free radical scavenging test. Fig.11 A in the figure shows that after the finished fish sauce is diluted with water to a 10% content, its DPPH free radical scavenging rate is 91.33±0.80%; after being diluted to a 1% content, the finished fish sauce still has a high antioxidant activity, and the DPPH free radical scavenging rate is 43.92±4.56%. The finished fish sauce is passed through a 1KDa nanofiltration membrane and divided into two components >1KDa and <1KDa. The finished fish sauce and its two components >1KDa and <1KDa are freeze-dried and diluted to 125ug / mL, 250ug / mL, 500ug / mL, 1000ug / mL, 2000ugmL, 4000ug / mL and 8000ug / mL concentrations, and the DPPH scavenging rate is measured. The IC50 half inhibition concentration of DPPH free radical scavenging of the finished fish sauce is 941.8ug / mL; the IC50 half inhibition concentration of DPPH free radical scavenging of the >1KDa component is 541.4ug / mL; the DPPH free radical scavenging rate of the <1KDa component is less than 50% at each concentration, and the results of DPPH scavenging rate at each dilution concentration are as follows Fig.11 As shown in B. Therefore, the finished fish sauce provided by the present invention has strong antioxidant activity, and its antioxidant activity mainly comes from the >1KDa component.

[0069] 11. Detection of uric acid activity in fish sauce: The xanthine oxidase inhibition test was used to evaluate the uric acid-lowering activity of soy sauce. The finished fish sauce was passed through a 1KDa nanofiltration membrane and divided into two components, >1KDa and <1KDa. The finished fish sauce and its two components, >1KDa and <1KDa, were freeze-dried and diluted to concentrations of 125ug / mL, 250ug / mL, 500ug / mL, 1000ug / mL, 2000ug / mL, 4000ug / mL and 8000ug / mL. 50uL of each component of the finished fish sauce with different concentrations and 50uL of 100U / L XOD xanthine oxidase were added to each well of a 96-well plate. The reaction was allowed to proceed for 30 minutes at room temperature. Then 100uL of 1000umol / L xanthine solution was added. The reaction was allowed to proceed for 30 minutes. The absorbance at 290nm was measured. Fig.12 From A in the figure, it can be seen that when the finished fish sauce is diluted to 10%, the inhibition rate of xanthine oxidase is 44.30±6.78%; when the finished fish sauce is diluted to 1%, the inhibition rate is 7.70±3.85%. Fig.12 From B, we can see that when the concentration of finished fish sauce is 8000ug / mL, the xanthine oxidase inhibition rate is 45.34±5.92%; the xanthine oxidase inhibition rate of the <1KDa component is 28.76±3.01%; the >1KDa component shows symptoms of promoting xanthine oxidase activity. The reason may be that the >1KDa component contains more xanthine formed by protein, which is decomposed by xanthine oxidase to form more uric acid levels.

[0070] 12. Sensory evaluation of fish sauce after nanofiltration desalination: Substances less than 300Da removed by nanofiltration have a strong fishy and ammonia smell, and also have a certain salty taste. Repeated deionized water was used to remove the fishy, ​​ammonia and sodium chloride smells in the finished fish sauce. After nanofiltration desalination, the fishy and salty scores of low-salt fish sauce were reduced in the sensory evaluation.

[0071] The embodiments described above are part of the embodiments of the present invention, but not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A method for preparing low-salt fish sauce by rapid fermentation of composite microorganisms, characterized in that: include: The marine fish is cleaned and the internal organs are removed to prepare minced meat; the minced meat is subjected to complex enzymatic hydrolysis and then the enzyme is inactivated at high temperature to obtain an enzymatic solution; seed koji is prepared using Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis, and the seed koji is used for fermentation of the enzymatic solution to obtain a fermented product; the fermented product is subjected to impurity removal and sterilization, ultrafiltration purification and nanofiltration desalination and fishy removal to obtain a low-salt fish sauce; The protease of the composite enzymatic hydrolysis is a combination of neutral protease and flavor protease, or a combination of neutral protease, flavor protease, pepsin, bromelain and papain; The seed koji is a mixture of Lactobacillus plantarum seed koji, Saccharomyces rouxii seed koji, Aspergillus oryzae seed koji and Bacillus subtilis seed koji in a mass ratio of 1:1:1:1; The ultrafiltration purification comprises: defatting and removing impurities from the fish sauce using a 10000Da ultrafiltration membrane to obtain a finished fish sauce; The nanofiltration desalination and fishy smell removal comprises: the finished fish sauce is mixed with water, passed through a 300Da nanofiltration membrane, concentrated, and mixed with water again, passed through a 300Da nanofiltration membrane, and concentrated.

2. The preparation method according to claim 1, characterized in that: The marine fish is sea tilapia; The composite enzymatic hydrolysis comprises: mixing minced meat with deionized water, adjusting the pH to 7 using NaOH and HCl, adding protease and shaking enzymatic hydrolysis at 50° C. for 8 hours; The high temperature enzyme inactivation includes: high temperature enzyme inactivation at 121° C. for 20 minutes.

3. The preparation method according to claim 1, characterized in that: The method for preparing seed koji comprises: uniformly mixing soybean meal, bran and water and sterilizing with high-pressure steam to obtain koji material; inoculating Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis at an inoculation rate of 1% on the koji material respectively, uniformly mixing, and culturing at 37°C for 3 days to obtain Lactobacillus plantarum, Saccharomyces rouxii, Aspergillus oryzae and Bacillus subtilis seed koji.

4. The preparation method according to claim 3, characterized in that: The preparation of the Lactobacillus plantarum liquid, the Saccharomyces rouxii liquid, the Aspergillus oryzae liquid and the Bacillus subtilis liquid comprises: culturing the Lactobacillus plantarum, the Saccharomyces rouxii, the Aspergillus oryzae and the Bacillus subtilis separately for 2 days.

5. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysate fermentation comprises: mixing the enzymatic hydrolysate, sodium chloride and koji, and fermenting at 37° C. and 60% humidity for 30 days.

6. The preparation method according to claim 5, characterized in that: The mass of sodium chloride is 8% of the mass of the enzymatic hydrolysate; The mass of the seed koji is 20% of the mass of the enzymatic hydrolysate.

7. The preparation method according to claim 1, characterized in that: The impurity removal and sterilization comprises: filtering the fermented product and sterilizing it in high pressure steam at 98° C. for 20 minutes.

8. The low-salt fish sauce prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The low-salt fish sauce has an amino acid nitrogen content of 1.051±0.004 g / 100 mL, a total acid content of 1.913±0.022 g / 100 mL, a total nitrogen content of 2.234±0.053 g / 100 mL, and a sodium chloride content of 10.15±0.10%.

9. Use of the low-salt fish sauce as claimed in claim 8 in the preparation of antioxidant products.

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