Fermentation method for reducing biogenic amine in fish sauce

Abstract

The invention relates to a fermentation method for reducing biogenic amine in fish sauce, which comprises the following steps: 1) taking fresh fish meat, adding 30-50% of sterile water, 18% of salt, 0.5-2% of neutral protease with activity of 2 * 10 < 4 > U / g and 0.03% of sodium pyrophosphate by mass, uniformly mixing and crushing, and bottling; (2) fermenting at the constant temperature of 35 DEG C for 72 days, determining the oxidation-reduction potential in the fermentation process of the fish sauce by using an oxidation-reduction potential depolarization method automatic determinator in the fermentation process, adding a proper amount of hydrogen peroxide solution with the mass fraction of 0.5% every three days, controlling the oxidation-reduction potential of the fish sauce fermentation liquid to be 300-340mV until the fermentation end point, and during the fermentation period, adding a proper amount of water into the fish sauce fermentation liquid, water is supplemented once every three days. The fermentation method is simple to operate and easy to implement, the content of biogenic amine, especially tyramine, of the fish sauce is remarkably reduced, and the quality of the obtained fish sauce is good.

Description

Technical Field

[0001] This invention relates to the field of fermented foods, and more particularly to a fermentation method for reducing biogenic amines in fish sauce. Background Technology

[0002] Fish sauce is a condiment popular in coastal areas of China, Southeast Asian countries, and countries and regions such as Japan and South Korea. Its traditional production process typically involves mixing low-value marine fish (such as anchovies, sardines, and anchovies) with salt in a specific ratio (20%-30%) and fermenting for 6-18 months. Fish sauce has a delicious flavor and is rich in nutrients, containing amino acids, polypeptides, minerals (mainly iron and calcium), and some B vitamins. It also possesses certain antioxidant, blood pressure-lowering, and anti-cancer properties.

[0003] Naturally fermented fish sauce often contains high levels of biogenic amines, exceeding FDA standards and posing a potential food safety risk. Biogenic amines are a collective term for a class of nitrogen-containing, low-molecular-weight organic compounds, widely found in fermented foods rich in protein and amino acids, such as fish sauce, ham, and cheese. Biogenic amines in food are difficult to eliminate through home cooking methods, and under certain conditions, amines can react with nitrites to form more toxic and carcinogenic N-nitroso compounds. Excessive accumulation of biogenic amines in the body can cause a range of adverse symptoms, including headaches, dizziness, and vomiting, especially since histamine and tyramine are highly toxic.

[0004] Biogenic amines in fish sauce are produced during fermentation by the decarboxylation of free amino acids catalyzed by amino acid decarboxylases, and this process is unavoidable. To reduce biogenic amines in fish sauce, existing research has focused on utilizing microorganisms capable of biogenic amine degradation. For example, Chinese patent CN118805866 A discloses the application of *Penicillium polonide* in the degradation of biogenic amines and the preparation of fish sauce. This patent describes how the use of microbial agents during the fermentation process of fish sauce can degrade histamine, tyramine, spermidine, and spermine to a certain extent. Chinese patent CN113265363 B discloses a strain of *Saccharomyces hominis* that reduces biogenic amines and its application. This strain was screened from wheat koji and can significantly reduce the content of biogenic amines in the fermentation environment or the fermented product without compromising the quality of the fermented product. While these microbial methods can achieve certain amine reduction effects, they all suffer from drawbacks such as cumbersome operating procedures, high technical difficulty, and high technical barriers. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention provides a fermentation method for reducing biogenic amines in fish sauce. The method is simple to operate and can not only significantly reduce the content of biogenic amines, especially tyramine, in fish sauce, but also produce fish sauce of better quality.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: A fermentation method for reducing biogenic amines in fish sauce includes the following steps: 1) Take fresh fish meat and add 30-50% of its weight in sterile water, 18% in salt, and 0.5-2% in an active ingredient at a concentration of 2x10. 4 U / g of neutral protease and 0.03% sodium pyrophosphate were mixed, crushed, and then bottled. 2) Ferment at a constant temperature of 35℃ for 72 days. During the fermentation process, the oxidation-reduction potential of the fish sauce was measured using an automatic oxidation-reduction potential depolarization method instrument. By adding an appropriate amount of 0.5% hydrogen peroxide solution every three days, the oxidation-reduction potential of the fish sauce fermentation liquid was controlled at 290-350mV until the end of fermentation. During the fermentation period, water was added every three days.

[0007] Preferably, in step 2), the oxidation-reduction potential of the fish sauce fermentation broth is controlled at 310-330mV.

[0008] Preferably, in step 2), the oxidation-reduction potential of the fish sauce fermentation liquid is controlled at 320mV.

[0009] Preferably, in step 1), fresh fish meat is mixed with 40% of its mass of sterile water, 18% of its mass of salt, and 1% of its mass of active ingredient (2x10). 4 The neutral protease at a concentration of U / g and sodium pyrophosphate at 0.03% were mixed, crushed, and then bottled.

[0010] Preferably, in step 2), an FJA-6 type automatic redox potential depolarization method measuring instrument is used.

[0011] The present invention employs the above-described structure and has the following advantages: 1. This fermentation method requires no microbial intervention, is simple to operate, and is easy to control.

[0012] 2. The fish sauce prepared using this fermentation method significantly reduces the content of biogenic amines, especially the highly toxic tyramine, which is reduced remarkably. Furthermore, the quality of the fish sauce produced by this method is comparable to commercially available fish sauce, indicating significant application potential. Attached Figure Description

[0013] Figure 1 A comparison chart of the biogenic amine content and reduction rate between the experimental group and control group 1; Figure 2 A comparison chart of fish sauce colors between the experimental group and control group 1; Figure 3 This is a comparison chart of the browning degree of fish sauce in the experimental group and control group 1.

[0014] Figure 4 This is a comparison chart of the fish sauce electronic nose analysis between the experimental group and control group 1.

[0015] Figure 5 This is a comparative analysis chart of the electronic tongue of fish sauce in the experimental group, control group 1, and commercially available fish sauce.

[0016] Figure 6 This is a comparison chart of the amino acid nitrogen content in fish sauce between the experimental group and control group 1. Detailed Implementation

[0017] To clearly illustrate the technical features of this solution, the invention will be described in detail below through specific implementation methods and in conjunction with the accompanying drawings.

[0018] (a) Fermentation method 1.1 Fermentation method of this application (experimental group) 1) Take 250g of fresh sand eel and add 30-50% of its weight in sterile water, 18% salt, and 0.5-2% of its active ingredient (2 x 10). 4 U / g of neutral protease and 0.03% sodium pyrophosphate were mixed, crushed, and then bottled. 2) Ferment at a constant temperature of 35℃ for 72 days. During the fermentation process, the oxidation-reduction potential of the fish sauce was measured using an FJA-6 type automatic oxidation-reduction potential depolarization method instrument. The oxidation-reduction potential of the fish sauce fermentation liquid was controlled at 310-330mV by adding 1-2mL of 0.5% hydrogen peroxide solution every three days until the end of fermentation. During the fermentation period, water was added every three days to maintain the total mass of the fermentation liquid.

[0019] 1.2 Control Experiment Control Experiment 1: The difference from the experimental group is that hydrogen peroxide was not added to regulate the redox potential. That is, the fish sauce fermentation liquid obtained without redox potential regulation was used as the sample of control group 1.

[0020] Control Experiment 2: The difference from the experimental group was that sodium pyrophosphate was not added. However, during fermentation in step 2) of 1.1, it was found that the redox potential of the fish sauce fermentation broth was difficult to maintain at the target level after being regulated with hydrogen peroxide solution. This indicates that sodium pyrophosphate plays an important role in the method established in this application. Due to the difficulty in regulating the redox potential, subsequent results were not measured.

[0021] 2. Measurement Method 2.1 Determination of biogenic amine content (1) Preparation of standard solutions: The eight biogenic amine stock solutions were diluted with ultrapure water to prepare gradient concentration solutions of 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 50 mg / L respectively. The eight biogenic amine stock solutions were histamine solution, tyramine solution, tryptamine solution, phenethylamine solution, cadaverine solution, putrescine solution, spermine solution, and spermidine solution; the concentration of each biogenic amine stock solution was 1 g / L.

[0022] (2) Preparation of detection solution: Take 1 mL of each of the above gradient concentration solutions and 1 mL of fish sauce fermentation broth, add 1 mL of Na2CO3 / NaHCO3 (pH=11) buffer solution and shake to mix, then add 1 mL of DNS-Cl and water bath for 60 min (40℃), then add 0.5 mL of 32% ammonia water and shake for 30 min (40℃), then add 3 mL of n-heptane for extraction, take 1 mL of the upper organic phase and blow dry with nitrogen at 40℃, finally redissolve with 1 mL of acetonitrile and filter with a 0.22 μm organic membrane to obtain filtrate.

[0023] (3) Determination of biogenic amine content: The biogenic amine content of the obtained filtrate was determined by high-performance liquid chromatography (HPLC). The determination was performed three times, and the average value was taken. The HPLC conditions were as follows: separation was performed at 35℃ using a C-18 column (5 μm, inner diameter 4.6 mm × 150 mm). A gradient elution program was used with ultrapure water as solvent A and acetonitrile as solvent B. The gradient elution conditions were as follows: 50% A and 50% B at 0 min; 30% A and 70% B at 6 min; 23% A and 77% B at 17 min; and 10% A and 90% B at 33 min. The flow rate was maintained at 1 mL / min, and the injection volume was 20 μL. Biogenic amines were detected at a wavelength of 254 nm.

[0024] Using the measured peak area as the x-axis and the content of each biogenic amine in the gradient concentration solution as the y-axis, standard curves were plotted for peak area versus biogenic amine (histamine, tyramine, tryptamine, phenylethylamine, cadaverine, putrescine, spermine, and spermidine) content. The content of each biogenic amine was obtained by substituting the measured peak area of ​​the fish sauce fermentation broth into the standard curves of each biogenic amine.

[0025] 2.2 Determination of the reduction rate of biogenic amines in fish sauce The reduction rate of biogenic amines was calculated based on the results of the biogenic amine content determination. The calculation formula is as follows: Reduction rate of biogenic amines (%) = (Biogenic amine content of control group - Biogenic amine content of experimental group) / Biogenic amine content of control group × 100.

[0026] 2.3 Determination of the degree of browning in fish sauce Assay method: Add 2 mL of fish sauce sample to 20 mL of methanol solution (50%, v / v) and extract continuously for 1 hour using a magnetic stirrer. After centrifuging the extract at 5000 RPM for 15 minutes, collect the supernatant and measure its absorbance at 420 nm. The absorbance of this supernatant is taken as the result (A). 420 () indicates the degree of browning in fish sauce.

[0027] 2.4 Detection of fish sauce odor Take 0.5 mL of fish sauce sample into a 15 mL injection bottle, seal it quickly, and let it stand for 30 min. Use the manual injection method, insert the electronic nose injection needle into the top of the injection bottle to draw up the volatile gases in the fish sauce for detection. Set the sample preparation time to 5 s, the gas flow rate to 400 mL / min, the sample detection time to 200 s, and the washing time to 120 s.

[0028] Table 1. Electronic nose sensor names and their corresponding sensitive gases

[0029] 2.5 Test of fish sauce flavor The taste of all fish sauce samples was detected using an electronic tongue device, and the default program was used to analyze the sourness, bitterness, umami, saltiness, and astringency of each sample. The control solution was a mixture of 30 mM potassium chloride and 0.3 mM tartaric acid, the positive electrode cleaning solution was a mixture of 10 mM potassium hydroxide, 100 mM potassium chloride, and 30% ethanol, and the negative electrode cleaning solution was a mixture of 100 mM hydrochloric acid and 30% ethanol.

[0030] 2.6 Detection of amino acid nitrogen The test was conducted using the colorimetric method as specified in the national food standard GB 5009.235-2016, "Determination of Amino Acid Nitrogen in Food".

[0031] 3. Measurement Results 3.1 Biogenic amine content and reduction rate (1) The biogenic amine content of the experimental group and control group 1 was determined by the biogenic amine content determination method in 2.1. Among them, the contents of histamine, phenylethylamine, spermine and spermidine are extremely low, less than 0.1% of the total biogenic amine, and are ignored here. The contents of tyramine, tryptamine, cadaverine and putrescine are shown in Table 1.

[0032] Table 2 Biogenic Amine Content

[0033] As shown in Table 2, compared with control group 1, the total biogenic amine content of the experimental group with redox potential regulation was significantly reduced, and the content of tyramine, which is more toxic, was greatly reduced, while the content of tryptamine, which is less toxic, was also slightly reduced. Although the contents of cadaverine and putrescine were slightly increased, these two amines are not very toxic.

[0034] (2) The reduction rate was calculated using the method described in 2.2 for determining the reduction rate of biogenic amines. The results of the reduction rates of biogenic amines (cadaverine, putrescine, tryptamine, tyramine) in the experimental group and control group 1 are shown in the figure. Figure 1 .

[0035] Depend on Figure 1 It can be seen that, compared with control group 1, the total biogenic amines in the experimental group of this application were significantly reduced, with a reduction rate of 26.10%. It is worth noting that the experimental group had the largest reduction in tyramine content, with a reduction rate of up to 92.40%.

[0036] 3.2 Color of fish sauce Figure 2 The fish sauces prepared for the control group 1 and the experimental group were compared. It can be seen that the fish sauce in the experimental group, which was regulated by redox potential, was orange-red in color, slightly lighter than that in the control group. This indicates that the fermentation method of the present invention is beneficial for producing light-colored fish sauce.

[0037] In addition, the degree of browning of the fish sauce in the experimental group and control group 1 was measured using the method in 2.3. The results are shown in [the table below]. Figure 3 .from Figure 3 It can also be seen that the browning degree of the experimental group was reduced, which also indicates that the method of this application is beneficial for the production of light-colored fish sauce.

[0038] 3.3 Fish sauce odor The fish sauce in the experimental group and control group 1 was tested using electronic nose assay according to method 2.4. The results are shown in [Figure 2]. Figure 4 .from Figure 4 As can be seen from the data, the sulfur odor detected by sensor W1W in the experimental group was significantly lower than that in the control group 1, indicating that the sulfur and nitrogen oxide content of the fish sauce obtained in the experimental group was reduced and the odor was better.

[0039] 3.4 Fish sauce flavor To further test the quality of the fish sauce described in this application, electronic tongue testing was performed on the experimental group, control group 1, and commercially available fish sauce (brand: Fengqiu Mai Chaoshan Fish Sauce) using method 2.5. The results are shown in [Figure 2.5]. Figure 5 .from Figure 5 As can be seen, there were no significant differences in various flavors between the experimental group and control group 1, indicating that the fish sauce in the experimental group was comparable in flavor to that in control group 1. However, compared with commercially available fish sauce, both the experimental group and control group 1 showed better performance in terms of umami richness.

[0040] 3.5 Amino acid nitrogen content Amino acid nitrogen content is also an indicator for evaluating the quality of fish sauce. The amino acid nitrogen content of the fish sauce in the experimental group and control group 1 was determined using method 2.6. The results are shown in [Figure 2.6]. Figure 6 .

[0041] from Figure 6 As can be seen, the amino acid nitrogen content of the control group was 0.59±0.01 g / 100mL, and the amino acid nitrogen content of the experimental group was 0.56±0.02 g / 100mL. There was no significant difference in the amino acid nitrogen content between the two groups, and the experimental group was better than the amino acid nitrogen content of Fengqiumai Chaoshan fish sauce on the market (approximately 0.50 g / 100mL).

[0042] In summary, the fermentation method for reducing biogenic amines in fish sauce presented in this application is not only simple to operate, but also significantly reduces the total biogenic amine content of the resulting fish sauce, especially the tyramine content. In addition, the fish sauce obtained in this application is of good quality, specifically in terms of aroma, taste, and amino acid nitrogen content.

[0043] The specific embodiments described above should not be construed as limiting the scope of protection of this invention. Any alternative modifications or variations made to the embodiments of this invention by those skilled in the art will fall within the scope of protection of this invention. All aspects not detailed in this invention are well-known to those skilled in the art.

Claims

1. A fermentation method for reducing biogenic amines in fish sauce, characterized in that, Includes the following steps: 1) Take fresh fish meat and add 30-50% of its weight in sterile water, 18% in salt, and 0.5-2% in an active ingredient at a concentration of 2x10. 4 U / g of neutral protease and 0.03% sodium pyrophosphate were mixed, crushed, and then bottled. 2) Ferment at a constant temperature of 35℃ for 72 days. During the fermentation process, the oxidation-reduction potential of the fish sauce was measured using an automatic oxidation-reduction potential depolarization method instrument. By adding an appropriate amount of 0.5% hydrogen peroxide solution every three days, the oxidation-reduction potential of the fish sauce fermentation liquid was controlled at 290-350mV until the end of fermentation. During the fermentation period, water was added every three days to keep the total mass of the fermentation liquid constant.

2. The fermentation method for reducing biogenic amines in fish sauce according to claim 1, characterized in that, In step 2), the oxidation-reduction potential of the fish sauce fermentation liquid is controlled between 310mV and 330mV.

3. The fermentation method for reducing biogenic amines in fish sauce according to claim 1, characterized in that, In step 1), fresh fish meat is mixed with 40% sterile water, 18% salt, and 1% active ingredient at a concentration of 2 x 10⁻⁶. 4 The neutral protease at a concentration of U / g and sodium pyrophosphate at 0.03% were mixed, crushed, and then bottled.

4. The fermentation method for reducing biogenic amines in fish sauce according to claim 1, characterized in that, In step 2), the redox potential of the fermentation broth is continuously detected using an FJA-6 type automatic redox potential depolarization method measuring instrument.

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