High-salt-resistant and high-nitrogen-resistant aspergillus and application thereof in fish sauce fermentation

Abstract

The invention discloses high-salt-resistant and high-nitrogen-resistant aspergillus and application thereof in fish sauce fermentation, and belongs to the technical field of microbial fermentation. Three strains of aspergillus domesticated in a high-salt and high-nitrogen environment are provided, non-domesticated strains are separated from koji essence, and the domesticated strains are inoculated to bran to serve as a leavening agent for preparing the fish sauce, so that the fermentation time of the fish sauce can be shortened, the content of total acid, amino acid nitrogen and flavor amino acid in the fish sauce is increased, the pH of the fish sauce is reduced, and the yield of the fish sauce is increased. The generated substances (3-methylbutyraldehyde, (E)-2-hexenal, 1-pentene-3-alcohol, 3-methyl-1-butanol, 2-ethylfuran and the like) such as aldehydes, alcohols and heterocyclic compounds can bring aroma of chocolate, vegetables, muskmelons and the like to the fish sauce; and secondary utilization of the mackerel viscera is also realized, so that the concepts of environmental protection and resource recycling are perfectly met. The method has important significance in improving the flavor of the fish sauce, increasing the utilization rate of byproducts and the like.

Description

Technical Field

[0001] The invention relates to high-salt and high-nitrogen resistant Aspergillus and application thereof in fish sauce fermentation, belonging to the technical field of microbial fermentation. Background Art

[0002] Fish sauce is a uniquely flavored aquatic condiment made from low-value fish or processing waste through natural fermentation. It is nutritious, delicious, and contains a variety of vitamins and minerals beneficial to the human body, making it a highly popular condiment. Traditionally, fish sauce production relies on natural fermentation, mixing low-value ingredients such as fish and shrimp with salt and allowing them to ferment in a natural environment for years. The enzymes inherent in the ingredients and microorganisms in the air work together to hydrolyze the proteins, resulting in a reddish-brown liquid. Each fermentation stage produces a specific microbial flora that influences the quality and flavor of the fish sauce. The interactions between these different microorganisms produce the characteristic flavor compounds of fish sauce.

[0003] Traditional fish sauce fermentation typically involves natural fermentation of low-value seafood (such as sardines, anchovies, and fish tails) for several years in high salt concentrations of 25%-50%. The distinctive aroma of fish sauce is gradually generated by the metabolic activity of halophilic microorganisms. However, these microorganisms are not dominant in the early stages of fish sauce fermentation, requiring a long time to adapt to the high-salt environment, which significantly prolongs the fermentation cycle. Using mackerel viscera as a raw material for fish sauce fermentation has garnered considerable attention in recent years. Firstly, mackerel viscera are rich in proteases (such as trypsin and lipase), which significantly accelerate the degradation of proteins and fats, thereby shortening the fermentation cycle, and also increase the content of umami amino acids such as aspartic acid and glutamic acid. Secondly, mackerel viscera are high in unsaturated fatty acids (such as EPA and DHA), which, upon oxidation, produce aldehydes and ketones, giving fish sauce its complex nutty aroma and richness. Finally, mackerel viscera account for 15%-20% of its body weight during processing and are traditionally discarded or used as feed. Using offal to produce fish sauce can increase added value, which is in line with the concept of circular economy. In addition, offal as a by-product is cheap and has a stable supply.

[0004] However, there is currently a lack of microbial agents that can tolerate high-salt, high-nitrogen fermentation environments, shorten the fish sauce fermentation cycle, and utilize mackerel viscera as a raw material. The advantages of using high-salt, high-nitrogen tolerant Aspergillus are primarily reflected in the following aspects: First, traditional fermentation relies on natural microbial communities, but many strains struggle to metabolize efficiently under these conditions. High-salt, high-nitrogen tolerant Aspergillus, through its own enzyme system and osmotic pressure regulation mechanism, can stably grow and dominate the fermentation process in extreme environments, improving efficiency. Second, the utilization rate of the nitrogen source (protein) in fish sauce fermentation directly affects product quality and yield. Aspergillus efficiently decomposes macromolecular nitrogen and converts it into soluble nitrogen-containing compounds (such as amino acids), reducing resource waste and lowering the amount of undecomposed protein in the fermentation broth, resulting in a clearer and more stable finished product. Finally, traditional fish sauce relies on environmental microorganisms, and its quality is significantly affected by region and season. The introduction of specific Aspergillus strains can achieve targeted fermentation. Combined with modern biotechnologies (such as immobilized cell fermentation), this can promote the standardization and industrialization of fish sauce production. Summary of the Invention

[0005] In response to the above-mentioned deficiencies in the prior art, the present invention provides a high-salt and high-nitrogen tolerant Aspergillus and its application in fish sauce fermentation, aiming to solve the technical problems in the prior art of lacking a fermentation environment that can tolerate high salt and high nitrogen, shortening the fish sauce fermentation cycle, and utilizing mackerel viscera as a raw material for microbial agents.

[0006] The first technical solution provided by the present invention is a strain of Aspergillus oryzae 3.042M1, which was deposited in the General Microbiology Center of the China Culture Collection Administration on January 3, 2025, with a deposit number of CGMCCNO.3.28627.

[0007] The second technical solution provided by the present invention is a strain of Aspergillus oryzae JY309M2, which was deposited in the General Microbiology Center of the China Culture Collection Administration on June 3, 2025, with a deposit number of CGMCCNO.3.28628.

[0008] The third technical solution provided by the present invention is a strain of Aspergillus niger 3.758H, which was deposited in the General Microbiology Center of the China Culture Collection Administration on June 3, 2025, with a deposit number of CGMCCNO.3.28629.

[0009] The present invention provides three strains of Aspergillus oryzae domesticated in a high-salt, high-nitrogen environment, with deposit numbers CGMCC NO.3.28627, CGMCC NO.3.28628, and CGMCC NO.3.28629. The two strains of Aspergillus oryzae and one strain of Aspergillus niger involved in the present invention were deposited on January 3, 2025, and June 3, 2025, respectively, at the General Microbiology Center of the China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Whether fermented alone or in combination, the Aspergillus oryzae 3.042M1 and Aspergillus niger 3.758H strains can lower the pH of the fish sauce and increase the content of total acid, amino acid nitrogen, umami amino acids, and sweet amino acids in the fish sauce. They also produce aldehydes such as 3-methylbutanal and (E)-2-hexenal, giving the fish sauce a chocolate and fat aroma.

[0011] The present invention provides three fermentation strains, which are extracted from three kinds of koji (Shanghai 3.042, JY309 and Shanghai 3.758) and then subjected to ten generations of high-salt and high-nitrogen acclimation, and the generations with the most suitable growth state are selected for late fermentation.

[0012] In one embodiment, the fermentation agent comprises acclimated Aspergillus oryzae 3.042M1, JY309M2 and Aspergillus niger 3.758H or bran inoculated with the three strains.

[0013] Preferably, Aspergillus oryzae 3.042M1, JY309M2 and Aspergillus niger 3.758H are present in the form of live bacteria in the starter.

[0014] The present invention provides a method for acclimating the fermentation strain and a method for preparing fermented bran. The method comprises the steps of obtaining Aspergillus oryzae 3.042M1, JY309M2 and Aspergillus niger 3.758H from koji essence and culturing them to obtain a bacterial liquid:

[0015] (1) Weigh 0.1 g of koji and dilute it with sterilized 0.9% saline (to 10%). 6 ), streak the dilution in a 250 mL eggplant-shaped culture flask with an inoculating loop, and culture in a constant temperature and humidity incubator at 28-30°C for 5 days. Elute the mature Aspergillus oryzae and Aspergillus niger with physiological saline and preserve the bacteria in glycerol to obtain unacclimated Aspergillus oryzae 3.042M1, JY309M2, and Aspergillus niger 3.758H.

[0016] (2) The resulting bacterial liquid was then inoculated into 50 mL PDA culture medium (containing 60 g mackerel visceral slurry, 30 g water, and 3 g edible salt) and cultured in a constant temperature and humidity incubator at 28-30°C for 5 days to obtain the first generation of domesticated strains.

[0017] (3) The domesticated first-generation bacterial liquid was inoculated into 50 mL PDA medium (containing 60 g mackerel visceral slurry, 30 g water and 6 g edible salt), and cultured in a constant temperature and humidity incubator at 28-30° C. for 5 days to obtain the domesticated second-generation strain.

[0018] (4) Similarly, the weight of mackerel viscera slurry added was 120g, 240g, 480g and 960g respectively; the weight of water added was 60g, 120g, 240g and 480g; the weight of edible salt added was 18g, 24g, 60g, 72g, 168g, 192g, 432g and 480g respectively. A total of ten generations of domestication were carried out.

[0019] (5) The optimal acclimation generation was determined by observing the growth rate and color of the acclimated strain, and the optimal generation strain was diluted with 0.9% physiological saline to prepare a bacterial suspension.

[0020] Preferably, the culture is carried out at 30°C.

[0021] The fourth technical solution provided by the present invention is a microbial preparation, which contains Aspergillus oryzae 3.042M1 described in the first technical solution, Aspergillus oryzae JY309M2 described in the second technical solution, or Aspergillus niger 3.758H described in the third technical solution.

[0022] The fifth technical solution provided by the present invention is a mixed bacterial agent, which includes the Aspergillus oryzae 3.042M1 described in the first technical solution and the Aspergillus niger 3.758H described in the third technical solution.

[0023] The sixth technical solution provided by the present invention is a starter culture, which contains the Aspergillus oryzae 3.042M1 described in the first technical solution, the Aspergillus oryzae JY309M2 described in the second technical solution, the Aspergillus niger 3.758H described in the third technical solution, the microbial preparation described in the fourth technical solution, or the mixed bacterial agent described in the fifth technical solution.

[0024] The seventh technical solution provided by the present invention is the use of Aspergillus oryzae 3.042M1 described in the first technical solution, Aspergillus oryzae JY309M2 described in the second technical solution, Aspergillus niger 3.758H described in the third technical solution, the microbial preparation described in the fourth technical solution, the mixed bacterial agent described in the fifth technical solution, or the starter described in the sixth technical solution in the preparation of fish sauce.

[0025] The eighth technical solution provided by the present invention is a fermentation method for preparing fish sauce, which uses mackerel viscera as raw materials and is prepared by fermentation using Aspergillus oryzae 3.042M1 described in the first technical solution, Aspergillus oryzae JY309M2 described in the second technical solution, Aspergillus niger 3.758H described in the third technical solution, the microbial preparation described in the fourth technical solution, the mixed bacterial agent described in the fifth technical solution, or the starter agent described in the sixth technical solution.

[0026] In one embodiment, bran inoculated with the strain is added to the fish sauce for fermentation before fermentation.

[0027] Preferably, the bacterial concentration of the strain in the initial fermentation system is 1×10 6 ~10 7 cfu / mL.

[0028] Preferably, when fermenting fish sauce with a mixed bacterial agent, Aspergillus oryzae 3.042M1 and Aspergillus niger 3.758H are inoculated at the same time; and the fermentation ratio is 3:1.

[0029] In one embodiment, the fermentation temperature is 32° C. and the fermentation time is 30 days.

[0030] The eighth technical solution provided by the present invention is the use of Aspergillus oryzae 3.042M1 described in the first technical solution, Aspergillus oryzae JY309M2 described in the second technical solution, Aspergillus niger 3.758H described in the third technical solution, the microbial preparation described in the fourth technical solution, the mixed bacterial agent described in the fifth technical solution, or the fermentation agent described in the sixth technical solution to increase the content of flavor substances in fish sauce.

[0031] In one embodiment, the flavor substances include volatile aldehydes such as 3-methylbutyraldehyde, (E)-2-hexenal, benzaldehyde, 2-furfural and (Z)-4-heptenal.

[0032] The technical effects of the present invention are as follows:

[0033] The present invention provides three strains of Aspergillus niger domesticated in a high-salt and high-nitrogen environment. Undomesticated strains are isolated and obtained from koji essence. The domesticated strains (single strain and mixed strain) are inoculated on bran as a starter for preparing fish sauce. Compared with fermentation by undomesticated Aspergillus niger, the strain is applied in fish sauce production to shorten the fermentation time of fish sauce, increase the content of total acid, amino acid nitrogen and umami amino acids in fish sauce, reduce the pH of fish sauce, and generate substances such as aldehydes, alcohols and heterocyclic compounds (3-methylbutanal, (E)-2-hexenal, 1-pentene-3-ol, 3-methyl-1-butanol and 2-ethylfuran, etc.) that can bring aromas such as chocolate, vegetables and melon to fish sauce, which will make the mouthfeel of fish sauce more harmonious; and mackerel viscera as a by-product also achieves secondary utilization, which perfectly meets the concept of environmental protection and resource recycling. It is of great significance for improving the flavor of fish sauce and increasing the utilization rate of by-products.

[0034] Biomaterial Deposit

[0035] Aspergillus oryzae 3.042M1, taxonomically named Aspergillus oryzae, was deposited in the General Microbiology Center of China Culture Collection Administration on January 3, 2025, with the deposit number CGMCC NO.3.28627. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0036] Aspergillus oryzae JY309M2, taxonomically named Aspergillus oryzae, was deposited in the General Microbiology Center of the China Culture Collection Administration on June 3, 2025, with the deposit number CGMCC NO.3.28628. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0037] Aspergillus niger 3.758H, taxonomically named Aspergillus niger, was deposited in the General Microbiology Center of China Culture Collection Administration on June 3, 2025, with the deposit number CGMCC NO.3.28629. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Figure 1 is the colony morphology of three Aspergillus strains;

[0039] Figure 2 The electrophoresis analysis of ITS PCR amplification of three strains;

[0040] Figure 3 This is a safety identification diagram for three strains;

[0041] Figure 4 It is the physicochemical diagram of fermentation performance of starter culture;

[0042] Figure 5 This is the finished product of fermented fish sauce;

[0043] Figure 6 This is a heat map of volatile flavor compounds in fish sauce;

[0044] Figure 7 This is the result of the fish sauce electronic nose.

[0045] Figure 8 This is the result of the fish sauce electronic tongue test. DETAILED DESCRIPTION

[0046] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0047] The raw materials used in the embodiment are:

[0048] 1. Mackerel viscera were donated by Qingdao Yihexing Food Co., Ltd.

[0049] 2. Bran, flour, koji extract, edible salt and flavor protease were purchased from Taobao;

[0050] 3. PDA culture medium was purchased from Qingdao Haibo Co., Ltd.

[0051] Example 1 Acclimation of strains

[0052] Aspergillus oryzae 3.042M1, JY309M2, and Aspergillus niger 3.758H isolated from koji essence were acclimated to high salt and high nitrogen conditions for ten generations. The optimal acclimation generation was determined by the growth rate and color change of the strains. The specific method is as follows:

[0053] (1) Weigh 0.1 g of koji and dilute it with sterilized 0.9% saline (to 10%). 6 ), streak the dilution in a 250 mL eggplant-shaped culture flask with an inoculating loop, and culture in a constant temperature and humidity incubator at 28-30°C for 5 days. Elute the mature Aspergillus oryzae and Aspergillus niger with physiological saline and preserve the bacteria in glycerol to obtain unacclimated Aspergillus oryzae 3.042M1, JY309M2, and Aspergillus niger 3.758H.

[0054] (2) The resulting bacterial liquid was then inoculated into 50 mL PDA culture medium (containing 60 g mackerel visceral slurry, 30 g water, and 3 g edible salt) and cultured in a constant temperature and humidity incubator at 28-30°C for 5 days to obtain the first generation of domesticated strains.

[0055] (3) The domesticated first-generation bacterial liquid was inoculated into 50 mL PDA medium (containing 60 g mackerel visceral slurry, 30 g water and 6 g edible salt), and cultured in a constant temperature and humidity incubator at 28-30° C. for 5 days to obtain the domesticated second-generation strain.

[0056] (4) Similarly, the weight of mackerel viscera slurry added was 120g, 240g, 480g and 960g respectively; the weight of water added was 60g, 120g, 240g and 480g; the weight of edible salt added was 18g, 24g, 60g, 72g, 168g, 192g, 432g and 480g respectively. A total of ten generations of domestication were carried out.

[0057] (5) The optimal acclimation generation was determined by observing the growth rate and color of the acclimated strain, and the optimal generation strain was diluted with 0.9% physiological saline to prepare a bacterial suspension.

[0058] Table 1 Domestication of Aspergillus oryzae Shanghai Brewing 3.042

[0059]

[0060]

[0061] Table 2 Aspergillus oryzae JY309 domestication

[0062]

[0063] Table 3 Domestication of Aspergillus niger Shanghai brewing 3.758

[0064]

[0065] It can be seen from Tables 1 to 3 that the best domestication generation is the sixth generation.

[0066] Example 2 Identification of strains

[0067] 1. Morphological identification

[0068] The acclimated 3.042M1, JY309M2 and 3.758H were spotted onto PDA culture plates and cultured. Their morphology was observed after 5 days. Figure 1As shown, the colonies of both strains of Aspergillus oryzae are yellow-green, but the color of the domesticated Aspergillus oryzae 3.042M1 and JY309M2 is relatively lighter than that of the undomesticated Aspergillus oryzae 3.042M1 and JY309M2. They grow radially from a white hyphae center, with a diameter of approximately 50-53 mm and a velvety texture. They produce no exudate or soluble pigments, and the reverse side is light yellow with distinct radial grooves (JY309M2 lacks them). It is difficult to observe the difference in colony morphology of Aspergillus niger 3.758H before and after domestication with the naked eye. The macroscopic colonies are dark brown and relatively regular in shape. The colonies are chrysanthemum-shaped, with hyphae radiating outward and smooth edges, forming a radial branching structure. The diameter is 60-65 mm, the texture is velvety, and the edges are smooth. Microscopic observation of bacterial morphology revealed that the acclimated strains of Aspergillus oryzae 3.042M1 and JY309M2 possessed conidiophores with numerous sustentacle cells at their bases. These conidiophores produced flask-shaped or subglobose apices with small stalks on their surfaces, upon which spherical conidia grew. The acclimated strain of Aspergillus niger 3.758H exhibited smooth conidiophores, spherical conidial heads measuring 3-6 μm in diameter, and subglobose apices measuring approximately 40-80 μm in diameter. Spores were arranged in two layers, with significantly fewer spores than in Aspergillus oryzae. Further scanning electron microscopic observation revealed that the spore heads of strain 3.042M1 were wheat-ear-shaped, with densely packed, largest spores. The conidiophores were abundant with small, spiny hairs and were relatively robust. The conidia were the largest in size, with a smoother surface. Strain JY309M2 had the most densely distributed spores, with spherical spore heads that were significantly smaller than those of strains 3.042M1 and 3.758H. Its conidia were thinner, with fewer spiny hairs, and its conidia were of moderate size. Strain 3.758H had a relatively densely distributed spores, with a chrysanthemum-shaped spore head, and a size intermediate between that of strains 3.042M1 and JY309M2. Its conidia were thicker, lacked visible spiny hairs, and its conidia were of moderate size.

[0069] 2. Molecular Biology Identification

[0070] ITS PCR amplification electrophoresis analysis and phylogenetic tree of three strains 3.042M1, JY309M2 and 3.758H Figure 2As shown in the figure, the universal primers ITS1 and ITS4-R were used to amplify the transcribed spacer region within the strain. Sequencing results showed that the PCR length of the 3.042M1 and JY309M2 strains was 570bp, while the PCR length of the 3.758H strain was 574bp, with clear and bright bands, indicating that the strains were of high purity and not contaminated. The obtained ITS sequences were subjected to Blast comparison analysis on NCBI, and a phylogenetic evolutionary tree was constructed using MEGA11.0 software and the NJ calculation method. As shown in the figure, the 3.042M1 and JY309M2 sequences have the highest homology with Aspergillus oryzae isolate YRA3 in the database, reaching 100%; the 3.758H sequence has the highest homology with Aspergillus niger strain KNUE 24S229 in the database, reaching 100%. Therefore, based on the analysis of the morphological structure, ITS region sequence and phylogenetic evolutionary tree of the three strains 3.042M1, JY309M2 and 3.758H, the domesticated strains 3.042M1 and JY309M2 were identified as Aspergillus oryzae, and the 3.758H strain was identified as Aspergillus niger.

[0071] 3. Safety assessment

[0072] In a cleanroom, add 5% sheep blood to sterilized Columbia medium. Shake the medium thoroughly, then streak the plate. Incubate in a 28°C incubator for 48 hours, observing for the presence of clearing zones. Use Staphylococcus aureus as a positive control.

[0073] Depend on Figure 3 It can be seen that Staphylococcus aureus, as the positive control strain of this experiment, has a clear transparent halo around the strain, indicating that it is β-hemolytic; while Aspergillus oryzae 3.042M1, JY309M2 and Aspergillus niger 3.758H do not have transparent or grass-green halo around them, indicating that they are not hemolytic and are safe.

[0074] Example 3 Performance Verification of Domesticated Aspergillus oryzae 3.042M1, JY309M2 and Aspergillus niger 3.758H

[0075] A spore suspension (10 μL) of acclimated Aspergillus oryzae 3.042M1 (M1), JY309M2 (M2), and Aspergillus niger 3.758H (H) strains was inoculated onto 10 g of sterilized wheat bran and incubated at 37°C in a constant temperature and humidity incubator for 5 days. Aspergillus utilizes the sugars in the system for growth and reproduction. Both Aspergillus oryzae and Aspergillus niger are high-enzyme-producing strains, so the fermentation capacity of these strains was investigated by measuring changes in enzyme activity in the finished yeast.

[0076] Physical and chemical indicators of finished song

[0077] After fermentation, the koji was centrifuged at 8000 rpm and 4°C for 10 minutes, and the supernatant was collected for analysis of neutral protease, acid protease, saccharifying enzyme, amino acid nitrogen, spore count, α-amylase, total acid, pH, and β-glucosidase. Neutral protease and acid protease were measured using GB / T 28715-2012; saccharifying enzyme was measured using GB 8276-2006; amino acid nitrogen was measured using GB 5009.235-2016; spore count was measured using SB / T 10315-1999; α-amylase was measured using Yoo's modified method; total acid was measured using GB 12456-2021; pH was measured using a pH meter; and β-glucosidase was measured using an enzyme-linked biological assay kit.

[0078] like Figure 4 As shown, koji made with Aspergillus oryzae (M1 and M2) had significantly higher neutral protease activity than the other groups, reaching 3321.69 U / g, but lower acid protease activity. Koji made with Aspergillus niger (H) had higher acid protease activity, reaching 1279.48 U / g, but its ability to produce neutral proteases was poor. Koji made with mixed Aspergillus species (M1+H and M2+H) had lower neutral and acid protease activity than those made with a single strain, but the combination was more harmonious and helped improve the flavor of the product.

[0079] The total acid content in the fermented koji is mainly affected by the metabolism and synthesis of organic acids by microorganisms, the metabolism of acid-producing microorganisms, and the degradation of substances such as proteins and starch. The changes in the pH of the fermented koji are relatively consistent with the changes in the total acid content. Figure 4 The pH of the koji fermented with Aspergillus niger was lower than that of the other four groups, at 5.21. Its total acid content was significantly higher, at 2.67 g / 100 mL. The remaining four groups all had pHs greater than 6 and total acid contents less than 1 g / 100 mL. Aspergillus niger has a strong acid-producing capacity and secretes large amounts of acidic proteases, which significantly lower the pH of the fermentation environment. The high total acid content in Group H is also due to the free amino acids, lactic acid, acetic acid, and other organic acids produced during fermentation.

[0080] The spore growth of single and mixed Aspergillus species was measured for 96 hours (as shown in the figure). The spore growth rate of group M1 and group H was significantly higher than that of the other three groups. At 96 hours, the spore number of the seed koji could reach 2.25×10 10 / g and 2.79×10 10 / g dry basis; although the spore count growth of the mixed Aspergillus group was lower than that of the single Aspergillus group, the spore count content of the seed Aspergillus had also reached the relevant requirements for seed Aspergillus in industrial production.

[0081] The fermentation degree of a product is generally determined by the amino acid nitrogen content of the fermented product. Figure 4 The amino acid nitrogen content in the mixed Aspergillus group was significantly higher than that in the single Aspergillus group, reaching 0.561 g / 100 mL and 0.589 g / 100 mL, respectively. This is because the acidic protease activity in the mixed Aspergillus group is higher than that in the single Aspergillus group. The activity of the acidic protease is positively correlated with the rate of amino acid release, significantly increasing the rate of protein hydrolysis in the raw material. Furthermore, acids are important flavor enhancers and precursors to the formation of various volatile flavor compounds.

[0082] Bran is a food mainly composed of starch. The α-amylase present in it will enzymatically hydrolyze starch. The activity of α-amylase will vary depending on the fermentation bacteria, which will directly affect the quality of the finished koji. Figure 4 As can be seen, the α-amylase activity in both the single-species Aspergillus oryzae group and the mixed Aspergillus group was significantly higher than that in the Aspergillus niger group, exceeding 200 U / mL. Because amylase is the primary enzyme secreted by Aspergillus oryzae, the α-amylase activity in group H was lower. The figure also shows that the saccharification enzyme activities in groups M1 and H were higher than those in the other three groups, exceeding 2000 U / g.

[0083] β-glucosidase activity primarily hydrolyzes glycosidic bonds in starch to promote glucose release, hydrolyzing the sugars in the finished koji into small polysaccharides and monosaccharides. These not only serve as energy for the initial growth of Aspergillus oryzae and Aspergillus niger but also significantly influence the sweetness of the fish sauce. As shown in the figure, the β-glucosidase activity in the M1 and M1+H groups was significantly higher than that in the other three groups, reaching 2172.39 nmol / min / g fresh weight and 1577.98 nmol / min / g fresh weight, respectively, reaching six and four times that of the H group.

[0084] Example 4 Low-salt fish sauce fermentation products made from different strains

[0085] The fermentation performance of combining different bacterial strains into song is viewed, and the bacterial strain that finally determines to carry out fish sauce fermentation is aspergillus oryzae 3.042M1, aspergillus niger H and mixed bacterial strain M1+H (rice: black=3:1), and the bacterial strain after determining is inoculated into wheat bran as the leavening agent of fish sauce. The preparation method of present embodiment fish sauce is: after mackerel viscera is thawed in flowing water, clean, boil and smash to pieces, get 180g mackerel viscera slurry+20g leavening agent+20g flour+360g water+1g flavor protease, then add 10% edible salt, seal with four layers of gauze, carry out 30d cultivation in 32 ℃ constant temperature and humidity incubator, stir every day and take samples every 10d, finished product is as shown in Figure 2. Figure 5 shown.

[0086] 1. Free amino acids in low-salt fish sauce fermented by different strains

[0087] A Waters ARC 2489 high performance liquid chromatograph was used for the determination of free amino acids. Column: Waterssymmetry C18 (4.6 x 250 mm 4.6 μm); Column temperature: 40°C; Detection wavelength: 360 nm; Injection volume: 10 μL; Flow rate: 1.5 mL / min; Injection time: 30 min; Mobile phase A: acetonitrile; Mobile phase B: Acetic acid-sodium acetate buffer: 2.5 g sodium acetate, 1.5 mL triethylamine, and 1.17 mL glacial acetic acid dissolved in 1 L of pure water. Seventeen amino acids (except tryptophan) were detected in the derivatized mixed standard working solution and sample solution, and the peak areas were integrated and recorded. The internal standard single-point quantitative method was used to calculate the content of each amino acid in the sample according to the following calculation formula:

[0088]

[0089] Tables 4 and 5 show that the free amino acid compositions of the three fish sauces were identical, including 10 essential amino acids and 7 nonessential amino acids, indicating a relatively rich variety of free amino acids in these three fish sauces. Compared with the fish sauce fermented for 0 days, the total free amino acid content of the fish sauce fermented for 30 days increased significantly, reaching (3594.117±43.577), (3684.339±84.249), and (3612.165±36.326) mg / 100 mL, respectively. This may be due to the fact that Aspergillus oryzae and Aspergillus niger promoted protein hydrolysis in the viscera as fermentation progressed. The free amino acid content of the fish sauces in different groups showed different patterns of change with increasing fermentation time, but the content of most free amino acids was positively correlated with time. Compared to fish sauce fermented for 0 days, the proportion of umami amino acids in fish sauce fermented for 30 days increased significantly, reaching 22.53%, 22.91%, and 21.00%, respectively. Glutamic acid was the highest content of umami amino acids, ranking first among the 17 amino acids. Among the three fermented fish sauces, bitter amino acids accounted for 44.18% of the total in M1-30, while sweet amino acids accounted for 52.59%. The amino acids that contributed most to the flavor were Glu (16.23), followed by Lys (6.91), Val (5.99), and Arg (5.12). Arginine, a representative bitter amino acid, can coordinate with other amino acids to enhance product abundance at low concentrations, but at high concentrations, it produces a bitter taste that reduces product acceptance. The proportion of bitter amino acids in the M1-30 group was higher than in the other two groups, which may contribute to its slightly inferior flavor. In the M1+H-30 group, Glu contributed most to the overall flavor, with a TAV value of 16.60, followed by Lys and Val (7.06 and 6.18). Furthermore, the essential amino acid content in the M1+H-30 group was significantly higher than that of the other two groups, at 52.64%, indicating its highest nutritional value. H-30 also had the highest proportion of sweet and umami amino acids, accounting for 56.57% of its total content, which may contribute to its superior flavor to the other two groups. The significantly higher proportion of sweet and umami amino acids in the H-30 group is primarily due to Aspergillus niger being considered a good source of serine proteases, and its ability to produce serine proteases is greater than that of Aspergillus oryzae, resulting in a Ser content four times and six times that of the other two groups. These results indicate that domesticated Aspergillus oryzae and Aspergillus niger strains not only accelerate protein hydrolysis but also ensure that the sweet and umami amino acid content in fermented fish sauce is relatively consistent with the bitter amino acid content, resulting in a harmonious flavor profile.

[0090] Table 4 Amino acid content

[0091]

[0092]

[0093]

[0094]

[0095] Table 5 TAV

[0096]

[0097]

[0098] 2. Volatile flavor compounds in low-salt fish sauce fermented by different strains

[0099] use Flavor compounds were determined by gas phase ion mobility spectrometry. Samples (1.0 g) were placed in 20 mL headspace vials, and each sample was measured in triplicate. Headspace injection conditions included the following: incubation temperature: 60°C; incubation for 10 min; injection volume: 500 μL; splitless injection; incubation speed: 500 rpm; injection needle temperature: 85°C; GC conditions included: column temperature: 60°C; carrier gas: high-purity nitrogen (purity ≥ 99.999%); programmed pressure increase: initial flow rate of 2.0 mL / min for 2 min, linearly increasing to 10.0 mL / min within 8 min, linearly increasing to 100.0 mL / min within 10 min, and linearly increasing to 150.0 mL / min within 10 min. Chromatographic run time: 30 min; Inlet temperature: 80°C; IMS conditions: Ionization source: Tritium (3H); Drift tube length: 53 mm; Electric field strength: 500 V / cm; Drift tube temperature: 45°C; Drift gas: High-purity nitrogen (≥99.999%); Flow rate: 75 mL / min; Positive ion mode. Based on the results of volatile flavor compound detection, qualitative analysis of the target compounds was performed using the VOCal software's built-in GC retention index (NIST 2020) database and the IMS migration time database for search and comparison.

[0100] The types of volatile compounds in each group are shown in Table 6. A total of 56 volatile flavor compounds were identified in the 12 samples, including 26 aldehydes, 18 alcohols, 6 ketones, 3 acids, 2 esters, 3 heterocyclic compounds, and 1 terpene, including monomers and dimers. Aldehydes, alcohols, and ketones were the main volatile compounds, accounting for 28.74%, 39.71%, and 14.99% of the total volatile compounds in the M1 group after 30 days of fermentation; 28.60%, 41.65%, and 13.41% of the total volatile compounds in the M1+H group; and 28.20%, 39.53%, and 13.82% of the total volatile compounds in the H group.

[0101] The proportion of volatile flavor compounds in fish sauce fermented by different low-salt strains Figure 6As shown: aldehydes are mainly derived from lipid metabolism. Because the fat content in fish viscera is relatively high, the aldehyde species are the most numerous; aldehydes usually have a pleasant taste, and their threshold value is relatively low, which will have a significant impact on the overall flavor of the fish sauce. The main aldehydes detected in this embodiment include volatile aldehydes such as 3-methylbutanal, (E)-2-hexenal, benzaldehyde, 2-furfural, and (Z)-4-heptenal. These substances will significantly improve the flavor of the fish sauce. For example, 3-methylbutanal helps the fish sauce form a chocolate flavor and fat aroma; (E)-2-hexenal brings the smell of grass and banana; and high levels of benzaldehyde bring a pleasant nutty and almond flavor to the fish sauce. When fermented for 30 days, the 3.042M1+H group contained the highest proportion of aldehydes, which indirectly proves that mixed strain fermentation is conducive to the production of aldehydes and can significantly improve the flavor of the fish sauce. Alcohols are typically derived from Stecker degradation of amino acids and lipid oxidation. Lipids in visceral organs are fermented into free amino acids, which are then further oxidatively converted into alcohols. Most alcohols contribute to a refreshing, fruity aroma, and unsaturated alcohols generally have a low threshold and contribute significantly to the overall flavor. During the fermentation of these three low-salt fish sauces, alcohols accounted for the highest percentage of alcohols. The 3.042M1 group showed an initial decrease followed by an increase in alcohol content, while the 3.042M1+H and H groups exhibited opposite trends. The unsaturated alcohols detected in this study primarily included 1-penten-3-ol, 3-methyl-1-butanol, 2-butanol, and 2-heptanol. 1-penten-3-ol is often described as having green and tropical fruit aromas; 3-methyl-1-butanol has whiskey and banana notes; and 2-heptanol imparts mushroom and melon aromas. At the end of 30 days of fermentation, 1-penten-3-ol accounted for the highest proportion in group H, while 3-methyl-1-butanol accounted for the highest proportion in group 3.042M1. Furthermore, as fermentation progressed, saturated alcohols with high thresholds, such as methanol, ethanol, propanol, and hexanol, were produced in the fish sauce, significantly contributing to the improved flavor. Ketones primarily originate from the cleavage of sugar molecules during the Maillard reaction. As fermentation progressed, the proportion of ketones in all three low-salt fish sauce groups increased and then decreased. The heterocyclic compounds detected in this study are formed by the reaction of carbon-based compounds with amino compounds. Three furan compounds, 2-ethylfuran, 2-pentylfuran, and tetrahydrofuran, were detected in all three low-salt fish sauce groups. 2-ethylfuran and 2-pentylfuran contribute caramel, bread, bean, and vegetable aromas to the fish sauce.

[0102] Table 6 Volatile matter content

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[0107]

[0108] 3. Electronic nose and electronic tongue of low-salt fish sauce fermented by different strains

[0109] Electronic nose assay method: Pipette 2 mL of fish sauce into a 20 mL headspace vial, immediately seal with three layers of parafilm, and incubate at 45°C for 10 minutes. Assay conditions: carrier gas flow rate of 300 mL / min, sample detection time of 130 seconds, and gas wash time of 180 seconds. Each sample was measured three times.

[0110] Electronic tongue measurement method: A SA402B electronic tongue was used. Balancing: The sensor was first cleaned in cleaning solution for 90 seconds, then in reference solution for 120 seconds, followed by another reference solution for another 120 seconds. The sensor was then reset to zero at the equilibrium position for 30 seconds. Testing: The test lasted 30 seconds, outputting the initial flavor value. Afterward, the sensor was rinsed with reference solution for 3 seconds, then inserted into a new reference solution for 30 seconds to test the aftertaste. The five food flavor sensors (C00, AE1, CA0, CT0, and AAE) were tested four times, with the first cycle excluded and the average of the last three cycles taken as the test result.

[0111] The fish sauce samples fermented by different strains at 0, 10, 20 and 30 days were analyzed by electronic nose. Figure 7 As shown, the fish sauce samples showed little response to the six sensors W1C, W3C, W6S, W5C, W2S, and W2W, indicating that little aromatic compounds, ammonia, hydrides, and organic sulfides were produced during the fermentation process. However, the responses to W5S, W1S, W1W, and W3S were high, indicating the production of nitrogen oxides, alkanes, sulfides, and aliphatic compounds in the fermentation broth. The principal component analysis plot shows that the principal component contribution rate for each group exceeded 90%, reflecting the majority of the sample information, and the three treatment groups were significantly differentiated. Among the fish sauce samples fermented for 30 days, the response values ​​for W1S (sensitive to alkanes) and W1W (sensitive to sulfides) in the H group were lower than those in the 3.042M1 and 3.042M1+H groups. This suggests that, under certain conditions, inoculation with Aspergillus niger can reduce the content of inorganic sulfides and alkanes in the fish sauce fermentation broth.

[0112] The response value of the electronic tongue is related to the strength of the taste. Generally, the stronger the taste, the greater the response value. Figure 8The effects of different fermentation strains on the electronic tongue taste of fish sauce at different fermentation stages were observed. The flavor of the fermented fish sauce broth was primarily umami, bitter, salty, and astringent, with no sourness. The bitterness and astringency sensor response values ​​were higher than those of the other sensors. By the end of the fermentation cycle (30 days), the bitterness and astringency response values ​​of group H were higher than those of the other two groups, while the umami and richness response values ​​were significantly lower than those of the other two groups. The astringent aftertaste of the three fish sauce samples also tended to decrease with increasing fermentation time. Furthermore, the umami value and richness of the 3.042M1 and 3.042M1+H groups were positively correlated with fermentation time, indicating that Aspergillus oryzae enriches the umami flavoring substances in the fermented fish sauce. Umami can generally be seen as a signal of protein; a higher umami response value indicates a higher protein content.

[0113] In summary, the present invention provides two strains of Aspergillus oryzae and one strain of Aspergillus niger, which have been deposited in the General Microbiology Center of the China Culture Collection Administration Committee on March 14, 2025, with the deposit numbers CGMCC3.28627, CGMCC3.28628 and CGMCC3.28629. The high-salt and high-nitrogen resistant Aspergillus oryzae and Aspergillus niger can tolerate the high-salt and high-nitrogen fermentation environment and improve the fermentation performance of the starter. The fish sauce fermented by the Aspergillus oryzae 3.042M1 and Aspergillus niger H has a relatively increased variety and content of the flavor compounds of the fermented fish sauce, and the obtained fish sauce has a deep and clear color, a rich aroma, and reduces resource waste.

[0114] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Aspergillus oryzae 3.042M1, characterized in that The Aspergillus oryzae 3.042M1 was deposited in the General Microbiology Center of the China Culture Collection Administration on January 3, 2025, with the deposit number CGMCCNO.3.28627.

2. A strain of Aspergillus oryzae JY309M2, characterized in that: The Aspergillus oryzae JY309M2 was deposited in the General Microbiology Center of the China Culture Collection Administration on June 3, 2025, with the deposit number CGMCCNO.3.28628.

3. A strain of Aspergillus niger 3.758H, characterized in that The Aspergillus niger 3.758H was deposited in the General Microbiology Center of the China Culture Collection Administration on June 3, 2025, with the deposit number CGMCCNO.3.28629.

4. A microbial preparation, characterized in that The microbial preparation contains the Aspergillus oryzae 3.042M1 described in claim 1, the Aspergillus oryzae JY309M2 described in claim 2, or the Aspergillus niger 3.758H described in claim 3.

5. A mixed bacterial agent, characterized in that: The mixed bacterial agent includes the Aspergillus oryzae 3.042M1 described in claim 1 and the Aspergillus niger 3.758H described in claim 3.

6. A leavening agent, characterized in that The fermentation agent contains the Aspergillus oryzae 3.042M1 according to claim 1, the Aspergillus oryzae JY309M2 according to claim 2, the Aspergillus niger 3.758H according to claim 3, the microbial preparation according to claim 4 or the mixed bacterial agent according to claim 5.

7. Use of the Aspergillus oryzae 3.042M1 according to claim 1, the Aspergillus oryzae JY309M2 according to claim 2, the Aspergillus niger 3.758H according to claim 3, the microbial preparation according to claim 4, the mixed bacterial agent according to claim 5, or the starter culture according to claim 6 in the preparation of fish sauce.

8. A fermentation method for preparing fish sauce, characterized in that: The method comprises the following steps: using mackerel viscera as raw material, and fermenting the mackerel viscera using the Aspergillus oryzae 3.042M1 described in claim 1, the Aspergillus oryzae JY309M2 described in claim 2, the Aspergillus niger 3.758H described in claim 3, the microbial preparation described in claim 4, the mixed bacterial agent described in claim 5, or the starter agent described in claim 6.

9. The method according to claim 8, characterized in that The bacterial concentration of the strain in the initial fermentation system was 1×10 6 ~10 7 cfu / mL, the fermentation temperature was 32°C, and the fermentation time was 30 days; Optionally, when using a mixed bacterial agent to ferment fish sauce, the inoculation ratio of Aspergillus oryzae 3.042M1 and Aspergillus niger 3.758H is 3:

1.

10. Use of the Aspergillus oryzae 3.042M1 according to claim 1, the Aspergillus oryzae JY309M2 according to claim 2, the Aspergillus niger 3.758H according to claim 3, the microbial preparation according to claim 4, the mixed bacterial agent according to claim 5, or the starter culture according to claim 6 to increase the content of flavor substances in fish sauce, characterized in that: The flavor substances include volatile aldehydes of 3-methylbutyraldehyde, (E)-2-hexenal, benzaldehyde, 2-furfural and (Z)-4-heptenal.

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