CN116941751A - Preparation method of tuna cooking liquor seasoning liquid - Google Patents

Preparation method of tuna cooking liquor seasoning liquid Download PDF

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Publication number
CN116941751A
CN116941751A CN202310734681.3A CN202310734681A CN116941751A CN 116941751 A CN116941751 A CN 116941751A CN 202310734681 A CN202310734681 A CN 202310734681A CN 116941751 A CN116941751 A CN 116941751A
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China
Prior art keywords
tuna
cooking liquor
yeast
liquor
tuna cooking
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梁璋成
林晓姿
何志刚
陈秉彦
李维新
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Institute of Agricultural Engineering Technology of Fujian Academy of Agricultural Sciences
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Institute of Agricultural Engineering Technology of Fujian Academy of Agricultural Sciences
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    • A—HUMAN NECESSITIES
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    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
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    • A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
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    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
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Abstract

The invention discloses a preparation method of a tuna cooking liquor seasoning liquid. The method comprises the following steps: 1) Aspergillus oryzae MJY2-5 is used as a strain to prepare a yeast; 2) Removing fishy smell of the tuna cooking liquid; 3) Aspergillus oryzae MJY2-5 is combined with the dilute enzymolysis of the protease preparation and combined with the post-salt fermentation technology to prepare the seasoning liquid. The invention screens out the excellent deodorization microorganism of the tuna cooking liquor, namely the Fisher-Tropsch microorganism, namely the Saccharomyces cerevisiae JGM-1 and the Lactobacillus plantarum RP26, so that the fishy smell of the tuna cooking liquor can be reduced, and unique fermentation fragrance can be generated; aspergillus oryzae combines with protease preparation dilute enzymolysis and combines with salt post fermentation technology, can utilize tuna cooking liquor protein as high as possible under the condition of controlling biogenic amine content to reach the standard, and prepares high-flavor amino acid seasoning liquor product.

Description

Preparation method of tuna cooking liquor seasoning liquid
Technical Field
The invention belongs to the field of food processing, and particularly relates to a preparation method of a tuna cooking liquor seasoning liquid.
Background
The tuna contains 8 amino acids necessary for human body, contains various nutritional ingredients and functional substances such as DHA, EPA and the like, and has the advantages of low fat, rich nutrition and the like. When processed into fish fillets and canned tunas, about 15% of cooking liquid byproducts are produced. At present, tuna cooking liquid is not well developed and utilized, most of the tuna cooking liquid is not treated as wastewater to be discharged, protein resources are greatly wasted, wastewater treatment cost is increased, and environmental pollution risks exist. The inventors have studied the quality of the tuna cooking liquid in the early stage test, and found that the solid substance of the tuna cooking liquid contains more than 80% of protein, the glutamic acid, aspartic acid and other flavor amino acids account for 52.91% of the total protein, and the glutamic acid, aspartic acid and other flavor amino acids are 2.32% higher than the soybean protein, so that the tuna cooking liquid is a high-quality raw material for producing foods such as seasonings. Therefore, the deep processing and comprehensive utilization of the tuna cooking liquid are developed, the tuna cooking liquid is developed into a seasoning liquid product with high added value, and the seasoning liquid has good market application prospect.
At present, research and development of foods by using cooking liquids of other raw materials are reported, and Zhao Huajie and the like are processed by using the cooking liquids of mussels to obtain fermented seasonings with good taste and unique flavor; zhang Lou and preparing bioactive peptide with antioxidant function by enzymolysis process with mackerel can decoction as raw material; zheng Xiaojie and the like are processed into the shrimp powder aquatic seasoning by utilizing the shrimp cooking liquid, and the spray drying process is optimized. Less research is done in the aspect of processing and utilizing the tuna cooking liquid, and the method mainly comprises the following steps: chen Qihang and the like, carrying out enzymolysis on the tuna cooking liquor by utilizing protease to prepare the flavor seafood seasoning liquor; yang Xiaoke and He Jiandong extracting fish oil from tuna cooking liquor by enzymolysis; li Kexin A tuna flavor salad dressing is prepared from the cooking juice of tuna. The protease hydrolysis has the advantages of mild condition, less side reaction, easy control of hydrolysis degree, better retention of nutrient components and the like, but also has the defects of unfixed degradation products of different protease preparations, easy generation of sulfur-containing amino acid to destroy the flavor of the product, high production cost and the like.
Disclosure of Invention
The invention aims to provide a preparation method of a tuna cooking liquor seasoning liquid. The method takes the tuna cooking liquor as a raw material, carries out the deep processing and comprehensive utilization of the tuna cooking liquor, and develops the tuna cooking liquor into a seasoning liquor base material product with high added value through microorganism deodorization and composite enzymolysis fermentation.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
aspergillus oryzae with high protein utilization rate, which is classified and named as Aspergillus oryzae MJY2-5, has a academic name of Aspergillus oryzae MJY-5 and is preserved in China center for type culture collection, and has a preservation number of CCTCC NO: m20221400, the preservation date is 09/2022.
The classification of the aroma-producing saccharomycete is named as Fisher-Corylus schizandrae JGM-1, the academic name is Cyberlindnera fabianii JGM-1, and the aroma-producing saccharomycete is preserved in China Center for Type Culture Collection (CCTCC) NO: m20221401, the preservation date is 09/2022.
The lactobacillus plantarum strain for producing the aroma is classified and named as lactobacillus plantarum RP26, the school name is Lactobacillus plantarum RP, and the lactobacillus plantarum strain is preserved in China Center for Type Culture Collection (CCTCC) NO: m2019298, the preservation date is 26 days of 2019, 04 and 26 months.
A preparation method of a tuna cooking liquor seasoning liquid comprises the following steps:
1) Preparing into yeast
1-1) activation of Aspergillus oryzae and preparation of spore suspension
Inoculating Aspergillus oryzae MJY2-5 onto a Bengalia red flat plate, separating and culturing for 70-74 h in a culture box at 31-33 ℃, inoculating single spores into an inclined plane wort agar culture medium by using an inoculating shovel under the aseptic condition, expanding and culturing for 70-74 h in the culture box at 31-33 ℃, completely stripping inclined plane spores by using the inoculating shovel, washing with aseptic water, shaking uniformly to obtain spore suspension, and refrigerating and preserving at 4 ℃ in a refrigerator;
1-2) preparation of a Yeast-forming culture Material
Mixing bran, bean pulp and water according to the mass ratio of 4:6-6.5:7-7.5, regulating the pH value to 6.5-6.6, slowly adding water, stirring while adding water, uniformly mixing by hand without dry powder, mixing until the color is uniform, sterilizing at 121 ℃ for 60 minutes, and cooling;
1-3) inoculating and culturing until obtaining the yeast
The inoculation amount of the seed is 7.0 to 9.0log according to spores 10 Inoculating cfu/100g spore suspension into yeast-forming culture material, spreading the culture material, making thickness 1-2 cm, at 32+ -1deg.C and humidity 90+ -2%, fermenting and culturing until yeast-forming growth is completed, drying the obtained yeast-forming material in oven at 40 deg.C until its water content is 10-15%, and making the yeast-forming material meet the national standard of brewed soy sauce [143 ]]Standard for the preparation of medium starter;
further, in the step 1-3), the fermentation culture time is 78-82 h, and the fermentation culture period is 2 times of stirring every 24 h;
2) Deodorization of tuna cooking liquor
Adding white granulated sugar and malic acid into the tuna cooking liquid, wherein the concentration of the white granulated sugar in the tuna cooking liquid is 18-22 g/L, the concentration of the malic acid is 2.8-3.2 g/L, and cooling after water bath sterilization; then inoculating composite bacteria, wherein the composite bacteria are lactobacillus plantarum RP26 and Fei Bien Szeberlin and Dener yeast JGM-1 with the volume ratio of 5:1, the inoculation amount of the composite bacteria is 4.8-5.2% of the volume of the tuna cooking liquor (the strain culture solution in the composite strain activation process is the tuna cooking liquor added with 18-22 g/L white granulated sugar and 2.8-3.2 g/L malic acid, and the biomass of lactobacillus plantarum RP26 in the culture solution is 1 multiplied by 10) 9 cfu/mL, fei Bien Seberlin Dener yeast JGM9-1 has a biomass of 1×10 8 cfu/mL), finally, fermenting for 65-75 hours at the temperature of 30+/-1 ℃, centrifuging the obtained fermentation liquor, and collecting supernatant fluid to obtain the deodorized tuna cooking liquor;
3) Aspergillus oryzae combined protease enzymolysis dilute fermentation preparation of seasoning liquid
3-1) taking a deodorized tuna cooking liquor supernatant, regulating the pH value of the supernatant to 5.8 by NaOH, mixing the finished starter with the deodorized tuna cooking liquor according to the mass ratio of 1:3.8-4.2 to obtain a mixture, adding compound protease with the mass ratio of 0.4% (collagenase: animal hydrolytic protease: bromelain=3:3:0.1) into the mixture, and carrying out enzymolysis for 4.8-5.2 hours at 48-52 ℃; adding salt according to the concentration of 20% (w/w) sodium chloride in the tuna cooking solution after enzymolysis is finished, stirring and mixing until the salt is completely dissolved, performing post fermentation at 39-40 ℃ for 28+/-1 days, and filtering to remove filter residues, wherein the obtained supernatant is the seasoning liquid base material;
3-2) Maillard flavoring
Regulating pH value of the seasoning liquid base material to 6.0+ -0.2 with food-grade citric acid and sodium hydroxide, reacting at 95+ -2deg.C for 90+ -5 min, filtering, and packaging to obtain the tuna cooking liquor seasoning liquid. The seasoning liquid is clear brown, has outstanding delicate flavor, is palatable, has outstanding sauce flavor and pleasant fragrance.
The tuna cooking liquor seasoning liquid prepared by the invention has higher amino acid content, mainly comprises glutamic acid, aspartic acid, alanine, glycine and other flavor amino acids, and accounts for more than 50 percent.
The invention takes the cooking liquor of the tuna processing byproducts as a research object, and screens out saccharomycetes and lactobacillus which have good deodorization effect and strong fragrance producing capability and are coordinated with the cooking liquor of the tuna and aspergillus oryzae which can metabolize to produce directional glycolysis protein to produce the protease system with strong flavor amino acid producing capability based on the self food fermentation microorganism germplasm resource library; the new product of the tuna seasoning liquid is developed by adopting the technology of biological deodorization, liquid fermentation and Maillard flavoring of the tuna cooking liquid developed by the optimized strain.
The invention has the advantages that:
1. the special seafood flavor of the tuna is a high-quality raw material for producing foods such as seasonings, protein nutrition enhancers and the like, but the fishy smell is heavy, so that the flavor of deep-processed products can be greatly influenced, and the high-value comprehensive development and utilization of high-quality tuna cooking liquid products are limited. The invention screens out the excellent deodorization microorganisms of the tuna cooking liquor, namely the Fisher-Tropsch yeast JGM-1 and the Lactobacillus plantarum RP26, can reduce the unpleasant flavor of the tuna cooking liquor and generate unique fermentation fragrance.
2. Aspergillus oryzae is a major microorganism for soy sauce brewing, and can secrete neutral protease, acidic protease, alkaline protease, aminopeptidase, carboxypeptidase, etc. and decompose protein raw material into sugar and amino acids, especially glutamic acid with delicate flavor. The invention adopts the combined enzymolysis of the high-yield glutaminase aspergillus oryzae and the enzyme preparation and combines the biological deodorization and Maillard flavoring technologies of the tuna cooking liquor to develop the tuna cooking liquor seasoning liquor.
3. The invention comprehensively considers the enzymolysis cost, enzymolysis condition and enzymolysis rate, takes the enzymolysis rate and ammonia nitrogen conversion rate as targets, and finally selects the combination of three proteases of collagenase, animal hydrolytic protease and bromelain, namely, the addition amount of collagenase is 0.3g/100g, the addition amount of animal hydrolytic protease is 0.3g/100g, the addition amount of bromelain is 0.01g/100g, thereby providing a certain theoretical basis for the deep enzymolysis development and utilization of tuna cooking liquor protein, but the ammonia nitrogen conversion rate is still in a lower level, so that the ammonia nitrogen conversion rate is improved by adopting the microbial fermentation enzyme production and the yeast-forming enzymolysis.
4. The invention adopts protease preparation combined with Aspergillus oryzae composite enzymolysis (only the enzyme preparation has low free amino acid nitrogen content and poor flavor, and only the Aspergillus oryzae protein utilization rate is inferior to that of the enzyme preparation), thereby effectively improving the protein utilization rate and the free amino acid nitrogen content of the seasoning liquid.
5. The invention adopts salt-free dilute enzymolysis and combines the post-salt fermentation technology to prepare the seasoning liquid base stock, which is a fermentation mode of salt-free enzymolysis firstly, is favorable for improving the proteolysis speed, and carries out post-fermentation by adding salt after the enzymolysis is finished, so that the protein utilization rate of the tuna cooking liquid can be improved as much as possible under the condition that the content of biogenic amine is controlled to reach the standard.
6. The implementation and the application of the invention not only can increase the added value of raw materials and realize the comprehensive utilization of the processing byproducts of the tuna, but also can solve the problems of cost pressure or environmental pollution caused by wastewater treatment.
Drawings
FIG. 1 shows the morphology and AFPA culture characteristics of strain MJY 2-5.
FIG. 2 is a phylogenetic tree of the ITS rDNA sequences of test strain MJY2-5 and related species constructed based on the orthotopic ligation method.
FIG. 3 is a phylogenetic tree of the β -BenA gene sequences of the test strain MJY2-5 and related species constructed based on the orthotopic ligation method.
FIG. 4 shows the PCR amplification product of toxigenic genes.
FIG. 5 is a colony morphology of strain JGM-1.
FIG. 6 is a microscopic morphology of strain JGM-1.
FIG. 7 is a 26S rDNAD1/D2 sequence phylogenetic tree of strain JGM-1 and related species.
FIG. 8 is a colony morphology of strain RP26.
FIG. 9 is a microscopic morphology of strain RP26.
FIG. 10 is a phylogenetic tree of 16S rDNA sequences of related species of strain RP26.
Detailed Description
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Example 1
Aspergillus oryzae MJY2-5 separation, purification, screening and identification
The soy sauce embryo with excellent fermentation flavor is taken as a breeding fungus source, is diluted in a gradient way and is coated on a tiger red culture medium plate, and purer strains are obtained through repeated lineation, separation and microscopic examination. And (3) observing culture characteristics of all the separated and purified strains after streaking flat-plate culture, selecting 4 strains with typical aspergillus oryzae colony characteristics, namely MDQ2-1, MDQ2-2, MDQ2-3 and MJY2-5, respectively inoculating the obtained strains on a wort slant culture medium, culturing at 28 ℃ for 3d, and preserving at 4 ℃ in a refrigerator for later use.
In order to improve the enzymolysis rate and the ammonia nitrogen conversion rate, aspergillus oryzae with excellent fermentation of tuna cooking liquor seasoning liquor is screened. In the embodiment, aspergillus oryzae MDQ2-1, MDQ2-2, MDQ2-3, MJY2-5, introduced and collected aspergillus oryzae 3.042, RIB40, AS3.863, CICC2022, 7801, ACCC30467 and the like are used AS strains, tuna cooking liquor seasoning liquor is used AS a culture material, and the aspergillus sporogenic quantity, glutaminase yield, acid protease, neutral protease, cellulase and saccharifying enzyme activity, proteolysis rate and ammonia nitrogen conversion rate are used AS indexes, so that aspergillus oryzae strains with high enzymolysis rate and ammonia nitrogen conversion rate are screened.
1. Materials and methods
Aspergillus oryzae MDQ2-1, MDQ2-2, MDQ2-3, and MJY2-5 were each isolated and stored in the laboratory, aspergillus oryzae 3.042 (Shanghai brewing one-plant Soy sauce koji), RIB40 (Ai Li biosciences (Shanghai) limited), AS3.863 (Ai Li biosciences (Shanghai), CICC2022 (Shanghai Industrial laboratory), 7801 (Shandong and Zhongkangyuan biosciences (Shandong)), ACCC30467 (Shanghai preservation Biotechnology center) were purchased from Shanghai brewing one-plant Soy koji, ai Li biosciences (Shanghai), shanghai Industrial laboratory, shandong and Zhongkangyuan biosciences (Shandong) limited, respectively. …
Tuna cooking liquor seasoning liquid: gu Tianlan distiller's grains of red rice wine brewed from red rice and supplied by red wine industry Co., ltd. Are dried by hot air in a granulating and boiling furnace, crushed and sieved to 80 meshes for standby. The water content was 11.13%, the crude protein (dry basis) was 36.42g/100g, and the acid-soluble protein was 2.06g/100g.
Bran: and is commercially available. The water content is 13.05%, and the bran crude protein is 17.39g/100g.
Bengalhong medium: purchased from maribo biotechnology limited for aspergillus oryzae activation and spore count. Sterilizing at 121deg.C for 20min for use.
Wort agar medium: purchased from Haibo biotechnology Co., ltd, used for the cultivation of Aspergillus oryzae for the enlarged cultivation. Sterilizing at 121deg.C for 20min for use.
Seasoning liquid culture material of tuna cooking liquid: according to the optimized proportioning ratio in the earlier stage, namely the tuna cooking liquor seasoning liquid: bran: mixing water at a ratio of 1:0.8:1.4, and sterilizing at 121 ℃ for 20min for later use.
Aspergillus oryzae spore liquid preparation
Inoculating Aspergillus oryzae strains onto Bengalum red plate, culturing at 30deg.C in incubator for 3d, inoculating spore shovel into slant malt wort agar medium under aseptic condition, culturing at 30deg.C in incubator for 3d, peeling all slant spores with inoculating loop, washing with aseptic water, shaking to obtain spore suspension, sampling, counting, and refrigerating at 4deg.C.
Preparation of finished yeast
Respectively collecting a certain amount of tuna cooking liquor seasoning liquor culture material, and inoculating 1.0X10 according to spore inoculum size 6 Inoculating 1.3 parts of the prepared Aspergillus oryzae spore liquid into each gram, and fermenting and culturing at 30deg.C with humidity of 90% for 72 hr. Detecting indexes such as the number of the aspergillus spores, acid protease, neutral protease, glutaminase, saccharifying enzyme, cellulase and the like.
Fermenting and enzymolysis process of yeast
Taking a certain amount of prepared yeast, inoculating 12% saline solution according to the solid-to-liquid ratio of 1:6, placing into a 500mL conical flask, and performing enzymolysis at 45 ℃. The enzymolysis process is to weigh and replenish water every day and detect the change of soluble solids, and the soluble solids are not changed any more after fermentation for 10 days. And detecting indexes such as enzymolysis rate and ammonia nitrogen conversion rate of fermentation liquor, sensory evaluation and the like.
2. Results and analysis
In the embodiment, 10 different aspergillus oryzae are used as microbial inoculum to prepare the koji, the MJY2-5 enzymolysis rate and the ammonia nitrogen conversion rate are highest, the spore yield of the koji is 10.13+/-0.23 lg/(g/g), the acid-producing protease is 782.07 +/-5.75U/g, the neutral-producing protease is 1250.90 +/-12.05U/g, the glutaminase is 15.67+/-0.43U/g, the saccharifying enzyme is 1800.20 +/-4.06U/g, and the cellulase is 91.79+/-1.94U/g. The enzymolysis rate and the ammonia nitrogen conversion rate in the seasoning liquid base material obtained by fermenting the yeast are 79.68+/-0.15 percent and 51.33+/-0.83 percent respectively, and the seasoning liquid base material has reddish brown luster, strong sauce flavor and typical sauce style.
3. Identification of Strain MJY2-5
(1) Morphological method identification result of MJY2-5
The color of bacterial colony of strain MJY2-5 growing on CYA culture medium and AFPA culture medium and the electron microscope image of spore-producing structure are shown in figure 1. The strain MJY2-5 is cultivated for 7d at 25 ℃ in a CYA culture medium, the colony diameter is 51-54mm, and the spore-forming structure is yellow-green and the edge is white; texture velvet shape; the reverse side of the bacterial colony is light yellow; no exudates are produced; the characteristic morphological features of aspergillus oryzae such as no soluble pigment is generated. The AFPA culture medium is cultured for 48 hours at 30 ℃, and the reverse side of the colony is yellow.
Microscopic morphology: the stems of the sporophores occur in the matrix, the walls are slightly rough and transparent; the conidiophore head is nearly spherical or radial, and the diameter is 25-80 μm; the top bag is flask-shaped, the diameter of the long shaft is 15-45 mu m, and three fourths of the surface is fertile; the spore-producing structure is mainly a single layer, the bottle stems are 5-15 multiplied by 3.5-5.5 mu m, occasionally have double layers, and also have single and double layers of small stems which exist in a top bag at the same time; conidia are nearly spherical or oval, with long axis diameters of 4-7 μm, smooth or slightly rough walls.
(2) Phylogenetic analysis results
The test strain ITS rDNA, beta-BenA gene sequence and phylogenetic tree of related kindred strain are shown in figure 2 and figure 3 respectively. The phylogenetic results show that 11 standard strains including the strain MJY2-5 are gathered into the same phylogenetic branch, and the sequence similarity is more than 97%. As can be seen from the phylogenetic results, the ITS rDNA sequence analysis can identify MJY2-5 as Aspergillus flavus group (Aspergillus section Flavi), and the beta-BenA gene sequence analysis can identify MJY2-5 as Aspergillus flavus (Aspergillus flavus) or Aspergillus oryzae (Aspergillus oryzae). Molecular biological identification has no way to fully distinguish between aspergillus flavus and aspergillus oryzae.
(3) Detection result of MJY2-5 toxigenic gene
The detection results of PCR amplification of 3 toxigenic genes are shown in FIG. 4, M represents DNAMaroer (D2000); 1 represents a positive control strain AS3.4408;2 represents a negative control strain cic c2487;3 represents the detection sample "MJY2-5";4 represents a PCR system blank; ITS rDNA is a positive reference for genomic DNA extraction and PCR reaction; af1R is one of the key genes for aflatoxin synthesis; omt-1 is one of key genes for synthesizing aflatoxin by transferring methoxylase gene to patulin; ver-1 is one of key genes for aflatoxin synthesis as a result of amplifying the versicolor A dehydrogenase gene ver-1. The af1R, omt-1 and ver-1 genes are 3 key genes in the aflatoxin synthesis pathway. Meanwhile, 3 genes are provided, and the aspergillus flavus is judged; 3 genes were not included, and Aspergillus oryzae was judged; 3 genes are deleted and are further confirmed according to a toxigenic test. As is clear from FIG. 4, MJY2-5 deleted the af1R gene, so that further toxicity test was required.
According to SH-QO01-044-2022 (quick detection method of aflatoxins B1, B2, G1 and G2) colloidal gold, the detection result shows that MJY2-5 does not produce aflatoxins B1 and B2 (less than 5.0 mug/kg) when cultured for 10d at 25 ℃ in a soybean meal culture medium.
The method is characterized in that the method combines the identification results of morphology, physiological chemistry, molecular biology, detection of the toxigenic genes of strains and the like, identifies the strain MJY2-5 as Aspergillus oryzae MJY2-5 (Aspergillus oryzae MJY 2-5), and is preserved in China center for type culture collection (CCTCC NO: m20221400, the preservation date is 09/2022.
EXAMPLE 2 isolation, screening, identification and preservation of Strain JGM-1
1. Isolation and purification of strains
Taking a honeycomb sample with excellent flavor as a breeding bacteria source, carrying out gradient dilution and coating on a yeast separation and purification culture medium plate, and carrying out repeated streak separation and microscopic examination to obtain a purer strain. According to TTC color change test, strain 12 with typical yeast colony characteristics is selected, and the strains are numbered JGM-1, JGM-2, JGM-3, JGM-4, JGM-9-1, JGM-2, JGM-9-3, JGM-4, JGM-5, JGM-6, JGM-7, JGM-9 and inoculated on malt wort slant culture medium, cultured at 28 ℃ for 1d and preserved at 4 ℃ in refrigerator for standby.
2. Screening of strains
The separated strains are respectively added into a rice saccharification liquid culture medium with an inoculum size of 2 percent, and are fermented for 5 days at a temperature of 20 ℃, and aroma-producing yeast strains are screened by taking fermentation flavor as an index.
The fermented samples were classified as 5-stage according to their flavor profile. The first level is that the fermented fragrance is rich, marked as "+"; ++ "; the second level is that the fermented fragrance is stronger, marked as' ++ "; the three stages are fermentation in general, and the fermentation is carried out, marked as' ++ "; the fourth level is that the fermented aroma is lighter and marked as "++"; grade five is non-fermented aroma, labeled "-". Please 20 trained professionals perform independent evaluation and take the average statistical analysis.
Fermentation flavor test results of 12 strains in rice saccharification liquid culture medium: the saccharomycete JGM-1 has strong fermentation aroma, the score is "+++++", yeast JGM9-5, JGM9-8, JGM9-2, JGM9-3, JGM9-4, JGM9-7 have fermenting aroma, the scores are "+++" and "+++", yeast JGM-2, JGM8-3, JGM-6 has lighter fermenting aroma, while none of yeasts JGM-1 and JGM8-4 produced fermented notes, scored "-". The results showed that the excellent yeast with unique fermentation flavor was JGM-9-1.
3. Identification of strains
Strain JGM-1 was inoculated into MEA medium and cultured at 28 ℃ for 72 hours, and colonies were observed as flat, white creamy, thick-textured, light-reflecting, and irregularly-edged colonies, as shown in fig. 5. As shown in FIG. 6, cells were observed under a microscope to be nearly spherical or elliptical, budding, single or twin, and were 1.5 to 6.5. Mu.m.times.1 to 6. Mu.m. .
26S rDNA detection was performed on strain JGM-1. The sequence alignment was as follows:
Alignment：JGM9-1
100.0％ Cyberlindnera fabianii CBS 5640T(KY107353)
99.3％ Cyberlindnera bimundalis NRRL Y-5343T(EF550329)
99.1％ Cyberlindnera veronae NRRL Y-7818T(EF550322)
98.9％ Cyberlindnera americana NRRL Y-2156T(EF550328)
98.9％ Candida stauntonica ATCC MYA-4699T(JQ812698)
98.7％ Cyberlindnera mississippiensis CBS 7023T(KY107376)
98.5％ Cyberlindnera xishuangbannaensis NYNU 16752T(KY213813)
98.4％ Candida pattaniensis JCM 12475T(NG 059424)
98.0％ Cyberlindnera amylophila CBS 7020T(KY107350)
98.0％ Cyberlindnera xylosilytica NRRL YB-2097T(NG 064313)
adopting MEGA software, constructing a phylogenetic tree by adopting an adjacent connection method, and carrying out similarity repeated calculation for 1000 times, wherein in the figure 7, the development tree nodes only show that the Bootstrap value is larger than 70%, and the superscript 'T' represents a model strain.
According to the gene sequencing result of the strain and the identification result such as the physiological and biochemical characteristics of the strain, the strain JGM-1 is identified as Fei Bien Siberian yeast JGM-1 (Cyberlindnera fabianii JGM 9-1) and is preserved in China center for type culture collection (CCTCC NO), wherein the preservation number is: m20221401, the preservation date is 09/2022.
EXAMPLE 3 isolation, screening, identification and preservation of Strain RP26
1. Isolation and purification of strains
The wine mash which is fermented by the malic acid is taken as a lactobacillus separation source, is diluted in a gradient way and is coated on a lactobacillus separation and purification culture medium flat plate, and purer bacterial strains are obtained through repeated scribing, separation and microscopic examination. Separating and purifying by plate scribing method, and selecting strain with typical lactobacillus colony characteristics, such as RP21, RP22, RP23, RP24, RP25, RP26, RP27, RP28, respectively inoculating the obtained strain on MRS inclined plane, culturing at 28deg.C for 3d, and preserving at 4deg.C in refrigerator.
2. Screening of strains
The separated strains are respectively added into a rice saccharification liquid culture medium with an inoculum size of 2 percent, and are fermented for 5 days at a temperature of 20 ℃, and the aroma-producing lactic acid strains are screened by taking the fermentation flavor as an index.
The fermented samples were classified into 5 grades according to their flavor profile (in particular, the examples). Fermentation flavor test results of 8 strains in rice saccharification liquid medium: lactic acid bacteria RP21 the lactic acid bacteria RP25 have a fermenting aroma, lactic acid bacteria RP21 and RP25 have fermenting fragrance, the scores are "+++" and "+++", lactic acid bacteria RP22, lactic acid bacteria RP23 and lactic acid bacteria RP24 have lighter fermented fragrance, while neither lactic acid bacteria RP27 nor lactic acid bacteria RP28 can produce fermented fragrance, and the score is "-". The results show that the excellent lactobacillus with unique fermentation flavor is lactobacillus RP26.
3. Identification of strains
Strain RP26 was inoculated on MRS plates, incubated at 30 ℃ for 2 days, and colony morphology was observed as follows: colonies were milky white, round, smooth, convex, moist, and clean-edged, as shown in fig. 8. As shown in fig. 9, the cell morphology was observed under the microscope as: the bacterial cells are in the shape of rod, the size is 0.5 μm multiplied by 1.0-1.67 μm, and are arranged singly or in short chain, and are free from spore generation and gram positive.
The strain RP26 was subjected to 16S rDNA detection. The similarity repeat calculation was performed 1000 times using MEGA4.1 software, orthotopic ligation showed 16S rDNA phylogenetic tree of strain RP26 and related species, and in fig. 10 the phylogenetic tree nodes only showed Bootstrap values greater than 50% values, with the superscript "T" indicating the model strain, (l.m., lactobacillus b., bifidobacterium).
Phylogenetic analysis showed that: the strain RP26 and the model strains of L.plantarum subsp.plantarum, L.plantarum subsp.argenturensis, L.paralytic and L.pentasus are gathered on one phylogenetic branch, and the sequence homology is more than 99.4 percent.
According to the gene sequencing result of the strain and the identification results of physiological and biochemical characteristics and the like of the strain, the strain RP26 is identified as lactobacillus plantarum RP26 (Lactobacillus plantarum RP), and the strain RP26 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: m2019298, the preservation date is 26 days of 2019, 04 and 26 months.
Example 4
A preparation method of a tuna cooking liquor seasoning liquid comprises the following steps:
1) Preparing into yeast
1-1) activation of Aspergillus oryzae and preparation of spore suspension
Inoculating Aspergillus oryzae MJY2-5 onto Bengalia red plate, separating and culturing at 32deg.C in incubator for 72 hr, inoculating single spore into slant malt wort agar medium under aseptic condition, culturing at 32deg.C in incubator for 72 hr, peeling slant spore with inoculating shovel, washing with aseptic water, shaking to obtain spore suspension, and refrigerating at 4deg.C in refrigerator;
1-2) preparation of a Yeast-forming culture Material
Mixing bran, bean pulp and water according to the mass ratio of 4:6:7, regulating the pH to 6.6, slowly adding water, stirring while adding water, mixing uniformly by hand without dry powder, mixing until the color is uniform, sterilizing at 121 ℃ for 60 minutes, and cooling;
1-3) inoculating and culturing until obtaining the yeast
The inoculation amount of the seed is 8.0log according to spores 10 Inoculating cfu/100g spore suspension into yeast-forming culture material, spreading the culture material to 1.5 cm in thickness, fermenting at 32deg.C with 90% humidity for 80 hr, stirring for 2 times every 24 hr during fermentation culture, and oven drying the obtained yeast in 40deg.C oven until water content is 12.5%, wherein the yeast-forming should meet national standard of brewed soy sauce [143 ]]Standard for the preparation of medium starter;
2) Deodorization of tuna cooking liquor
Adding white granulated sugar and malic acid into the tuna cooking liquid, wherein the concentration of the white granulated sugar in the tuna cooking liquid is 20g/L, the concentration of the malic acid is 3.0g/L, and cooling after water bath sterilization; then inoculating a composite bacterium which is lactobacillus plantarum RP26 and Fei Bien with the volume ratio of 5:1The inoculation amount of the composite bacteria of the Saberlin and the Nardostachys chinensis JGM-1 is 5.0 percent of the volume of the cooking liquid of the tuna, wherein the biomass of the lactobacillus plantarum RP26 is 1 multiplied by 10 9 cfu/mL, fei Bien Seberlin Dener yeast JGM9-1 has a biomass of 1×10 8 cfu/mL; finally, fermenting for 70 hours at the temperature of 30 ℃, centrifuging the obtained fermentation liquor, and collecting supernatant fluid to obtain the tuna cooking liquor after deodorization;
3) Aspergillus oryzae combined protease enzymolysis dilute fermentation preparation of seasoning liquid
3-1) taking a deodorized tuna cooking liquor supernatant, regulating the pH value of the supernatant to 5.8 by NaOH, mixing the finished starter with the deodorized tuna cooking liquor according to a mass ratio of 1:4 to obtain a mixture, adding compound protease with a mass ratio of 0.4% (collagenase: animal hydrolytic protease: bromelain=3:3:0.1) into the mixture, and carrying out enzymolysis for 5 hours at 50 ℃; adding salt according to the concentration of 20% (w/w) sodium chloride in the tuna cooking solution after enzymolysis is finished, stirring and mixing until the salt is completely dissolved, performing post fermentation at 40 ℃ for 28 days, and filtering to remove filter residues to obtain supernatant which is the seasoning liquid base material;
3-2) Maillard flavoring
Regulating pH value of the seasoning liquid base material to 6.0 by using food-grade citric acid and sodium hydroxide, reacting for 90min at 95 ℃, filtering and filling to obtain the tuna cooking liquor seasoning liquid. The seasoning liquid is clear brown, has outstanding delicate flavor, is palatable, has outstanding sauce flavor and pleasant fragrance.
Example 5
A preparation method of a tuna cooking liquor seasoning liquid comprises the following steps:
1) Preparing into yeast
1-1) activation of Aspergillus oryzae and preparation of spore suspension
Inoculating Aspergillus oryzae MJY2-5 onto Bengalia red plate, separating and culturing at 31deg.C in incubator for 74 hr, inoculating single spore into slant malt wort agar medium under aseptic condition, culturing at 31deg.C in incubator for 74 hr, peeling slant spore with inoculating shovel, washing with aseptic water, shaking to obtain spore suspension, and refrigerating at 4deg.C in refrigerator;
1-2) preparation of a Yeast-forming culture Material
Mixing bran, bean pulp and water according to the mass ratio of 4:6.5:7.5, regulating the pH value to 6.5, slowly adding water, stirring while adding water, mixing uniformly by hand without dry powder, mixing until the color is uniform, sterilizing at 121 ℃ for 60 minutes, and cooling;
1-3) inoculating and culturing until obtaining the yeast
The inoculation amount of the seed is 7.0log 10 Inoculating cfu/100g spore suspension into yeast-forming culture material, spreading the culture material to 1 cm in thickness, fermenting at 31deg.C and 88% humidity for 82 hr, stirring for 2 times every 24 hr during fermentation culture, and oven drying the obtained yeast in 40deg.C oven until water content is 10%, wherein the yeast-forming should meet national standard of brewed soy sauce [143 ]]Standard for the preparation of medium starter;
2) Deodorization of tuna cooking liquor
Adding white granulated sugar and malic acid into the tuna cooking liquid, wherein the concentration of the white granulated sugar in the tuna cooking liquid is 18g/L, the concentration of the malic acid is 3.2g/L, and cooling after water bath sterilization; then inoculating a composite bacterium, wherein the composite bacterium is lactobacillus plantarum RP26 and Fei Bien Szechwan yeast JGM-1 with the volume ratio of 5:1, the inoculation amount of the composite bacterium is 4.8% of the volume of the tuna cooking liquid, and the biomass of the lactobacillus plantarum RP26 is 1 multiplied by 10 9 cfu/mL, fei Bien Seberlin Dener yeast JGM9-1 has a biomass of 1×10 8 cfu/mL; finally, fermenting for 75 hours at 29 ℃ under the control of temperature, centrifuging the obtained fermentation liquor, and collecting supernatant to obtain the deodorized tuna cooking liquor;
3) Aspergillus oryzae combined protease enzymolysis dilute fermentation preparation of seasoning liquid
3-1) taking a deodorized tuna cooking liquor supernatant, regulating the pH value of the supernatant to 5.8 by NaOH, mixing the finished starter with the deodorized tuna cooking liquor according to the mass ratio of 1:3.8 to obtain a mixture, adding compound protease with the mass ratio of 0.4% (collagenase: animal hydrolytic protease: bromelain=3:3:0.1) into the mixture, and carrying out enzymolysis for 5.2 hours at 48 ℃; adding salt according to the concentration of 20% (w/w) sodium chloride in the tuna cooking solution after enzymolysis is finished, stirring and mixing until the salt is completely dissolved, performing post fermentation at 39 ℃ for 291 days, and filtering to remove filter residues to obtain supernatant as a seasoning liquid base material;
3-2) Maillard flavoring
Regulating pH value of the seasoning liquid base material to 5.8 by using food-grade citric acid and sodium hydroxide, reacting for 95min at 93 ℃, filtering and filling to obtain the tuna cooking liquor seasoning liquid. The seasoning liquid is clear brown, has outstanding delicate flavor, is palatable, has outstanding sauce flavor and pleasant fragrance.
Example 6
A preparation method of a tuna cooking liquor seasoning liquid comprises the following steps:
1) Preparing into yeast
1-1) activation of Aspergillus oryzae and preparation of spore suspension
Inoculating Aspergillus oryzae MJY2-5 onto Bengalia red plate, separating and culturing at 33deg.C in incubator for 70 hr, inoculating single spore into slant malt wort agar medium under aseptic condition, culturing at 33deg.C in incubator for 70 hr, peeling slant spore with inoculating shovel, washing with aseptic water, shaking to obtain spore suspension, and refrigerating at 4deg.C in refrigerator;
1-2) preparation of a Yeast-forming culture Material
Mixing bran, bean pulp and water according to the mass ratio of 4:6:7.5, regulating the pH value to 6.6, slowly adding water, stirring while adding water, mixing uniformly by hand without dry powder, mixing until the color is uniform, sterilizing at 121 ℃ for 60 minutes, and cooling;
1-3) inoculating and culturing until obtaining the yeast
The inoculation amount of the seed is 9.0log according to spores 10 Inoculating cfu/100g spore suspension into yeast-forming culture material, spreading the culture material, making the thickness 2 cm, fermenting and culturing at 33 deg.C and humidity 902% for 78-h, stirring 2 times every 24h during fermentation and culturing, baking the obtained yeast in oven at 40 deg.C until its water content is 15%, and making the yeast meet national standard of brewed soy sauce [143 ]]Standard for the preparation of medium starter;
2) Deodorization of tuna cooking liquor
Adding white granulated sugar and malic acid into the tuna cooking liquid, wherein the concentration of the white granulated sugar in the tuna cooking liquid is 22g/L, the concentration of the malic acid in the tuna cooking liquid is 3.2g/L, and cooling after water bath sterilization; then inoculating the composite bacteria to the strain,the composite bacteria are lactobacillus plantarum RP26 and Fei Bien Szechwan yeast JGM-1 with the volume ratio of 5:1, the inoculation amount of the composite bacteria is 5.2 percent of the volume of the tuna cooking liquor (the strain culture solution in the composite strain activation process is the tuna cooking liquor added with 18-22 g/L white granulated sugar and 2.8-3.2 g/L malic acid, and the biomass of lactobacillus plantarum RP26 in the culture solution is 1 multiplied by 10) 9 cfu/mL, fei Bien Seberlin Dener yeast JGM9-1 has a biomass of 1×10 8 cfu/mL); finally, fermenting for 65 hours at the temperature of 31 ℃, centrifuging the obtained fermentation liquor, and collecting supernatant fluid to obtain the tuna cooking liquor after deodorization;
3) Aspergillus oryzae combined protease enzymolysis dilute fermentation preparation of seasoning liquid
3-1) taking a deodorized tuna cooking liquor supernatant, regulating the pH value of the supernatant to 5.8 by NaOH, mixing the finished starter with the deodorized tuna cooking liquor according to the mass ratio of 1:4.2 to obtain a mixture, adding compound protease with the mass ratio of 0.4% (collagenase: animal hydrolytic protease: bromelain=3:3:0.1) into the mixture, and carrying out enzymolysis for 4.8 hours at 52 ℃; adding salt according to the concentration of 20% (w/w) sodium chloride in the tuna cooking solution after enzymolysis is finished, stirring and mixing until the salt is completely dissolved, performing post fermentation at 40 ℃ for 27 days, and filtering to remove filter residues to obtain supernatant as a seasoning liquid base material;
3-2) Maillard flavoring
Regulating pH value of the seasoning liquid base material to 6.2 by using food-grade citric acid and sodium hydroxide, reacting for 85min at 97 ℃, filtering and filling to obtain the tuna cooking liquor seasoning liquid. The seasoning liquid is clear brown, has outstanding delicate flavor, is palatable, has outstanding sauce flavor and pleasant fragrance.
Comparative example 1
Step 3-1) of examples 4-6 adopts the process of salt-free dilute enzymolysis and then salt fermentation.
Comparative example 1 differs from example 4 in that step 3-1) the salt-first dilute fermentation enzymatic hydrolysis process is carried out, and the other steps are the same as example 4.
The step 3-1) is specifically as follows:
3-1) taking a deodorized tuna cooking liquor supernatant, regulating the pH value of the supernatant to 5.8 by NaOH, mixing the finished starter with the deodorized tuna cooking liquor according to the mass ratio of 1:4 to obtain a mixture, adding salt according to the concentration of 20% (w/w) sodium chloride in the tuna cooking liquor, stirring and mixing until the salt is completely dissolved, adding 0.4% (mass ratio collagenase: animal hydrolytic protease: bromelain=3:3:0.1) of compound protease, and carrying out enzymolysis for 5 hours at 50 ℃; post-fermentation is carried out for 28 days at 40 ℃, filter residues are removed by filtration, and the obtained supernatant is the base material of the seasoning liquid.
The following compares the products prepared by the two processes of comparative example 1 and example 4
(1) The products obtained in comparative example 1 and example 4 were brown in color, exhibited a soy sauce aroma, were salty and delicious enough, and had a typical style of soy sauce. The comparative example product had a stronger soy sauce aroma, and the example 4 product had a weak fish aroma, all without bad flavor.
(2) Influence of two fermentation processes on physicochemical indexes of products
The main physical and chemical index measurement results of the seasoning liquid base materials prepared by two different processes are shown in table 1. The sample of comparative example 1 had an amino acid nitrogen of 0.82.+ -. 0.01g/100g, the sample of example 4 had an amino acid nitrogen of 0.89.+ -. 0.02g/100g; comparative example 1 sample ammonium salt was (0.22.+ -. 0.01) g/100g, ammonium salt was 26.83% and example 4 sample ammonium salt was 0.23.+ -. 0.01g/100g, ammonium salt was 25.84%; comparative example 1 sample total nitrogen was 2.07.+ -. 0.05g/100g, example 4 sample total nitrogen was 2.10.+ -. 0.05g/100g; the sample of comparative example 1 has a soluble salt-free solid content of 20.60 + -0.05 g/100g, the sample of example 4 has a soluble salt-free solid content of 21.00+ -0.1 g/100g, and the index measurement results of the two processes are significantly different. Both process samples reach the first level standard of soy sauce brewing and meet the safety standard of soy sauce brewing.
TABLE 1 physical and chemical index results of products
(3) Free amino acid test results of two fermentation processes on product to product
The free amino acid content of the base stock of the seasoning liquid has an important effect on the flavor impact produced by the base stock of the seasoning liquid, 18 free amino acids are detected in both process samples, and the results are shown in Table 2. Amino acids that are sweet in taste include alanine, threonine, glycine, and serine; the amino acids with delicate flavor include glutamic acid and aspartic acid; bitter taste includes phenylalanine, isoleucine, leucine, methionine, valine, histidine and arginine. The total amino acid content of the sample of comparative example 1 was 3.40g/100g, wherein the fresh, sweet amino acid was 1.69g/100g, the proportion was 49.71%, the total bitter amino acid was 1.16g/100g, the proportion was 34.12%, the total hydrophobic amino acid was 1.56g/100g, and the proportion was 45.88%; the sample of example 4 had a total amino acid content of 3.80g/100g, with a fresh, sweet amino acid content of 1.97g/100g, a proportion of 51.84%, a total bitter amino acid content of 1.24g/100g, a proportion of 32.63%, a total hydrophobic amino acid content of 1.70g/100g, and a proportion of 44.74%. The fermentation temperature of the sample of example 4 was lower than that of the sample of comparative example 1, and the content of free amino acids produced was also higher, which suggests that salt-free dilute enzymatic hydrolysis followed by salt addition and fermentation favors the accumulation of free amino acids in the flavor base. However, comparative example 1 was more fragrant, probably due to the higher temperature to accelerate Maillard reaction, forming a richer aroma flavor, while also consuming some of the amino acids. The salt-free dilute enzymolysis and salt adding post-fermentation process of the embodiment 4 is adopted to prepare the seasoning base material, the fermentation mode of salt-free enzymolysis is firstly utilized, the fermentation enzymolysis speed is improved, more organic acid, amino acid and antioxidant substances are generated, salt is added for post-fermentation after the enzymolysis is finished, the content of biogenic amine is reduced, the osmotic pressure can be increased by adding salt, and the pollution caused by mixed bacteria is prevented, and experiments prove that the salt-free dilute enzymolysis and salt adding post-fermentation process of the embodiment 4 is more excellent, so that the method has important significance in guiding the actual production of dilute fermentation seasonings.
Table 2 comparison of free amino acid content of the two processes
Note that: "ND" means undetected, "sweet" means amino acids exhibiting sweetness, and "fresh" means amino acids exhibiting umami.
In conclusion, although the ammonium salt of the seasoning liquid base material prepared in example 4 has relatively low content of free amino acid and high content of flavor-developing amino acid from the physical and chemical indexes, has sauce flavor and fish flavor, has more abundant flavor and has the problem of insufficient typical flavor of soy sauce, the problem of insufficient flavor can be overcome by using Maillard reaction on the basis of the seasoning liquid base material later. Compared with example 4, the method of comparative example 1 was simpler, but the enzymolysis efficiency was lower, and the aroma and flavor of the sauce after the fermentation was over was more pronounced, but the amino acid nitrogen content was slightly lower. Therefore, the method for preparing the flavoring base material by adopting the salt-free dilute enzymolysis and the post-salt fermentation in the embodiment 4 is more preferable.

Claims (9)

1. The preparation method of the tuna cooking liquor seasoning liquor is characterized by comprising the following steps:
1) Preparing into yeast
Inoculating Aspergillus oryzae MJY2-5 as strain into culture material to obtain yeast;
2) Deodorization treatment for tuna cooking liquor
Adding white granulated sugar and malic acid into the tuna cooking liquid, sterilizing in a water bath, cooling, inoculating composite bacteria which are lactobacillus plantarum RP26 and Fei Bien Szechwan scholaris JGM-1, wherein the inoculation amount of the composite bacteria is 4.8-5.2% of the volume of the tuna cooking liquid, performing temperature control fermentation for 65-75 hours at 30+/-1 ℃, centrifuging the obtained fermentation liquid, and collecting supernatant to obtain the tuna cooking liquid after deodorization;
the Fei Bien Saberlin Dener-yeast JGM-1 has the academic name ofCyberlindnera fabianiiJGM9-1, which is preserved in China Center for Type Culture Collection (CCTCC) NO: m20221401, the date of preservation is 2022, 09;
the plant milk stalkBacteria RP26, school nameLactobacillus plantarum RP26, which is preserved in China Center for Type Culture Collection (CCTCC) NO: m2019298, date of preservation 2019, 04, 26;
3) Aspergillus oryzae combined protease enzymolysis dilute fermentation preparation of seasoning liquid
3-1) taking a supernatant of the deodorized tuna cooking liquor, regulating the pH value of the supernatant to be 5.0+/-0.2, mixing the finished starter with the deodorized tuna cooking liquor according to the mass ratio of 1:3.8-4.2 to obtain a mixture, adding compound protease accounting for 0.4% of the mass of the mixture, and carrying out enzymolysis for 4.0-5.0 h at 48-52 ℃; adding salt according to the concentration of 20% (w/w) sodium chloride in the tuna cooking solution after enzymolysis is finished, stirring and mixing until the salt is completely dissolved, performing after-fermentation at 40+/-2 ℃ for 28+/-2 days, and filtering to remove filter residues, wherein the obtained supernatant is the seasoning liquid base material;
the Aspergillus oryzae MJY2-5 has the academic name ofAspergillus oryzae MJY2-5, which is preserved in China center for type culture collection, has the preservation number of CCTCC NO: m20221400, the date of preservation is 2022, 09;
3-2) Maillard flavoring
Regulating pH value of seasoning liquid base material to 6.0+ -0.2, reacting at 95+ -2deg.C for 90+ -5 min, filtering, and packaging to obtain tuna cooking liquor seasoning liquid.
2. The method for preparing a seasoning liquid for a tuna cooking liquid according to claim 1, wherein the method for preparing the yeast in step 1) comprises the following steps:
1-1) activation of Aspergillus oryzae and preparation of spore suspension
Inoculating Aspergillus oryzae MJY2-5 onto a Bengalia red flat plate, separating and culturing for 70-74 h in a 31-33 ℃ incubator, inoculating single spores into an inclined plane wort agar medium by using an inoculating shovel under the aseptic condition, expanding and culturing for 70-74 h in the 31-33 ℃ incubator, completely stripping inclined plane spores by using the inoculating shovel, washing with aseptic water, shaking uniformly to obtain spore suspension, and refrigerating and preserving at 4 ℃;
1-2) preparation of a Yeast-forming culture Material
Mixing bran, bean pulp and water according to the mass ratio of 4:6-6.5:7-7.5, adjusting the pH value to 6.5-6.6, sterilizing, and cooling;
1-3) inoculating and culturing until obtaining the yeast
The inoculation amount of the spores is 7.0-9.0 log 10 Inoculating the spore suspension into a yeast-forming culture material by cfu/100g, paving the culture material to a thickness of 1-2 cm, fermenting and culturing at 32+/-1 ℃ and a humidity of 90+/-2% until the yeast-forming growth is finished, and drying the obtained yeast until the moisture content is 10-15%.
3. The method for preparing a tuna cooking liquor seasoning liquor according to claim 2, wherein in the step 1-3), the fermentation culture time is 78-82 h, and the fermentation culture is performed for 2 times every 24 h.
4. The method for preparing the tuna cooking liquor seasoning liquor according to claim 1, wherein in the step 2), the concentration of white granulated sugar in the tuna cooking liquor is 18-22 g/L, and the concentration of malic acid is 2.8-3.2 g/L.
5. The method for preparing a tuna cooking liquor seasoning liquor according to claim 1, wherein in the step 2), the lactobacillus plantarum RP26 and the Fei Bien Szechwan der's yeast JGM-1 have a volume ratio of 5:1.
6. The method for preparing a seasoning liquid for a cooking liquor of tuna according to claim 1, wherein in the step 3-1), the complex protease is composed of collagenase, animal hydrolytic protease, bromelain.
7. The method for preparing the tuna cooking liquor seasoning liquor according to claim 6, wherein the mass ratio of collagenase, animal hydrolytic protease and bromelain in the composite protease is 3:3:0.1.
8. A tuna cooking liquor seasoning liquor obtainable by the process of any one of claims 1 to 7.
9. The tuna cooking liquor seasoning liquor of claim 8 having a flavor amino acid content of more than 50%, comprising glutamic acid, aspartic acid, alanine, glycine.
CN202310734681.3A 2023-06-20 2023-06-20 Preparation method of tuna cooking liquor seasoning liquid Pending CN116941751A (en)

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