A method for removing fishy smell from tuna cooking liquid using microorganisms

Abstract

The invention discloses a method for removing the fishy smell of tuna cooking liquid by microorganisms. The method comprises the following steps: 1) adding white sugar and malic acid to the tuna cooking liquid, sterilizing in a water bath and cooling the mixture; 2) inoculating composite bacteria, wherein the composite bacteria are Lactobacillus plantarum RP26 and Fabianseberlindnerella JGM9‑1, wherein the preservation number of Fabianseberlindnerella JGM9‑1 is CCTCC NO: M 20221401, and the preservation number of Lactobacillus plantarum RP26 is CCTCC NO: M 2019298; 3) fermenting the mixture at a temperature controlled temperature of 30±1°C for 65 to 75 h, centrifuging the fermented liquid, and collecting the supernatant, which is the tuna cooking liquid without fishy smell. The present invention adopts Fabianseber Lindner yeast JGM9-1 and Lactobacillus plantarum RP26 to compound ferment tuna cooking liquid, which can metabolize and consume fishy smell substances such as fishy smell, oily smell and sour smell, and metabolize to produce grassy, ​​floral and fruity smell substances with characteristics of both lactic acid bacteria and yeast.

Description

Technical Field

[0001] The invention relates to the field of aquatic product processing, and in particular to a method for removing fishy smell from tuna cooking liquid using microorganisms. Background Art

[0002] Tuna can be processed into raw fish fillets, canned fish, fish floss and other products. Among them, when tuna is processed into fish fillets and canned tuna, 1000kg of tuna produces about 300-500kg of by-products - tuna cooking liquid. The cooking liquid is rich in nutrients such as amino acids, soluble proteins, nucleotides, unsaturated fatty acids, etc. that are needed by the human body and easy to absorb. The cooking liquid tastes delicious and has the unique seafood flavor of tuna. It is a high-quality raw material for producing condiments, protein nutritional enhancers and other foods, but it has a strong fishy smell, which will have a great impact on the taste of deep-processed products, limiting the high-value comprehensive development and utilization of high-quality tuna cooking liquid products. At present, tuna cooking liquid in my country has not been well developed and utilized. Most of it is discharged as wastewater without treatment, which not only greatly wastes protein resources, but also increases the cost of wastewater treatment and poses an environmental pollution risk.

[0003] At present, there are mainly physical deodorization, chemical deodorization and biological deodorization. Physical deodorization generally involves adding a certain amount of adsorbent to the raw materials so that the odorous substances are adsorbed by the adsorbent to achieve the effect of deodorization. However, it has the disadvantages of incomplete deodorization and difficulty in industrial application. Chemical deodorization uses the compounds with unpleasant flavors in aquatic products to react with chemical substances to remove them. However, it has the disadvantages of easily producing odors and difficulty in industrial application. Biological deodorization does not mix with other additives or chemical substances and will not cause harm to the human body. Microbial fermentation can not only effectively reduce the unpleasant flavor of fermented products, but also inject a unique and pleasant flavor into the products, thereby improving the overall flavor value of the food. Gu Saiqi and others used yeast to deodorize kelp, with a removal rate of up to 39.75% and good deodorization effect; Zhang Haiyan and others used yeast extract to deodorize sea bass. The processed fish fillets had no fishy smell and had no effect on the color, texture and freshness of the fish fillets. Yeast increased the content of aromatic compounds to give the fermented liquid a special flavor and flavor. Zhao et al. studied naturally fermented tilapia sausage, in which lactic acid bacteria played an important role in the formation of volatile flavor compounds and flavor.

[0004] Different aquatic products have different fishy substances, and the deodorizing microorganisms used are also different. The Chinese patent "A method for deodorizing kelp with composite microorganisms ZL201710981772.1" discloses kelp microbial deodorizing strains and deodorizing methods, the Chinese patent "A seaweed fermented algae cake ZL201810898673.1" discloses Gracilaria microbial deodorizing strains and deodorizing methods, and the Chinese patent application "A biological deodorizing method for shellfish meat by ozone combined with oxygen-insulated microbial fermentation" discloses scallop microbial deodorizing bacteria and deodorizing methods. However, these strains are not effective in deodorizing tuna cooking liquid. Summary of the invention

[0005] The purpose of the present invention is to provide a method for removing the fishy smell of tuna cooking liquid by microorganisms.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A strain of aroma-producing yeast, whose classification name is Cyberlindnera fabianii JGM9-1, and whose Latin name is Cyberlindnera fabianii JGM9-1, has been deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China. Its deposit number is CCTCC NO: M 20221401, and its deposit date is September 9, 2022.

[0008] A fragrance-producing Lactobacillus plantarum strain, whose classification name is Lactobacillus plantarum RP26, whose Latin name is Lactobacillus plantarum RP26, has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit number is CCTCC NO: M 2019298, and the deposit date is April 26, 2019.

[0009] A method for removing fishy smell from tuna cooking liquid using microorganisms comprises the following steps:

[0010] 1) Add sugar and malic acid to the tuna cooking liquid, wherein the concentration of sugar in the tuna cooking liquid is 18-22 g / L, and the concentration of malic acid is 2.8-3.2 g / L, sterilize in a water bath, and then cool;

[0011] 2) inoculating composite bacteria, wherein the composite bacteria are Lactobacillus plantarum RP26 and Fabianseberlindner yeast JGM9-1, and the inoculation amount of the composite bacteria is 4.8-5.2% of the volume of the tuna cooking liquid; wherein the bacterial culture liquid in the composite bacteria activation culture process is the tuna cooking liquid added with 18-22 g / L white sugar and 2.8-3.2 g / L malic acid, and the biomass of Lactobacillus plantarum RP26 is 1×10 9cfu / mL, the biomass of Fabianseber Lindnerella JGM9-1 was 1×10 8 cfu / mL;

[0012] 3) Ferment at a controlled temperature of 30±1°C for 65-75h, centrifuge the resulting fermentation liquid, and collect the supernatant, which is odorless tuna cooking liquid, which can be used to further process high-value products such as seasoning liquid, peptide liquid, and amino acid oral liquid.

[0013] Furthermore, the concentration of white sugar in the tuna cooking liquid is 20 g / L, and the concentration of malic acid is 3.0 g / L.

[0014] Furthermore, the inoculation amount of the composite bacteria is 5% of the volume of the tuna cooking liquid.

[0015] Furthermore, the biomass ratio of the Lactobacillus plantarum RP26 to the Fabian Seberlindner yeast JGM9-1 is 5:1.

[0016] Preferably, the concentration of white sugar in the tuna cooking liquid is 20 g / L, the concentration of malic acid is 3 g / L, and the inoculation amount of the composite bacteria is 5% of the volume of the tuna cooking liquid.

[0017] Furthermore, the fermentation temperature is 30°C and the fermentation time is 72h.

[0018] The flavor substances of tuna cooking liquid are mainly decanal, hexanal, 1-octen-3-ol with fishy smell, 2-nonanal with oily smell, heptanal, trimethylamine, dimethyl trisulfide with sour smell, which are the main components of the fishy smell substances of tuna cooking liquid. These fishy smell substances can be degraded by fermentation with Fabianseber Lindner yeast JGM9-1 and Lactobacillus plantarum RP26. Among them, the flavor characteristic substances consumed by Lactobacillus plantarum RP26 are 2-nonanal with oily smell, trimethylamine and dimethyl trisulfide with sour smell; the characteristic flavor substances produced are 2-butyl-2-octenal and 2-dodecenal with green grass fragrance, and 2-undecanone with fruity fragrance. The characteristic flavor substances consumed by Fabianseberlindner yeast JGM9-1 are decanal, hexanal, and 1-octen-3-ol with fishy smell; trimethylamine and dimethyl trisulfide with sour smell; the characteristic flavor substances produced are β-phenylethanol, ethyl tetradecanoate, and ethyl phenylacetate with floral flavor, and ethyl pyruvate, ethyl propionate, ethyl laurate, ethyl caprylate, octanol, ethyl acetate, and isopentanol with fruity flavor. The present invention adopts Fabianseberlindner yeast JGM9-1 and Lactobacillus plantarum RP26 to compound ferment tuna cooking liquid, which can simultaneously metabolize and consume odor substances with fishy smell, greasy smell, and sour smell, and metabolize and produce grassy, ​​floral, and fruity aroma substances with characteristics of both lactic acid bacteria and yeast, and the deodorization effect is better than that of single bacteria treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the colony morphology of strain JGM9-1.

[0020] Figure 2 This is the microscopic morphology of strain JGM9-1.

[0021] Figure 3 This is the phylogenetic tree of 26S rDNA D1 / D2 sequences of strain JGM9-1 and related species.

[0022] Figure 4 This is the colony morphology of strain RP26.

[0023] Figure 5 This is the microscopic morphology of strain RP26.

[0024] Figure 6 The phylogenetic tree of 16S rDNA sequences of strain RP26 and related species.

[0025] Figure 7 This is a graph showing the effects of fermentation with different bacterial strains on the sensory evaluation of tuna cooking liquid.

[0026] Figure 8 This is a graph showing the effect of fermentation with different bacterial species ratios on the sensory evaluation of tuna cooking liquid.

[0027] Fig. 9 The following are the changes in the components and contents of volatile flavor substances during the fermentation process of different microorganisms in Example 4; (a) comparative analysis of the number of volatile flavor substances among groups; (b) comparative analysis of the concentration of volatile flavor substances among types; (c) the content concentration of each type of substance.

[0028] Fig.10 This is the HCA tree cluster analysis of tuna cooking liquid before and after fermentation.

[0029] Fig.11 is the average variable importance projection (VIP) value. Red bars indicate VIP values ​​> 1, and green indicates VIP values ​​< 1. (A) VIP-CK is the VIP value of the tuna cooking liquid stock solution group (CK treatment), (B) VIP-R corresponds to the VIP value of the lactic acid bacteria fermentation group (R treatment), (C) VIP-RJ is the VIP value of the lactic acid bacteria-dominated fermentation group (RJ1, RJ2, and RJ3 treatments), (D) is the VIP value of the yeast-dominated fermentation JR group (RJ4 and RJ5 treatments); (E) VIP-J corresponds to the VIP value of the yeast-dominated fermentation J group (J treatment).

[0030] Fig.12 Correlation analysis of characteristic flavor substances in different strains; (A) lactic acid bacteria; (B) yeast.

[0031] Fig.13 This is a network diagram showing significant correlations between characteristic aroma substances and microorganisms.

[0032] Fig.14 Wayne consumption metabolic diagram; (A) consumption (B) production.

[0033] Fig.15 To study the effect of the amount of added white sugar on the deodorization of tuna cooking liquid.

[0034] Fig.16 The effect of malic acid addition on the deodorization of tuna cooking liquid.

[0035] Fig.17 This study is the effect of bacterial inoculation amount on the deodorization effect of tuna cooking liquid.

[0036] Fig.18 The effect of fermentation time on the deodorization effect of tuna cooking liquid.

[0037] Fig.19 To investigate the effect of fermentation temperature on the deodorization of tuna cooking liquid. DETAILED DESCRIPTION

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0039] Example 1 Isolation, screening, identification and preservation of strain JGM9-1

[0040] 1. Isolation and purification of strains

[0041] Honeycomb samples with good flavor were used as the breeding source, gradient dilution and coating on yeast isolation and purification medium plates, and purer strains were obtained after multiple streaking separation and microscopic examination. According to the TTC color change test, 12 strains with typical yeast colony characteristics were selected, numbered JGM8-1, JGM8-2, JGM8-3, JGM8-4, JGM9-1, JGM9-2, JGM9-3, JGM9-4, JGM9-5, JGM9-6, JGM9-7, and JGM9-8. The obtained strains were inoculated on malt juice slant medium, cultured at 28℃ for 1d, and stored in a refrigerator at 4℃ for later use.

[0042] 2. Screening of strains

[0043] The isolated strains were added into rice saccharification liquid medium at an inoculation rate of 2%, and fermented at 20°C for 5 days. The aroma-producing yeast strains were screened based on the fermentation flavor.

[0044] Fermented samples are divided into 5 levels according to their flavor characteristics. Level 1 is strong fermentation aroma, marked as "+++++"; Level 2 is relatively strong fermentation aroma, marked as "++++"; Level 3 is general fermentation, marked as "+++"; Level 4 is relatively light fermentation aroma, marked as "++"; Level 5 is no fermentation aroma, marked as "-". 20 trained professionals were invited to conduct independent evaluations, and the average value was taken for statistical analysis.

[0045] The results of the fermentation flavor test of 12 strains in rice saccharification liquid medium showed that yeast JGM9-1 had a strong fermentation aroma, with a score of "+++++", yeast JGM9-5, JGM9-8, JGM9-2, JGM9-3, JGM9-4, and JGM9-7 had fermentation aroma, with scores of "++++" and "+++", yeast JGM8-2, JGM8-3, and JGM9-6 had a weak fermentation aroma, and yeast JGM8-1 and JGM8-4 could not produce fermentation aroma, with a score of "-". The results showed that the excellent yeast with unique fermentation flavor was JGM9-1.

[0046] 3. Identification of strains

[0047] The strain JGM9-1 was inoculated into MEA medium and cultured at 28°C for 72 h. The colonies were observed to be flat, creamy, sticky, shiny, and with irregular edges. Figure 1 As shown. Figure 2 Under the microscope, the cells were observed to be nearly spherical or elliptical, budding, solitary or opposite, and 1.5-6.5μm×1-6μm in size.

[0048] The strain JGM9-1 was tested for 26S rDNA. The sequence comparison results are as follows:

[0049] Alignment:JGM9-1

[0050] 100.0% Cyberlindnera fabianii CBS 5640T(KY107353)

[0051] 99.3% Cyberlindnera bimundalis NRRL Y-5343T(EF550329)

[0052] 99.1% Cyberlindnera veronae NRRL Y-7818T(EF550322)

[0053] 98.9% Cyberlindnera americana NRRL Y-2156T(EF550328)

[0054] 98.9% Candida stauntonica ATCC MYA-4699T(JQ812698)

[0055] 98.7% Cyberlindnera mississippiensis CBS 7023T (KY107376)

[0056] 98.5% Cyberlindnera xishuangbannaensis NYNU 16752T(KY213813)

[0057] 98.4% Candida pattaniensis JCM 12475T(NG 059424)

[0058] 98.0% Cyberlindnera amylophila CBS 7020T (KY107350)

[0059] 98.0% Cyberlindnera xylosilytica NRRL YB-2097T(NG 064313)

[0060] The phylogenetic tree was constructed using MEGA software and the neighbor-joining method, and the similarity was calculated 1000 times. Figure 3 Only the Bootstrap values ​​greater than 70% are shown in the nodes of the developmental tree, and the superscript "T" indicates the model strain.

[0061] Based on the gene sequencing results of the strain, which have a high homology with yeast, combined with the physiological and biochemical characteristics of the strain and other identification results, the strain JGM9-1 was identified as Cyberlindnerafabianii JGM9-1, which has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M20221401 and the deposit date is September 9, 2022.

[0062] Example 2 Isolation, screening, identification and preservation of strain RP26

[0063] 1. Isolation and purification of strains

[0064] The mash that had undergone malolactic fermentation was used as the source of lactic acid bacteria isolation, and the mixture was gradiently diluted and spread on the lactic acid bacteria isolation and purification medium plate. After multiple streaking separation and microscopic examination, a relatively pure strain was obtained. The plate streaking method was used for separation and purification. After Gram staining, catalase reaction, indole test, and sugar fermentation test, strains with typical lactic acid bacteria colony characteristics were selected and numbered as RP21, RP22, RP23, RP24, RP25, RP26, RP27, and RP28. The obtained strains were inoculated on MRS slants, cultured at 28°C for 3 days, and stored in a refrigerator at 4°C for later use.

[0065] 2. Screening of strains

[0066] The isolated strains were added into rice saccharification liquid medium at an inoculation rate of 2%, and fermented at 20°C for 5 days. The fermentation flavor was used as an indicator to screen the fragrant lactic acid-producing strains.

[0067] The fermented samples were divided into 5 grades (specifically the same as in the embodiment) according to their flavor characteristics. The fermentation flavor test results of 8 strains in rice saccharification liquid medium: lactic acid bacteria RP26 has a strong fermentation fragrance, which is scored as "+++++", lactic acid bacteria RP21 and lactic acid bacteria RP25 have fermentation fragrance, which are scored as "++++" and "+++", lactic acid bacteria RP22, lactic acid bacteria RP23, and lactic acid bacteria RP24 have a lighter fermentation fragrance, while lactic acid bacteria RP27 and lactic acid bacteria RP28 can not produce fermentation fragrance, which are scored as "-". The result shows that the excellent lactic acid bacteria with unique fermentation flavor is lactic acid bacteria RP26.

[0068] 3. Identification of strains

[0069] The strain RP26 was inoculated on an MRS plate and cultured at 30°C for 2 days. The colony morphology was observed to be milky white, round, smooth, convex, moist, and with neat edges. Figure 4 As shown. Figure 5 The cell morphology observed under a microscope was: rod-shaped, 0.5μm×1.0~1.67μm in size, arranged singly or in short chains, non-spore-forming, and Gram-positive.

[0070] The strain RP26 was tested for 16S rDNA. The MEGA4.1 software was used to display the 16S rDNA phylogenetic tree of strain RP26 and related species by the proximity joining method, and the similarity was repeated 1000 times. Figure 6 Only the Bootstrap values ​​greater than 50% are displayed in the nodes of the developmental tree. The superscript "T" indicates the model strain (L., Lactobacillus B., Bifidobacterium).

[0071] Phylogenetic analysis showed that strain RP26 and the model strains of L. plantarum subsp. plantarum, L. plantarum subsp. argentoratensis, L. paraplantarum, and L. pentosus were clustered in a phylogenetic branch, and the sequence homology was greater than 99.4%.

[0072] According to the gene sequencing results of the strain, combined with the physiological and biochemical characteristics of the strain, the strain RP26 was identified as Lactobacillus plantarum RP26, which has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2019298 and the deposit date is April 26, 2019.

[0073] Example 3 Breeding of superior strains for removing fishy smell from tuna cooking liquid

[0074] 1 Materials and methods

[0075] 1.1 Materials

[0076] 1.1.1 Strains

[0077] (1) Yeast: Lindnerella fabianseberii JGM9-1, Saccharomyces cerevisiae J4 (an excellent strain for removing the fishy smell of kelp, disclosed in application No. ZL201010534961.2), Pichia guilliermondii 31485 (CICC 31485), Saccharomyces cerevisiae JJ4 (an excellent strain for removing the fishy smell of scallop and Agaricus lemaneiformis, disclosed in application No. ZL201810898674.6), Saccharomyces cerevisiae JH301 (disclosed in application No. ZL201510411228.4).

[0078] (2) Lactic acid bacteria: Lactobacillus paracasei RYPC301 (CICC 22709), Lactobacillus paracasei subsp. paracasei RP38 (disclosed in Chinese patent application CN110656061A) and Lactobacillus plantarum RP26.

[0079] 1.1.2 Tuna cooking liquid

[0080] Tuna cooking liquid: provided by Fujian Haihui Biotechnology Industry Co., Ltd., filtered, packaged and stored in a -20℃ refrigerator for later use. Cooking liquid pH 6.67; soluble solids 3.75%; total acid 0.238g / 100g, calculated as lactic acid (0.090); amino acid nitrogen 0.07g / kg; reducing sugar 0.032g / 100g; total sugar 0.089g / 100g; protein 2.84g / 100g; crude polysaccharide 0.0184g / 100g; hydroxyproline 0.236g / 100g.

[0081] 1.2 Test methods

[0082] 1.2.1 Preparation of culture medium

[0083] YPD liquid medium: used for yeast culture; sterilize at 121℃ for 30min, cool and set aside.

[0084] MRS liquid culture medium: used for lactic acid bacteria culture; sterilize at 121℃ for 20 minutes and set aside.

[0085] 1.2.2 Seed solution preparation

[0086] After the strains are activated, they are inoculated into appropriate culture media and cultured until the yeast count reaches 10 8 cfu / mL, lactic acid bacteria count up to 10 9 cfu / mL, then centrifuge at 5500r / min and 4℃ for 10min, take the bacterial mud and add an equal volume of sterile water for standby use.

[0087] 1.2.3 Fermentation process

[0088] Take 100 mL of cooking liquid and place it in a 250 mL conical flask, add 2% (w / w) white sugar and 3 g / L malic acid, sterilize in a 100°C water bath for 10 min, and cool. 7 cfu / mL, lactic acid bacteria 10 8 cfu / mL were inoculated with the strains selected in 1.1.1, and fermented at 30°C for 72 h. The fermentation broth was collected and centrifuged for later use. Each treatment was repeated 3 times.

[0089] 1.2.4 Sensory evaluation of fermentation flavor of different strains

[0090] The fermentation liquid obtained in 1.2.3 was used as the object, the original liquid of tuna cooking liquid without added bacteria was used as the control (CK group), and the sensory evaluation was used as the measurement index to screen the yeast strain suitable for removing the fishy smell of tuna cooking liquid.

[0091] 1.2.5 Evaluation of deodorization effect

[0092] Take 100 mL of cooking liquid and place it in a 250 mL conical flask, add 20 g / L white sugar and 3 g / L malic acid, sterilize in a 100°C water bath for 10 min, and cool. 7 cfu / mL was inoculated with yeast JGM9-1 and fermented at 30±1℃ for 72h. The fermentation broth was centrifuged at 8000r / min for 5min, and the supernatant was collected. The components and contents of characteristic volatile flavor substances of each treatment were detected by HS-SPME-GC-MS, and the flavor sensory evaluation was performed. Each treatment was repeated 3 times.

[0093] 1.2.6 Sensory evaluation method

[0094] Thirteen trained professionals conducted a profile description of tuna cooking liquid according to "GB / T 10220 General Theory of Sensory Analysis Methodology", and selected fishy smell, sour smell, oily smell, green smell, floral smell, fruity smell, mellow smell, and lactic acid fermentation smell as flavor sensory descriptors. The evaluation team used a scale of "0-5" to quantitatively describe the flavor intensity, and drew sensory profile diagrams to compare the deodorization effects after fermentation with different bacteria.

[0095] 2 Results and analysis

[0096] The flavor intensity was quantitatively described on a scale of "0-5" and the tuna cooking liquor was scored after fermentation for 72 h with a single strain, see Figure 7 . Y1 (Fabianseberlindner's yeast JGM9-1) produces more obvious floral and fruity aromas, Y2 (J4 Saccharomyces cerevisiae) produces obvious mellow aromas; Y5 (31485 Pichia guillermong), Y6 (JJ4 Saccharomyces cerevisiae), and Y7 (JH301 Saccharomyces cerevisiae) produce unharmonious odors. The sour and greasy smells of M6 (RP26 Lactobacillus plantarum) decreased, but there was no obvious change in M3 (RYPC301 Lactobacillus paracasei) and M5 (RP38 Lactobacillus paracasei subsp. paracasei). Considering the three aspects of deodorization, aroma production, and stability, Y1 (Fabianseberlindner's yeast JGM9-1) and M6 (RP26 Lactobacillus plantarum) are the best strains for deodorization of tuna cooking liquid fermentation.

[0097] Example 4: Deodorization of tuna cooking liquid by composite fermentation using lactic acid bacteria and yeast

[0098] YPD liquid medium: used for yeast culture; sterilize at 121℃ for 30min, cool and set aside.

[0099] MRS liquid culture medium: used for lactic acid bacteria culture; sterilize at 121℃ for 20 minutes and set aside.

[0100] Seed solution preparation: After the strains are activated, they are inoculated into appropriate culture media and cultured until the yeast count reaches 10 8 cfu / mL, lactic acid bacteria count up to 10 9 cfu / mL, then centrifuge at 5500r / min and 4℃ for 10min, take the bacterial mud and add an equal volume of sterile water for standby use.

[0101] Fermentation process: 100 mL of cooking liquid was placed in a 250 mL conical flask, 20 g / L white sugar and 3 g / L malic acid were added, sterilized in a 100 °C water bath for 10 min, and cooled. RP26 and JGM9-1 were inoculated according to the design scheme in Table 1, and the temperature was controlled and fermented at 30 ± 1 °C for 72 h. The fermentation liquid was centrifuged at 8000 r / min for 5 min, and the supernatant was collected. The volatile flavor components and contents of each treatment were detected by HS-SPME-GC-MS, and the flavor sensory evaluation was performed. Each treatment was repeated 3 times.

[0102] Table 1 Vaccination scheme

[0103]

[0104] (1) Evaluation of the effect of microbial deodorization on tuna cooking liquid:

[0105] The sensory evaluation was conducted with reference to the methods of Wang et al. and GB / T 10220 General Introduction to Sensory Analysis Methodology. Thirty panelists (20-60 years old, 15 males and 15 females) were selected for training to ensure that each panelist could correctly describe the aromatic flavor. Finally, 20 panelists (10 males and 10 females, aged from 20 to 60 years old) were selected by the laboratory to profile the tuna cooking liquid. Fishy, ​​sour, greasy, grassy, ​​floral, and fruity flavors were selected as flavor sensory descriptors. The evaluation team used a scale of "0-5" to quantitatively describe the flavor intensity, and drew sensory profiles to compare the deodorization effects of single bacteria and compound bacteria fermentation.

[0106] After tuna cooking liquid is fermented and deodorized by different treatments, the sensory flavors vary. See the flavor radar chart. Figure 8 . The main flavors of tuna cooking liquid are fishy, ​​sour and greasy. Fermentation with Fabian Seber Lindner's yeast JGM9-1 can significantly reduce the fishy and sour smells of the cooking liquid, and produce pleasant floral and fruity aromas, but there is still a small amount of fishy and sour smells. Fermentation with Lactobacillus plantarum RP26 can significantly reduce the sour and greasy smells of the cooking liquid, and produce a good grassy aroma, but there is still a small amount of fishy and sour smells. The deodorization effect of yeast-lactic acid bacteria composite fermentation is better than that of single bacteria treatment, and the fermentation aroma produced has the style of lactic acid bacteria and yeast; the higher the yeast addition ratio, the lower the fishy and sour smells of the fermentation liquid, and the higher the floral and fruity aromas produced; the higher the lactic acid bacteria addition ratio, the more conducive it is to remove the sour and greasy smells, and the higher the grassy aroma produced. The results showed that the two treatment groups RJ4 (R:J=5:1) and RJ5 (R:J=1:1) were the most popular. Their fermentation liquid had no obvious fishy smell and had floral, fruity and grassy aromas.

[0107] (2) Detection of volatile flavor substances in marlin cooking liquid: refer to the detection method of Liang et al.:

[0108] A total of 108 volatile flavor substances were detected in the 8 treatments, including hydrocarbons, alcohols, aldehydes, acids, esters, etc. See Fig. 9 (a), (b), (c). Among them, 59 volatile flavor substances were detected in the CK group, mainly hydrocarbons and aldehydes, with contents of 267.48u / g and 135.93u / g, accounting for 80.68% of the total, respectively. After pure bacterial fermentation of lactic acid bacteria RP26 (R group), a total of 66 volatile flavor substances were detected, mainly ketones and aldehydes, with contents of 62.77ug / g and 157.27ug / g, which were 390.23% and 15.67% higher than before fermentation. Through pure bacterial fermentation of yeast JGM9-1 (J group), a total of 65 volatile flavor substances were detected, mainly alcohols and esters, with contents of 174.29ug / g and 192.68ug / g, which were 930.08% and 4017.09% higher than before fermentation. The fermentation aroma produced by yeast-lactic acid bacteria composite fermentation has the styles of both lactic acid bacteria and yeast, and is mainly composed of ketones, aldehydes, alcohols and esters; the higher the proportion of yeast added, the higher the content of alcohol and ester substances in the fermentation liquid; the higher the proportion of lactic acid bacteria added, the higher the content of ketones and aldehydes in the fermentation liquid.

[0109] (3) Succession of characteristic flavor substances in tuna cooking liquid before and after deodorization

[0110] The OPLS-DA method was used to perform cluster analysis on the eight treatments. Based on the similarities between the volatile substances in the fermentation broth, a dendrogram was drawn using the connections and distances between them. Fig.10 At a relative distance of 200, the similarity of volatile flavor substances in the fermentation broth of each treatment can be divided into five categories. The first category: CK group (CK); the second category: lactic acid bacteria group (R); the third category: lactic acid bacteria dominant group (RJ1, RJ2, RJ3); the fourth category: yeast dominant group (RJ4, RJ5); the fifth category: yeast group (J).

[0111] Based on the reported threshold values, the OAV values ​​of the flavor components of tuna fermented liquid were calculated, and the results are shown in Table 2. The VIP value analysis results of the main volatile flavor substances in the five treatments are shown in Fig.11. Based on the analysis of VIP and OAV (OAV value ≥ 1 and VIP value > 1), combined with the description of flavor substances, the main flavor substances of the five types of fermentation broth analyzed by HCA are as follows: Category I: The flavor substances of the CK group (CK treatment) are mainly decanal (6.35), hexanal (3.62), 1-octen-3-ol (3.18) with fishy smell, 2-nonanone (26.10) with oily smell, heptaldehyde (2.30), trimethylamine (6.62), and dimethyl trisulfide (3.41) with sour smell, which are the main components of the fishy smell of tuna cooking liquid. In addition, the CK group also contains some flavor substances, such as 2-ethyl-furan (11.03) and 2-pentyl-furan (14.91) with fishy smell. The second category: The main flavor substances of the R group (R treatment) are 2-dodecenal (2.01) and 2-butyl-2-octenal (1.52) with green grass aroma; 2-undecanone (5.32) with fruity aroma; 2-ethyl-furan (21.47) and 2-pentyl-furan (15.61) with fishy aroma. The third category: The flavor substances of the RJ group (RJ1, RJ2, and RJ3 treatments) are mainly β-phenylethanol (4.64) and ethyl phenylacetate (3.70) with floral aroma; 2-undecanone (7.05) and ethyl pyruvate (7.45) with fruity aroma; 2-butyl-2-octenal (1.07) with green grass aroma; 2-ethyl-furan (10.37) and 2-pentyl-furan (6.60) with fishy aroma. The fourth category: The flavor substances of the JR group (RJ4 and RJ5 treatments) are mainly β-phenylethanol (8.94), ethyl tetradecanoate (1.50), and ethyl phenylacetate (5.07) with floral aroma; ethyl octanoate (10.54), octanol (7.60), 2-undecanone (1.08), ethyl propionate (1.53), and ethyl acetate (2.30) with fruity aroma; 2-dodecenal (2.18) and 2-butyl-2-octenal (1.47) with grassy flavor; 2-ethyl-furan (1.64) and 2-pentyl-furan (2.38) with fishy aroma. The fifth category: The flavor substances of group J (J treatment) are mainly fruity isopentanol (1.28), octanol (2.74), ethyl pyruvate (24.74), ethyl propionate (1.76), ethyl octanoate (6.37), and ethyl acetate (2.86); floral β-phenylethanol (11.85), ethyl phenylacetate (5.57), and ethyl tetradecanoate (1.08); and fishy 2-ethyl-furan (1.50) and 2-pentyl-furan (3.23).

[0112] Table 2 Analysis results of volatile flavor substances OVA≥1 in tuna cooking liquid

[0113]

[0114]

[0115] (4) Contribution of lactic acid bacteria and yeast to the evolution of characteristic flavor substances in tuna cooking liquid

[0116] The spermman correlation analysis method was used to analyze the characteristic flavor substances fermented by Lactobacillus plantarum RP26 and Fabianseberlindnerella JGM9-1 (P<0.05 and correlation |r|>0.5). The results are shown in Fig.12 Based on the comprehensive analysis of Opls-da, spearman correlation, OAV and related flavor description, it is clear that the flavor substances with VIP value>1.0, correlation|r|>0.5 and OAV>1 are the characteristic flavor substances of tuna cooking liquid microbial fermentation. The results are shown in Fig.13 . The results showed that the characteristic flavor substances consumed by Lactobacillus plantarum RP26 were 2-nonanone with an oily smell, trimethylamine and dimethyl trisulfide with a sour smell; the characteristic flavor substances produced were 2-butyl-2-octenal and 2-dodecenal with a grassy aroma, and 2-undecanone with a fruity aroma. The characteristic flavor substances consumed by Fabianseber Lindnerella JGM9-1 were decanal, hexanal, and 1-octen-3-ol with a fishy smell; trimethylamine and dimethyl trisulfide with a sour smell; the characteristic flavor substances produced were β-phenylethanol, ethyl myristate, and ethyl phenylacetate with a floral flavor, and ethyl pyruvate, ethyl propionate, ethyl laurate, ethyl caprylate, octanol, ethyl acetate, and isopentanol with a fruity flavor. Fig.14 It can be seen that the fermented tuna cooking liquid of RP26 and JGM9-1 can jointly consume trimethylamine and dimethyl trisulfide with sour and smelly taste, and the characteristic flavor substances produced are specific.

[0117] The composite fermentation of JGM9-1 and RP26 can simultaneously metabolize and consume decanal, hexanal, 1-octen-3-ol with fishy smell, 2-nonanone with oily smell, and trimethylamine and dimethyl trisulfide with sour smell, and metabolize to produce flavor substances with the characteristics of both lactic acid bacteria and yeast, including 2-butyl-2-octenal with grassy aroma, 2-dodecenal, 2-undecanone, octanol, ethyl octanoate, ethyl propionate, ethyl acetate with fruity flavor, and β-phenylethanol, ethyl tetradecanoate, and ethyl phenylacetate with floral aroma.

[0118] Example 5

[0119] A method for removing fishy smell from tuna cooking liquid by microorganisms comprises the following steps:

[0120] 100 mL of tuna cooking liquid was added with 20 g / L white sugar and 3 g / L malic acid, sterilized in a water bath at 100 °C, cooled for 10 min, and inoculated with RP26 and JGM9-1, with the inoculation amount being 5% of the tuna cooking liquid. The bacterial culture liquid in the composite bacteria activation culture process was tuna cooking liquid with 18-22 g / L white sugar and 2.8-3.2 g / L malic acid added, and the biomass of plant lactobacillus RP26 was 1×10 9 cfu / mL, the biomass of Fabianseber Lindnerella JGM9-1 was 1×10 8 cfu / mL, mixed at a biomass ratio of 5:1. Then, the fermentation was carried out at 30±1℃ for 72h, the fermentation liquid was centrifuged at 8000r / min for 5min, and the supernatant was collected, which was the odorless tuna cooking liquid, which can be used to further process high-value products such as seasoning liquid, peptide liquid, and amino acid oral liquid.

[0121] In this embodiment, the process parameters are obtained through the following creative test screening:

[0122] (1) Single factor test on biological deodorization of tuna cooking liquid:

[0123] 100 mL of cooking liquid was placed in a 250 mL conical flask, and after adjusting the sugar and acid, sterilized in a water bath at 100 ° C for 10 min, and inoculated for fermentation after cooling. The amount of white sugar added in the single factor was 10, 20, 30, 40, 50 g / L, the amount of malic acid added was 0, 1, 2, 3, 4 g / L, and the inoculation amount (the bacterial culture liquid in the composite bacteria activation culture process was tuna cooking liquid with 18-22 g / L white sugar and 2.8-3.2 g / L malic acid, and the biomass of Lactobacillus plantarum RP26 was 1×10 9 cfu / mL, the biomass of Fabianseber Lindnerella JGM9-1 was 1×10 8 cfu / mL, mixed at a biomass ratio of 5:1) was 1%, 3%, 5%, 7%, and 9%, the fermentation temperature was 21°C, 24°C, 27°C, 30°C, and 33°C, and the fermentation time was 48h, 60h, 72h, 84h, and 96h. The fixed processing parameters were 20g / L of white sugar, 3g / L of malic acid, 5% inoculation, 30°C, and 72h of fermentation temperature. The fermentation broth was centrifuged at 8000r / min for 5min, and the supernatant was collected. HS-SPME-GC-MS was used to detect the components and content of the characteristic volatile flavor substances of each treatment, and the flavor sensory evaluation was performed. Each treatment was repeated 3 times.

[0124] (2) Sensory fuzzy comprehensive evaluation

[0125] 15 teachers and students of food majors who have received relevant training were organized to form an evaluation team. The analytic hierarchy process was used to establish the evaluation domain of tuna cooking liquid U = {fishy smell U1, aroma U2, taste U3, color U4}, and the fuzzy weight vector A = {0.35, 0.35, 0.25, 0.05} of each evaluation index was set, and the evaluation level S = {excellent S1, good S2, qualified S3, unqualified S4} was set. The evaluation criteria are shown in Table 3. The M (-, +) operator was used to establish a fuzzy comprehensive evaluation model. The standards for the values ​​of each level are shown in Table 4. Each sample constitutes a common set (domain): F = {F1, F2, ..., F 19}, Fi{i=1, 2, …19}, Fi represents the i-th type of Agaricus lemaneiformis sample, and the rank-weighted average principle is adopted according to the membership degree for sensory fuzzy comprehensive evaluation.

[0126] The calculation formula is as follows: 15 evaluators evaluate the smell, aroma, taste and color of the sample, where the value of the number of people evaluating each level divided by the total number of people is the fuzzy weight vector of each indicator: U1 = {a1, b1, c1, d1}, U2 = {a2, b2, c2, d2}, U3 = {a3, b3, c3, d3}, U4 = {a4, b4, c4, d4}. The four indicators are written in matrix form: Through matrix multiplication, we get the comprehensive evaluation set F1=A·y={S1S2S3S4}. Finally, we multiply the insignificant value of each level by each vector of the comprehensive evaluation set and add them up, that is, the total score H=S1×90+S2×70+S3×50+S4×20.

[0127] Table 3 Sensory scoring standards

[0128]

[0129] Table 4: Outlier values ​​of each level

[0130]

[0131] (3) Effect of the amount of added white sugar on the deodorization of tuna cooking liquid

[0132] Effects of different amounts of sugar added on the deodorization of tuna cooking liquid Fig.15 As shown in the figure, with the increase of the amount of white sugar added, the sensory score of the fermentation liquid first increased and then tended to be flat. When the amount of white sugar added was 20g / L, the sensory score was the highest, and there was no significant difference in the sensory score with the treatment of continuing to increase the amount of sugar (P>0.05). The results show that considering cost savings, the appropriate amount of white sugar added is 20g / L.

[0133] (4) Effect of malic acid addition on the deodorization of tuna cooking liquid

[0134] Effect of malic acid addition on the deodorization of tuna cooking liquid by Agaricus lemaneiformis Fig.16 As shown in the figure, with the increase of malic acid addition, the fermentation sensory score first increased and then decreased, indicating that too high or too low malic acid addition will affect the deodorization effect, and adding an appropriate amount of malic acid is conducive to improving the fermentation flavor. The results showed that the optimal malic acid addition was 3g / L, and the difference with other treatments reached an extremely significant level (P<0.01).

[0135] (5) Effect of bacterial inoculation amount on the deodorization effect of tuna cooking liquid

[0136] Effect of different inoculation amounts on the deodorization of tuna cooking liquid Fig.17 . As can be seen from the figure: with the increase of bacterial inoculum, the sensory score of the fermentation liquid first increases and then tends to be flat. When the inoculum amount is 5%, the sensory score is the highest, and further increasing the inoculum amount has no significant effect on the sensory score (P>0.05). The results show that from the perspective of cost saving, the appropriate inoculum amount is 5%.

[0137] (6) Effect of fermentation time on the deodorization effect of tuna cooking liquid

[0138] Effects of different fermentation times on the deodorization of tuna cooking liquid Fig.18 As shown in the figure, with the increase of fermentation time, the sensory score of the fermentation liquid first increased and then tended to be flat. The sensory score was the highest when the fermentation time was 72h, and the difference with 84h and 96h was not significant (P>0.05). The results showed that the appropriate fermentation time was 72h.

[0139] (7) Effect of fermentation temperature on the deodorization of tuna cooking liquid

[0140] Effects of different fermentation temperatures on the deodorization of tuna cooking liquid Fig.19 As shown in the figure, with the increase of fermentation temperature, the sensory score of the fermentation liquid first increased and then decreased. When the fermentation temperature was 30℃, the sensory score of tuna cooking liquid was the highest, and the difference with other treatments reached a very significant level (P<0.01). The results showed that the suitable fermentation temperature was 30℃.

[0141] Example 6

[0142] A method for removing fishy smell from tuna cooking liquid by microorganisms comprises the following steps:

[0143] Take 100mL of tuna cooking liquid, add 20g / L white sugar, 3g / L malic acid, sterilize in 100℃ water bath, cool for 10min, inoculate RP26 and JGM9-1 (the bacterial culture liquid in the composite bacteria activation culture process is tuna cooking liquid with 18-22g / L white sugar and 2.8-3.2g / L malic acid, and the biomass of Lactobacillus plantarum RP26 is 1×10 9 cfu / mL, the biomass of Fabianseber Lindnerella JGM9-1 was 1×10 8 cfu / mL, mixed at a biomass ratio of 5:1), the inoculation amount was 5% of the tuna cooking liquid, the biomass ratio of RP26 and JGM9-1 was 5:1, and then the temperature was controlled and fermented at 30±1℃ for 72h, the fermentation liquid was centrifuged at 8000r / min for 5min, and the supernatant was collected, which was the tuna cooking liquid without fishy smell, and could be used for further processing of high-value products such as seasoning liquid, peptide liquid, and amino acid oral liquid.

[0144] In this embodiment, tuna cooking liquid is directly used for bacterial activation culture, so that it is an edible culture medium during inoculation, and there is no need to discard the culture medium by centrifugation, thus simplifying the process.

Claims

1. A method for removing fishy smell from tuna cooking liquid by microorganisms, characterized in that: The following steps are involved: 1) Add sugar and malic acid to the tuna cooking liquid. The concentration of sugar in the tuna cooking liquid is 20 g / L and the concentration of malic acid is 3 g / L. Sterilize in a water bath and then cool. 2) inoculating a composite bacteria, wherein the composite bacteria are Lactobacillus plantarum RP26 and Fabianseberlindner yeast JGM9-1, the inoculation amount of the composite bacteria is 5% of the volume of the tuna cooking liquid, and the biomass ratio of Lactobacillus plantarum RP26 to Fabianseberlindner yeast JGM9-1 in the composite bacteria is 5:1; The Fabianseber Lindner yeast JGM9-1, scientific name Cyberlindnera fabianii JGM9-1 has been deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 20221401 and the deposit date being September 9, 2022; The Lactobacillus plantarum RP26, whose scientific name is Lactobacillus plantarum RP26, has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 2019298 and a deposit date of April 26, 2019; The culture medium for the composite bacteria activation culture process was tuna cooking liquid with 18-22 g / L white sugar and 2.8-3.2 g / L malic acid added. The biomass of Lactobacillus plantarum RP26 was 1×10 9 cfu / mL, the biomass of Fabianseber Lindnerella JGM9-1 was 1×10 8 cfu / mL; 3) Ferment at 30±1℃ for 65-75 h, centrifuge the fermentation liquid and collect the supernatant, which is the odorless tuna cooking liquid.

2. The method for removing fishy smell from tuna cooking liquid by microorganisms according to claim 1, characterized in that: The fermentation temperature is 30°C and the fermentation time is 72h.

3. The method for removing fishy smell from tuna cooking liquid by microorganisms according to claim 1, characterized in that: The tuna cooking liquid is fermented with Fabianseber Lindner yeast JGM9-1 and Lactobacillus plantarum RP26 to simultaneously metabolize and consume decanal, hexanal, 1-octen-3-ol, 2-nonanone, trimethylamine, and dimethyl trisulfide, which have fishy, ​​sour, and greasy smells, and metabolize to produce flavor substances with the characteristics of both lactic acid bacteria and yeast, including 2-butyl-2-octenal, 2-dodecenal, 2-undecanone, octanol, ethyl octanoate, ethyl propionate, ethyl acetate, β-phenylethanol, ethyl tetradecanoate, and ethyl phenylacetate, which have grassy, ​​fruity, and floral aromas.

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