Method and application for preparing oyster fermentation broth by using compound strains

Abstract

The present invention provides a method for preparing oyster fermentation broth using composite strains and its application, and the steps are as follows: pretreatment of oyster meat, enzymatic hydrolysis, enzyme inactivation, and fermentation with composite strains to obtain oyster fermentation broth. The present invention inoculates the oyster autogenous strains into the oyster enzymatic hydrolysate, and Lactobacillus pentosus 7-K and Pediococcus argentinicus 7-I are co-fermented to give full play to the high adaptability and optimized metabolic mechanism among the same organisms, and jointly act on the nutrient components of the oyster enzymatic hydrolysate, promoting decomposition and conversion into small molecular substances that are more easily absorbed by the human body. At the same time, the bitter and fishy smell substances in the oyster enzymatic hydrolysate are metabolized and decomposed, improving the flavor quality of the oyster fermentation broth and overcoming the original flavor limitation of the oyster enzymatic hydrolysate.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermentation products, and particularly relates to a method for preparing oyster fermentation broth using a composite strain and its application. Background Art

[0002] Oysters, also known as sea oysters and raw oysters, belong to the phylum Mollusca, class Bivalvia, order Ostreoida, family Ostreidae, and genus Ostrea. Fresh oyster meat is soft and tender in texture, rich in nutrients, containing proteins, polysaccharide substances, and a small amount of lipid components (DHA and EPA), minerals, and trace elements, endowing it with various health-beneficial effects. It is one of the first batches of "medicinal and edible homologous" aquatic products approved in China.

[0003] In order to fully utilize oyster meat and form high-value products, an enzymatic hydrolysis method is selected to treat whole oyster meat to prepare oyster enzymatic hydrolysate. The enzymatic hydrolysis method uses different types of proteases to hydrolyze protein raw materials, with advantages such as mild conditions and easy control, and has become one of the main production methods of oyster protein peptides. Research shows that the oyster enzymatic hydrolysis products obtained by protease hydrolysis may have various physiological functions, such as anti-photoaging, anti-fatigue, blood sugar lowering, and blood pressure lowering functions. However, the oyster smell and bitterness are obvious after enzymatic hydrolysis. Through SPME-GC-MS detection and analysis, it is found that the main volatile components of oyster meat include alcohol substances (such as 1-octen-3-ol), aldehyde substances (such as 2-nonenal), furan substances (such as 2-ethylfuran), ketone substances (such as 2,3-octanedione), etc. These compounds are the main flavor substances that give oyster meat pleasant odors such as fruity and grassy scents. After oysters are enzymatically hydrolyzed, the main flavor substances become aldehydes (mainly C3-C6) and thioether compounds (dimethyl sulfide) that present a fishy smell. The presence of these substances makes the flavor of oyster enzymatic hydrolysate worse, and the overall performance is fishy, stinky, and rancid smells that are not easily acceptable to people. Summary of the Invention

[0004] Technical problems to be solved: Aiming at the above technical problems, the purpose of the present invention is to provide a method for preparing oyster fermentation broth using a composite strain and its application, and the steps are as follows: pretreatment of oyster meat, enzymatic hydrolysis, enzyme inactivation, and fermentation with a composite strain to obtain oyster fermentation broth. The present invention inoculates the oyster autogenous strain into the oyster enzymatic hydrolysate, and Lactobacillus pentosus 7-K and Pediococcus argentinicus 7-I co-ferment, giving full play to the high adaptability and optimized metabolic mechanism among the same species of organisms, jointly acting on the nutrient components of the oyster enzymatic hydrolysate, promoting decomposition and conversion into small molecular substances that are more easily absorbed by the human body, and at the same time metabolizing and decomposing the bitter and fishy substances in the oyster enzymatic hydrolysate, improving the flavor quality of the oyster fermentation broth, and overcoming the original flavor limitations of the oyster enzymatic hydrolysate.

[0005] Technical solution: A method for preparing oyster fermentation broth using a composite strain, comprising the following steps:

[0006] S1. Select oysters with clean outer shells, thin and closed shells, and relatively heavier weight of the same size. Remove the oyster shells, add water and homogenize to obtain oyster homogenate.

[0007] S2. Mix and homogenize the oyster homogenate with water at a volume ratio of 1:2. The homogenization speed is 8000 - 10000 r / min, and the homogenization time is 2 - 3 min to obtain oyster homogenized liquid.

[0008] S3. Add animal protease to the oyster homogenized liquid for enzymatic hydrolysis, inactivate the enzyme and sterilize to obtain oyster enzymatic hydrolysate.

[0009] S4. Inoculate the oyster enzymatic hydrolysate with the activated complex bacterial liquid for fermentation to obtain oyster fermented liquid.

[0010] The complex bacterial liquid is a composition of Lactobacillus pentosus 7-K and Pediococcus argentinicus 7-I; or a composition of Lactobacillus pentosus 7-K, Pediococcus argentinicus 7-I and commercial bacterial agent C356-F.

[0011] Further, in step S3, the addition amount of animal protease is 3000 - 4000 U / g, the enzymatic hydrolysis temperature is 50 - 55 °C, and the enzymatic hydrolysis time is 4 - 5 h.

[0012] Further, the method for inactivating the enzyme and sterilizing in step S3 is to hold the pressure at 600 MPa for 10 - 15 min under ultra-high pressure.

[0013] Further, in step S4, the addition amount of the complex bacterial liquid is 4 - 6%.

[0014] Further, the ratio of Lactobacillus pentosus 7-K to Pediococcus argentinicus 7-I is 2:1, or the ratio of Lactobacillus pentosus 7-K, Pediococcus argentinicus 7-I to commercial bacterial agent C356-F is 2:1:1.

[0015] Further, the activation method of Lactobacillus pentosus 7-K, Pediococcus argentinicus 7-I, and commercial bacterial agent C356-F is to inoculate them in MRS medium, culture at 37 °C for 24 h, and then activate and culture continuously for two more times to make the final concentration of the strain reach 10 4 -10 5 CFU / mL. Centrifuge the bacterial liquid at 4 °C and 8000 r / min for 10 min, wash the bacteria twice with the same volume of physiological saline, and resuspend them in physiological saline to obtain the activated bacterial liquid.

[0016] Further, the fermentation conditions in step S4 are fermentation temperature 37 - 40 °C and fermentation time 36 - 40 h.

[0017] Furthermore, the preservation unit of Lactobacillus pentosus 7-K is the China Center for Type Culture Collection, with the preservation number CCTCC NO: M2023711 and the preservation date of May 9, 2023.

[0018] Beneficial effects:

[0019] 1. The present invention utilizes the co-fermentation of Lactobacillus pentosus 7-K and Pediococcus argentinicus 7-I on the oyster hydrolysate. They occupy different ecological niches in the fermentation system and, through the complementarity of metabolic pathways, achieve the maximum utilization of each component in the oyster hydrolysate and the diversification of metabolites. It significantly reduces the content of bitter amino acids such as tyrosine and arginine in the oyster hydrolysate, increases the content of free fatty acids in the oyster fermentation broth, improves the bitter and fishy smell generated after oyster hydrolysis, enhances the flavor quality of the oyster fermentation broth. Moreover, Lactobacillus pentosus 7-K and Pediococcus argentinicus 7-I are autochthonous fermentation strains of oysters, having a good positive regulation effect on flavor, enhancing the unique pleasant flavor substances of the oyster fermentation broth, and being able to efficiently mask the generation and increase of off-flavor substances.

[0020] 2. The present invention adopts the co-fermentation of Lactobacillus pentosus 7-K and Pediococcus argentinicus 7-I on the oyster hydrolysate. Based on the unique metabolic ability of autochthonous strains of oysters during fermentation, it can better adapt to the reaction environment of the oyster fermentation broth, improve the microbial diversity in the oyster fermentation broth, and at the same time significantly inhibit the growth of pathogenic bacteria and spoilage bacteria by producing antibacterial substances or competing for nutrient substrates and adhesion sites, ensuring the excellent quality of the oyster fermentation broth. Description of the drawings

[0021] Figure 1 are the sensory scores of Example 2, Example 3, and Comparative Examples 2-6;

[0022] Figure 2 are the total amount change of free amino acids (A) and the heat map of specific components change of free amino acids (B) of Example 2, Example 3, Comparative Example 1, and Comparative Example 2;

[0023] Figure 3 are the taste radar maps of electronic tongue analysis of Example 2, Example 3, Comparative Example 1, and Comparative Example 2;

[0024] Figure 4 are the evaluation results of taste acceptance of Example 2, Example 3, Comparative Example 1, and Comparative Example 2;

[0025] Figure 5 are the GC-MS heat map analysis of Example 2, Example 3, Comparative Example 1, and Comparative Example 2;

[0026] Figure 6 are the flavor fingerprint maps of Example 2, Example 3, Comparative Example 1, and Comparative Example 2;

[0027] Figure 7 are the odor evaluation radar charts (A) and acceptance (B) of Example 2, Example 3, Comparative Example 1 and Comparative Example 2;

[0028] Figure 8 are the distribution and differences of the microbial diversity of Example 2, Example 3, Comparative Example 1 and Comparative Example 2 at the phylum level;

[0029] Figure 9 are the distribution and differences of the microbial diversity of Example 2, Example 3, Comparative Example 1 and Comparative Example 2 at the genus level. Detailed implementation manners

[0030] The present invention will be further described below in conjunction with embodiments. The following embodiments are explanations of the present invention, and the present invention is not limited to the following embodiments:

[0031] The strains used in the present invention: C356-F (composed of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus), purchased from Hebei Yiran Biotechnology Co., Ltd.; Lactobacillus pentosus 7-K (Lactobacillus pentosus 7-K, preservation number CCTCC NO: M2023711, extracted from oysters), preserved in the China Center for Type Culture Collection (the preservation date is May 9, 2023, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University); The reference for the source of Pediococcus argentinicus strain 7-I is Liu L, Liu T, Wang H, et al. Identification and validation of core microbes for the formation of the characteristic flavor of fermented oysters (Crassostrea gigas). [J]. Food chemistry, 2024, 449, 138970 (DOI: 10.1016 / j.foodchem.2024.138970)

[0032] Example 1

[0033] A method for activating a composite strain, comprising the following steps:

[0034] Step 1. Inoculate C356-F in MRS medium, culture at 37°C for 24 h, and then continuously activate and culture twice to make the final concentration of the strain reach 10 5CFU / mL. The bacterial solution was centrifuged at 8000 r / min for 10 min at 4°C, washed twice with the same volume of physiological saline, and resuspended in physiological saline to obtain an activated C356-F bacterial solution;

[0035] Step 2. Lactobacillus pentosus 7-K was inoculated in MRS medium and cultured at 37°C for 24 h, and then continuously activated and cultured twice to make the final concentration of the strain reach 10 5 CFU / mL. The bacterial solution was centrifuged at 8000 r / min for 10 min at 4°C, washed twice with the same volume of physiological saline, and resuspended in physiological saline to obtain an activated Lactobacillus pentosus 7-K bacterial solution;

[0036] Step 3. Pediococcus argentinicus 7-I was inoculated in MRS medium and cultured at 37°C for 24 h, and then continuously activated and cultured twice to make the final concentration of the strain reach 10 5 CFU / mL. The bacterial solution was centrifuged at 8000 r / min for 10 min at 4°C, washed twice with the same volume of physiological saline, and resuspended in physiological saline to obtain an activated Pediococcus argentinicus 7-I bacterial solution.

[0037] Example 2

[0038] A method for preparing oyster fermentation broth using a composite strain, comprising the following steps:

[0039] S1. Select oysters with clean outer shells, thin shells and closed mouths, and of the same size and heavier weight. Remove the oyster shells, add water and homogenize to obtain oyster homogenate;

[0040] S2. Weigh 100 mL of oyster homogenate, add 200 mL of water, mix and place in a dispersing homogenizer, and homogenize at a speed of 10000 r / min for 2 min to obtain oyster homogenized liquid;

[0041] S3. Add 3000 U / g of animal protease to the oyster homogenized liquid, enzymatically hydrolyze at 50°C for 4 h, and inactivate enzymes and sterilize under hydrostatic pressure at 600 MPa for 10 min to obtain oyster enzymatically hydrolyzed liquid;

[0042] S4. Inoculate 12 mL of the activated composite bacterial solution (8 mL of activated Lactobacillus pentosus 7-K bacterial solution, 4 mL of activated Pediococcus argentinicus 7-I bacterial solution) into the oyster enzymatically hydrolyzed liquid, and ferment at 37°C for 36 h to obtain oyster fermentation broth, denoted as 7KI.

[0043] Example 3

[0044] A method for preparing oyster fermentation broth using a composite strain, comprising the following steps:

[0045] S1. Select oysters with clean outer shells, thin and closed shells, and heavier weight for the same size. Remove the oyster shells, add water and homogenize to obtain oyster homogenate;

[0046] S2. Weigh 100 mL of oyster homogenate, add 200 mL of water, mix and place it in a dispersing homogenizer, and homogenize for 2 min at a rotation speed of 10000 r / min to obtain oyster homogenized liquid;

[0047] S3. Add 3000 U / g of animal protease to the oyster homogenized liquid, enzymatically hydrolyze at 50 °C for 4 h, and inactivate enzymes and sterilize under hydrostatic pressure at 600 MPa for 10 min to obtain oyster enzymatically hydrolyzed liquid;

[0048] S4. Inoculate 12 mL of activated complex bacterial liquid (6 mL of activated Lactobacillus pentosus 7-K bacterial liquid, 3 mL of activated Pediococcus argentinicus 7-I bacterial liquid, 3 mL of activated C356-F bacterial liquid) into the oyster enzymatically hydrolyzed liquid, and ferment at 37 °C for 36 h to obtain oyster fermented liquid, denoted as 7KIC.

[0049] Comparative Example 1

[0050] The difference between this comparative example and Example 2 is that the activated complex bacterial liquid is not added.

[0051] A method for preparing oyster fermented liquid, comprising the following steps:

[0052] S1. Select oysters with clean outer shells, thin and closed shells, and heavier weight for the same size. Remove the oyster shells, add water and homogenize to obtain oyster homogenate;

[0053] S2. Weigh 100 mL of oyster homogenate, add 200 mL of water, mix and place it in a dispersing homogenizer, and homogenize for 2 min at a rotation speed of 10000 r / min to obtain oyster homogenized liquid;

[0054] S3. Add 3000 U / g of animal protease to the oyster homogenized liquid, enzymatically hydrolyze at 50 °C for 4 h, and inactivate enzymes and sterilize under hydrostatic pressure at 600 MPa for 10 min to obtain oyster enzymatically hydrolyzed liquid;

[0055] S4. Ferment the oyster enzymatically hydrolyzed liquid at 37 °C for 36 h to obtain oyster fermented liquid, denoted as UF.

[0056] Comparative Example 2

[0057] The difference between this comparative example and Example 3 is that Lactobacillus pentosus 7-K and Pediococcus argentinicus 7-I in the complex bacterial species are replaced by C356-F.

[0058] A method for preparing oyster fermented liquid using C356-F bacteria, comprising the following steps:

[0059] S1. Select oysters with clean outer shells, thin and closed shells, and of the same size but heavier in weight. Remove the oyster shells, add water and homogenize to obtain oyster homogenate.

[0060] S2. Weigh 100 mL of oyster homogenate, add 200 mL of water, mix and place it in a dispersing homogenizer. Homogenize at a speed of 10,000 r / min for 2 min to obtain oyster homogenized liquid.

[0061] S3. Add 3000 U / g of animal protease to the oyster homogenized liquid, enzymatically hydrolyze at 50 °C for 4 h, and inactivate enzymes and sterilize under hydrostatic pressure of 600 MPa for 10 min to obtain oyster enzymatically hydrolyzed liquid.

[0062] S4. Inoculate 12 mL of activated C356-F bacterial liquid into the oyster enzymatically hydrolyzed liquid, ferment at 37 °C for 36 h to obtain oyster fermented liquid, denoted as CF.

[0063] Comparative Example 3

[0064] The difference between this comparative example and Example 2 is that Pediococcus argentinicus 7-I in the composite strain is replaced by Lactobacillus pentosus 7-K. A method for preparing oyster fermented liquid using Lactobacillus pentosus 7-K includes the following steps:

[0065] S1. Select oysters with clean outer shells, thin and closed shells, and of the same size but heavier in weight. Remove the oyster shells, add water and homogenize to obtain oyster homogenate.

[0066] S2. Weigh 100 mL of oyster homogenate, add 200 mL of water, mix and place it in a dispersing homogenizer. Homogenize at a speed of 10,000 r / min for 2 min to obtain oyster homogenized liquid.

[0067] S3. Add 3000 U / g of animal protease to the oyster homogenized liquid, enzymatically hydrolyze at 50 °C for 4 h, and inactivate enzymes and sterilize under hydrostatic pressure of 600 MPa for 10 min to obtain oyster enzymatically hydrolyzed liquid.

[0068] S4. Inoculate 12 mL of activated Lactobacillus pentosus 7-K bacterial liquid into the oyster enzymatically hydrolyzed liquid, ferment at 37 °C for 36 h to obtain oyster fermented liquid, denoted as 7K.

[0069] Comparative Example 4

[0070] The difference between this comparative example and Example 2 is that Lactobacillus pentosus 7-K in the composite strain is replaced by Pediococcus argentinicus 7-I. A method for preparing oyster fermented liquid using Pediococcus argentinicus 7-I includes the following steps:

[0071] S1. Select oysters with clean outer shells, thin and closed shells, and of the same size but heavier in weight. Remove the oyster shells, add water and homogenize to obtain oyster homogenate.

[0072] S2. Weigh 100 mL of oyster homogenate, add 200 mL of water, mix and place it in a dispersing homogenizer. Homogenize it for 2 min at a rotational speed of 10,000 r / min to obtain oyster homogenized liquid;

[0073] S3. Add 3000 U / g of animal protease to the oyster homogenized liquid, enzymatically hydrolyze it at 50 °C for 4 h, and inactivate enzymes and sterilize it by hydrostatic pressure at 600 MPa for 10 min to obtain oyster enzymatically hydrolyzed liquid;

[0074] S4. Inoculate 12 mL of activated Pediococcus argentinicus 7-I bacterial liquid into the oyster enzymatically hydrolyzed liquid, ferment it at 37 °C for 36 h to obtain oyster fermented liquid, denoted as 7I.

[0075] Comparative Example 5

[0076] The difference between this comparative example and Example 2 is that Pediococcus argentinicus 7-I in the composite strain is replaced by C356-F.

[0077] A method for preparing oyster fermented liquid using a composite strain, comprising the following steps:

[0078] S1. Select oysters with clean outer shells, thin and closed shells, of the same size and relatively heavy weight. Remove the oyster shells, add water and homogenize to obtain oyster homogenate;

[0079] S2. Weigh 100 mL of oyster homogenate, add 200 mL of water, mix and place it in a dispersing homogenizer. Homogenize it for 2 min at a rotational speed of 10,000 r / min to obtain oyster homogenized liquid;

[0080] S3. Add 3000 U / g of animal protease to the oyster homogenized liquid, enzymatically hydrolyze it at 50 °C for 4 h, and inactivate enzymes and sterilize it by hydrostatic pressure at 600 MPa for 10 min to obtain oyster enzymatically hydrolyzed liquid;

[0081] S4. Inoculate 12 mL of activated composite bacterial liquid (8 mL of activated Lactobacillus pentosus 7-K bacterial liquid, 4 mL of activated C356-F bacterial liquid) into the oyster enzymatically hydrolyzed liquid, ferment it at 37 °C for 36 h to obtain oyster fermented liquid, denoted as 7KC.

[0082] Comparative Example 6

[0083] The difference between this comparative example and Example 2 is that Lactobacillus pentosus 7-K in the composite strain is replaced by C356-F.

[0084] A method for preparing oyster fermented liquid using a composite strain, comprising the following steps:

[0085] S1. Select oysters with clean outer shells, thin and closed shells, of the same size and relatively heavy weight. Remove the oyster shells, add water and homogenize to obtain oyster homogenate;

[0086] S2. Weigh 100 mL of oyster homogenate, add 200 mL of water and mix, then place it in a dispersing and homogenizing machine and homogenize for 2 min at a rotation speed of 10,000 r / min to obtain an oyster homogenate solution;

[0087] S3. Add 3000 U / g of animal protease to the oyster homogenate solution, enzymatically hydrolyze it at 50 °C for 4 h, and then sterilize it by hydrostatic pressure at 600 MPa for 10 min to obtain an oyster enzymatically hydrolyzed solution;

[0088] S4. Inoculate 12 mL of the activated complex bacterial solution (6 mL of the activated C356-F bacterial solution and 6 mL of the activated Pediococcus argentinicus 7-I bacterial solution) into the oyster enzymatically hydrolyzed solution, and ferment it at 37 °C for 36 h to obtain an oyster fermented solution, denoted as 7IC.

[0089] Performance test

[0090] (1) Sensory evaluation

[0091] A 40-person sensory evaluation panel was established to conduct sensory evaluations on the oyster fermented solution, and the scores were given from 0 (very dislike) to 5 (very like).

[0092] It can be seen from Figure 1 that the sensory scores of 7KI and 7KIC are both higher than those of CF, 7K, 7I, 7KC, and 7IC, that is, the sensory scores of Example 2 and Example 3 are both higher than those of Comparative Examples 2-6. This shows that the compounding of Lactobacillus pentosus 7-K and Pediococcus argentinicus 7-I from the oyster's own origin can metabolize and decompose the bitter and fishy smell substances in the oyster enzymatically hydrolyzed solution, reduce the content, and the further compounding of C356-F bacteria can positively regulate the generation of flavor substances in the oyster fermented solution to the greatest extent and improve the flavor quality of the oyster fermented solution.

[0093] (2) Free amino acids

[0094] Accurately weigh 2 mL of the oyster enzymatically hydrolyzed solution into a test tube, then add 15 mL of HCl solution with a concentration of 0.02 mol / L and homogenize for 1 min, then ultrasonically treat for 5 min, centrifuge at 4 °C and 8000 r / min for 10 min, and collect the supernatant; add another 10 mL of HCl solution with a concentration of 0.02 mol / L to the residue, homogenize for 1 min, centrifuge at 4 °C and 8000 r / min for 6 min, combine the supernatants and make up the volume to 50 mL; after volume making, take 2 mL of the extract, add 2 mL of sulfosalicylic acid (5%, v / v), centrifuge at 4 °C and 10,000 r / min for 10 min, and pass the supernatant through a 0.22 μm water-based filter membrane and determine the free amino acid concentration by a HITACHI L-8900 amino acid analyzer.

[0095] It can be seen from Figure 2It can be seen that free amino acids are classified into umami amino acids, sweet amino acids, and bitter amino acids according to their types, which endow oyster fermentation broth with different flavors. By fermenting oyster hydrolysate, it was found that the content of bitter amino acids decreased significantly after fermentation of CF, 7KI, and 7KIC (p<0.05). Among them, the amino acids with obvious decrease in content were tyrosine and arginine, and the two could approach 0 in 7KI and 7KIC, indicating that the addition of autochthonous oyster strains could significantly reduce Tyr and Arg, and the bitterness of oyster hydrolysate was significantly improved after fermentation.

[0096] (3) Free fatty acids

[0097] Sample pretreatment: The oyster fermentation broth prepared in Example 2, Example 3, Comparative Example 1, and Comparative Example 2 was freeze-dried to obtain a freeze-dried powder. 1.0 g of the freeze-dried powder was weighed, 50 mL of chloroform-methanol (2:1, v / v) was added, and it was left standing at 4 °C for 2 h and then filtered. Then, 5 mL of sodium chloride solution (0.9%, w / v) was added to the filtrate, and it was centrifuged at 4 °C and 4000 r / min for 10 min. The lower layer solution was taken for nitrogen blowing until constant weight; 2 mL of potassium hydroxide methanol solution with a concentration of 0.5 mol / L and 3 mL of boron trifluoride methanol solution with a mass fraction of 14% were added thereto, and it was heated in a water bath at 60 °C for 20 min. After cooling, 2 mL of n-hexane and 1 mL of ultrapure water were mixed, and the operation was repeated three times. It was blown to constant weight with nitrogen and then made up to 10 mL with n-hexane.

[0098] Sample detection: A Shimadzu QP2010-SE gas chromatography-mass spectrometry (Japan) was used. The chromatographic column was an HP-5MS chromatographic column (Agilent, USA). The injection port temperature was 250 °C, the carrier gas was helium, the flow rate was 1.5 mL / min, and the injection volume was 1 μL.

[0099] Gas chromatography conditions: The initial temperature was maintained at 60 °C for 1 min, then it was heated to 160 °C at a rate of 10 °C / min and maintained for 5 min, then it was heated to 200 °C at a rate of 3 °C / min and maintained for 10 min, and finally it was heated to 280 °C at a rate of 6 °C / min and maintained for 5 min; Mass spectrometry conditions: The ion source temperature was 250 °C, the scanning range was 35 - 500 m / z, and the electron energy was 70 eV.

[0100] Table 1 Free fatty acid content (FFAs) of Example 2, Example 3, Comparative Example 1, and Comparative Example 2

[0101]

[0102]

[0103] Note: Different letters of the same sample represent significance (p<0.05), and ND indicates not detected.

[0104] Free fatty acids play important flavor and nutritional roles. From a flavor perspective, free fatty acids are precursors of various alcohols and aldehydes, and their content affects the flavor of food. The oyster hydrolysate after fermentation is mainly composed of saturated and unsaturated fatty acids with more than 12 carbon atoms. The composition and content of free amino acids in the oyster fermentation broth prepared in Example 2, Example 3, Comparative Example 1, and Comparative Example 2 are shown in Table 1.

[0105] As can be seen from Table 1, the content of saturated fatty acids (SFA) in the oyster hydrolysate after fermentation showed a significant increasing trend (p<0.05) except for 7KI (Example 2). Among them, the contents of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) were significantly higher than those of UF (Comparative Example 1) (p<0.05). Compared with the comparative examples, the total free fatty acid content of 7KI (Example 2) and 7KIC (Example 3) increased significantly. Especially, the content of oleic acid (C18:1, cis-9) increased significantly after fermentation, from 0.695 mg / g to 1.000 mg / g and 0.927 mg / g respectively, an increase of about 43.88% and 33.38%, which has a certain positive regulatory effect on the oxidation of oleic acid to octanol.

[0106] (4) Electronic tongue

[0107] The oyster fermentation broths prepared in Example 2, Example 3, Comparative Example 1, and Comparative Example 2 were diluted 4 times respectively, centrifuged at 4 °C and 8000 r / min for 10 min, and the supernatant was placed in a sample cup, about 35 mL per cup. The Insent SA402B electronic tongue was used for analysis, and the measurement was carried out according to its default program to detect its sourness, bitterness, astringency, umami, saltiness, and sweetness, and analyze its aftertaste - astringency, aftertaste - bitterness, and richness.

[0108] The taste characteristics of the oyster fermentation broths prepared in Example 2, Example 3, Comparative Example 1, and Comparative Example 2 were analyzed using an electronic tongue. The electronic tongue is an intelligent taste recognition system that converts electrical signals into taste characteristics and further analyzes them into more objective taste characteristics. From Figure 3 it can be seen that the tastes of the oyster fermentation broths prepared with 7KI and 7KIC changed significantly. Among them, the sweetness, bitterness, and umami all decreased significantly, indicating that the autochthonous bacteria Pediococcus argentinicus 7-I and Lactobacillus pentosus 7-K in oysters can utilize the bitter and umami amino acids in the oyster hydrolysate during the lactic acid bacteria metabolism process, resulting in a decrease in bitterness and umami. At the same time, the acidity increased significantly in 7KI and 7KIC, which is the only taste characteristic that increased after fermentation. This is due to the production of organic acids and other acidic substances by lactic acid bacteria during fermentation. The astringency did not change significantly during fermentation.

[0109] (5) Taste acceptance

[0110] The acceptability of the oyster fermentation broth prepared in Example 2, Example 3, Comparative Example 1 and Comparative Example 2 was evaluated. The acceptability was judged according to the intensity from 0 (very dislike) to 5 (very like).

[0111] It can be seen from Figure 4 that the acceptable degrees of 7KIC and 7KI can reach 4.14 and 4.38 respectively, with good acceptability, which is better than UF and CF. This shows that fermentation has the effect of improving the flavor of oyster enzymolysis broth. The reason why the improvement effect of CF is lower than that of 7KI and 7KIC is the difference in the microbial community structure. Due to the better adaptability and metabolism of the autogenous strains of oysters, the synergistic effect with Pediococcus argentinensis makes the flavors of 7KI and 7KIC after fermentation significantly improved.

[0112] (6) Volatile flavor substances

[0113] Add 2 mL of oyster fermentation broth and 20 μL of standard product (2-methyl-3-heptanone, 1.0 mg / mL) into a 20 mL headspace vial, incubate at 50 °C and 250 r / min for 30 min, insert the SPME extraction head into the headspace vial, and then desorb the extraction head at 250 °C in the injection port for 5 min.

[0114] Detection and analysis were carried out using Agilent GC8890-MS / MS 7000D (USA) on an Agilent 19091N-133I J&W HP-INNOWax chromatographic column (30 m × 0.25 mm, 0.25 μm) (USA). Helium (99.999%) was used as the carrier gas with a flow rate of 1.0 mL / min and splitless injection. The temperature programming procedure: the column oven was initially maintained at 50 °C for 3 min, then heated to 160 °C at a rate of 3 °C / min and maintained for 3 min, and then heated to 230 °C at a rate of 10 °C / min and maintained for 10 min; the mass spectrometry conditions: the ion source temperature was 230 °C, the scanning range was 33 - 450 m / z, and the electron energy was 70 eV.

[0115] It can be seen from Figure 5It can be seen that all samples were divided into two groups. One group consisted of 7KI and 7KIC, and the other group consisted of CF and UF. The flavor quality of 7KI and 7KIC was better than that of CF and UF, indicating that the compound addition of autogenous lactic acid bacteria from oysters and Pediococcus argentinus could improve the degree of metabolic reactions and produce more flavor substances with a positive regulatory effect on the flavor of oyster hydrolysate and stability. The oyster fermentation broth prepared from 7KI and 7KIC was rich in 5-nonanone, 2-undecanone, 3-octanol, 3-methylthiopropanol, 1-nonanol, phenethyl alcohol, and γ-decalactone, jointly presenting a strong tropical fruit fragrance, and the off-flavor compounds with negative impacts such as 1-hepten-3-one, 1-octen-3-one, and 1-octen-3-ol basically disappeared, indicating that the autogenous lactic acid bacteria from oysters had an excellent positive regulatory effect on the flavor regulation of oyster hydrolysate, improved the unique and pleasant flavor of oyster fermentation broth, and could efficiently mask the generation and increase of off-flavor substances.

[0116] (7) Flavor fingerprint

[0117] Add 2 mL of oyster fermentation broth into a 20 mL headspace vial, incubate it at 50 °C and 250 r / min for 30 min, and then inject the sample aspirated from its headspace into G.A.S FlavourSpec GC-IMS (Germany) for analysis in a G.A.S MXT-WAX chromatographic column (15 m × 0.53 mm) (Germany).

[0118] Analysis conditions: The injection volume was 500 μL of gas sample, nitrogen was used as the carrier gas, and the IMS drift tube temperature was 45 °C; flow rate program: The initial flow rate was maintained at 2.0 mL / min for 2 min, then increased gradientally to 10 mL / min and maintained for 10 min, then increased gradientally to 100 mL / min and maintained for 20 min, and increased gradientally to 150 mL / min and maintained for 25 min; all experiments were repeated three times, and the fingerprint was analyzed and plotted using the G.A.S Laboratory Analyzer Viewer (VOCal 0.4.03, Germany).

[0119] The flavor fingerprint can more intuitively and clearly display the changes of each flavor substance in different combinations. Each row represents its corresponding sample combination, and each column represents the type and relative content of its corresponding flavor substance. The combination of the two can describe the changes of the same flavor substance in different combinations. Dark colors indicate a higher concentration of the flavor substance, with obvious flavor characteristics; lighter colors indicate a lower concentration of the flavor substance, and its characteristic flavor may be masked; black indicates that the flavor substance was not detected.

[0120] From Figure 6It can be seen that the flavor fingerprint formed by 7KI, 7KIC, UF, and CF was divided into three parts, namely A, B, and C, for analysis. Region A is mainly the region where the substances detected in UF itself have increased in content after fermentation by the autogenous bacteria combination 7KI and 7KIC of oysters. It can be clearly seen that the corresponding substance concentrations have increased significantly. These substances include isovaleraldehyde presenting tropical fruit flavor; decanal with flower fragrance, cucumber flavor, orange peel flavor, and butter flavor; butanol with fruit flavor; isobutanol with apple flavor, cocoa flavor, and wine flavor; 2-nonanone with fruit flavor, grass flavor, and hot milk flavor; methyl octanoate with fruit flavor, orange flavor, and wine flavor, etc.

[0121] Region B is mainly the region where the flavor substances have relatively high concentrations in 7KI and 7KIC, but relatively low concentrations or are not detected in the UF and CF groups. These include propyl butyrate presenting fruit flavor, apricot flavor, and pineapple flavor; linalool with flower fragrance, lavender flavor, lemon flavor, and rose flavor; 2-heptanone with fruit flavor, grass flavor, nut flavor, and spice flavor; isoamyl acetate with apple flavor and fruit flavor; 2-hexanol with fruit flavor; ethyl propionate with apple flavor, pineapple flavor, strawberry flavor, and rum flavor; 3-ethyl-2,6-dimethylpyrazine with grape juice flavor, nut flavor, and potato flavor; 2-pentanone with fruit flavor, etc. Analyzing regions A and B comprehensively, the fermentation of the autogenous oyster strains plays a positive regulatory role in the compounds that can improve the bad flavor of the oyster hydrolysate, significantly increasing the generation of fruit flavor and flower fragrance in the sample; while the flavor substances corresponding to region B are the specific flavor substances produced by the autogenous oyster strains, thus making the flavor of the oyster hydrolysate more excellent after the combined fermentation of 7KI and 7KIC.

[0122] Region C is mainly the region where the flavor substances significantly present in UF have decreased significantly or disappeared after fermentation. These include 1-octen-3-one presenting fishy and earthy flavors (oyster characteristic flavor); 1-octen-3-ol with mushroom flavor, fishy flavor, and cucumber flavor (oyster characteristic flavor); isobutyric acid with burnt flavor, sweat flavor, and cheese flavor; hydroxyacetone with spicy flavor, butter flavor, and greasy flavor; hexanal with raw oil flavor and pungent flavor; furfural with burnt flavor, roasted potato flavor, and bitter almond flavor; acetal with earthy flavor, etc. Region C mainly corresponds to some of the bad flavors originally present in UF. After fermentation, the influence of these substances on the flavor of the oyster fermentation broth can be reduced or eliminated, indicating that the addition of autogenous oyster strains can play a more obvious subtractive role, and at the same time proving its superiority in fermenting homologous substrates.

[0123] Based on the favorable flavor enhancement effects in regions A and B and the unfavorable flavor reduction effect in region C, it can be seen that the fermentation effect of adding autogenous oyster strains and Pediococcus argentinicus has obvious advantages both in terms of the increase in the types and contents of positive flavors and the reduction of off-flavor substances. This indicates that the combined fermentation of autogenous oyster strains and Pediococcus argentinicus can better utilize the off-flavor alcohols and acids in the substrate, metabolize and combine them into esters, thereby greatly generating ester flavor substances with positive and active regulatory effects.

[0124] (8) Odor evaluation

[0125] Fifteen professionally trained flavor assessors (8 males and 7 females, aged between 20 - 30 years old) were selected for the experiment. The assessors smelled the UF, CF, 7KI, and 7KIC samples to determine the odor descriptors, and all assessors needed to reach an agreement on the selected characteristic flavor descriptors; the selected odor descriptors (sweet, fruity, fermented, fragrant, fishy, and putrid) were evaluated according to the intensity values from 0 (not detectable) to 5 (very strong); the acceptability of the odors was evaluated separately, and the acceptability was judged according to the intensity from 0 (very dislike) to 5 (very like).

[0126] As can be seen from Figure 7 A, sweet, fruity, fermented, fragrant, fishy, and putrid are the most important flavor attributes in UF, CF, 7KI, and 7KIC, obtained through free-choice profiling analysis. The sweet and fruity flavors are significantly higher in 7KI and 7KIC than in UF and CF, while in UF, putrid and fishy flavors are dominant. Although CF has been improved to a certain extent, the effect is not ideal. The fruity and sweet flavors come from substances such as 5-nonanone, 2-undecanone, 3-octanol, 3,6-nonadien-1-ol, and γ-decalactone; as can be seen from Figure 7 B, the acceptability of 7KI is the highest, superior to the 7KIC group, because the fruity and sweet flavors in the 7KI group are more mellow and do not have the strong stimulation of 7KIC.

[0127] (9) Microbial colony diversity

[0128] The V4 region of the 16S rRNA gene of the microorganisms was extracted from the oyster fermentation broths of UF, CF, 7KI, and 7KIC respectively for high-throughput sequencing. The sequences obtained from high-throughput sequencing were compared with the Silva database to obtain the taxonomic information of the sequences and compare the differences in the microbial flora structures.

[0129] As can be seen from Figure 8It can be seen that at the phylum level, Firmicutes and Proteobacteria are the dominant phyla in UF, while in CF, 7KI, and 7KIC, only Firmicutes are present, indicating that inoculation fermentation can inhibit the growth of irrelevant bacteria, even harmful bacteria. In particular, the inhibitory effects of 7KI and 7KIC are significantly better than that of CF. Due to the interaction between the autochthonous bacteria of oysters and Pediococcus argentinicus and various substances in the oyster hydrolysate, the growth of strains in Proteobacteria can be better inhibited.

[0130] It can be seen from Figure 9 that at the genus level, a total of 381 bacterial genera were identified in UF, CF, 7KI, and 7KIC. Among them, there are 11 core bacterial genera with a relative abundance greater than 1% in UF, namely Staphylococcus, Arcobacter, Synechococcus, Shewanella, Corynebacterium, Vibrio, Syntrophobacteraceae, and Pseudoalteromonas, etc.; in the CF group, only Streptococcus has a relative abundance greater than 1%, while in the 7KI and 7KIC groups, they are both f_Lactobacillaceae and Lactobacillus, and Streptococcus can reach 0.9% in 7KIC, indicating that the colony structure has changed greatly after the fermentation of the oyster hydrolysate. CF and 7KI are basically composed of only the added strains themselves, while although there are certain dominant strains in the CF group in 7KIC, the autochthonous strains of oysters and Pediococcus argentinicus still play a dominant role. This is because of the special metabolic ability of the autochthonous bacteria of oysters during the fermentation process and the synergistic effect with Pediococcus argentinicus, which can better adapt to the substrate environment, quickly reproduce, and become the dominant flora. Microorganisms have a significant positive regulatory effect on increasing the volatile flavor substances in the oyster fermentation broth. 7KIC prepared by co-fermentation of C356-F, Lactobacillus pentosus 7-K, and Pediococcus argentinicus 7-I makes the flavor of 7KIC softer and more acceptable, has the effect of improving the volatile flavor in the oyster fermentation broth, and can achieve the purpose of removing fishy smell and enhancing fragrance at the same time. Moreover, the inoculation of Lactobacillus pentosus 7-K (autochthonous bacteria of oysters) and Pediococcus argentinicus 7-I is beneficial to changing the microbial flora structure in the substrate and has a significant effect on inhibiting the growth of pathogenic and spoilage bacteria.

[0131] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above, or modify it into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing oyster fermentation broth using a composite strain, characterized in that, It includes the following steps: S1. Select oysters with clean outer shells, thin and closed shells, and heavier weight of the same size. Remove the oyster shells, add water and homogenize to obtain oyster homogenate. S2. Mix the oyster homogenate with water at a volume ratio of 1:2 and homogenize. The homogenization speed is 8000 - 10000 r / min, and the homogenization time is 2 - 3 min to obtain oyster homogenized liquid. S3. Add animal protease to the oyster homogenized liquid for enzymatic hydrolysis, inactivate the enzyme and sterilize to obtain oyster enzymatic hydrolysate. The addition amount of the animal protease is 3000 - 4000 U / g, the enzymatic hydrolysis temperature is 50 - 55 °C, and the enzymatic hydrolysis time is 4 - 5 h. S4. Inoculate the activated complex bacterial liquid into the oyster enzymatic hydrolysate for fermentation to obtain oyster fermented liquid. The addition amount of the complex bacterial liquid is 4 - 6%. The composite bacterial liquid is a composition of Lactobacillus pentosus ( Lactobacillus pentosus ) 7-K and Pediococcus argentinicus ( Pediococcus argentinicus strain ) 7-I; or Lactobacillus pentosus( Lactobacillus pentosus ) 7-K, Pediococcus argentinicus( Pediococcus argentinicus strain ) 7-I and a composition of commercial microbial agent C356-F; the commercial microbial agent C356-F is purchased from Hebei Yiran Biotechnology Co., Ltd.; The preservation unit of Lactobacillus pentosus 7-K is the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M2023711. The preservation date is May 9, 2023.

2. The method for preparing oyster fermentation broth using a composite bacterial strain according to claim 1, characterized in that: The method of inactivating the enzyme and sterilizing in step S3 is to hold the pressure at 600 MPa for 10 - 15 min under ultra-high pressure.

3. A method for preparing oyster fermentation broth using a composite strain, as described in claim 1, characterized in that: The ratio of Lactobacillus pentosus 7-K to Pediococcus argentinicus 7-I is 2:1, or the ratio of Lactobacillus pentosus 7-K, Pediococcus argentinicus 7-I to commercial bacterial agent C356-F is 2:1:

1.

4. A method for preparing oyster fermentation broth using a composite strain according to claim 1, characterized in that: The activation method of Lactobacillus pentosus 7-K, Pediococcus argentinicus 7-I, and commercial bacterial agent C356-F is to inoculate them in MRS medium and culture at 37 °C for 24 h, then continuously activate and culture twice more to make the final concentration of the strain reach 10 4 -10 5 CFU / mL. Centrifuge the bacterial solution at 4 °C and 8000 r / min for 10 min, wash the bacteria twice with the same volume of physiological saline, and resuspend them in physiological saline to obtain the activated bacterial solution.

5. A method for preparing oyster fermentation broth using a composite strain according to claim 1, characterized in that: The fermentation conditions in step S4 are a fermentation temperature of 37 - 40 °C and a fermentation time of 36 - 38 h.

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