Extraction method of stinky mandarin fish fermented bacterial colony

Abstract

The invention relates to the technical field of fermentation processing of aquatic products, discloses an extraction method of stinky mandarin fish fermentation colonies, and provides a technical scheme for extracting composite flora in stages aiming at the bottlenecks of large inter-batch flavor difference, long production period and the like caused by uncontrollable flora succession in traditional natural fermentation. Aiming at three different fermentation periods of mandarin fish, respectively designing a bacterial colony extraction scheme and an inspection step before extraction; the extracted composite flora can be re-fed into subsequent batches of fermentation according to a time sequence, so that a fermentation process is rebuilt, and the consistency of a flavor precursor synthesis path is ensured from the source; meanwhile, the fermentation time of the subsequent composite flora can be greatly shortened through the composite flora, the production efficiency of the smelly mandarin fish is improved, the method is suitable for large-scale production of the smelly mandarin fish, and the industry is promoted to be upgraded from experience driving to precise regulation and control of microbial metabolism.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic product fermentation processing, and specifically is a method for extracting stinky mandarin fish fermentation colonies. Background Art

[0002] As a quintessential Anhui cuisine, stinky mandarin fish, with its unique "garlic meat" texture and "stinky smell, fragrant taste" flavor, has earned it a prominent place in the culinary world. However, its production currently relies primarily on natural fermentation, a traditional process with numerous bottlenecks that need to be addressed.

[0003] First, the fermentation cycle is lengthy. Natural fermentation typically takes nearly a month, and the process is significantly affected by fluctuations in environmental factors such as temperature and humidity. This prolonged fermentation not only reduces production efficiency but also increases production costs and management complexity.

[0004] Secondly, natural fermentation suffers from a lack of standardization. Due to over-reliance on experience, salinity control is unstable, and bacterial succession is difficult to effectively regulate, resulting in significant flavor differences between different batches.

[0005] To overcome these limitations, a technology for extracting stinky mandarin fish fermentation colonies is urgently needed. This technology should be time-matched, enabling the targeted extraction of fermentation colonies from the early, middle, and late stages of stinky mandarin fish fermentation. These colonies can then be applied to subsequent fermentation processes, fundamentally addressing the issue of synergistically improving flavor stability, safety, and production efficiency across batches of stinky mandarin fish. This is of great significance to the development of the stinky mandarin fish industry. Summary of the Invention

[0006] In order to solve the problems in the background technology, the present invention provides a method for extracting stinky mandarin fish fermentation bacteria to solve the above problems.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for extracting stinky mandarin fish fermentation bacteria colonies, which is divided into three stages according to the fermentation cycle of mandarin fish;

[0009] (1) Extraction of initial colonies: After fermentation, the pH of the fish tissue was monitored in real time. When the pH dropped to 4.5-5.0, the initial fermentation stage was completed. The abdominal mucosal layer and fish tissue were washed with physiological saline and crushed to form a slurry. The slurry was spread on a solid plate rich in culture medium and cultured anaerobically at 30°C for 24 hours. Milky white, smooth, raised, and 1-2 mm in diameter circular colonies were picked and inoculated on an MRS slant and refrigerated at 4°C.

[0010] (2) Extraction of mid-term colonies: After the initial fermentation stage, the ethyl acetate concentration in the mandarin fish meat tissue was monitored in real time. When the concentration change rate between two consecutive times was ≤5%, the mid-term fermentation stage was completed. The fish meat tissue was washed with physiological saline and crushed to form a slurry. The slurry was spread on a solid plate rich in culture medium and cultured aerobically at 30°C for 48 hours. Milky white colonies with a diameter of 2–4 mm and smooth edges were picked and inoculated on an MRS slant and refrigerated at 4°C.

[0011] The formula for calculating the rate of change is as follows:

[0012] in 、 The values ​​of two adjacent measurements

[0013] (3) Late colony extraction: After the mid-term fermentation stage, the indole concentration in the flesh of mandarin fish was monitored in real time. When the concentration change rate between two consecutive times was ≤5%, 3 g of gill and abdominal mucus was collected and homogenized with PBS buffer. The slurry was spread on a solid plate rich in culture medium and cultured aerobically at 30°C for 72 hours. The target colonies were picked and inoculated on LB slant or TSB slant; and refrigerated at 4°C.

[0014] The formula for calculating the rate of change is as follows:

[0015] in 、 These are the values ​​of two adjacent measurements.

[0016] As a preference, the culture medium used for initial colony extraction is culture medium A, and its specific configuration method is as follows:

[0017] Step 1: Weigh 5 g glucose, 1 g peptone, 0.9 g NaCl, 0.1 g potassium sorbate, and 0.6 g calcium carbonate powder;

[0018] Step 2: Prepare dilute hydrochloric acid solution: Take 16.7 ml of concentrated hydrochloric acid (36%) and add distilled water to make up to 100 ml;

[0019] Step 3: Dissolve NaCl in 100 ml of distilled water, add glucose and peptone to the NaCl solution, and stir until completely dissolved; add potassium sorbate and continue stirring for 5 minutes to mix evenly;

[0020] Step 4: Use dilute hydrochloric acid solution to adjust the pH of the NaCl solution to 6.0-6.5; then add 0.6g of CaCO3 powder and stir for ten minutes to form a suspension;

[0021] Step 5: The suspension is sterilized to obtain culture medium A.

[0022] As a preference, the culture medium used for mid-term colony extraction is culture medium B, and its specific configuration method is as follows:

[0023] Step 1: Weigh 3 g glucose, 2 g yeast extract, 1.5 ml ethanol, sodium acetate, 0.8 g Na2HPO4·12H2O, 0.2 g KH2PO4, 0.05 g MgSO4·7H2O, and 0.05 g potassium sorbate;

[0024] Step 2: Take 80ml of distilled water, add 0.8g Na2HPO4·12H2O, 0.2g KH2PO4, and 0.05g MgSO4·7H2O; stir until completely dissolved;

[0025] Step 3: Add 3.0g glucose, 2.0g yeast extract, and 0.5g sodium acetate and continue stirring until completely dissolved;

[0026] Step 4: Add 1.5 ml of ethanol and 0.05 g of potassium sorbate and stir to dissolve;

[0027] Step 5: Add distilled water to 100 ml and measure the pH value, with a target pH of 6.0–6.5. If the pH is < 6.0, add 0.1 mol / L NaOH solution to adjust the pH. If the pH is > 6.5, add 0.1 mol / L HCl solution to adjust the pH.

[0028] Step 6: The above solution is sterilized to obtain culture medium B.

[0029] As a preference, the culture medium used for the later colony extraction is culture medium C, and its specific configuration method is as follows:

[0030] Step 1: Weigh 4.0 g peptone, 1.0 g yeast extract, 0.3 g tryptophan, 0.2 g cysteine·HCl, 1.5 g NaCl, 0.6 g Tris base, 0.03 g MgSO4·7H2O, and 0.02 g potassium sorbate.

[0031] Step 2: Take 80 ml of distilled water, add 0.6 g of Tris base, 1.5 g of NaCl, and 0.03 g of MgSO4·7H2O, and stir to dissolve;

[0032] Step 3: Add 4.0 g peptone, 1.0 g yeast extract, and 0.3 g tryptophan, and stir in a 60°C water bath for 15 minutes;

[0033] Step 4: Add 0.2 g cysteine·HCl and 0.02 g potassium sorbate and stir for 5 minutes until dissolved;

[0034] Step 5: Dilute to 100 ml with distilled water, target pH 7.2–7.5; if pH < 7.2, add 0.1 mol / L Tris base solution; if pH > 7.8, add 0.1 mol / L HCl solution;

[0035] Step 6: After the solution is sterilized for 15 min, culture medium C is obtained.

[0036] Preferably, after the mandarin fish has been fermented for 36 hours, the fish tissue from the inner wall of the abdominal cavity is taken every 2 to 3 hours for pH testing. The pH testing method of mandarin fish is as follows: 3-5 g of fish tissue from the inner wall of the abdominal cavity is taken and the fish meat is crushed; after crushing, pre-cooled sterile distilled water (the distilled water temperature is below 5°C) is added at a ratio of 1:9 (mass:volume); centrifugal stirring is performed for 5 minutes; after standing, the supernatant is taken for pH determination; a calibrated pH meter electrode is used to insert the supernatant, and the reading is recorded after it stabilizes; the measurement is repeated 3 times, and a deviation of >0.2 requires re-measurement; the average value of the three times is calculated as the measured pH value.

[0037] As a preference, after the total fermentation time of mandarin fish is full, ie 144 hours, samples are taken every 6 hours to detect the ethyl acetate concentration of the fish meat.

[0038] Preferably, the ethyl acetate content in the fish meat is detected by gas chromatography; the fish meat is pretreated as follows before detection: 3–5 g of fish meat tissue is taken; pre-cooled ultrapure water (e.g., 5 g of meat + 25 ml of water) is added at a ratio of 1:5 (fish meat mass: ultrapure water volume); the fish meat is centrifuged for 15 minutes (4°C); the fish meat is then allowed to stand, the supernatant is taken, and filtered; the filtered supernatant is detected by gas chromatography.

[0039] As a preferred method, after the total fermentation time of mandarin fish is 192 hours, samples are taken every 6 hours to detect the indole content of the fish gills and abdominal mucus.

[0040] As a preference;

[0041] The ethyl acetate content in fish meat was determined using gas chromatography. Before indole analysis, the samples underwent the following pretreatment steps:

[0042] Gill and peritoneal mucus samples (approximately 3 g) were collected, avoiding contact with metal tools to prevent indole oxidation; the samples were ground to a particle size ≤ 0.5 mm, and a 1:10 (fish mass: phosphate buffer volume) volume of pre-chilled phosphate buffer (PBS, pH 7.0) was added to obtain a homogenate; the homogenate was centrifuged at 4°C for 20 minutes, and the supernatant was filtered to obtain the supernatant. The samples were extracted twice with an equal volume of dichloromethane, and the organic phases were combined; the samples were dehydrated with anhydrous sodium sulfate, concentrated to dryness under reduced pressure at 40°C, and reconstituted with 1 mL of methanol.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. Traditional natural fermentation relies on the random succession of environmental microorganisms, resulting in significant differences in bacterial flora structure between different batches, which in turn causes fluctuations in the content of key flavor compounds such as esters and indoles. The breakthrough of this invention is to capture bacterial flora in a sequential manner: colonies are extracted at different fermentation cycles. These three stages of colonies can be used to replicate the fermentation process of previous batches of mandarin fish in subsequent batches, ensuring the stability of the flavor of different fermented mandarin fish batches to the greatest extent.

[0045] 2. The complex bacterial flora extracted at each stage of the present invention contains multiple microorganisms with complementary functions, fully recreating the synergistic metabolic network of natural fermentation. Compared with commercial single-species bacterial strains (such as Lactobacillus alone), the complex bacterial flora is more adaptable to high-salt, low-temperature, and anaerobic environments, and the fermented mandarin fish has a better flavor.

[0046] 3. During the fermentation process of subsequent batches of mandarin fish, adding corresponding fermentation bacteria at different stages can increase the speed of subsequent mandarin fish fermentation and ultimately improve the fermentation efficiency of mandarin fish. DETAILED DESCRIPTION

[0047] To make the purpose, features, and advantages of this application more apparent and understandable, the technical solutions in the embodiments of this application are described clearly and completely below. Obviously, the embodiments described below are only some of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0048] 1. Fermentation culture of mandarin fish and extraction of microorganisms

[0049] 1. Pretreatment of raw mandarin fish

[0050] Step 1: Select fresh mandarin fish with shiny scales, bright red gills and no fishy smell.

[0051] Step 2: Clean, remove scales and internal organs, flush blood from the abdominal cavity with running water, and soak in 10% salt water for 10 minutes to sterilize.

[0052] Step 3: Ventilate and drain, then weigh to the nearest gram.

[0053] 2. Prepare the culture medium

[0054] 2.1. There are three types of culture medium: A, B, and C, which correspond to the three fermentation cycles of mandarin fish (early, middle, and late).

[0055] The specific fermentation cycle of mandarin fish is as follows:

[0056] Initial stage (approximately 0–2 days): Lactic acid bacteria rapidly produce acid, and the pH drops significantly.

[0057] Middle stage (about 3-7 days): Ester synthesis peaks. Turning the fish over during this period can promote uniform fermentation of the mandarin fish.

[0058] Late stage (about 8–14 days): Odor-smelling substances such as indole are produced.

[0059] 2.2. The preparation steps of culture medium A (corresponding to initial fermentation) are as follows:

[0060] Weigh the raw materials (accurate to 0.01g): 5g glucose, 1g peptone, 0.9g NaCl, 0.1g potassium sorbate, 0.6g calcium carbonate powder.

[0061] Glucose serves as the primary carbon source for bacterial growth, providing energy. Peptone provides nitrogen and growth factors, supporting bacterial proliferation. NaCl is dissolved in 100ml of distilled water to simulate an isotonic environment and inhibit bacteria. Potassium sorbate (a preservative) inhibits mold and yeast, ensuring that lactic acid bacteria dominate fermentation.

[0062] Preparation of dilute hydrochloric acid solution: Take 16.7 ml of concentrated hydrochloric acid (concentration is 36%) and add distilled water to make up to 100 ml.

[0063] Mixing and dissolving: Add glucose and peptone to the NaCl solution and stir magnetically for 20 minutes until completely dissolved. Add potassium sorbate and continue stirring for 5 minutes to mix thoroughly.

[0064] Adjust pH: Use a dropper to slowly add dilute hydrochloric acid to the NaCl solution, stirring continuously and monitoring the pH. Target pH is 6.0–6.5 (you can add two to three drops at a time, waiting 30 seconds before checking the pH). Add 0.6g of CaCO3 powder and stir for 10 minutes to form a suspension.

[0065] Sterilization: The suspension was placed into a heat-resistant glass bottle, sealed, and sterilized by high pressure (121°C, 0.1 MPa, 15–20 min) to obtain culture medium A.

[0066] 2.2. The preparation steps of culture medium B (corresponding to mid-term fermentation) are as follows:

[0067] Weigh the raw materials (accurate to 0.01g): 3g glucose, 2g yeast extract, 1.5ml ethanol, sodium acetate, 0.8g Na2HPO4·12H2O; 0.2g KH2PO4; 0.05g MgSO4·7H2O; 0.05g potassium sorbate.

[0068] Prepare basic solution: take 80ml of distilled water and add , 0.05gMgSO4·7H2O; stir until completely dissolved.

[0069] Add glucose and yeast extract: Add 3.0g glucose, 2.0g yeast extract, and 0.5g sodium acetate and continue stirring until completely dissolved.

[0070] Mix in ethanol and potassium sorbate: Add 1.5 ml of 95% ethanol and 0.05 g of potassium sorbate and stir to dissolve.

[0071] Adjust volume and pH: Dilute to 100 ml with distilled water and measure pH, aiming for a pH of 6.0–6.5. If pH < 6.0, add 0.1 mol / L NaOH solution, one drop at a time, checking every 30 seconds. If pH > 6.5, adjust with 0.1 mol / L HCl solution.

[0072] Packaging and sterilization: The solution was placed in a heat-resistant glass bottle and the bottle mouth was sealed; sterilized at 121°C for 15 minutes to obtain culture medium B.

[0073] 2.3. The preparation steps of culture medium C (corresponding to the later fermentation) are as follows:

[0074] Weigh the raw materials (accurate to 0.01g): 4.0g peptone, 1.0g yeast extract, 0.3g tryptophan, 0.2g cysteine·HCl, 1.5g NaCl, 0.6g Tris base, 0.03g MgSO4·7H2O, and 0.02g potassium sorbate.

[0075] Dissolve buffer salts and osmotic pressure agent: Take 80 ml of distilled water, add 0.6 g Tris base, 1.5 g NaCl, and 0.03 g MgSO4·7H2O, and stir magnetically for 20 minutes until transparent.

[0076] Add nitrogen source and precursor: add 4.0 g peptone, 1.0 g yeast extract, 0.3 g tryptophan, and stir in a 60°C water bath for 15 minutes.

[0077] Add reducing agent and antibacterial agent: add 0.2g cysteine·HCl and 0.02g potassium sorbate and stir for 5 minutes until dissolved.

[0078] Volume and pH adjustment: Add distilled water to 100 ml, target pH 7.2–7.5. If pH < 7.2, add 0.1 mol / L Tris base solution dropwise. If pH > 7.8, adjust by adding 0.1 mol / L HCl solution dropwise.

[0079] Packaging: The solution was placed into a heat-resistant glass bottle, the bottle mouth was sealed, and sterilized at 121°C for 15 minutes to obtain culture medium C.

[0080] 3. Marinade and fermentation of mandarin fish

[0081] Remove the internal organs of the mandarin fish and pat it dry. Rub the fish inside and out with 20-30g of iodine-free salt for every 500g of fish, focusing on massaging the joints between the bones. Then, lay the fish flat in a container, place a 1kg weight on top of it, and seal the container for fermentation. Fermentation should last no longer than 14 days at a temperature between 15°C and 25°C.

[0082] 4. Colony Extraction and Cultivation during the Fermentation Process of Mandarin Fish

[0083] 4.1 Extraction and cultivation of fermentation colonies during the initial fermentation cycle

[0084] After the 36th hour of fermentation, fish tissue from the inner wall of the abdominal cavity was sampled every 2 to 3 hours for pH testing, with 3–5 g of fish tissue sampled each time. When the detected pH value was between 4.5 and 5.0, the initial fermentation cycle of the mandarin fish was completed.

[0085] The pH detection method for mandarin fish is as follows: take 3-5g of fish meat from the abdominal wall and crush the fish meat; after crushing, add pre-cooled sterile distilled water (distilled water temperature below 5°C) at a ratio of 1:9 (mass: volume); centrifuge and stir for 5 minutes; after standing, take the supernatant for pH measurement.

[0086] Use a calibrated pH meter electrode to insert the supernatant and record the reading once it stabilizes. Repeat the measurement three times. If the deviation is greater than 0.2, repeat the measurement. Calculate the average of the three readings as the measured pH value.

[0087] When the pH drops to 4.5-5.0, the extraction step of the bacterial colonies on the surface of the fish tissue in the initial fermentation cycle can be carried out.

[0088] The steps for extracting bacterial colonies from the surface of fish tissue during the initial fermentation cycle are as follows:

[0089] Step 1: Take the mucosal layer of the abdominal cavity of the mandarin fish and the fish tissue close to it (about 5g)

[0090] Step 2: Wash the fish tissue with 0.85% sterile saline for 3 times to remove surface salt and free impurities;

[0091] Step 3: The fish tissue was ground into granules and then added to 20 mL of distilled water to obtain a fish slurry;

[0092] Step 4: Take 1 ml of fish slurry and smear it on a solid plate of enriched culture medium A (culture medium A is solidified by adding agar)

[0093] Step 5: Incubate at 30°C under anaerobic conditions for 24 hours and pick milky white, smooth, raised, circular colonies with a diameter of 1–2 mm.

[0094] Step 6: Inoculate the colony onto an MRS slant (stored at 4°C for 1 month).

[0095] 4.2 Extraction and cultivation of fermentation colonies during the mid-fermentation cycle

[0096] After the total fermentation time of mandarin fish reaches 6 days (i.e. 144 hours), samples are taken every 6 hours to test the ethyl acetate concentration of the fish meat (detected by gas chromatography). When the difference between two adjacent ethyl acetate concentrations is ≤5%, it means that the mid-term fermentation cycle is over and the extraction step of the surface colonies of the fish meat tissue in the mid-term fermentation cycle can be carried out.

[0097] The calculation formula is as follows:

[0098] in 、 These are the values ​​of two adjacent measurements.

[0099] The fish meat was pretreated before gas chromatography detection as follows:

[0100] Take 3–5 g of fish tissue; add pre-cooled ultrapure water (e.g., 5 g of fish + 25 ml of water) at a ratio of 1:5 (fish mass: ultrapure water volume); centrifuge for 15 minutes (4°C); then let it stand, collect the supernatant, and filter it; the filtered supernatant is detected by gas chromatography.

[0101] The steps for extracting bacterial colonies from the surface of fish tissue during the mid-fermentation period are as follows:

[0102] Step 1: Take the mucosal layer of the abdominal cavity of the mandarin fish and the fish tissue close to it (about 5g)

[0103] Step 2: Wash the fish tissue with 0.85% sterile saline for 3 times to remove surface salt and free impurities;

[0104] Step 3: The fish tissue was crushed into granules and then added to 20 mL of distilled water to obtain a fish slurry;

[0105] Step 4: Take 1 ml of the fish slurry and spread it on a solid plate of enriched culture medium B (culture medium B is solidified by adding agar to form a solid medium, containing 2% agar);

[0106] Step 5: Incubate the culture medium aerobically at 30°C for 48 hours and pick milky white colonies with a diameter of 2–4 mm and smooth edges.

[0107] Step 5: Inoculate the colony onto an MRS slant (stored at 4°C for 1 month).

[0108] 4.3 Extraction and cultivation of fermentation colonies in the late fermentation cycle

[0109] Sampling Timing and Criteria: After a total fermentation time of 14 days (192 hours), gill and peritoneal mucus samples (approximately 3g) should be collected and tested every 6 hours. The core metric for testing is indole concentration, which is analyzed by gas chromatography. A change in indole concentration of ≤5% between two consecutive tests (12 hours apart) indicates that the late fermentation cycle has reached its end point, and the extraction and culture of fermentation colonies within this period can be performed.

[0110] The calculation formula is as follows:

[0111] in 、 These are the values ​​of two adjacent measurements.

[0112] Before indole detection, samples underwent the following pretreatment steps:

[0113] Gill and peritoneal mucus samples (approximately 3 g) were collected, avoiding contact with metal tools to prevent indole oxidation. Grind to a particle size ≤ 0.5 mm and add a 1:10 (fish mass:phosphate buffer) volume of pre-chilled phosphate buffer (PBS, pH 7.0) to create a homogenate. Centrifuge at 4°C for 20 minutes and filter the supernatant. Extract twice with equal volumes of dichloromethane, combining the organic phases.

[0114] Anhydrous sodium sulfate was added for dehydration, and the mixture was concentrated to dryness under reduced pressure at 40°C, and re-dissolved in 1 mL of methanol.

[0115] The steps for extracting bacterial colonies from the surface of fish tissue in the late fermentation cycle are as follows:

[0116] Step 1: Take the residual mucus tissue (about 3g) from the gills and abdominal cavity of the mandarin fish and place it in a sterile sampling tube.

[0117] Step 2: Add 10 mL of 0.85% sterile saline, vortex for 5 minutes, and repeat 3 times.

[0118] Step 3: After the fish tissue is pulverized, add 20 mL of pre-cooled sterile PBS buffer (pH 7.0) and process in a homogenizer for 2 minutes (keep in an ice bath to prevent overheating).

[0119] Step 4: Take 1 mL of the slurry and spread it on a solid plate rich in culture medium C (containing 2% agar).

[0120] Step 5: Incubate at 30°C under aerobic conditions for 72 h.

[0121] Step 6: Pick a colony and inoculate it on LB slant or TSB slant (stored frozen at 4°C).

[0122] 2. Application of the extracted bacterial colonies in subsequent batches of mandarin fish fermentation

[0123] Step 1: Salt the mandarin fish according to the original process (20-30g salt / 500g fish); take the colonies extracted during the initial fermentation period and resuspend them in 0.85% sterile saline to .

[0124] Step 2: Spray the bacterial suspension from the initial fermentation period in step 1 evenly on the abdominal cavity and body surface of the mandarin fish (dosage: 5 mL / 500 g fish)

[0125] Step 3: Place the mandarin fish in a fermentation tank and seal it with a heavy object;

[0126] Step 4: After the total fermentation time reaches 48 hours, open the lid and wash the mandarin fish with salt water.

[0127] Step 5: Take the colonies extracted in the mid-fermentation period and resuspend them in 1% trehalose solution. ; Spray the bacterial suspension on the abdominal cavity of the fish and the surface of the fish body after turning it over (3mL / 500g fish), and then press it with a heavy object to seal it for fermentation.

[0128] Step 6: After the total fermentation time reaches 7 days (168h), spray the fish with 0.05% lysozyme solution (2mL / 500g fish); take the colonies extracted in the late fermentation cycle and resuspend them in PBS containing 0.1% tryptophan. Obtain bacterial suspension.

[0129] Step 7: Inject the bacterial suspension in step 6 into the fish abdominal cavity (1 mL / 500 g fish), seal and ferment for 10 days.

[0130] As described above, the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for extracting stinky mandarin fish fermentation bacteria, characterized in that: According to the fermentation cycle of mandarin fish, the extraction of bacterial colonies was carried out in three stages; (1) Extraction of initial colonies: After fermentation, the pH of the fish tissue was monitored in real time. When the pH dropped to 4.5-5.0, the initial fermentation stage was completed. The abdominal mucosal layer and fish tissue were washed with physiological saline and crushed to form a slurry. The slurry was spread on a solid plate rich in culture medium and cultured anaerobically at 30°C for 24 hours. Milky white, smooth, raised, and 1-2 mm in diameter circular colonies were picked and inoculated on an MRS slant and refrigerated at 4°C. (2) Extraction of mid-term colonies: After the initial fermentation stage, the ethyl acetate concentration in the mandarin fish meat tissue was monitored in real time. When the concentration change rate between two consecutive times was ≤5%, the mid-term fermentation stage was completed. The fish meat tissue was washed with physiological saline and crushed to form a slurry. The slurry was spread on a solid plate rich in culture medium and cultured aerobically at 30°C for 48 hours. Milky white colonies with a diameter of 2–4 mm and smooth edges were picked and inoculated on an MRS slant and refrigerated at 4°C. The formula for calculating the rate of change is as follows: in 、 The values ​​of two adjacent measurements (3) Late colony extraction: After the mid-term fermentation stage, the indole concentration in the flesh of mandarin fish was monitored in real time. When the concentration change rate between two consecutive times was ≤5%, 3 g of gill and abdominal mucus was collected and homogenized with PBS buffer. The slurry was spread on a solid plate rich in culture medium and cultured aerobically at 30°C for 72 hours. The target colonies were picked and inoculated on LB slant or TSB slant; and refrigerated at 4°C. The formula for calculating the rate of change is as follows: in 、 These are the values ​​of two adjacent measurements.

2. The method for extracting a stinky mandarin fish fermentation colony from the energy storage cabinet according to claim 1, characterized in that: The culture medium used for initial colony extraction is culture medium A, and its specific preparation method is as follows: Step 1: Weigh 5 g glucose, 1 g peptone, 0.9 g NaCl, 0.1 g potassium sorbate, and 0.6 g calcium carbonate powder; Step 2: Prepare dilute hydrochloric acid solution: Take 16.7 ml of concentrated hydrochloric acid (36%) and add distilled water to make up to 100 ml; Step 3: Dissolve NaCl in 100 ml of distilled water, add glucose and peptone to the NaCl solution, and stir until completely dissolved; add potassium sorbate and continue stirring for 5 minutes to mix evenly; Step 4: Use dilute hydrochloric acid solution to adjust the pH of the NaCl solution to 6.0-6.5; then add 0.6g of CaCO3 powder and stir for ten minutes to form a suspension; Step 5: The suspension is sterilized to obtain culture medium A.

3. The method for extracting stinky mandarin fish fermented bacteria colonies according to claim 1, wherein: The culture medium used for mid-term colony extraction is culture medium B, and its specific preparation method is as follows: Step 1: Weigh 3 g glucose, 2 g yeast extract, 1.5 ml ethanol, sodium acetate, 0.8 g Na2HPO4·12H2O, 0.2 g KH2PO4, 0.05 g MgSO4·7H2O, and 0.05 g potassium sorbate; Step 2: Take 80ml of distilled water, add 0.8g Na2HPO4·12H2O, 0.2g KH2PO4, and 0.05g MgSO4·7H2O; stir until completely dissolved; Step 3: Add 3.0g glucose, 2.0g yeast extract, and 0.5g sodium acetate and continue stirring until completely dissolved; Step 4: Add 1.5 ml of ethanol and 0.05 g of potassium sorbate and stir to dissolve; Step 5: Add distilled water to 100 ml and measure the pH value, with a target pH of 6.0–6.

5. If the pH is less than 6.0, add 0.1 mol / L NaOH solution to adjust the pH. If the pH is greater than 6.5, add 0.1 mol / L HCl solution to adjust the pH. Step 6: The above solution is sterilized to obtain culture medium B.

4. The method for extracting stinky mandarin fish fermented bacteria colonies according to claim 1, wherein: The culture medium used for colony extraction in the later stage is culture medium C, and its specific preparation method is as follows: Step 1: Weigh 4.0 g peptone, 1.0 g yeast extract, 0.3 g tryptophan, 0.2 g cysteine·HCl, 1.5 g NaCl, 0.6 g Tris base, 0.03 g MgSO4·7H2O, and 0.02 g potassium sorbate. Step 2: Take 80 ml of distilled water, add 0.6 g of Tris base, 1.5 g of NaCl, and 0.03 g of MgSO4·7H2O, and stir to dissolve; Step 3: Add 4.0 g peptone, 1.0 g yeast extract, and 0.3 g tryptophan, and stir in a 60°C water bath for 15 minutes; Step 4: Add 0.2 g cysteine·HCl and 0.02 g potassium sorbate and stir for 5 minutes until dissolved; Step 5: Dilute to 100 ml with distilled water, target pH 7.2–7.5; if pH < 7.2, add 0.1 mol / L Tris base solution; if pH > 7.8, add 0.1 mol / L HCl solution; Step 6: After the solution is sterilized for 15 min, culture medium C is obtained.

5. The method for extracting stinky mandarin fish fermented bacteria colonies according to claim 1, wherein: After fermenting for 36 hours, the fish tissue from the abdominal cavity was collected every 2-3 hours for pH testing. The pH testing method for mandarin fish is as follows: 3-5g of fish tissue from the abdominal cavity was collected and crushed. After crushing, pre-cooled sterile distilled water (distilled water temperature below 5°C) was added at a ratio of 1:9 (mass:volume). The mixture was centrifuged and stirred for 5 minutes. After standing, the supernatant was collected for pH measurement. A calibrated pH meter electrode was inserted into the supernatant and the reading was recorded after it stabilized. The pH value was calculated by repeating the measurement three times if the deviation was greater than 0.

2.

6. The method for extracting stinky mandarin fish fermented bacteria colonies according to claim 1, wherein: After the total fermentation time of mandarin fish reached 144 h, samples were taken every 6 h to detect the ethyl acetate concentration in the fish meat.

7. The method for extracting stinky mandarin fish fermented bacterial colonies according to claim 6, wherein: The ethyl acetate content in fish meat was determined using gas chromatography. The fish meat was pretreated as follows before testing: 3–5 g of fish tissue was collected; pre-cooled ultrapure water (e.g., 5 g of fish + 25 ml of water) was added at a ratio of 1:5 (fish mass: ultrapure water volume); the fish meat was centrifuged for 15 minutes (4°C); the fish meat was allowed to stand, and the supernatant was collected and filtered. The filtered supernatant was then tested by gas chromatography.

8. The method for extracting stinky mandarin fish fermented bacteria colonies according to claim 1, wherein: After the total fermentation time of mandarin fish reached 192 h, samples were taken every 6 h to detect the indole content in the gills and abdominal mucus.

9. The method for extracting stinky mandarin fish fermented bacterial colonies according to claim 8, wherein: The ethyl acetate content in fish meat was determined using gas chromatography. Before indole analysis, the samples underwent the following pretreatment steps: Gill and peritoneal mucus samples (approximately 3 g) were collected, avoiding contact with metal tools to prevent indole oxidation. Grind to a particle size ≤ 0.5 mm, add 1:10 (fish mass: phosphate buffer volume) volume of pre-chilled phosphate buffer (PBS, pH 7.0) to obtain a homogenate. Centrifuge at 4°C for 20 min, filter to obtain the supernatant, extract twice with an equal volume of dichloromethane, and combine the organic phases. Dehydrate with anhydrous sodium sulfate, concentrate to dryness under reduced pressure at 40°C, and reconstitute with 1 mL of methanol.