Staphylococcus carnosus color development strengthening method based on exogenous additive regulation and application of staphylococcus carnosus color development strengthening method in salted dried fish

Abstract

The invention discloses a staphylococcus carnosus chromophore strengthening method based on regulation and control of exogenous additives and application of the staphylococcus carnosus chromophore strengthening method in salting dried fish. The staphylococcus carnosus chromophore strengthening method comprises the following steps: mixing a methemoglobin solution with broth, adding the exogenous additives, and constructing a simulation system; inoculating staphylococcus carnosus into the simulation system for culturing, and observing the color change of the simulation system; wherein the exogenous additive comprises one or more of L-arginine, tea polyphenol, ascorbic acid or tocopherol. One or more of L-arginine, tea polyphenols, ascorbic acid or tocopherol are used as exogenous additives for synergistic fermentation with staphylococcus carnosus, so that the redness value of the salted dried fish is remarkably increased.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic product processing, and particularly relates to a method for enhancing the color development of Staphylococcus carnosus based on the regulation of exogenous additives and an application of the method in salted and dried fish. Background Art

[0002] Salted and dried fish, a traditional aquatic product with typical Chinese characteristics, is highly favored by consumers for its unique flavor and storability. Color is a key indicator of product quality and market value. Traditional processes often rely on nitrite for color development to impart an attractive bright red color to the product, but the resulting food safety risks need to be addressed urgently. Microbial fermentation color development technology, as a green and safe alternative to nitrite, has attracted considerable attention in the meat processing industry. Research has shown that certain microorganisms can convert L-arginine into nitric oxide (NO) via the nitric oxide synthase (NOS) pathway. NO then binds to myoglobin to form nitrosylmyoglobin, a vibrant red substance that imparts a stable bright red color to the product. However, the ability of microbial NOS to produce nitrosylmyoglobin is limited, and NOS activity in microorganisms is generally weak, resulting in a weaker color development effect than nitrite.

[0003] In the published literature reports, it was found that the patent application with publication number CN114568644A disclosed a processing method for promoting the improvement of the red color of fermented sausages. The method uses low-intensity high hydrostatic pressure treatment to increase the expression of nitric oxide synthase in coagulase-negative Staphylococcus, thereby promoting the production of nitrosylmyoglobin in fermented sausages and improving the color of fermented sausages; the patent application with publication number CN115232830A disclosed a meat coloring agent based on recombinant bacterial nitric oxide synthase, a method and its application. The method uses recombinant expression technology to express nitric oxide synthase in a large amount in an edible-grade Bacillus subtilis expression system, and the recombinant bacterial nitric oxide synthase and coagulase-negative Staphylococcus are jointly applied to fermented sausages, obtaining a coloring effect equivalent to that of adding sodium nitrite; the patent application with publication number CN115232830A disclosed a meat coloring agent based on recombinant bacterial nitric oxide synthase, a method and its application. Patent application No. 18252232A discloses a colorant based on a nitric oxide-producing fusion enzyme and its application. This method combines nitric oxide synthase, flavoprotein, and flavoprotein reductase in sequence, two by two, through a connecting peptide to prepare a fusion enzyme. The fusion enzyme has high enzymatic activity, can catalyze the production of a large amount of nitric oxide, and can effectively combine with myoglobin in meat products to produce nitrosylmyoglobin, effectively improving the red color of the meat products and obtaining a coloring effect equivalent to that of sodium nitrite; Patent application No. CN118109367A discloses a hydrogen peroxide-treated veal Staphylococcus aureus colorant and its preparation method and application. This method uses low-intensity hydrogen peroxide to treat veal Staphylococcus aureus to increase the nitric oxide synthase activity of the veal Staphylococcus aureus, and then inoculates it into the meat product to be colored to achieve a better coloring effect.

[0004] Although these methods can, to a certain extent, enhance nitric oxide synthase activity in microorganisms, thereby enhancing their color development capabilities, they are currently limited by high production costs and limited scalable processing capabilities, making them difficult to meet the actual needs of industrial production. Therefore, it is necessary to develop cost-effective, easily scalable, and food-grade microbial color enhancement technologies to overcome the multiple limitations of existing processes in terms of effectiveness, cost, and operability. Summary of the Invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for enhancing the color development of Staphylococcus carnosus based on the regulation of exogenous additives.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for enhancing the color development of Staphylococcus carnosus based on the regulation of exogenous additives, comprising:

[0009] The methemoglobin solution was mixed with broth and exogenous additives were added to construct a simulation system;

[0010] Inoculate Staphylococcus carnosus into the simulation system and culture it, and observe the color change of the simulation system;

[0011] Wherein, the exogenous additives include one or more of L-arginine, tea polyphenols, ascorbic acid or tocopherol.

[0012] As a preferred embodiment of the method of the present invention, the content of L-arginine in the simulation system is 40 to 60 mmol / L.

[0013] As a preferred embodiment of the method of the present invention, the content of L-arginine in the simulation system is 40 to 60 mmol / L.

[0014] As a preferred embodiment of the method of the present invention, the content of tea polyphenols in the simulation system is 200-400 μmol / L.

[0015] As a preferred embodiment of the method of the present invention, the content of ascorbic acid in the simulation system is 200-400 μmol / L.

[0016] As a preferred embodiment of the method of the present invention, the content of tocopherol in the simulation system is 100-300 μmol / L.

[0017] As a preferred embodiment of the method of the present invention, the concentration of methemoglobin in the simulation system is 2-4 mg / mL, the inoculation amount of Staphylococcus carnosus is 10 5 ~10 7 CFU / mL.

[0018] As a preferred embodiment of the method of the present invention, the culture temperature of the simulation system is 35-40° C., and the culture time is 24 hours.

[0019] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for coloring salted and dried fish, comprising:

[0020] mixing the fish pieces with blood residues with salt, glucose and exogenous additives and curing the pieces to obtain cured fish pieces;

[0021] The pickled fish blocks are inoculated with bacteria and then fermented at a constant temperature, and the fermented fish is dried to obtain a pickled dried fish product;

[0022] Wherein, the exogenous additives include one or more of L-arginine, tea polyphenols, ascorbic acid or tocopherol;

[0023] The amount of the exogenous additive added is 50 mmol / kg to 300 μmol / kg relative to the mass of the fish block.

[0024] As a preferred embodiment of the method of the present invention, the mixed pickling time is 2 to 4 days and the temperature is 1 to 5°C;

[0025] The constant temperature fermentation temperature is 20°C and the fermentation time is 3 days;

[0026] The added amount of L-arginine is 50 mmol / kg, the added amount of tea polyphenols is 300 μmol / kg, the added amount of ascorbic acid is 300 μmol / kg, and the added amount of tocopherol is 200 μmol / kg.

[0027] Beneficial effects of the present invention:

[0028] (1) In the present invention, one or more of L-arginine, tea polyphenols, ascorbic acid or tocopherol are mixed and cultured with Staphylococcus carnosus as exogenous additives in a simulation system, which significantly improves the nitric oxide synthase activity in Staphylococcus carnosus (46.36% to 84.09%), thereby enhancing the color development ability of Staphylococcus carnosus, providing a feasible solution for enhancing the color development ability of Staphylococcus carnosus in salted and dried fish by regulating the activity of nitric oxide synthase.

[0029] (2) The present invention significantly improves the redness value of salted dried fish by using one or more of L-arginine, tea polyphenols, ascorbic acid or tocopherol as exogenous additives for synergistic fermentation with Staphylococcus carnosus. Compared with the fermentation method of inoculating Staphylococcus carnosus alone, the synergistic fermentation process increases the content of nitrosohemoglobin in salted dried fish by 19.91% to 48.08%, and the product presents an excellent red color. The reason is that the exogenous additives can effectively enhance the nitric oxide synthase activity of Staphylococcus carnosus, thereby compensating for the technical problem of poor color development caused by insufficient natural nitric oxide synthase activity of the strain.

[0030] (3) The present invention provides a new color development process based on the non-bleeding slaughtering method and combined with the synergistic effect of Staphylococcus aureus fermentation and exogenous additives. Compared with the traditional nitrite color development process, the new color development process reduces the residual nitrite in the salted and dried fish (87.16% to 91.88%) while maintaining the excellent red color of the salted and dried fish, overcoming the food safety hazard caused by the excessively high residual nitrite in the traditional process, and achieving a dual improvement in the color quality and food safety of the salted and dried fish products; the new color development process replaces chemical additives by biological fermentation, providing a green, safe and efficient color development technology solution for salted and dried fish products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0032] Figure 1 This is the effect of L-arginine concentration on the redness value of the simulation system;

[0033] Figure 2 This is the effect of tea polyphenols concentration on the redness value of the simulation system;

[0034] Figure 3 is the effect of ascorbic acid concentration on the redness value of the simulation system;

[0035] Figure 4 This is the effect of tocopherol concentration on the redness value of the simulation system;

[0036] Figure 5 This is a picture of the salted and dried fish product to which L-arginine is added in Example 15;

[0037] Figure 6 This is a picture of the salted and dried fish product with added tea polyphenols in Example 16;

[0038] Figure 7 This is a diagram of the salted and dried fish product to which ascorbic acid is added in Example 17;

[0039] Figure 8 This is a picture of the salted and dried fish product with added tocopherol in Example 18;

[0040] Figure 9 This is a picture of the salted and dried fish product to which compound L-arginine and tocopherol are added in Example 19;

[0041] Figure 10 This is a diagram of the salted and dried fish product to which composite L-arginine, ascorbic acid, and tocopherol are added in Example 20;

[0042] Figure 11 This is a picture of the bloodletting and pickling control product in Comparative Example 2;

[0043] Figure 12 This is a picture of the salted and dried fish product in Comparative Example 3 with 0.15g / kg nitrite added;

[0044] Figure 13 This is a picture of the salted and dried fish product fermented by inoculating only Staphylococcus carnosus in Comparative Example 4;

[0045] Figure 14 This is a diagram showing the effect of different exogenous additives on the redness value of the simulation system;

[0046] Figure 15 This is a diagram showing the effects of different exogenous additives on the UV-visible absorption spectrum of the simulation system;

[0047] Figure 16 This figure shows the effects of different exogenous additives on the activity of nitric oxide synthase in the simulation system. DETAILED DESCRIPTION

[0048] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0049] Unless otherwise specified, the raw materials used in the present invention are all commercially available.

[0050] The Staphylococcus carnosus used in the present invention is from Shanghai Collection Biotechnology Center, with the collection number being ATCC 51365.

[0051] The total nitric oxide synthase assay kit was purchased from Nanjing Jiancheng Bioengineering Institute and used to determine the activity of nitric oxide synthase.

[0052] Hemoglobin and sodium phosphate were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0053] The residual nitrite content in salted and dried fish products was determined with reference to the national standard GB 5009.33-2016.

[0054] The UV-visible absorption spectra of the materials prepared in the present invention were measured as follows:

[0055] The model solution was appropriately diluted and scanned using a UV-visible spectrophotometer in the range of 450 nm to 650 nm at intervals of 1 nm.

[0056] The material prepared in the embodiment of the present invention was subjected to the determination of nitrosohemoglobin content according to the following method:

[0057] Take 5g of fish meat in a centrifuge tube, add 21.5mL of extract (acetone: water volume ratio = 40:3), homogenize at 10000r / min for 1min, then stand at 4℃ in the dark for 1h, take out, centrifuge at 3000r / min for 10min, take the supernatant and filter, and measure the absorbance of the filtrate at 540nm. 540 ;

[0058] Nitrosohemoglobin (mg / kg) = A 540 ×290.

[0059] The materials prepared in the present invention were subjected to nitric oxide synthase activity assay as follows:

[0060] The simulated system solution was centrifuged (10,000 rpm for 5 minutes), and the precipitate was resuspended in sodium phosphate buffer (20 mmol / L, pH 7.2). The cells were disrupted on ice using an ultrasonic disruptor for 10 minutes, with a duration of 2 seconds and an interval of 2 seconds. After centrifugation, the supernatant was collected as the crude enzyme solution. The NOS enzyme activity was determined using a total nitric oxide synthase assay kit, with 1 nmol of nitric oxide produced per minute per milliliter of bacteria being defined as one unit of activity.

[0061] Example 1

[0062] This embodiment provides a simulation system construction method, specifically:

[0063] (1) Preparation of methemoglobin (MetHb) solution: A 10 mg / mL hemoglobin solution was prepared using 50 mmol / L sodium phosphate buffer (pH 6.0), heated at 50°C for 30 min, and centrifuged (4°C, 10,000 rpm, 5 min) to obtain the supernatant.

[0064] The supernatant was sterilized by filtration through a 0.45 μm water membrane and stored at 4°C until use.

[0065] (2) Construction of simulation system: MetHb solution was added to sterile NB broth to a final concentration of 3 mg / mL, and L-arginine was added to a final concentration of 50 mmol / L to obtain a simulation system.

[0066] (3) Inoculation: Staphylococcus carnosus was inoculated into the simulation system to a final cell concentration of 10 6 CFU / mL.

[0067] (4) Cultivation: The simulation system was cultured at 37°C for 24 h.

[0068] Example 2

[0069] The difference between this embodiment and embodiment 1 is that:

[0070] The concentration of L-arginine in step (2) was adjusted to 40 mmol / L.

[0071] Example 3

[0072] The difference between this embodiment and embodiment 2 is that:

[0073] The concentration of L-arginine in step (2) was adjusted to 60 mmol / L.

[0074] The redness a* value of the simulation system constructed in Examples 1 to 3 was measured, and the results were as follows: Figure 1It can be seen that the redness value of Example 1 with an L-arginine concentration of 50 mmol / L in the simulation system is 23.94, which is higher than that of Example 2 with an L-arginine concentration of 40 mmol / L and Example 3 with an L-arginine concentration of 60 mmol / L.

[0075] Therefore, based on the consideration of the color enhancement effect of L-arginine, the best technical effect can be obtained when the L-arginine concentration used in the present invention is 50mmol / L.

[0076] Example 4

[0077] (1) Preparation of methemoglobin (MetHb) solution: A 10 mg / mL hemoglobin solution was prepared using 50 mmol / L sodium phosphate buffer (pH 6.0), heated at 50°C for 30 min, and centrifuged (4°C, 10,000 rpm, 5 min) to obtain the supernatant.

[0078] The supernatant was sterilized by filtration through a 0.45 μm water membrane and stored at 4°C until use.

[0079] (2) Construction of simulation system: MetHb solution was added to sterile NB broth to a final concentration of 3 mg / mL, and tea polyphenols were added to a final concentration of 300 μmol / L to obtain a simulation system.

[0080] (3) Inoculation: Staphylococcus carnosus was inoculated into the simulation system to a final cell concentration of 10 6 CFU / mL.

[0081] (4) Cultivation: The simulation system was cultured at 37°C for 24 h.

[0082] Example 5

[0083] The difference between this embodiment and embodiment 4 is that the concentration of tea polyphenols in step (2) is adjusted to 200 μmol / L.

[0084] Example 6

[0085] The difference between this embodiment and embodiment 2 is that the concentration of tea polyphenols in step (2) is adjusted to 400 μmol / L.

[0086] The redness a* value of the simulation system constructed in Examples 4 to 6 was measured, and the results were as follows: Figure 2As shown in the figure, it can be seen that in the simulation system, the redness values ​​of Example 4, where the tea polyphenol concentration was 300 μmol / L, and Example 6, where the tea polyphenol concentration was 400 μmol / L, were 22.74 and 22.56, respectively, which were higher than those of Example 5, where the tea polyphenol concentration was 200 μmol / L. Therefore, based on a comprehensive consideration of the color enhancement effect of tea polyphenols and application costs, the best technical effect can be achieved when the tea polyphenol concentration used in the present invention is 300 μmol / L.

[0087] Example 7

[0088] (1) Preparation of methemoglobin (MetHb) solution: A 10 mg / mL hemoglobin solution was prepared using 50 mmol / L sodium phosphate buffer (pH 6.0), heated at 50°C for 30 min, and centrifuged (4°C, 10,000 rpm, 5 min) to obtain the supernatant.

[0089] The supernatant was sterilized by filtration through a 0.45 μm water membrane and stored at 4°C until use.

[0090] (2) Construction of simulation system: MetHb solution was added to sterile NB broth to a final concentration of 3 mg / mL, and ascorbic acid was added to a final concentration of 300 μmol / L to obtain a simulation system.

[0091] (3) Inoculation: Staphylococcus carnosus was inoculated into the simulation system to a final cell concentration of 10 6 CFU / mL.

[0092] (4) Cultivation: The simulation system was cultured at 37°C for 24 h.

[0093] Example 8

[0094] The difference between this embodiment and embodiment 7 is that the concentration of ascorbic acid in step (2) is adjusted to 200 μmol / L.

[0095] Example 9

[0096] The difference between this embodiment and embodiment 2 is that the concentration of ascorbic acid in step (2) is adjusted to 400 μmol / L.

[0097] The redness a* value of the simulation system constructed in Examples 7 to 9 was measured, and the results were as follows: Figure 3As shown in the figure, it can be seen that the redness value of Example 7, in which the ascorbic acid concentration in the simulation system was 300 μmol / L, was 23.83, which is higher than that of Example 8, in which the ascorbic acid concentration was 200 μmol / L, and Example 9, in which the ascorbic acid concentration was 400 μmol / L. Therefore, based on the consideration of the color enhancement effect of ascorbic acid, the best technical effect can be achieved when the ascorbic acid concentration used in the present invention is 300 μmol / L.

[0098] Example 10

[0099] (1) Preparation of methemoglobin (MetHb) solution: A 10 mg / mL hemoglobin solution was prepared using 50 mmol / L sodium phosphate buffer (pH 6.0), heated at 50°C for 30 min, and centrifuged (4°C, 10,000 rpm, 5 min) to obtain the supernatant.

[0100] The supernatant was sterilized by filtration through a 0.45 μm water membrane and stored at 4°C until use.

[0101] (2) Construction of simulation system: MetHb solution was added to sterile NB broth to a final concentration of 3 mg / mL, and tocopherol was added to a final concentration of 200 μmol / L to obtain a simulation system.

[0102] (3) Inoculation: Staphylococcus carnosus was inoculated into the simulation system to a final cell concentration of 10 6 CFU / mL.

[0103] (4) Cultivation: The simulation system was cultured at 37°C for 24 h.

[0104] Example 11

[0105] The difference between this embodiment and embodiment 7 is that the concentration of tocopherol in step (2) is adjusted to 100 μmol / L.

[0106] Example 12

[0107] The difference between this embodiment and embodiment 2 is that the concentration of tocopherol in step (2) is adjusted to 300 μmol / L.

[0108] The redness a* value of the simulation system constructed in Examples 10 to 12 was measured, and the results were as follows: Figure 4 As shown in the figure, it can be seen that the redness value of Example 10, in which the tocopherol concentration in the simulation system was 200 μmol / L, was 23.96, which was higher than that of Example 11, in which the tocopherol concentration was 100 μmol / L, and Example 12, in which the tocopherol concentration was 300 μmol / L. Therefore, based on the consideration of the color enhancement effect of tocopherol, the best technical effect can be achieved when the tocopherol concentration used in the present invention is 200 μmol / L.

[0109] Example 13

[0110] (1) Preparation of methemoglobin (MetHb) solution: A 10 mg / mL hemoglobin solution was prepared using 50 mmol / L sodium phosphate buffer (pH 6.0). The solution was heated at 50°C for 30 min and then centrifuged (4°C, 10,000 rpm, 5 min) to obtain a supernatant. The supernatant was sterilized by filtration through a 0.45 μm water filter and stored at 4°C until use.

[0111] (2) Construction of simulation system: MetHb solution was added to sterile NB broth to a final concentration of 3 mg / mL, and 25 mmol / L L-arginine + 100 μmol / L tocopherol were added to obtain a simulation system.

[0112] (3) Inoculation: Staphylococcus carnosus was inoculated into the simulation system to a final cell concentration of 10 6 CFU / mL.

[0113] (4) Cultivation: The simulation system was cultured at 37°C for 24 h.

[0114] Example 14

[0115] (1) Preparation of methemoglobin (MetHb) solution: A 10 mg / mL hemoglobin solution was prepared using 50 mmol / L sodium phosphate buffer (pH 6.0), heated at 50°C for 30 min, and centrifuged (4°C, 10,000 rpm, 5 min) to obtain the supernatant.

[0116] The supernatant was sterilized by filtration through a 0.45 μm water membrane and stored at 4°C until use.

[0117] (2) Construction of simulation system: MetHb solution was added to sterile NB broth to a final concentration of 3 mg / mL, and 16.7 mmol / L L-arginine + 100 μmol / L ascorbic acid + 66.7 μmol / L tocopherol were added to obtain a simulation system.

[0118] (3) Inoculation: Staphylococcus carnosus was inoculated into the simulation system to a final cell concentration of 10 6 CFU / mL.

[0119] (4) Cultivation: The simulation system was cultured at 37°C for 24 h.

[0120] Example 15

[0121] (1) Live fish slaughter: Fresh grass carp of about 2 kg were slaughtered by decapitation, scaled and eviscerated without bleeding, cleaned and cut into 6 cm × 4 cm × 4 cm pieces;

[0122] (2) Curing: Mix 60 g / kg of table salt, 10 g / kg of glucose, and 50 mmol / kg of L-arginine (based on the weight of the fish pieces) and rub it all over the fish body. Place the fish in a container with one layer of salt and marinate at 4°C for 3 days.

[0123] (3) Inoculation: The culture of Staphylococcus carnosus was centrifuged, the bacterial sludge was washed three times with sterile water and then resuspended to obtain a concentration of 10 7 CFU / mL of microbial fermentation culture;

[0124] The pickled fish pieces in step (2) are inoculated with bacteria, and the fish pieces are soaked in the fermentation agent at room temperature (20-25° C.) for 30 minutes, and then taken out and drained;

[0125] (4) Fermentation: The inoculated fish pieces were placed in a constant temperature of 20°C for fermentation for 3 days;

[0126] (5) Drying: The fermented fish pieces were dried at 43°C for 16 h;

[0127] (6) Packaging: After drying, the fish pieces are vacuum packed to obtain salted and dried fish.

[0128] See the product diagram in this embodiment. Figure 5 .

[0129] Example 16

[0130] (1) Live fish slaughter: Fresh grass carp of about 2 kg were slaughtered by decapitation, scaled and eviscerated without bleeding, cleaned and cut into 6 cm × 4 cm × 4 cm pieces;

[0131] (2) Curing: Mix 60 g / kg of table salt, 10 g / kg of glucose, and 300 μmol / kg of tea polyphenols (based on the weight of the fish) and rub them all over the fish body. Place the fish in a container with one layer of salt and marinate at 4°C for 3 days.

[0132] (3) Inoculation: The culture of Staphylococcus carnosus was centrifuged, the bacterial sludge was washed three times with sterile water and then resuspended to obtain a concentration of 10 7 CFU / mL of microbial fermentation agent. The marinated fish pieces in step (2) are inoculated with bacteria, and the fish pieces are soaked in the fermentation agent at room temperature (20-25°C) for 30 minutes, then removed and drained;

[0133] (4) Fermentation: The inoculated fish pieces were placed in a constant temperature of 20°C for fermentation for 3 days;

[0134] (5) Drying: The fermented fish pieces were dried at 43°C for 16 h;

[0135] (6) Packaging: After drying, the fish pieces are vacuum packed to obtain salted and dried fish. Figure 6 .

[0136] Example 17

[0137] (1) Live fish slaughter: Fresh grass carp of about 2 kg were slaughtered by decapitation, scaled and eviscerated without bleeding, cleaned and cut into 6 cm × 4 cm × 4 cm pieces;

[0138] (2) Curing: Mix 60 g / kg of table salt, 10 g / kg of glucose, and 300 μmol / kg of ascorbic acid (based on the weight of the fish pieces) and rub it all over the fish body. Place the fish in a container with one layer of salt and marinate at 4°C for 3 days.

[0139] (3) Inoculation: The culture of Staphylococcus carnosus was centrifuged, the bacterial sludge was washed three times with sterile water and then resuspended to obtain a concentration of 10 7 CFU / mL of microbial fermentation agent. The marinated fish pieces in step (2) are inoculated with bacteria, and the fish pieces are soaked in the fermentation agent at room temperature (20-25°C) for 30 minutes, then removed and drained;

[0140] (4) Fermentation: The inoculated fish pieces were placed in a constant temperature of 20°C for fermentation for 3 days;

[0141] (5) Drying: The fermented fish pieces were dried at 43°C for 16 h;

[0142] (6) Packaging: After drying, the fish pieces are vacuum packed to obtain salted and dried fish. Figure 7 .

[0143] Example 18

[0144] (1) Live fish slaughter: Fresh grass carp of about 2 kg were slaughtered by decapitation, scaled and eviscerated without bleeding, cleaned and cut into 6 cm × 4 cm × 4 cm pieces;

[0145] (2) Curing: Mix 60 g / kg of table salt, 10 g / kg of glucose, and 200 μmol / kg of tocopherol (based on the weight of the fish) and rub it all over the fish body. Place the fish in a container with one layer of salt and marinate at 4°C for 3 days.

[0146] (3) Inoculation: The culture of Staphylococcus carnosus was centrifuged, the bacterial sludge was washed three times with sterile water and then resuspended to obtain a concentration of 10 7CFU / mL of microbial fermentation agent. The marinated fish pieces in step (2) are inoculated with bacteria, and the fish pieces are soaked in the fermentation agent at room temperature (20-25°C) for 30 minutes, then removed and drained;

[0147] (4) Fermentation: The inoculated fish pieces were placed in a constant temperature of 20°C for fermentation for 3 days;

[0148] (5) Drying: The fermented fish pieces were dried at 43°C for 16 h;

[0149] (6) Packaging: After drying, the fish pieces are vacuum packed to obtain salted and dried fish. Figure 8 .

[0150] Example 19

[0151] (1) Live fish slaughter: Fresh grass carp of about 2 kg were slaughtered by decapitation, scaled and eviscerated without bleeding, cleaned and cut into 6 cm × 4 cm × 4 cm pieces;

[0152] (2) Curing: Mix 60 g / kg of table salt, 10 g / kg of glucose, and 25 mmol / kg of L-arginine + 100 μmol / kg of tocopherol (based on the weight of the fish pieces) and rub it all over the fish body. Place the fish in a container with one layer of salt on top of the other and marinate at 4°C for 3 days.

[0153] (3) Inoculation: The culture of Staphylococcus carnosus was centrifuged, the bacterial sludge was washed three times with sterile water and then resuspended to obtain a concentration of 10 7 CFU / mL of microbial fermentation agent. The marinated fish pieces in step (2) are inoculated with bacteria, and the fish pieces are soaked in the fermentation agent at room temperature (20-25°C) for 30 minutes, then removed and drained;

[0154] (4) Fermentation: The inoculated fish pieces were placed in a constant temperature of 20°C for fermentation for 3 days;

[0155] (5) Drying: The fermented fish pieces were dried at 43°C for 16 h;

[0156] (6) Packaging: After drying, the fish pieces are vacuum packed to obtain salted and dried fish. Figure 9 .

[0157] Example 20

[0158] (1) Live fish slaughter: Fresh grass carp of about 2 kg were slaughtered by decapitation, scaled and eviscerated without bleeding, cleaned and cut into 6 cm × 4 cm × 4 cm pieces;

[0159] (2) Curing: Mix 60 g / kg of table salt, 10 g / kg of glucose, and 16.7 mmol / kg of L-arginine + 100 μmol / kg of ascorbic acid + 66.7 μmol / kg of tocopherol (based on the weight of the fish pieces) and rub it all over the fish body. Place the mixture in a container with one layer of fish and one layer of salt, and curing at 4°C for 3 days.

[0160] (3) Inoculation: The culture of Staphylococcus carnosus was centrifuged, the bacterial sludge was washed three times with sterile water and then resuspended to obtain a concentration of 10 7 CFU / mL of microbial fermentation agent. The marinated fish pieces in step (2) are inoculated with bacteria, and the fish pieces are soaked in the fermentation agent at room temperature (20-25°C) for 30 minutes, then removed and drained;

[0161] (4) Fermentation: The inoculated fish pieces were placed in a constant temperature of 20°C for fermentation for 3 days;

[0162] (5) Drying: The fermented fish pieces were dried at 43°C for 16 h;

[0163] (6) Packaging: After drying, the fish pieces are vacuum packed to obtain salted and dried fish. Figure 10 .

[0164] Comparative Example 1

[0165] This comparative example differs from Example 1 in that L-arginine is not added, specifically:

[0166] (1) Preparation of methemoglobin (MetHb) solution: Prepare a 10 mg / mL hemoglobin solution using 50 mmol / L sodium phosphate buffer (pH 6.0). Heat the solution at 50°C for 30 min and centrifuge (4°C, 10,000 rpm, 5 min) to obtain a supernatant. Sterilize the supernatant by filtration through a 0.45 μm water filter and store at 4°C until use.

[0167] (2) Simulation system construction: MetHb solution was added to sterile NB broth to a final concentration of 3 mg / mL to obtain a simulation system;

[0168] (3) Inoculation: Staphylococcus carnosus was inoculated into the simulation system to a final cell concentration of 10 6 CFU / mL;

[0169] (4) Cultivation: The simulation system was cultured at 37°C for 24 h.

[0170] Comparative Example 2

[0171] This comparative example differs from Example 15 in that the live fish are slaughtered in a different manner in step (1), L-arginine is not added during the pickling process in step (2), and the inoculation and fermentation step is omitted. Specifically,

[0172] (1) Live fish slaughter: Fresh grass carp weighing about 2 kg were knocked unconscious and their tails removed. The fish were placed in ice water, with the water changed every 10 minutes until no obvious bleeding occurred. After descaling and evisceration, the fish were cut into 6 cm × 4 cm × 4 cm pieces.

[0173] (2) Curing: Mix 60 g / kg of table salt and 10 g / kg of glucose (based on the weight of the fish) and rub it all over the fish body. Place the fish in a container with one layer of salt on top of the other and marinate at 4°C for 6 days.

[0174] (3) Drying: Wipe off the surface moisture of the marinated fish pieces and dry them at 43°C for 16 h;

[0175] (4) Packaging: After drying, the fish pieces are vacuum packed to obtain salted and dried fish. Figure 11 .

[0176] Comparative Example 3

[0177] This comparative example differs from Example 15 in that the live fish are slaughtered in a different manner in step (1), sodium nitrite is added in the pickling process of step (2) but L-arginine is not added, and the inoculation and fermentation step is omitted. Specifically,

[0178] (1) Live fish slaughter: Fresh grass carp weighing about 2 kg were knocked unconscious and their tails removed. The fish were placed in ice water, with the water changed every 10 minutes until no obvious bleeding occurred. After descaling and evisceration, the fish were cut into 6 cm × 4 cm × 4 cm pieces.

[0179] (2) Curing: Mix 60 g / kg of table salt, 10 g / kg of glucose, and 0.15 g / kg of sodium nitrite (based on the weight of the fish pieces) and rub it all over the fish body. Place the fish in a container with one layer of salt and marinate at 4°C for 6 days.

[0180] (3) Drying: Wipe off the surface moisture of the marinated fish pieces and dry them at 43°C for 16 h;

[0181] (4) Packaging: After drying, the fish pieces are vacuum packed to obtain salted and dried fish. Figure 12 .

[0182] Comparative Example 4

[0183] This comparative example differs from Example 15 in that L-arginine is not added during the pickling process of step (2), specifically:

[0184] (1) Live fish slaughter: Fresh grass carp of about 2 kg were slaughtered by decapitation, scaled and eviscerated without bleeding, cleaned and cut into 6 cm × 4 cm × 4 cm pieces;

[0185] (2) Curing: Mix 60 g / kg of table salt and 10 g / kg of glucose (based on the weight of the fish) and rub it all over the fish body. Place the fish in a container with one layer of salt on top of the other and marinate at 4°C for 3 days.

[0186] (3) Inoculation: The culture of Staphylococcus carnosus was centrifuged, the bacterial sludge was washed three times with sterile water and then resuspended to obtain a concentration of 10 7 CFU / mL of microbial fermentation agent. The marinated fish pieces in step (2) are inoculated with bacteria, and the fish pieces are soaked in the fermentation agent at room temperature (20-25°C) for 30 minutes, then removed and drained;

[0187] (4) Fermentation: The inoculated fish pieces were placed in a constant temperature of 20°C for fermentation for 3 days;

[0188] (5) Drying: The fermented fish pieces were dried at 43°C for 16 h;

[0189] (6) Packaging: After drying, the fish pieces are vacuum packed to obtain salted and dried fish. Figure 13 .

[0190] The redness a* value of the simulated systems prepared in Examples 1, 4, 7, 10, 13, 14 and Comparative Example 1 was measured. The results are as follows: Figure 14 As shown. It can be seen that compared with the comparative example 1 in which only Staphylococcus carnosus was inoculated, the redness values ​​of Example 1 with the addition of 50 mmol / L L-arginine, Example 4 with the addition of 300 μmol / L tea polyphenols, Example 7 with the addition of 300 μmol / L ascorbic acid, and Example 10 with the addition of 200 μmol / L tocopherol increased significantly, reaching 23.94, 22.74, 23.83, and 23.96, respectively, indicating that L-arginine, tea polyphenols, ascorbic acid, and tocopherol can effectively promote the color development effect of Staphylococcus carnosus. In addition, Example 13, in which 25 mmol / L L-arginine + 100 μmol / L tocopherol were added, and Example 14, in which 16.7 mmol / L L-arginine + 100 μmol / L ascorbic acid + 66.7 μmol / L tocopherol were added, showed a significant synergistic effect, and their redness values ​​were increased to 24.65 and 24.51, respectively, which were higher than the redness values ​​when each exogenous additive was added alone, indicating that the compound combination of exogenous additives showed a better synergistic color enhancement effect.

[0191] The UV-visible absorption spectra of the simulated systems prepared in Examples 1, 4, 7, 10, 13, 14 and Comparative Example 1 were measured. The results are as follows: Figure 15 As shown. Each embodiment and comparative example showed maximum absorption peaks at 542nm and 574nm, which are typical absorption peaks of nitrosohemoglobin, indicating that nitrosohemoglobin is formed in a simulation system without the addition of nitrite. The formation of nitrosohemoglobin is related to the action of nitric oxide synthase in Staphylococcus aureus. Nitric oxide synthase can decompose L-arginine to produce nitric oxide, which can further combine with hemoglobin to form red nitrosohemoglobin. It can be seen from the absorption peak of nitrosohemoglobin that the absorption peak of comparative example 1 inoculated with Staphylococcus aureus alone is the lowest, while the absorption peaks of Examples 1, 4, 7, 10, 13, and 14 to which various exogenous additives are added are all higher than those of comparative example 1, which is consistent with the changing trend of the redness value, indicating that exogenous additives can enhance the ability of Staphylococcus aureus to promote the production of nitrosohemoglobin.

[0192] The nitric oxide synthase activity of the simulated systems prepared in Examples 1, 4, 7, 10, 13, 14 and Comparative Example 1 was determined. The results are as follows: Figure 16 As shown. In Comparative Example 1 in which only Staphylococcus carnosus was inoculated, the nitric oxide synthase activity was 2.20 U / mL, while in Examples 1, 4, 7, 10, 13, and 14 to which exogenous additives were added, the nitric oxide synthase activity was increased by 46.36% to 84.09% compared with Comparative Example 1, indicating that exogenous additives can enhance the nitric oxide synthase activity in Staphylococcus carnosus and thereby enhance its color development effect. This may be because tea polyphenols, ascorbic acid, and tocopherol can increase the gene expression of nitric oxide synthase and protect tetrahydrobiopterin in cells from oxidation, thereby providing optimal conditions for the production of nitric oxide synthase and improving enzyme activity. L-arginine is the substrate of nitric oxide synthase, and a moderate increase in its content can increase enzyme activity by increasing the substrate metabolism rate.

[0193] The redness a* value, nitrosohemoglobin, and nitrite residue of the salted and dried fish products prepared in Examples 15 to 20 were measured and compared with Comparative Examples 2 to 4. The results are shown in the following table.

[0194]

[0195]

[0196] Comparative Examples 2 and 3 exhibited lower a* values ​​after bleeding. Although the addition of nitrite improved the red color of Comparative Example 3, its a* value remained negative, indicating that the myoglobin content in the fish was low after bleeding, making it difficult to achieve a vibrant red color. Compared to Comparative Example 4, which was fermented solely with Staphylococcus carnosus, Examples 15-20, which were fermented with exogenous additives, exhibited higher redness values, demonstrating that exogenous additives can enhance the color-forming ability of Staphylococcus carnosus, resulting in a superior red color for the salted and dried fish.

[0197] The nitrosohemoglobin content in Comparative Example 3 was 3.29 mg / kg, which was significantly improved compared with Comparative Example 2, but still at a low level, indicating that the fish meat lacked sufficient heme substrate after bleeding, limiting the ability of nitrite to react and generate nitrosohemoglobin. The nitrosohemoglobin content in Examples 15 to 20, which were fermented after adding exogenous additives, was higher, and increased by 19.91% to 48.08% compared with Comparative Example 4, which was fermented by inoculating only Staphylococcus carnosus, indicating that exogenous additives can enhance the ability of Staphylococcus carnosus to promote the production of nitrosohemoglobin, which also corresponds to the result of enhanced characteristic absorption peak of nitrosohemoglobin in the simulation system ( Figure 15 This may be because the exogenous additives increased the activity of nitric oxide synthase in Staphylococcus carnosus, thereby increasing the production of NO and promoting the production of nitrosoheme in the salted and dried fish. In addition, the nitrosoheme contents in Examples 19 and 20, in which the exogenous additives were added in combination, were 9.67 mg / kg and 9.68 mg / kg, respectively, significantly higher than the nitrosoheme contents when each exogenous additive was added alone, indicating that the combined addition had a more significant effect on enhancing the ability of Staphylococcus carnosus to promote nitrosoheme production.

[0198] The residual nitrite in Comparative Example 3 was 62.77 mg / kg, which was 10.26 times that of Comparative Example 2. This indicates that although the addition of nitrite can improve the red color of salted and dried fish prepared by the traditional bleeding method to a certain extent, it will also cause a significant increase in the residual nitrite in the salted and dried fish, posing a major food safety risk. The nitrite content in Examples 15 to 20, which were fermented after adding exogenous additives, was low, decreasing by 87.16% to 91.88% compared to Comparative Example 3. It can be seen that compared with the traditional method of enhancing the red color of salted and dried fish by adding nitrite after bleeding and slaughtering, the new color development process using the non-bleeding slaughtering method and combining the fermentation of Staphylococcus carnosus with the synergistic effect of exogenous additives can not only significantly improve the redness value of salted and dried fish, but also effectively control the nitrite content, thereby significantly reducing the safety risks caused by the addition of nitrite.

[0199] In summary, the present invention proposes a method for enhancing the color development of Staphylococcus carnosus based on the regulation of exogenous additives and its application in salted and dried fish. In a simulated system, the present invention co-cultures Staphylococcus carnosus with one or more of L-arginine, tea polyphenols, ascorbic acid, or tocopherol as exogenous additives. This significantly increases the nitric oxide synthase activity in Staphylococcus carnosus (46.36% to 84.09%), thereby enhancing the color development ability of Staphylococcus carnosus. This provides a feasible solution for enhancing the color development ability of Staphylococcus carnosus in salted and dried fish by regulating nitric oxide synthase activity.

[0200] The present invention significantly improves the redness of salted and dried fish by incorporating one or more of L-arginine, tea polyphenols, ascorbic acid, or tocopherol as exogenous additives into a synergistic fermentation with Staphylococcus carnosus. Compared to fermentation with Staphylococcus carnosus alone, this synergistic fermentation process increases the nitrosoheme content in the salted and dried fish by 19.91% to 48.08%, resulting in an excellent red color. This is because the exogenous additives effectively enhance the nitric oxide synthase activity of Staphylococcus carnosus, thereby compensating for the poor color development caused by insufficient natural nitric oxide synthase activity in this strain.

[0201] The present invention provides a novel color development process based on a non-bleeding slaughtering method combined with the synergistic effects of Staphylococcus carnosus fermentation and exogenous additives. Compared with the traditional nitrite color development process, the novel color development process reduces the residual nitrite in the salted and dried fish (87.16% to 91.88%) while maintaining the excellent red color of the salted and dried fish. This overcomes the food safety hazard caused by excessive nitrite residues in the traditional process, achieving a dual improvement in the color quality and food safety of the salted and dried fish products. By replacing chemical additives with biological fermentation, the novel color development process provides a green, safe, and efficient color development technology solution for salted and dried fish products.

[0202] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. A method for enhancing the color development of Staphylococcus carnosus based on the regulation of exogenous additives, characterized by: include, The methemoglobin solution was mixed with broth and exogenous additives were added to construct a simulation system; Inoculate Staphylococcus carnosus into the simulation system and culture it, and observe the color change of the simulation system; Wherein, the exogenous additives include one or more of L-arginine, tea polyphenols, ascorbic acid or tocopherol.

2. The method according to claim 1, wherein: The content of L-arginine in the simulation system is 40-60 mmol / L.

3. The method according to claim 1 or 2, wherein: The content of L-arginine in the simulation system is 40-60 mmol / L.

4. The method according to claim 3, wherein: The content of the tea polyphenols in the simulation system is 200-400 μmol / L.

5. The method according to claim 4, wherein: The content of ascorbic acid in the simulation system is 200-400 μmol / L.

6. The method according to claim 5, wherein: The content of the tocopherol in the simulation system is 100-300 μmol / L.

7. The method according to any one of claims 1, 2, 4 to 6, characterized in that: The concentration of methemoglobin in the simulation system is 2-4 mg / mL, and the inoculation amount of Staphylococcus carnosus is 10 5 ~10 7 CFU / mL.

8. The method according to claim 7, wherein: The culture temperature of the simulation system is 35-40° C., and the culture time is 24 hours.

9. A method for coloring salted and dried fish, characterized in that: include, mixing the fish pieces with blood residues with salt, glucose and exogenous additives and curing the pieces to obtain cured fish pieces; The pickled fish blocks are inoculated with bacteria and then fermented at a constant temperature, and the fermented fish is dried to obtain a pickled dried fish product; Wherein, the exogenous additives include one or more of L-arginine, tea polyphenols, ascorbic acid or tocopherol; The amount of the exogenous additive added is 50 mmol / kg to 300 μmol / kg relative to the mass of the fish block.

10. The method according to claim 9, wherein: The mixed pickling time is 2 to 4 days at a temperature of 1 to 5°C; The constant temperature fermentation temperature is 20°C and the fermentation time is 3 days; The added amount of L-arginine is 50 mmol / kg, the added amount of tea polyphenols is 300 μmol / kg, the added amount of ascorbic acid is 300 μmol / kg, and the added amount of tocopherol is 200 μmol / kg.

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