Marine natural umami seasoning containing shrimps and crabs and preparation method of marine natural umami seasoning

Abstract

The invention discloses a shrimp and crab type marine natural umami seasoning and a preparation method thereof, and belongs to the technical field of food processing. The method comprises the following steps: 1) preparing a suspension from shrimp and crab aquatic products, inoculating activated compound bacteria, and fermenting to obtain a fermented suspension; adding a compound enzyme into the fermentation turbid liquid, and carrying out enzymolysis, enzyme deactivation, filtration centrifugation and graded ultrafiltration to obtain supernate; (2) adsorbing amino acid by using a polyvinyl alcohol pre-membrane, compounding the pre-membrane subjected to saturated adsorption of the amino acid with a bottom membrane by using a cross-linking agent, performing electrostatic spinning to obtain an amino acid embedded electrostatic spinning membrane, and eluting the embedded amino acid to obtain an electrostatic spinning membrane containing amino acid holes; the supernate obtained in the third step is purified and then eluted, an eluted solution is obtained, and the seasoning is obtained after spray drying; the amino acid in the step 4 is selected from Ala, Asp, Asn, Glu and Gln. The obtained seasoning is high in sensitization reduction rate, sufficient in delicate flavor and free of fishy smell and peculiar smell.

Description

Technical Field

[0001] The present application relates to a natural marine umami seasoning for shrimps and crabs and a preparation method thereof, belonging to the technical field of food processing. Background Art

[0002] Shrimp and crab are important economic aquatic products in my country. They are not only delicious but also extremely nutritious, and are deeply loved by people. The seafood seasoning prepared from them contains rich flavor components such as amino acids, organic acids, sugars, peptides, nucleotides, etc., and is widely favored by people. However, it is also one of the foods that cause allergic reactions. Some people will have skin allergies after eating them. Unlike egg and milk allergies, aquatic product allergies usually accompany lifelong. Therefore, food allergies caused by aquatic products deserve great attention.

[0003] In the prior art, CN 109363107 A uses a method combining secondary fermentation, lactic acid bacteria and yeast composite strains synergistic fermentation and ultrasonic treatment to reduce the allergenicity of crab meat. However, there are the following problems: 1) the degree of degradation and allergenicity needs to be improved; and fermentation may cause the texture of the aquatic product to become soft, rotten and loose, affecting the taste and edible quality; 2) the fishy smell of shrimp and crab products themselves is difficult to remove.

[0004] In addition, the products after fermentation / enzymatic hydrolysis in the prior art contain many impurities and other "peculiar smells", which will affect the taste and flavor of shrimp and crab processed products.

[0005] Therefore, a new preparation process is needed to prepare a shrimp and crab marine natural umami seasoning with low allergenicity, no fishy smell and good quality. Summary of the invention

[0006] In order to solve the above problems, the present application provides a shrimp and crab marine natural umami seasoning and a preparation method thereof. The seasoning obtained by the method has a high allergenicity reduction rate, is full of umami, and has no fishy smell or peculiar smell.

[0007] The specific technical solutions of this application are as follows:

[0008] The present application provides a method for preparing a shrimp and crab marine natural umami seasoning, comprising:

[0009] Step 1: Prepare a suspension of shrimp and crab aquatic products;

[0010] Step 2, fermentation: sterilizing the suspension and inoculating the activated composite bacteria for fermentation to obtain a fermented suspension;

[0011] Step 3: Enzymatic hydrolysis: adding compound enzyme to the fermentation suspension, performing enzymatic hydrolysis, enzyme inactivation, filtration centrifugation, and graded ultrafiltration to obtain a supernatant;

[0012] Step 4: Purification and elution

[0013] 1) After the corresponding amino acids are adsorbed by a polyvinyl alcohol pre-membrane, the pre-membrane saturated with the amino acids is mixed with the base membrane by a cross-linking agent, and then an amino acid embedded electrospinning membrane is obtained by an electrospinning process, and the embedded amino acids are eluted to obtain an electrospinning membrane containing amino acid holes; 2) The supernatant obtained in step 3 is purified and eluted by the electrospinning membrane containing amino acid holes to obtain an elution solution, and the seasoning is obtained after spray drying;

[0014] The composite bacteria is selected from Staphylococcus xylosus, Acetobacter, Pentosaccharococcus; the composite enzyme is selected from alkaline protease, bromelain, and ficin;

[0015] The amino acid in step 4 is selected from Ala, Asp, Asn, Glu, and Gln.

[0016] Optionally, the composite bacteria is obtained by mixing Staphylococcus xylosus, acetic acid bacteria, and Pentosaccharococcus in a mass ratio of (1-3): (0.5-6): 1;

[0017] Optionally, the fermentation conditions are: fermentation temperature 20-40° C., fermentation time 20-72 h, fermentation pH: 3-7.5, and the amount of bacteria added is 0.5-10% of the weight of the suspension in step one.

[0018] Optionally, the composite enzyme is prepared by alkaline protease: bromelain: ficin according to the mass ratio of (0.5-3): (0.1-2): (1-5); the amount of the composite enzyme added is 0.25-0.5% of the weight of the fermentation suspension in step 2; and the enzymatic hydrolyzate obtained after enzymatic hydrolysis in step 3 is filtered, centrifuged, and preliminarily purified;

[0019] Preferably, the filter mesh number is 50-120 mesh, the centrifugal rate is 6000-12000r / min; the centrifuge is preliminarily purified by graded ultrafiltration, the ultrafiltration membrane molecular weight cutoff is >10kDa, 3-5kDa, <3kDa, the ultrafiltration pressure is 0.02-0.1MPa, the ultrafiltration temperature is 25-40°C, and the amount of water added for ultrafiltration is 0.5-5 times the mass of the enzymatic hydrolyzate;

[0020] More preferably, the filter mesh number is 60-90 mesh; the centrifugal rate is 8000-10000 r / min, the ultrafiltration pressure is 0.02 MPa, 0.03 MPa and 0.05 MPa, the ultrafiltration temperature is 30°C, and the ultrafiltration water addition amount is 2-3.5 times.

[0021] Optionally, in step 4, the amino acid in the electrospinning membrane with amino acid holes is any one of 1) Ala, Asp and Asn, 2) Glu and Gln, 3) Ala, Asp, Asn, Glu and Gln, 4) Ala, Asp and Glu, 5) Glu, Asp and Gln, 6) Asn and Gln, 7) Ala, Asn, Glu and Gln, 8) Ala, Asp, Asn, Glu;

[0022] Preferably, it is any one of Ala, Asp and Asn; Glu and Gln; Ala, Asp, Asn, Glu and Gln; Ala, Asp and Glu.

[0023] Optionally, the base film in step 4 is a polymer material, including any one of amino-functionalized polyacrylonitrile, amino-functionalized polysiloxane, polyvinyl alcohol, and polyaspartic acid;

[0024] Any one of amino-functionalized polyacrylonitrile and amino-functionalized polysiloxane is preferred.

[0025] Optionally, in step 4 1) the solution for eluting the amino acid embedded in the amino acid embedded electrospinning membrane is a sodium hydroxide solution with a concentration of 0.2-3M / L; preferably, 0.5-1.5M / L;

[0026] Optionally, in step 41), the adsorption capacity of amino acids by the electrospinning membrane is 45-90 mg / g.

[0027] Optionally, the shrimps include prawns, lobsters, and river shrimps; the crabs include swimming crabs, blue crabs, and river crabs.

[0028] Further, the specific steps of the purification and elution treatment in step 4 are as follows:

[0029] 1) Stirring a polyvinyl alcohol aqueous solution with a mass concentration of 1-6% until uniform, and then standing, degassing, and drying to obtain a pre-film; taking the pre-film and placing it in a 0.2-1g / L amino acid aqueous solution for adsorption for 12-54h and then vacuum drying; the mass volume ratio is: m 前置膜 / V 氨基酸水溶液 =(1mg / 2500mL)~

[0030] (1mg / 1500mL).

[0031] Preferably, the amino acid mass concentration is 0.5%; the static adsorption time is any one of 18, 24, and 32 hours; according to the mass volume ratio, m 前置膜 / V 氨基酸水溶液 Any one of 1 mg / 1200 mL, 1 mg / 1500 mL and 1 mg / 1600 mL.

[0032] Preferably, the electrospinning membrane has an adsorption capacity of 45-90 mg / g for amino acids. Preferably, the mass concentration of the polyvinyl alcohol aqueous solution is any one of 2.5%, 3%, and 3.5%.

[0033] 2) According to the mass volume ratio m 饱和吸附氨基酸后的前置膜 / V 混合溶液 =(1mg / 800mL)~

[0034] (1 mg / 1500 mL), placing the pre-film after saturated amino acid adsorption in a mixed solution of water and ethanol, adding a cross-linking agent accounting for 0.5%-3% of the mass fraction of the pre-film after saturated amino acid adsorption, stirring at pH less than 7 and 40-60° C. to obtain a mixed solution A;

[0035] Preferably, the cross-linking agent is any one of glutaraldehyde, polyethylene glycol diglycidyl ether, and EDC / NHS, and the added amount is any one of 0.5%, 1.5%, and 2%.

[0036] 3) dissolving the polymer base film material and mixing it with the mixed solution A to obtain a mixed solution B, performing electrospinning treatment to obtain an amino acid embedded electrospinning membrane, and eluting to obtain an electrospinning membrane containing amino acid holes; taking out the electrospinning membrane containing amino acid holes and soaking it in the supernatant after centrifugation in step three, then taking it out and eluting it in a sodium hydroxide solution, and obtaining the seasoning after spray drying.

[0037] The present application also provides a seasoning prepared by the above preparation method.

[0038] The beneficial effects of this application include but are not limited to:

[0039] 1. This application uses a combination of specific fermentation bacteria (Staphylococcus xylosus, acetic acid bacteria, Pentosaccharococcus) and complex enzymes (alkaline protease, bromelain, ficin) (fermentation first and then enzymolysis) to process shrimps and crabs. Compared with enzymolysis first and then fermentation, and the use of other fermentation bacteria and complex enzymes for process treatment, the product has better effects in reducing allergenicity and removing odor and fishy smell, and has good stability and a long shelf life. The reason is that the combination of specific fermentation bacteria and complex enzymes in this application (fermentation first and then enzymolysis) can produce better synergistic effects and effectively decompose the macromolecular proteins in the substrate into small molecular umami peptides with a relatively high proportion; it greatly reduces the allergenicity of the seasoning product.

[0040] 2. The present application utilizes an electrospinning membrane with specific amino acid cavities (selected from a specific combination of Ala, Asp, Asn, Glu, and Gln) to perform specific purification and elution treatment on a mixture of umami peptides to obtain specific target peptides. Compared with the peptides separated by liquid chromatography technology in the prior art, this greatly saves time and effort, has high purification efficiency, and is easy to obtain target peptides without impurities and with higher purity.

[0041] Furthermore, the present application also screened the materials of the electrospinning membrane: the preferred base membrane material was amino-functionalized polyacrylonitrile and amino-functionalized polymethylsiloxane, for the best adsorption effect. The reason is that the surfaces of the above two electrospinning membrane substrates are positively charged under the pH (pH < 7) environment, which is conducive to the adsorption of umami peptides in the holes through strong electrostatic adsorption, and is conducive to the smooth progress of the purification process.

[0042] 3. The specific fermentation + enzymatic hydrolysis + purification treatment combined method of the present application is more targeted to shrimps. The reason may be that shrimps / crabs from different sources may have certain changes in protein structure and composition due to differences in living environment and evolution, and their allergens may also have certain differences. DETAILED DESCRIPTION

[0043] The present application is described in detail below in conjunction with the examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials in the examples are all commercially available.

[0044] Example 1 A natural marine flavor seasoning for shrimps and crabs is prepared by the following method:

[0045] Step 1: Raw material preparation

[0046] Soak shrimp (Chinese prawn) and crab (Chinese mitten crab, also known as hairy crab, river crab) in light salt water for 3 times (20 minutes / time), then wash with clean water, mash and take 80g (40g each of shrimp and crab), add 3 times the weight of distilled water, and then pass through high-pressure homogenization (working pressure: 18Mpa, speed: 6000r / min, time: 15min), and then pass through a colloid mill (working pressure: 2Mpa, speed: 2000r / min, time: 30min) to make a uniform suspension.

[0047] Since the high-pressure homogenizer can provide higher shear force and pressure, using the high-pressure homogenizer first can better crush and homogenize the material, and then using the colloid mill for further refinement and homogenization, which can achieve better results.

[0048] Step 2: Fermentation

[0049] The suspension prepared in step 1 is first sterilized, and then activated composite bacteria (obtained by mixing Staphylococcus xylosus, acetic acid bacteria, and Pentosaccharococcus in a mass ratio of 1:1:1) are added for fermentation to obtain a fermentation suspension.

[0050] The fermentation temperature was 25° C., the fermentation time was 36 h, the fermentation pH was 4.0, and the amount of bacteria added was 3.5% of the weight of the suspension.

[0051] Step 3: Enzymatic hydrolysis

[0052] The fermentation suspension obtained in step 2 is adjusted to 6.0, and a composite enzyme accounting for 0.25% of the weight of the fermentation suspension is added (obtained by compounding alkaline protease: bromelain: fig protease in a mass ratio of 1:1:1); the enzyme is hydrolyzed for 5.5 hours at 45°C and 260W power in an ultrasonic water bath; then the enzyme is inactivated in a 100°C water bath. The enzyme is inactivated for 10 minutes; after the temperature drops to room temperature, it is filtered through an 80-mesh filter, and the filtrate is centrifuged at 4°C and 10,000 r / min for 10 minutes;

[0053] The centrifuge liquid is initially purified by graded ultrafiltration, so that the graded ultrafiltration membrane cutoff molecular weight is >10kDa, 3-5kDa, <3kDa (specifically 12kDa, 5kDa, 2kDa in this embodiment), three-stage ultrafiltration; the ultrafiltration pressure is 0.02MPa, the ultrafiltration temperature is 30°C, and the amount of water added for ultrafiltration is twice the mass of the enzymatic hydrolyzate; and the centrifuged supernatant is obtained.

[0054] Step 4: Purification and elution

[0055] Purification and elution are performed to overcome the problem in the prior art that products after fermentation or enzymatic hydrolysis have other odors and the freshness is not significantly improved.

[0056] 4.1) Preparation of electrospun membranes with amino acid cavities

[0057] 4.1.1) Stir the polyvinyl alcohol aqueous solution with a mass concentration of 2.5% until uniform, and then stand, degas, and dry to obtain the pre-film. Take the pre-film and place it in a 0.5g / L amino acid aqueous solution (amino acids are Ala, Asp, and Asn added in a mass ratio of 1:1:1) for adsorption (mass to volume ratio: m 前置膜 / V 氨基酸水溶液 =1mg / 1500mL) for 12 hours, and then take it out and vacuum dry it at 60℃ for 24 hours to obtain the pre-film after saturated adsorption of amino acids. The pre-film after saturated adsorption of amino acids was placed in a water / ethanol (V / V=7 / 3) mixture (m 饱和吸附氨基酸后的前置膜 / V 混合溶液 =1 mg / 800 mL), add 0.5% of the mass fraction of the pre-membrane after saturated adsorption of amino acids (glutaraldehyde), stir for 12 h at pH = 6 and 50 ° C to obtain a mixed solution A.

[0058] Select 1 g of polymer base film material - amino-modified polyacrylonitrile (PAN), dissolve it in 16 ml of DMF solvent, put a magnetic rotor into the mixed solution and seal it with plastic wrap to prevent interference from the external environment, and stir it magnetically until it dissolves; then add the prepared mixed solution A, continue magnetic stirring for 12 hours, and obtain mixed solution B.

[0059] 4.1.2) Introduce 3 ml of mixed solution B into a 5 ml plastic syringe equipped with a 22-gauge needle, and perform electrospinning at a feed rate of 1 ml / h and a spinning voltage of 18 kV for 6 h to obtain a nanofiber membrane (amino acid embedded electrospinning membrane). Dry the membrane in a vacuum at 40 °C for 12 h and place it in a ziplock bag for later use (electrospinning process).

[0060] 4.1.3) Take a piece of prepared nanofiber membrane and place it in 50mL of 1.0mol / L sodium hydroxide solution for elution for 3h. Since sodium hydroxide is a strong base, when sodium hydroxide comes into contact with amino acids, it will destroy the chemical structure of amino acids, and the hydrolysis of sodium hydroxide can also destroy the connection between amino acids and the membrane, causing the amino acids to fall off the membrane, thereby forming specific amino acid (Ala, Asp, Asn) holes on the amino acid embedded electrospinning membrane (there are several peptides composed of different amino acids in the supernatant after centrifugation. The formation of specific amino acid holes can be used to lock / screen the umami peptides composed of the specific amino acids to be adsorbed on the fiber membrane, thereby obtaining the target peptide), and obtaining an electrospinning membrane containing amino acid holes.

[0061] 4.1.4) Then take out the membrane and soak it in the supernatant after centrifugation in step 3 for 12 hours. During this process, the three amino acids Ala, Asp, and Asn in the supernatant are adsorbed into the amino acid holes by the membrane (greatly saving time and energy, and improving purification efficiency, making it easier to obtain the target peptide). After the adsorption is completed, take out the membrane and put it in 1.0 mol / L sodium hydroxide solution for 3 hours to elute the target peptide adsorbed on the membrane.

[0062] The final prepared electrospinning membrane with specific amino acid cavities has an adsorption capacity of 60 mg / g for the corresponding amino acid.

[0063] Step 5: spray-drying the eluted solution to obtain seasoning.

[0064] Example 2

[0065] A natural marine flavor seasoning for shrimps and crabs is prepared by the following method:

[0066] Step 1: Raw material preparation

[0067] Soak shrimp (Chinese prawn) and crab (swimming crab) in light salt water for 4 times (20 minutes / time), then wash with clean water, mash and take 80g (40g each of shrimp and crab), add 3 times the weight of distilled water, and then pass through high-pressure homogenization (working pressure: 25Mpa, speed: 8000r / min, time: 30min), and then pass through a colloid mill (working pressure: 6Mpa, speed: 1500r / min, time: 20min) to make a uniform suspension.

[0068] Step 2: Fermentation

[0069] The suspension prepared in step 1 is first sterilized, and then activated composite bacteria (obtained by mixing Staphylococcus xylosus, acetic acid bacteria, and Pentosaccharococcus in a mass ratio of 3:3:1) are added for fermentation to obtain a fermentation suspension.

[0070] The fermentation temperature is 40° C., the fermentation time is 20 h, the fermentation pH is 6.0, and the added amount of the strain is 7% of the weight of the suspension.

[0071] Step 3: Enzymatic hydrolysis

[0072] Adjust the fermentation suspension obtained in step 2 to 6.0, and add 0.25% of the weight of the fermentation suspension (obtained by compounding alkaline protease: bromelain: fig protease in a mass ratio of 0.5:2:1); use an ultrasonic water bath to enzymolyze for 5.5 hours at 45°C and 260W power; then inactivate the enzyme in a 100°C water bath. Inactivate the enzyme for 10 minutes. After the temperature drops to room temperature, filter through an 80-mesh filter, and centrifuge the filtrate at 4°C and 10,000 r / min for 10 minutes;

[0073] The centrifuge liquid is initially purified by graded ultrafiltration, so that the molecular weight cutoffs of the three-stage ultrafiltration membrane are >10kDa, 3-5kDa, and <3kDa, respectively (specifically 12kDa, 5kDa, and 2kDa in this embodiment), the ultrafiltration pressure is 0.02MPa, the ultrafiltration temperature is 30°C, and the amount of water added for ultrafiltration is twice the mass of the enzymatic hydrolyzate; and the centrifuge supernatant is obtained.

[0074] Step 4: Purification and elution

[0075] Purification and elution are performed to overcome the problem in the prior art that products after fermentation or enzymatic hydrolysis have other odors and the freshness is not significantly improved.

[0076] 4.1) Preparation of electrospun membranes with amino acid cavities

[0077] 4.1.1) Stir a 6% polyvinyl alcohol aqueous solution until uniform, and after standing, degassing, and drying, obtain a pre-film; take the pre-film and place it in a 0.2g / L amino acid aqueous solution (amino acids are Ala, Asp, and Asn added in a mass ratio of 1:1:1) for adsorption (mass to volume ratio: m 前置膜 / V 氨基酸水溶液 =1mg / 1500mL) for 12 hours and then taken out and dried in vacuum at 60°C for 24 hours. The membrane after saturated adsorption of amino acids was placed in a water / ethanol (V / V=7 / 3) mixture (m 饱和吸附氨基酸后的前置膜 / V 混合溶液

[0078] =1 mg / 800 mL), add 2% of the mass fraction of the pre-membrane after saturated adsorption of amino acids (polyethylene glycol diglycidyl ether), stir for 12 h at pH = 6 and 50 ° C to obtain a mixed solution A.

[0079] Select 1 g of polymer base film material - amino polysiloxane, dissolve it in 16 ml of DMF solvent, put a magnetic rotor into the mixed solution and seal it with plastic wrap to prevent interference from the external environment, and stir it magnetically until it dissolves; then add the prepared mixed solution A, continue magnetic stirring for 12 hours, and obtain mixed solution B.

[0080] 4.1.2)-4.1.3) are the same as in Example 1.

[0081] Step 5: Same as Example 1.

[0082] Example 3

[0083] The difference from Example 1 is that only shrimp (Penaeus chinensis) is used as the raw material, and 80 g is taken after being crushed; the rest is the same as Example 1.

[0084] Example 4

[0085] The difference from Example 1 is that only crab (Chinese mitten crab, also known as hairy crab, river crab) is used as the raw material, and 80 g is taken after being crushed; the rest is the same as Example 1.

[0086] The difference between Example 5-13 and Example 1 is that in step 4, the amino acids added in the purification and elution treatment are different (in each example, the added mass fractions of different amino acids are the same), and the remaining steps are the same as Example 1. See Table 1 below for details.

[0087] Table 1 Different amino acid addition settings

[0088]

[0089]

[0090] Note: Ala (Ala here represents β-alanine), Asp (Asp here represents DL-aspartic acid), Asn (Asn here represents D-asparagine), Glu (Glu here represents L-glutamic acid), Gln (Gln here represents L-glutamine), these five amino acids were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0091] Compared with Example 1, Comparative Example 1 did not perform purification elution.

[0092] Comparative Example 2

[0093] Compared with Example 1, the composite bacteria used in step 2 is different, and is prepared from Bacillus licheniformis: Candida utilis: Lactobacillus plantarum in a mass ratio of 1:1:1; the addition amount and other steps are the same as in Example 1.

[0094] Comparative Example 3

[0095] Compared with Example 1, the composite enzyme used in step three is different, and is prepared from chymosin, proteinase P, pepsin, and trypsin in a mass ratio of 1:1:1:1; the amount added and other steps are the same as in Example 1.

[0096] Comparative Example 4: Enzymatic hydrolysis followed by fermentation

[0097] The only difference from Example 1 is that the order of step (2) and step (3) is exchanged, that is, enzymatic hydrolysis is performed before fermentation. Specifically, after step (2), enzyme is first added to the sterilized suspension for enzymatic hydrolysis, and then the composite bacteria is inoculated into the supernatant after enzymatic hydrolysis for fermentation.

[0098] Comparative Example 5

[0099] Compared with Example 1, the material of the electrospinning membrane is different: 1 g of the polymer material polypropylene (PP) is selected, and the rest is the same as Example 1.

[0100] Embodiment 14

[0101] Compared with Example 1, the source of the crab is different, and the crab is commercially available king crab.

[0102] The hydrolysis degree, peptide yield and umami intensity of the supernatant prepared in the above examples and comparative examples 2-4 (i.e., after enzymatic hydrolysis + fermentation treatment in step 3) were tested. The results are shown in the table below.

[0103] The peptide is detected by ultraviolet spectrophotometry in the prior art. The peptide has a certain absorbance at a specific wavelength. By measuring the absorbance of the sample at this wavelength, the peptide content can be calculated, and then the peptide yield can be calculated.

[0104] Table 2 Supernatant test results

[0105]

[0106] (Note: Umami intensity is 0-10 points, 0 means no taste, 10 means strong umami, and 5 means the umami intensity of 0.35% MSG)

[0107] Conclusion: According to the data in the above table, compared with Comparative Example 2 with different composite bacteria, Comparative Example 3 with different composite enzymes, and Comparative Example 4 with enzymatic hydrolysis followed by fermentation, the hydrolysis degree and peptide yield of the supernatant in the examples are significantly higher than those of the comparative examples, and the umami intensity is more obvious. Among them, Examples 1, 2 and 5 have the best effects.

[0108] Higher hydrolysis degree and peptide yield means that more proteins are hydrolyzed into small molecule peptides, which greatly reduces the allergenicity. Moreover, these peptides are not only more easily absorbed by the human body, but also retain more biological activity, thus exerting more health benefits.

[0109] The reason why the embodiments have good effects may be that the specific composite bacteria of the present application will produce some metabolites during the growth process, and these metabolites can change the structure of the protein substrate, making it easier to be hydrolyzed by the specific enzyme preparation in the embodiments.

[0110] The seasonings prepared in the above embodiments and comparative examples were tested for indicators, and the results are shown in Table 3. The main allergen proteins (tropomyosin) of shrimp and crab were directly detected and analyzed by mass spectrometry technology (tandem mass spectrometry MS / MS) ion monitoring mass spectrometry in the prior art, and the detection method was referenced from (Zhao Xin, 2022).

[0111] Allergenicity reduction rate (%) = (allergen content in the suspension without any treatment - allergen content in the seasoning product after treatment) / allergen content in the suspension without any treatment.

[0112] Table 3 Effects of different treatment processes on seasoning products

[0113]

[0114]

[0115]

[0116] Conclusion: According to the data in the above table, compared with the comparative example, the allergenicity reduction rate of the seasoning products obtained in Examples 1-13 is high, which can reach 65%; even after storage for 6 months, the allergenicity reduction rate can still reach more than 60%. Among them, the comprehensive effect of Examples 1-4 is relatively the best.

[0117] The seasoning obtained in the comparative example not only has a relatively low allergenicity reduction rate, but also has undesirable flavors such as fishy smell, and the umami intensity is also relatively weak.

[0118] Compared with Comparative Example 2 with different composite strains, Comparative Example 3 with different composite enzymes, and Comparative Example 4 with enzymatic hydrolysis followed by fermentation, the reason is that the specific fermentation bacteria in the examples will produce a series of enzymes and metabolites during the growth and metabolism process. When used in combination with the enzyme preparation added in the examples, the enzymes produced by the strains and the added enzyme preparations can work synergistically to transform and decompose the substances that produce the fishy smell, thereby significantly reducing the fishy smell; and in the process, the macromolecular proteins in the substrate are effectively decomposed into small molecular umami peptides with a relatively high proportion; greatly reducing the allergenicity of the seasoning product.

[0119] In the prior art, liquid chromatography is mostly used for elution, and the allergenicity reduction rate of the obtained seasoning is generally about 15%, and the product has an unpleasant smell and the umami taste is not obvious. This is because liquid chromatography elution not only separates umami peptides but also other peptides (including peptides with unpleasant smell and high allergenicity), and it is impossible to separate the umami peptides separately, so the product has an obvious unpleasant smell and the allergenicity reduction rate is also low.

[0120] Compared with Example 14, it may be that the specific enzymes and strains in the present application cannot effectively remove the fishy smell of the umami peptide of the king crab species because crabs from different sources will also affect the odor of the umami peptide due to their different water quality and food sources.

[0121] In addition, after the products prepared in Examples 1-5 of the present application were packaged, a distinct fresh aroma could be smelled within 2 seconds after opening. Examples 6-14 could also smell a distinct fresh aroma within 3-5 seconds.

[0122] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

[0123] References:

[0124] Zhao, X. (2022). Detection and identification of aquatic product allergens based on mass spectrometry. (Master). Retrieved from https: / / link.cnki.net / doi / 10.27314 / d.cnki.gsscu.2022.000918 Available from Cnki.

Claims

1. A method for preparing a natural marine flavor seasoning of shrimps and crabs, characterized in that: include: Step 1: Prepare a suspension of shrimp and crab aquatic products; Step 2, fermentation: sterilizing the suspension and inoculating the activated composite bacteria for fermentation to obtain a fermented suspension; Step 3: Enzymatic hydrolysis: adding compound enzyme to the fermentation suspension, performing enzymatic hydrolysis, enzyme inactivation, filtration centrifugation, and graded ultrafiltration to obtain a supernatant; Step 4: Purification and elution 1) After the corresponding amino acids are adsorbed by a polyvinyl alcohol pre-membrane, the pre-membrane saturated with the amino acids is mixed with a base membrane by a cross-linking agent, and then an amino acid embedded electrospinning membrane is obtained by an electrospinning process, and the embedded amino acids are eluted to obtain an electrospinning membrane containing amino acid cavities; 2) purifying and eluting the supernatant obtained in step 3 using an electrospinning membrane containing amino acid holes to obtain an elution solution, and spray-drying the elution solution to obtain a seasoning; The composite bacteria is selected from Staphylococcus xylosus, Acetobacter, Pentosaccharococcus; the composite enzyme is selected from alkaline protease, bromelain, and ficin; The amino acid in step 4 is selected from Ala, Asp, Asn, Glu, and Gln.

2. The preparation method according to claim 1, characterized in that: The composite bacteria is obtained by mixing Staphylococcus xylosus, acetic acid bacteria and Pentosaccharococcus in a mass ratio of (1-3): (0.5-6): 1; The fermentation conditions are: fermentation temperature 20-40°C, fermentation time 20-72h, fermentation pH: 3-7.5, and the amount of bacteria added is 0.5-10% of the weight of the suspension in step one.

3. The preparation method according to claim 1, characterized in that: The composite enzyme is prepared by alkaline protease: bromelain: ficin according to the mass ratio of (0.5-3): (0.1-2): (1-5); the amount of the composite enzyme added is 0.25-0.5% of the weight of the fermentation suspension in step 2; and the enzymatic hydrolyzate obtained after enzymolysis in step 3 is filtered, centrifuged, and preliminarily purified; Preferably, the filter mesh number is 50-120 mesh, the centrifugal rate is 6000-12000r / min; the centrifuge is preliminarily purified by graded ultrafiltration, the ultrafiltration membrane molecular weight cutoff is >10kDa, 3-5kDa, <3kDa, the ultrafiltration pressure is 0.02-0.1MPa, the ultrafiltration temperature is 25-40°C, and the amount of water added for ultrafiltration is 0.5-5 times the mass of the enzymatic hydrolyzate; More preferably, the filter mesh number is 60-90 mesh; the centrifugal rate is 8000-10000 r / min, the ultrafiltration pressure is 0.02 MPa, 0.03 MPa and 0.05 MPa, the ultrafiltration temperature is 30°C, and the ultrafiltration water addition amount is 2-3.5 times.

4. The preparation method according to claim 1, characterized in that: In step 4, the amino acid in the electrospun membrane with amino acid holes is any one of 1) Ala, Asp and Asn, 2) Glu and Gln, 3) Ala, Asp, Asn, Glu and Gln, 4) Ala, Asp and Glu, 5) Glu, Asp and Gln, 6) Asn and Gln, 7) Ala, Asn, Glu and Gln, 8) Ala, Asp, Asn, Glu; Preferably, it is any one of Ala, Asp and Asn; Glu and Gln; Ala, Asp, Asn, Glu and Gln; Ala, Asp and Glu.

5. The preparation method according to claim 1, characterized in that: The base film in step 4 is a polymer material, including any one of amino polyacrylonitrile, amino functionalized polysiloxane, polyvinyl alcohol, and polyaspartic acid; Any one of amino-functionalized polyacrylonitrile and amino-functionalized polysiloxane is preferred.

6. The preparation method according to claim 1, characterized in that: Step 4 1) embedding the amino acid into the amino acid embedded electrospinning membrane; the solution for eluting the amino acid is a sodium hydroxide solution with a concentration of 0.2-3 M / L; preferably 0.5-1.5 M / L; In step 4 1), the adsorption capacity of amino acids by the electrospun membrane is 45-90 mg / g.

7. The preparation method according to claim 1, characterized in that: The shrimps include prawns, lobsters, and river shrimps; the crabs include swimming crabs, blue crabs, and river crabs.

8. The preparation method according to claim 1, characterized in that: The specific steps of step 4 purification and elution treatment are as follows: The polyvinyl alcohol aqueous solution with a mass concentration of 1-6% is stirred until uniform, and the pre-film is obtained after standing, degassing and drying; the pre-film is taken and adsorbed in a 0.2-1g / L amino acid aqueous solution for 12-54h and then vacuum dried; the mass volume ratio is: m 前置膜 / V 氨基酸水溶液 =(1mg / 2500mL)~(1mg / 1500mL); According to the mass-to-volume ratio m 饱和吸附氨基酸后的前置膜 / V 混合溶液 = (1 mg / 800 mL) ~ (1 mg / 1500 mL), placing the pre-film after saturated amino acid adsorption in a mixed solution of water and ethanol, adding a cross-linking agent accounting for 0.5%-3% of the mass fraction of the pre-film after saturated amino acid adsorption, stirring at pH less than 7 and 40-60° C. to obtain a mixed solution A; The polymer base film material is dissolved and mixed with the mixed solution A to obtain the mixed solution B, and the electrospinning treatment is performed to obtain the amino acid embedded electrospinning membrane, and the electrospinning membrane containing amino acid holes is obtained after elution; the electrospinning membrane containing amino acid holes is taken out and immersed in the supernatant after centrifugation in step three, and then taken out and eluted in the sodium hydroxide solution, and the seasoning is obtained after spray drying.

9. The preparation method according to claim 8, characterized in that: The cross-linking agent is any one of glutaraldehyde, polyethylene glycol diglycidyl ether, hexamethylene diisocyanate, EDC / NHS, and toluene diisocyanate, and the added amount is 0.5-2%; preferably, any one of 0.5%, 1.5%, and 2%.

10. The seasoning obtained by the preparation method according to any one of claims 1 to 9.

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