Shrimp, crab marine natural fresh taste seasoning and its preparation method
By combining a specific compound microbial strain and enzyme with a pre-fermentation and enzymatic hydrolysis process and electrospinning membrane purification technology, the allergenicity and fishy smell problems of shrimp and crab seasonings have been solved, resulting in high-purity, fishy-smelling umami seasonings that improve the taste and stability of the products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QILU UNIVERSITY OF TECHNOLOGY (SHANDONG ACADEMY OF SCIENCES)
- Filing Date
- 2024-10-18
- Publication Date
- 2026-06-12
AI Technical Summary
Existing technologies for shrimp and crab marine seasonings are insufficient in terms of degradation and allergenicity, removal of fishy odor, and improvement of taste. Furthermore, the fermentation process may cause the product to soften, affecting its taste and flavor.
A process involving fermentation followed by enzymatic hydrolysis using specific compound microbial strains (Staphylococcus xylose, acetic acid bacteria, and Staphylococcus pentosaceus) and compound enzymes (alkaline protease, bromelain, and fig protease), combined with a polyvinyl alcohol pre-membrane and electrospinning technology for purification and elution, was used to prepare an amino acid-embedded electrospinned membrane. The amino acid mixture was then purified and eluted to obtain a high-purity natural umami seasoning for shrimp and crab.
It significantly reduces the allergenicity of shrimp and crab seasonings, removes fishy and off-flavors, improves the umami and stability of products, has high purification efficiency, and produces products of excellent quality.
Smart Images
Figure SMS_1 
Figure SMS_3 
Figure SMS_4
Abstract
Description
Technical Field
[0001] This application relates to a natural marine umami seasoning for shrimp and crab and its preparation method, belonging to the field of food processing technology. Background Technology
[0002] Shrimp and crab are important economic aquatic products in my country, not only delicious but also extremely nutritious, making them very popular. Seafood seasonings made from them are rich in flavor components such as amino acids, organic acids, sugars, polypeptides, and nucleotides, and are widely favored. However, they are also among the foods that can trigger allergic reactions. Some people experience skin allergies after consuming them. Unlike egg or milk allergies, seafood allergies are usually lifelong; therefore, food allergies caused by seafood deserve serious attention.
[0003] In the existing technology, CN 109363107 A uses a combination of secondary fermentation, synergistic fermentation of lactic acid bacteria and yeast, and ultrasonic treatment to reduce the allergenicity of crab meat. However, the following problems exist: 1) The degree of degradation and allergen reduction needs to be improved; and fermentation may cause the texture of aquatic products to become soft and loose, affecting the taste and edibility; 2) The fishy smell of shrimp and crab products is difficult to remove.
[0004] In addition, the products produced by existing fermentation / enzymatic hydrolysis technologies contain many impurities and other "off-flavors," which can affect the taste and flavor of processed shrimp and crab products.
[0005] Therefore, a new preparation process is needed to produce a natural umami seasoning for shrimp and crab that is low in allergens, odorless, and of high quality. Summary of the Invention
[0006] To address the aforementioned issues, this application provides a natural marine umami seasoning for shrimp and crab, and its preparation method. The seasoning obtained by this method exhibits a high rate of reduction in allergenicity, is rich in umami flavor, and is free of fishy or off-flavors.
[0007] The specific technical solution of this application is as follows:
[0008] This application provides a method for preparing a natural umami seasoning from shrimp and crab, comprising:
[0009] Step 1: Prepare a suspension from shrimp and crab seafood products;
[0010] Step 2, Fermentation: After sterilizing the suspension, inoculate it with the activated compound bacteria for fermentation to obtain a fermented suspension;
[0011] Step 3, Enzymatic hydrolysis: Add a compound enzyme to the fermentation suspension, and obtain the supernatant by enzymatic hydrolysis, enzyme inactivation, filtration, centrifugation, fractionation and ultrafiltration.
[0012] Step 4: Purification and Elution Process
[0013] 1) After adsorbing the corresponding amino acids using a polyvinyl alcohol pre-membrane, the pre-membrane saturated with adsorbed amino acids is mixed with the base membrane using a crosslinking agent and then electrospinned to obtain an amino acid-embedded electrospun membrane. The embedded amino acids are then eluted to obtain an electrospun membrane containing amino acid cavities. 2) The supernatant obtained in step 3 is purified and eluted using the electrospun membrane containing amino acid cavities to obtain an eluent solution, which is then spray-dried to obtain a seasoning.
[0014] The compound bacteria are selected from Staphylococcus xylose, acetic acid bacteria, and Staphylococcus pentosaceus; the compound enzyme is selected from alkaline protease, bromelain, and fig protease.
[0015] The amino acids mentioned in step four are selected from Ala, Asp, Asn, Glu, and Gln.
[0016] Optionally, the compound bacteria are obtained by mixing Staphylococcus xylose, acetic acid bacteria, and Staphylococcus pentosaceus in a mass ratio of (1-3):(0.5-6):1;
[0017] Optionally, the fermentation conditions are: fermentation temperature 20-40℃, fermentation time 20-72h, fermentation pH 3-7.5, and the amount of inoculum added is 0.5-10% of the weight of the suspension in step one.
[0018] Optionally, the complex enzyme is prepared from alkaline protease: bromelain: fig protease in a mass ratio of (0.5-3):(0.1-2):(1-5); the amount of complex enzyme added is 0.25-0.5% of the weight of the fermentation suspension in step two; and the enzymatic hydrolysate obtained after enzymatic hydrolysis in step three is filtered, centrifuged, and preliminarily purified.
[0019] Preferably, the filter sieve mesh size is 50-120 mesh, the centrifugation rate is 6000-12000 r / min; the centrifuged liquid is initially purified by fractional ultrafiltration, with the ultrafiltration membrane retaining molecular weights of >10kDa, 3-5kDa, and <3kDa respectively, the ultrafiltration pressure is 0.02-0.1MPa, the ultrafiltration temperature is 25-40℃, and the amount of water added for ultrafiltration is 0.5-5 times the mass of the enzymatic hydrolysate;
[0020] More preferably, the filter screen mesh size is 60-90 mesh; the centrifugation 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℃; and the ultrafiltration water addition is 2-3.5 times.
[0021] Optionally, in step four, the amino acids in the electrospun membrane with amino acid holes are any one of the following: 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, and 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 substrate in step four is a polymer material, including any one of aminated polyacrylonitrile, amino-functionalized polysiloxane, polyvinyl alcohol, and polyaspartic acid.
[0024] Preferably, it is any one of aminated polyacrylonitrile or amino-functionalized polysiloxane.
[0025] Optionally, in step four 1), the solution used for eluting the embedded amino acids in the amino acid-embedded electrospun membrane is a sodium hydroxide solution with a concentration of 0.2-3 M / L; preferably, it is 0.5-1.5 M / L.
[0026] Optionally, in step 4(1), the adsorption capacity of the electrospun membrane for amino acids is 45-90 mg / g.
[0027] Optionally, the shrimp include prawns, lobsters, and river shrimp; the crabs include swimming crabs, mud crabs, and river crabs.
[0028] Furthermore, the specific steps for the purification and elution process in step four are as follows:
[0029] 1) A 1-6% (w / w) polyvinyl alcohol aqueous solution is stirred until homogeneous, and after standing, degassing, and drying, a pre-film is obtained; the pre-film is placed in a 0.2-1 g / L amino acid aqueous solution for adsorption for 12-54 h, and then vacuum dried; the mass-to-volume ratio is: m 前置膜 / V 氨基酸水溶液 = (1mg / 2500mL) ~
[0030] (1 mg / 1500 mL).
[0031] Preferably, the amino acid mass concentration is 0.5%; the static adsorption time is any one of 18, 24, or 32 hours; and the mass-volume ratio is m 前置膜 / V 氨基酸水溶液 It is any one of 1 mg / 1200 mL, 1 mg / 1500 mL and 1 mg / 1600 mL.
[0032] Preferably, the electrospun membrane adsorbs 45-90 mg / g of amino acids. Preferably, the polyvinyl alcohol aqueous solution has a mass concentration of any one of 2.5%, 3%, and 3.5%.
[0033] 2) 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 = (1mg / 800mL) ~
[0034] (1mg / 1500mL) The pre-membrane after saturation adsorption of amino acids is placed in a mixture of water and ethanol, and a crosslinking agent of 0.5%-3% of the mass fraction of the pre-membrane after saturation adsorption of amino acids is added. The mixture is stirred at pH less than 7 and 40-60℃ to obtain mixture A.
[0035] Preferably, the crosslinking agent is any one of glutaraldehyde, polyethylene glycol diglycidyl ether, and EDC / NHS, and the addition amount is any one of 0.5%, 1.5%, and 2%.
[0036] 3) After dissolving the polymer substrate material, mix it with mixture A to obtain mixture B. Perform electrospinning treatment to obtain an amino acid embedded electrospinned membrane. After elution, obtain an electrospinned membrane containing amino acid cavities. Take out the electrospinned membrane containing amino acid cavities and soak it in the supernatant after centrifugation in step 3. Then take it out and place it in sodium hydroxide solution for elution. After spray drying, obtain the seasoning.
[0037] This application also provides a seasoning prepared by the method described above.
[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 xylose, acetic acid bacteria, and Staphylococcus pentosacchari) and complex enzymes (alkaline protease, bromelain, and fig protease) to process shrimp and crab using a method of fermentation followed by enzymatic hydrolysis. Compared to enzymatic hydrolysis followed by fermentation, or using other fermentation bacteria and complex enzymes, the product exhibits better reduction of allergens and removal of off-odors and fishy smells, as well as better stability and a longer shelf life. This is because the combination of specific fermentation bacteria and complex enzymes in this application (fermentation followed by enzymatic hydrolysis) produces a better synergistic effect, effectively breaking down large protein molecules in the substrate into a relatively high proportion of small umami peptides; thus greatly reducing the allergenicity of the seasoning product.
[0040] 2. This application utilizes an electrospun membrane with specific amino acid holes (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, thereby obtaining specific target peptides. Compared with peptides obtained by using liquid chromatography in the prior art, this method greatly saves time and effort, has high purification efficiency, is easy to obtain target peptides, and is free of impurities with higher purity.
[0041] Furthermore, this application also screened the materials of the electrospun membrane: the adsorption effect was best when the substrate material was amino-functionalized polyacrylonitrile or amino-functionalized polymethylsiloxane. The reason is that the surface of the above two electrospun membrane substrates is positively charged in this pH (pH < 7) environment, which is conducive to the adsorption of umami peptides into the cavities by strong electrostatic adsorption, which is conducive to the smooth progress of the purification process.
[0042] 3. The specific method of combining fermentation, enzymatic hydrolysis and purification in this application is more targeted to shrimp. The reason may be that shrimp / crab from different sources may have certain changes in protein structure and composition due to differences in living environment and evolution, and the allergens may also be different. Detailed Implementation
[0043] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials in the embodiments are all commercially available.
[0044] Example 1: A natural marine umami seasoning for shrimp and crab, 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 or river crab) in lightly salted water three times (20 min each time), then rinse with clean water, crush them and take 80g (40g each of shrimp and crab), add 3 times the weight of distilled water, then first homogenize under high pressure (working pressure: 18 MPa, speed: 6000 r / min, time: 15 min), and then make a uniform suspension by using a colloid mill (working pressure: 2 MPa, speed: 2000 r / min, time: 30 min).
[0047] Because high-pressure homogenizers can provide higher shear force and pressure, using a high-pressure homogenizer first can better crush and homogenize materials, and then using a colloid mill for further refinement and homogenization can achieve better results.
[0048] Step 2: Fermentation
[0049] The suspension prepared in step one is first sterilized, and then inoculated with activated compound bacteria (obtained by mixing Staphylococcus xylose, acetic acid bacteria and pentosococcus in a mass ratio of 1:1:1) for fermentation to obtain a fermentation suspension.
[0050] The fermentation temperature was 25℃, the fermentation time was 36h, the fermentation pH was 4.0, and the amount of inoculum added was 3.5% of the weight of the suspension.
[0051] Step 3: Enzymatic hydrolysis
[0052] Adjust the fermentation suspension obtained in step two to 6.0, and add 0.25% of the weight of the fermentation suspension with a compound enzyme (obtained by mixing alkaline protease, bromelain, and fig protease in a mass ratio of 1:1:1). Enzymatic hydrolysis is performed in an ultrasonic water bath at 45℃ and 260W for 5.5 hours; then, the enzyme is inactivated in a 100℃ water bath for 10 minutes. After the temperature drops to room temperature, the solution is filtered through an 80-mesh sieve, and the filtrate is centrifuged at 4℃ and 10000 r / min for 10 minutes.
[0053] The centrifuged liquid was initially purified by fractional ultrafiltration, with the molecular weight cutoffs of the fractional ultrafiltration membranes being >10kDa, 3-5kDa, and <3kDa (specifically 12kDa, 5kDa, and 2kDa in this embodiment), respectively. The ultrafiltration pressure was 0.02MPa, the ultrafiltration temperature was 30℃, and the amount of water added during ultrafiltration was twice the mass of the enzymatic hydrolysate. The centrifuged supernatant was then obtained.
[0054] Step 4: Purification and Elution Process
[0055] Purification and elution are intended to overcome the problems in existing technologies where fermented or enzymatically hydrolyzed products have other off-flavors and the freshness is not significantly improved.
[0056] 4.1) Preparation of electrospun membranes with amino acid holes
[0057] 4.1.1) A 2.5% (w / w) polyvinyl alcohol aqueous solution was stirred until homogeneous, and after standing, degassing, and drying, a pre-film was obtained. The pre-film was then placed in a 0.5 g / L amino acid aqueous solution (amino acids Ala, Asp, and Asn were added sequentially in a 1:1:1 mass ratio) for adsorption (mass-volume ratio: m...). 前置膜 / V 氨基酸水溶液 =1mg / 1500mL) After 12 hours, the membrane was removed and vacuum dried at 60℃ for 24 hours to obtain a pre-membrane saturated with amino acids. The pre-membrane saturated with amino acids was then 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 =1mg / 800mL), add 0.5% of the crosslinking agent (glutaraldehyde) as the mass fraction of the pre-membrane after saturation adsorption of amino acids, and stir for 12h at pH=6 and 50℃ to obtain mixture A.
[0058] Select 1g of the polymer base film material - aminated polyacrylonitrile (PAN) - and dissolve it in 16ml of DMF solvent. Place a magnetic rotor in the mixed solution and seal it with plastic wrap to prevent interference from the external environment. Stir magnetically until dissolved. Then add the prepared mixture A and continue stirring magnetically for 12h to obtain mixture B.
[0059] 4.1.2) Introduce 3 ml of mixture B into a 5 ml plastic syringe equipped with a No. 22 needle. 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 electrospinned membrane). Vacuum dry at 40 °C for 12 h and place in a self-sealing bag for later use (electrospinning process).
[0060] 4.1.3) Take a piece of the prepared nanofiber membrane and elute it in 50 mL of 1.0 mol / L sodium hydroxide solution for 3 h. Since sodium hydroxide is a strong base, when sodium hydroxide comes into contact with amino acids, it will destroy the chemical structure of the amino acids. The hydrolysis of sodium hydroxide can also destroy the linkage between the amino acids and the membrane, causing the amino acids to fall off the membrane. This forms specific amino acid (Ala, Asp, Asn) cavities on the amino acid embedded electrospun membrane (the supernatant after centrifugation contains several peptides composed of different amino acids. The formation of specific amino acid cavities can be used to lock / screen the umami peptides composed of the specific amino acids to be adsorbed onto the fiber membrane, thereby obtaining the target peptide), and obtain an electrospun membrane containing amino acid cavities.
[0061] 4.1.4) Then, remove the membrane and immerse it in the supernatant after centrifugation in step three for 12 hours. During this process, the three amino acids Ala, Asp, and Asn present in the supernatant are adsorbed into the amino acid cavities by the membrane (greatly saving time and effort, improving purification efficiency, and making it easier to obtain the target peptide). After adsorption is complete, remove the membrane and elute it in 1.0 mol / L sodium hydroxide solution for 3 hours to elute the target peptide adsorbed on the membrane.
[0062] The final electrospun membrane with specific amino acid cavities had an adsorption capacity of 60 mg / g for the corresponding amino acid.
[0063] Step 5: Spray dry the eluted solution to obtain the seasoning.
[0064] Example 2
[0065] A natural umami seasoning for shrimp and crab is prepared by the following method:
[0066] Step 1: Raw material preparation
[0067] Soak shrimp (Chinese prawn) and crab (swimming crab) in lightly salted water four times (20 min each time), then rinse with clean water, crush them and take 80g (40g each of shrimp and crab), add 3 times their weight of distilled water, then first homogenize under high pressure (working pressure: 25 MPa, speed: 8000 r / min, time: 30 min), and then make a uniform suspension by using a colloid mill (working pressure: 6 MPa, speed: 1500 r / min, time: 20 min).
[0068] Step 2: Fermentation
[0069] The suspension prepared in step one is first sterilized, and then inoculated with activated compound bacteria (obtained by mixing Staphylococcus xylose, acetic acid bacteria and pentosococcus in a mass ratio of 3:3:1) for fermentation to obtain a fermentation suspension.
[0070] The fermentation temperature was 40℃, the fermentation time was 20h, the fermentation pH was 6.0, and the amount of inoculum added was 7% of the weight of the suspension.
[0071] Step 3: Enzymatic hydrolysis
[0072] Adjust the fermentation suspension obtained in step two to 6.0, and add 0.25% of the weight of the fermentation suspension with a compound enzyme (obtained by mixing alkaline protease: bromelain: fig protease in a mass ratio of 0.5:2:1). Enzymatically hydrolyze the enzyme in an ultrasonic water bath at 45℃ and 260W for 5.5 hours; then inactivate the enzyme in a 100℃ water bath for 10 minutes. After the temperature drops to room temperature, filter the solution through an 80-mesh sieve, and centrifuge the filtrate at 4℃ and 10000 rpm for 10 minutes.
[0073] The centrifuged liquid was initially purified by staged ultrafiltration, with the three ultrafiltration membranes having molecular weight cutoffs of >10kDa, 3-5kDa, and <3kDa (specifically 12kDa, 5kDa, and 2kDa in this embodiment). The ultrafiltration pressure was 0.02MPa, the ultrafiltration temperature was 30℃, and the amount of water added during ultrafiltration was twice the mass of the enzymatic hydrolysate; the centrifuged supernatant was obtained.
[0074] Step 4: Purification and Elution Process
[0075] Purification and elution are intended to overcome the problems in existing technologies where fermented or enzymatically hydrolyzed products have other off-flavors and the freshness is not significantly improved.
[0076] 4.1) Preparation of electrospun membranes with amino acid holes
[0077] 4.1.1) A 6% (w / w) polyvinyl alcohol aqueous solution was stirred until homogeneous, and after standing, degassing, and drying, a pre-film was obtained; the pre-film was then placed in a 0.2 g / L amino acid aqueous solution (amino acids Ala, Asp, and Asn were added in a mass ratio of 1:1:1) for adsorption (mass-volume ratio: m... 前置膜 / V 氨基酸水溶液 =1mg / 1500mL) After 12 hours, the membrane was removed and vacuum dried at 60℃ for 24 hours. The membrane saturated with amino acid adsorption was then 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] =1mg / 800mL), add 2% of the mass fraction of crosslinking agent (polyethylene glycol diglycidyl ether) to the pre-membrane after saturation adsorption of amino acids, and stir for 12h at pH=6 and 50℃ to obtain mixture A.
[0079] Select 1g of the polymer base film material - aminated polysiloxane, dissolve it in 16ml of DMF solvent, place a magnetic rotor in the mixed solution and then seal it with plastic wrap to prevent interference from the external environment, and magnetically stir until dissolved; then add the prepared mixture A, and continue magnetic stirring for 12h to obtain mixture B.
[0080] 4.1.2)-4.1.3) Same as Example 1.
[0081] Step 5: Same as Example 1.
[0082] Example 3
[0083] The difference from Example 1 is that only shrimp (Chinese shrimp) was selected as the raw material, and 80g was taken after crushing; the rest is the same as in Example 1.
[0084] Example 4
[0085] The difference from Example 1 is that only crab (Chinese mitten crab, also known as hairy crab or river crab) is selected as the raw material, and 80g is taken after crushing; the rest is the same as in Example 1.
[0086] The difference between Examples 5-13 and Example 1 is that the amino acids added in step four, purification and elution, are different (in each example, the mass fractions of different amino acids added are equal), while the remaining steps are the same as in Example 1. See Table 1 below for details.
[0087] Table 1. Different amino acid addition settings
[0088]
[0089]
[0090] Note: The five amino acids Ala (where Ala represents β-alanine), Asp (where Asp represents DL-aspartic acid), Asn (where Asn represents D-asparagine), Glu (where Glu represents L-glutamic acid), and Gln (where Gln represents L-glutamine) were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
[0091] Compared with Example 1, Comparative Example 1 did not undergo purification elution.
[0092] Comparative Example 2
[0093] Compared with Example 1, the compound strain used in step two is different. It is prepared by Bacillus licheniformis: Candida utilis: Lactobacillus plantarum in a mass ratio of 1:1:1; the amount added and other steps are the same as in Example 1.
[0094] Comparative Example 3
[0095] Compared with Example 1, the compound enzyme used in step three is different. It is prepared by chymotrypsin, 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 steps (2) and (3) is changed, that is, the enzyme is first hydrolyzed and then fermented. Specifically, after step (2), the enzyme is first added to the sterilized suspension for hydrolysis, and then the compound bacteria are inoculated into the supernatant after hydrolysis for fermentation.
[0098] Comparative Example 5
[0099] Compared with Example 1, the material of the electrospun film is different: 1 g of polymer material polypropylene (PP) was selected, and the rest is the same as in Example 1.
[0100] Example 14
[0101] Compared to Example 1, the source of the crab is different; the crab described is a commercially available king crab.
[0102] The degree of hydrolysis, peptide yield, and umami intensity of the supernatant (i.e., the supernatant after enzymatic hydrolysis and fermentation in step three) prepared in the above embodiments and comparative examples 2-4 were tested, and the results are shown in the table below.
[0103] The existing ultraviolet spectrophotometry method is used to detect peptides. Peptides have a certain absorbance at a specific wavelength. By measuring the absorbance of the sample at that wavelength, the content of peptides can be calculated, and then the peptide yield can be calculated.
[0104] Table 2. Detection results of supernatant
[0105]
[0106] (Note: Umami intensity is scored from 0 to 10, where 0 indicates no taste, 10 indicates strong umami, and 5 indicates the umami intensity of 0.35% MSG.)
[0107] Conclusion: According to the data in the table above, compared with Comparative Example 2 (different strains), Comparative Example 3 (different enzymes), and Comparative Example 4 (enzymatic hydrolysis followed by fermentation), the hydrolysis degree and peptide yield of the supernatant in the examples were significantly higher than those of the comparative examples, and the umami intensity was more pronounced. Among them, Examples 1, 2, and 5 showed the best results.
[0108] Higher degree of hydrolysis and peptide yield mean that more protein is hydrolyzed into smaller peptide molecules, significantly reducing allergenicity. Furthermore, these peptides are not only more easily absorbed by the body but also retain more biological activity, thus providing greater health benefits.
[0109] The good results of the examples may be due to the fact that the specific compound strains of this application produce some metabolites during the growth process. These metabolites can change the structure of the protein substrate, making it easier for it to be hydrolyzed by the specific enzyme preparations in the examples.
[0110] The seasonings prepared in the above embodiments and comparative examples were subjected to index testing, and the results are shown in Table 3 below. Among them, the main shrimp and crab allergen protein (tropomyosin) was directly detected and analyzed by mass spectrometry (tandem mass spectrometry MS / MS) ion monitoring mass spectrometry, which is a technique in the prior art. The detection method is referenced from (Zhao Xin, 2022).
[0111] Allergen reduction rate % = (Allergen content in the untreated suspension - Allergen content in the treated seasoning product) / Allergen content in the untreated suspension.
[0112] Table 3. Effects of different treatment processes on condiment products
[0113]
[0114]
[0115]
[0116] Conclusion: According to the data in the table above, compared with the comparative examples, the seasoning products obtained in Examples 1-13 have a higher allergenicity reduction rate, reaching 65%; even after 6 months of storage, the allergenicity reduction rate can still reach more than 60%. Among them, Examples 1-4 have the best overall effect.
[0117] The seasonings obtained in the comparative ratio not only had a relatively low rate of reduction in allergenicity, but also had unpleasant flavors such as fishiness, and the umami intensity was also relatively weak.
[0118] Compared to Comparative Example 2 with different compound microbial strains, Comparative Example 3 with different compound enzymes, and Comparative Example 4 with enzymatic hydrolysis followed by fermentation, the reason for this difference is that the specific fermentation bacteria in the examples produce a series of enzymes and metabolites during their growth and metabolism. When used in combination with the enzyme preparations added in the examples, the enzymes produced by the microbial strains and the added enzyme preparations can work synergistically to transform and decompose substances that produce a fishy smell, thereby significantly reducing the fishy smell; and in this process, they effectively decompose large molecular proteins in the substrate into relatively high proportions of small molecular umami peptides; greatly reducing the allergenicity of the seasoning products.
[0119] In existing technologies, liquid chromatography is often used for elution. The resulting seasonings typically have a sensitization reduction rate of around 15%, and the products have off-flavors and a weak umami taste. This is because liquid chromatography elution not only separates umami peptides but also other peptides (including those with off-flavors and high sensitization rates). It is impossible to specifically separate umami peptides individually, resulting in products with noticeable off-flavors and a low sensitization reduction rate.
[0120] Compared to Example 14, the specific enzymes and strains used in this application may not be as effective at removing the fishy odor of umami peptides from king crab. This is because different crab sources, due to variations in water quality and food sources, can influence the off-flavor of umami peptides.
[0121] Furthermore, the products prepared in Examples 1-5 of this application, after packaging, all exhibit a distinct fresh and fragrant aroma within 2 seconds of opening. Examples 6-14 also exhibit a distinct fresh and fragrant aroma within 3-5 seconds.
[0122] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.
[0123] References:
[0124] Zhao Xin. (2022). Detection and identification of aquatic product allergens based on mass spectrometry. (Master's thesis). Retrieved from https: / / link.cnki.net / doi / 10.27314 / d.cnki.gsscu.2022.000918 Available from Cnki.
Claims
1. A method for preparing a shrimp, crab marine natural fresh flavor seasoning, characterized in that, include: Step 1: Prepare a suspension from shrimp and crab seafood products; Step 2, Fermentation: After sterilizing the suspension, inoculate it with the activated compound bacteria for fermentation to obtain a fermented suspension; Step 3, Enzymatic hydrolysis: Add a compound enzyme to the fermentation suspension, and obtain the supernatant by enzymatic hydrolysis, enzyme inactivation, filtration, centrifugation, fractionation and ultrafiltration. Step 4: Purification and Elution Process 1) After adsorbing the corresponding amino acids using a polyvinyl alcohol pre-film, the pre-film after saturation adsorption of amino acids is mixed with the base film using a crosslinking agent and then obtained by electrospinning to obtain an amino acid embedded electrospun film. The embedded amino acids are eluted, and the elution solution is a sodium hydroxide solution, to obtain an electrospun film containing amino acid cavities. 2) The supernatant obtained in step 3 is purified and eluted using an electrospun membrane containing amino acid holes to obtain an eluent solution, which is then spray-dried to obtain a seasoning. The substrate is either aminated polyacrylonitrile or amino-functionalized polysiloxane. The crosslinking agent is either glutaraldehyde or polyethylene glycol diglycidyl ether. The compound bacteria are Staphylococcus xylose, acetic acid bacteria, and Pentosacchariformis, with a mass ratio of (1-3):(0.5-6):1; the compound enzyme is alkaline protease, bromelain, and fig protease, with a mass ratio of (0.5-3):(0.1-2):(1-5). The amino acids mentioned in step four are selected from Ala, Asp, Asn, Glu, and Gln.
2. The preparation method according to claim 1, characterized in that, The fermentation conditions are as follows: fermentation temperature 20-40℃, fermentation time 20-72h, fermentation pH 3-7.5, and the amount of inoculum 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 amount of compound enzyme added is 0.25-0.5% of the weight of the fermentation suspension in step two.
4. The preparation method according to claim 1, characterized in that, The filtration process uses a filter sieve with a mesh size of 50-120 mesh and a centrifugation rate of 6000-12000 r / min. The centrifuged liquid is then preliminarily purified by fractional ultrafiltration, with the ultrafiltration membranes retaining molecular weights of >10 kDa, 3-5 kDa, and <3 kDa respectively. The ultrafiltration pressure is 0.02-0.1 MPa, the ultrafiltration temperature is 25-40℃, and the amount of water added during ultrafiltration is 0.5-5 times the mass of the enzymatic hydrolysate.
5. The preparation method according to claim 1, characterized in that, The filter screen has a mesh size of 60-90; the centrifugation rate is 8000-10000 r / min; the ultrafiltration pressure is 0.02 MPa, 0.03 MPa or 0.05 MPa; the ultrafiltration temperature is 30℃; and the ultrafiltration water addition is 2-3.5 times.
6. The preparation method according to claim 1, characterized in that, In step four, the amino acids in the electrospun membrane containing amino acid holes are any one of the following: 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, and Glu.
7. The preparation method according to claim 1, characterized in that, The amino acids in the electrospun membrane containing amino acid holes are any one of Ala, Asp, and Asn; Glu and Gln; Ala, Asp, Asn, Glu, and Gln; or Ala, Asp, and Glu.
8. The preparation method according to claim 1, characterized in that, In step 4, 1), the concentration of the sodium hydroxide solution is 0.2-3 mol / L. In step 4, the electrospun membrane adsorbs 45-90 mg / g of amino acids.
9. The preparation method according to claim 1, characterized in that, The concentration of sodium hydroxide solution is 0.5-1.5 mol / L.
10. The preparation method according to claim 1, characterized in that, The shrimp mentioned include prawns, lobsters, and river shrimp; the crabs mentioned include swimming crabs, mud crabs, and river crabs.
11. The preparation method according to claim 1, characterized in that, The specific steps for the purification and elution process in step four are as follows: A 1-6% (w / v) polyvinyl alcohol aqueous solution was stirred until homogeneous, and then allowed to stand, degas, and dry to obtain a pre-film. The pre-film was placed in a 0.2-1 g / L amino acid aqueous solution for adsorption for 12-54 h and then vacuum dried. The mass-volume ratio was: m pre-film / V amino acid aqueous solution = (1 mg / 2500 mL) ~ (1 mg / 1500 mL). According to the mass-volume ratio m of the pre-membrane after saturation adsorption of amino acids / V of the mixture of water and ethanol = (1 mg / 800 mL) ~ (1 mg / 1500 mL), the pre-membrane after saturation adsorption of amino acids is placed in the mixture of water and ethanol, and a crosslinking agent accounting for 0.5%-3% of the mass fraction of the pre-membrane after saturation adsorption of amino acids is added. The mixture is stirred at pH less than 7 and 40-60℃ to obtain mixture A. The polymer substrate material is dissolved and mixed with mixture A to obtain mixture B. Electrospinning is then performed to obtain an amino acid embedded electrospinned membrane. After elution, an electrospinned membrane containing amino acid cavities is obtained. The electrospinned membrane containing amino acid cavities is then taken out and immersed in the supernatant after centrifugation in step three. It is then taken out and eluted in sodium hydroxide solution. After spray drying, the seasoning is obtained.
12. The preparation method according to claim 11, characterized in that, The amount of crosslinking agent added is 0.5-2%.
13. The preparation method according to claim 11, characterized in that, The amount of crosslinking agent added is any one of 0.5%, 1.5%, or 2%.
14. The seasoning prepared by the preparation method according to any one of claims 1-13.