Salt denitrifying mycobacillus capable of improving quality of low-salt shrimp paste and producing high-yield protease

Abstract

The invention discloses a high-yield protease salt denitration mycobacillus for improving the quality of low-salt shrimp paste, and belongs to the field of food biotechnology and application. The salt denitration mycobacillus XJ-229 provided by the invention has relatively good salt tolerance (the tolerance range is 1%-22%) and enzyme production (the protease production is 15.38 U / mL) capacity, and a proper fermentation agent is provided for fermentation of low-salt shrimp paste. The cladobacillus sp. XJ-229 is used for preparing shrimp paste, the content of volatile basic nitrogen, malondialdehyde and biogenic amine can be reduced, the content of free amino acid can be increased, the flavor of the shrimp paste can be improved, and the prepared shrimp paste can be accepted by people more easily. Therefore, the salt denitration mycobacillus XJ-229 provided by the invention is beneficial to improving the quality of the shrimp paste and improving the market acceptability of the shrimp paste.

Description

Technical Field

[0001] The invention belongs to the field of food biotechnology and application, and in particular relates to a high-yield protease-producing bacillus denitrificans for improving the quality of low-salt shrimp paste. Background Art

[0002] Shrimp paste is a seasoning made from shrimp and salt in a certain proportion and fermented, and is very popular among the public. The types of shrimp paste in different regions are different, and their production processes and flavors vary. Traditional shrimp paste not only has a long fermentation cycle and low production efficiency, but also has a high salt content, generally higher than 25%, which does not meet people's needs for a healthy diet. In recent years, in order to solve the limitations of traditional shrimp paste, a low-salt shrimp paste has emerged. Studies have reported a low-salt shrimp paste whose salt content can be reduced to about 12%. Compared with traditional shrimp paste, although the fermentation cycle and salt content of low-salt shrimp paste are significantly reduced, it may have problems such as poor flavor, texture and palatability.

[0003] The unique flavor of shrimp paste is not only one of the important reasons for its popularity, but also an important parameter for evaluating its quality. The unique flavor of shrimp paste is related to a variety of enzymes, especially proteases, which can convert proteins into peptides, amino acids and small molecular nitrogen-containing substances, thus giving shrimp paste a unique flavor and aroma. Proteases in the fermentation process of shrimp paste are often believed to come from two pathways, one is the endogenous enzymes of shrimp, and the other is from enzyme-producing salt-tolerant (halophilic) microorganisms. Since shrimp paste fermentation is a high osmotic pressure system, the activity of endogenous enzymes is limited or even inactive, resulting in low efficiency in the hydrolysis of proteins and fats. Compared with endogenous enzymes, these resistant (halophilic) microorganisms can grow, metabolize and reproduce in this high-salt system, and continuously provide proteases and other enzymes, thereby shortening the fermentation cycle of shrimp paste, improving the quality of shrimp paste and changing the overall flavor of shrimp paste.

[0004] Based on omics sequencing technology, some salt-tolerant (halophilic) microorganisms, such as Staphylococcus, Bacillus, Tetragenococcus, Salimicrobium, Salinicoccus, Virgibacillus and Lactobacillus, are believed to exist in shrimp paste of different types or fermentation stages, and play a dominant role in the fermentation process of shrimp paste. These tolerant (halophilic) microorganisms have been confirmed to have a certain correlation with alcohols, pyrazines, aldehydes, ketones and nitrogen-containing compounds in shrimp paste, and have an important impact on the nutritional quality and flavor of shrimp paste. Halodenitrifying Cladosporium is a group of halophilic microorganisms with good salt tolerance and protease production ability. It can grow at a salt concentration of 15% or higher, and the extracellular protease produced can catalyze the hydrolysis of shrimp protein. "Characteristics and Applications of Bioamine Degrading Bacteria in Grasshopper Shrimp Paste" clearly pointed out that Halodenitrifying Cladosporium can be used as a starting starter for fermenting aquatic products. In the prior art, Bacillus halodenitrificans has been widely reported to be used in the production of shrimp paste. For example, the document "Virgibacillus halodenitrificans MSK-10P, a Potential Protease-producing Starter Culture for Fermented Shrimp Paste (kapi) Production" reports a strain of Bacillus halodenitrificans MSK-10P with high protease production, and the protease production reaches 9.47U / mL. However, the protease production of the strain is still low and cannot meet the needs of industrial production.

[0005] The prior art has not reported a salt-denitrifying Cladosporium that can produce high levels of protease and is suitable for rapid fermentation of low-salt shrimp paste. Therefore, making full use of this bacterium as a starter for shrimp paste and finding a salt-denitrifying Cladosporium that is suitable for low-salt shrimp paste fermentation and produces high levels of protease is of great significance for shortening the fermentation cycle of shrimp paste and improving the flavor quality. Summary of the invention

[0006] To solve the above problems, the object of the present invention is to provide a protease-producing halophilic bacteria strain Virgibacillus halodenitrificans XJ-229, which has been deposited in the China Center for Type Culture Collection on July 22, 2024, with a deposit number of CCTCC NO: M 20241629, and a deposit address of Wuhan University, Wuhan, China.

[0007] The monoclonal colony of the salt-denitrifying Cladosporium of the present invention is characterized by milky white, round or oval, smooth and opaque colony surface, neat strain edges, and easy to pick. Figure 1The salt-denitrifying Cladosporium XJ-229 of the present invention has the following excellent properties:

[0008] (1) Derived from traditional high-salt food samples, such as shrimp paste, salted fish, ham, etc.;

[0009] (2) When applied to high-salt fermented food systems, it will not affect the normal fermentation of high-salt fermented foods;

[0010] (3) It grows vigorously in high-pressure, high-salt fermentation food systems and has excellent protease production capabilities;

[0011] (4) It has a good ability to degrade large molecular proteins in the raw materials of the fermentation system.

[0012] In order to achieve the above object, the present invention adopts the following technical measures:

[0013] The present invention also provides a method for screening and identifying salt-denitrifying Cladosporium XJ-229, the specific steps of which are as follows:

[0014] Transfer 2 g shrimp paste to liquid TSB medium containing 5% to 15% sodium chloride and culture on a shaker at 37°C and 120 to 150 rpm for 5 to 8 h, then dilute to 10 -2 ~10 -5 The dilution was then spread on a solid plate medium with the corresponding salt concentration and cultured at 37°C for 24 to 48 hours. A single colony was picked for purification and separation, and the 16Sr RNA molecular sequence was used to identify a strain of Bacillus halodenitrificans XJ-229. The screened strain was streaked and inoculated in different TSB media to verify its tolerance. The screened strain was inoculated on skim milk medium to verify its protease production ability.

[0015] The invention also provides a microbial preparation containing the salt-denitrifying Cladosporium XJ-229.

[0016] In one embodiment of the present invention, the microbial preparation comprises one or more of living cells, inactivated cells, fermentation broth, lysate or extract of the salt-denitrifying Cladosporium XJ-229.

[0017] The invention also provides a product containing the salt-denitrifying Cladosporium XJ-229 or the microbial preparation.

[0018] In one embodiment of the present invention, the product includes food, medicine or health care product.

[0019] In one embodiment of the present invention, the food comprises fermented food; the fermented food comprises fruit and vegetable products, dairy products, bean products, and meat products; optionally, the fermented food comprises shrimp paste.

[0020] In one embodiment of the present invention, the dosage form of the drug includes powder, granules, capsules, tablets, pills or oral liquid.

[0021] The invention also provides a meat sauce starter, which contains the salt-denitrifying Cladosporium XJ-229.

[0022] In one embodiment of the present invention, the number of the salt-denitrifying Cladosporium XJ-229 in the meat sauce starter is ≥1×10 6 CFU / mL or 1×10 6 CFU / g.

[0023] The invention also provides an application method of the salt-denitrifying Cladosporium XJ-229 in the fermentation of low-salt shrimp paste.

[0024] In one embodiment of the present invention, the salt-denitrifying Cladosporium XJ-229 or the microbial preparation is added to shrimp products for fermentation.

[0025] In one embodiment of the present invention, the shrimp products include but are not limited to shrimp minced meat, shrimp meat chunks, and shrimp meat extracts.

[0026] In one embodiment of the present invention, the application method of the salt-denitrifying Cladosporium XJ-229 is to inoculate the strain into a liquid TSB medium containing 10% to 18% NaCl and activate it twice continuously, and use the same medium to adjust the strain to 10 6 ~10 8 CFU / mL, added into shrimp paste at a certain mass percentage and fermented under certain conditions.

[0027] In one embodiment of the present invention, the salt content of the low-salt shrimp paste is 10% to 18% (w / w).

[0028] In one embodiment of the present invention, the certain mass percentage is 1% to 6% (v / w).

[0029] In one embodiment of the present invention, the fermentation under certain conditions is a fermentation temperature of 10 to 40° C. and a fermentation time of 15 to 30 days.

[0030] In one embodiment of the present invention, the shrimp paste added includes adding salt-denitrifying Bacillus bacteria liquid alone, or adding a mixed bacterial liquid of salt-denitrifying Bacillus XJ-229 in combination with one or more other strains.

[0031] In one embodiment of the present invention, the combined use is addition, formulation or compounding.

[0032] In one embodiment of the present invention, the mixed bacterial solution contains ≥1×10 6 CFU / mL.

[0033] The present invention also provides the use of the salt-denitrifying Cladosporium XJ-229 or the microbial preparation in preparing shrimp paste or products containing shrimp paste.

[0034] Beneficial effects:

[0035] (1) The present invention provides and confirms a salt-denitrifying Bacillus XJ-229 through experiments such as salt tolerance comparison, enzyme production ability comparison, morphological characteristics and molecular biological sequence analysis. The strain has good salt tolerance (tolerance range is 1%-22%) and enzyme production (protease production 15.38U / mL), providing a suitable starter for low-salt shrimp paste fermentation.

[0036] (2) The salt-denitrifying Cladosporium XJ-229 of the present invention was inoculated into low-salt shrimp paste at a 2% inoculation rate and fermented at 37°C for 30 days. Compared with the uninoculated shrimp paste, the volatile basic nitrogen content was reduced from 135.13±6.65 mg / 100 g to 118.04±6.31 mg / 100 g, the malondialdehyde content was reduced from 4.62±0.22 mg / kg to 3.90±0.24 mg / kg, the biogenic amine content was reduced from 432.34±13.34 mg / kg to 326.14±5.85 mg / kg, and the free amino acid content was increased from 8295.65±13.45 mg / 100 g to 8506.71±13.16 mg / kg. Sensory analysis showed that the shrimp paste fermented with low salt enhanced by the bacteria was more acceptable to humans. Therefore, the fermentation of low-salt shrimp paste with salt-denitrifying Cladosporium XJ-229 can promote the fermentation of low-salt shrimp paste by improving the nutritional content of shrimp paste, enhancing the flavor quality and reducing harmful substances.

[0037] Biomaterial Deposit

[0038] A strain of Virgibacillus halodenitrificans XJ-229, taxonomically named Virgibacillus halodenitrificans, was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number CCTCCNO: M 20241629, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Colony morphology of Cladobacillus halodenitrificans XJ-229;

[0040] Figure 2Phylogenetic tree of Cladobacillus halodenitrificans XJ-229 constructed using MEGA11;

[0041] Figure 3 OD values ​​of strains under different salt concentrations;

[0042] Figure 4 OD values ​​of strains at different pH values;

[0043] Figure 5 OD values ​​of strains at different temperatures;

[0044] Figure 6 Hydrolysis zones of different halophilic strains from shrimp paste on skimmed milk powder plates;

[0045] Figure 7 Comparison of protease production ability of different halophilic (halophilic) strains from shrimp paste;

[0046] Figure 8 Types and contents of biogenic amines in shrimp paste inoculated and uninoculated with XJ-229;

[0047] Fig. 9 Radar chart of sensory scores in shrimp paste inoculated and not inoculated with XJ-229. DETAILED DESCRIPTION

[0048] In order to fully understand the present invention, the inventors describe it in detail with reference to the following specific examples. Obviously, the specific embodiments described below are only some embodiments of the present invention. The present invention can be implemented in many other ways different from those described herein, and is therefore not limited by the embodiments disclosed below. Those skilled in the art can make similar promotions without violating the connotation of the invention. However, all other embodiments obtained by ordinary technicians in the field of the present invention without creative work are within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, culture media and equipment used in the present invention can be obtained commercially.

[0049] Terminology explanation:

[0050] The terms "comprising", "including", "having", and "containing" used in the present invention mean "including but not limited to", "including but not limited to", "having but not limited to", and "containing but not limited to", and can be used interchangeably with corresponding phrases. Unless the context clearly indicates otherwise, the term "or" used in this article is used in this article to mean the term "and / or" and can be used interchangeably with it. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, the examples of suitable methods and materials described below are illustrative only and do not limit the present invention in any way.

[0051] The term "culturing" as used herein refers to the cultivation of the microorganisms for a period of time until a desired goal is achieved.

[0052] The term "fermentation broth" used in this article refers to liquid culture medium inoculated with microbial strains. After a period of cultivation, the microorganisms use the nutrients in the culture medium to synthesize bacteria and secrete products. This liquid metabolized by the microorganisms is called fermentation broth.

[0053] The term "strain extract" as used herein refers to an extract obtained by fermentation of the Cladosporium halodenitrificans strain, which is inoculated into a suitable medium, fermented under conventional fermentation conditions to synthesize and secrete the product into the medium, and then purified.

[0054] The term "strain lysate" used herein refers to the lysate of the strain obtained by inoculating cultured Cladosporium halodenitrificans into bacterial lysis solution, lysing under conventional conditions, and then centrifuging and purifying.

[0055] The term "low-salt shrimp paste fermentation" used in the present invention refers to obtaining shrimp paste by fermentation under the condition of less than 18% NaCl.

[0056] The term "microbial preparation" as used in the present invention refers to a solid, liquid or semisolid preparation containing one or more of living cells, inactivated cells, fermentation broth, lysate or extract of a microorganism.

[0057] In the following examples, the liquid TSB culture medium formula involved is as follows:

[0058] Tryptone 17.0g / L, soytone 3.0g / L, glucose 2.5g / L, K2HPO4 2.5g / L, pH 6.8. Solid culture medium needs to add 20g / L agar. Add sodium chloride according to the specific situation and sterilize at 121℃ for 20min.

[0059] In the following examples, the skimmed milk powder culture medium formula involved is as follows:

[0060] Yeast extract powder 2g / L, skim milk powder 10g / L, glucose 2g / L, NaCl 60g / L, agar 20g / L, pH 7.0. Skim milk powder and other reagents should be sterilized separately at 115℃ for 20min.

[0061] Example 1 Screening and Identification of Salt-Denitrifying Cladosporium XJ-229

[0062] (1) Screening and colony morphology observation

[0063] 2 g shrimp paste was transferred to liquid TSB medium containing 10% sodium chloride and cultured on a shaker at 37°C and 150 rpm for 8 h, and then diluted to 10 -3 , 10 -4 , 10 -5 The dilution was then spread on solid TSB medium with the corresponding salt concentration and cultured at 37°C for 36 hours. The strain was purified to a single colony on the same medium and the colony morphology was observed.

[0064] A single colony of XJ-229 was obtained by purification and isolation, such as Figure 1 As shown, its morphological characteristics are milky white, round or oval, smooth and opaque, with neat edges in TSB solid culture medium.

[0065] (2) Molecular Biological Identification

[0066] The isolated single strain colony of XJ-229 was mixed evenly with 100uL sterilized distilled water. 1uL of bacterial solution was taken and added to the PCR reaction system (12.5uL Taq enzyme solution, 1uL upstream primer, 1uL downstream primer, 9.5uL sterile water) and mixed evenly before PCR amplification. The upstream and downstream primers were 27F (nucleotide sequence 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (nucleotide sequence 5'-TACGGCTACCTTGTTACGACTT-3'). Agarose gel electrophoresis was used to detect whether the PCR product was qualified, and the qualified product was subjected to 16Sr RNA molecular sequence identification. The phylogenetic tree was constructed using the adjacent connection method in MEGA11 software.

[0067] The sequence length of the XJ-229 strain obtained by Shanghai Biotechnology is 1467 bp, and its nucleotide sequence is shown in SEQ ID No: 1. The phylogenetic tree shows that the similarity between XJ-229 and Virgibacillus halodenitrificans strain SF-121 is 99.79%, and they are in the same branch, such as Figure 2As shown. Therefore, combining the morphological characteristics, XJ-229 was identified as Virgibacillus halodenitrificans, and XJ-229 was named Virgibacillus halodenitrificans XJ-229. SEQ ID No.1:

[0068] GCCATGCGGCGTGCTATACATGCAGTCGAGCGCGGGAAGCAAGCTGATCCTCTTCGGAG

[0069] GTGACGCTTGTGGAACGAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTA

[0070] AGACTGGGATAACCCCGGGAAACCGGGGCTAATACCGGATAATACTTTTCATCACCTGAT

[0071] GGAAAGTTGAAAGGTGGCTTCTTGCTACCACTTACAGATGGGCCCGCGGCGCATTAGCT

[0072] AGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGAT

[0073] CGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAAT

[0074] CTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCG

[0075] GATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCGAATAGGGCGGCACCTTG

[0076] ACGGTACCTAACCAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG

[0077] GGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTC

[0078] TGATGTGAAAGCCCACGGCTTAACCGTGGAGGGTCATTGGAAACTGGAGGACTTGAGT

[0079] ACAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGA

[0080] ACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTG

[0081] GGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTG

[0082] TTAGGGGGTTTCCGCCCCTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAG

[0083] TACGGCCGCAAGGCTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAG

[0084] CATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGCAAT

[0085] CGGTAGAGATACCGAGTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTC

[0086] AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGT

[0087] TGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTG

[0088] GGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGAT

[0089] GGAACAAAGGGAAGCAAAACCGCGAGGTCAAGCAAATCCCATAAAACCATTCTCAGTT

[0090] CGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAG

[0091] CATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGT

[0092] TGGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGTGACAAGTT.

[0093] The Virgibacillus halodenitrificans XJ-229 has been deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number CCTCC NO: M 20241629, and the deposit address is Wuhan University, Wuhan, China.

[0094] Example 2 Comparison of tolerance and enzyme production ability of salt-denitrifying Cladosporium XJ-229

[0095] This example compares the salt tolerance and enzyme production capacity of the salt-denitrifying Cladosporium XJ-229 isolated in Example 1, as follows:

[0096] (1) Strain tolerance growth experiment

[0097] a) Salt tolerance

[0098] TSB medium (pH 7.0) containing 0%, 1%, 2%, 3%, 6%, 9%, 12%, 15%, 18%, 21%, 22%, 23%, 24% and 27% salt (NaCl) concentrations were prepared respectively, and the bacterial solution activated at 37°C and 150 rpm for 18 h was inoculated into the medium at an inoculum size of 3% (v / v). The initial OD 600 0.03, 37°C, 150 rpm, culture for 24 h. Pipette 0.2 mL of bacterial solution and measure the OD of the bacterial solution with a spectrophotometer. 600 .

[0099] After implementation, if Figure 3As shown in the figure, with the increase of salt content in the culture medium, the OD value of the bacteria first increased and then decreased. At 0% salt content, the strain OD value was 0.03, and the strain did not grow; at 1% salt concentration, the strain OD value was 0.43, which shows that the strain can only grow slowly in the presence of salt; when the salt concentration increased from 2% to 6%, the strain OD value increased from 0.89 to 1.36; above 6% salt concentration, the strain OD value decreased, indicating that the growth of the strain may be inhibited by the salt concentration; at 22% salt concentration, the strain OD value was 0.19, which shows that the growth of the strain was severely inhibited; above 22% salt concentration, the strain OD value was lower than 0.1, and the strain could not grow. Therefore, the salt tolerance range of this bacterium is 1%-22%.

[0100] b) Acid resistance

[0101] TSB medium (pH 7.0) containing 3% salt concentration was prepared, and the bacterial solution activated at 37°C and 150 rpm for 18 h was inoculated into medium with pH values ​​of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5 and 11.0, respectively, at an initial OD 600 0.03, culture at 37°C, 150 rpm for 24 h. Pipette 0.2 mL of bacterial solution and measure the OD of the bacterial solution using a spectrophotometer 600 .

[0102] After implementation, if Figure 4 As shown in the figure, the OD value of the strain first increased and then decreased with the increase of pH value. When the pH is lower than 5.0 or higher than 10.5, the OD value of the strain is lower than 0.1, which means that the strain cannot grow; when the pH increases from 5.0 to 7.0, the OD value of the strain increases from 0.19 to 1.26; with the increase of pH value, the OD value of the strain decreases, and the growth of the strain is inhibited; when the pH is 10.5, the OD value of the strain is 0.15, and the growth of the strain is severely inhibited. Therefore, the pH tolerance range of the growth of this bacterium is 5.0-10.5.

[0103] c) Temperature resistance

[0104] Prepare TSB medium (pH 7.0) containing 3% salt concentration, and inoculate the culture medium with 3% (v / v) bacterial solution activated at 37°C and 150 rpm for 18 h. The initial OD 600 The concentration of the culture medium was 0.03, and the culture temperature was set to 0℃, 3℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 47℃, 49℃ and 50℃, respectively, and cultured at 150rpm for 24h. 0.2mL of the bacterial solution was taken and the OD of the bacterial solution was measured with a spectrophotometer. 600 .

[0105] After implementation, if Figure 5 As shown in the figure, the OD value of the strain first increases and then decreases with the increase of temperature. When the temperature is lower than 3℃ or higher than 47℃, the OD value of the strain is lower than 0.1, which means that the strain cannot grow; when the temperature increases from 3℃ to 40℃, the OD value of the strain increases from 0.1 to 1.27; when the temperature is higher than 40℃, the OD value of the strain decreases, and the growth of the strain is inhibited; when the temperature is 47℃, the OD value of the strain is 0.35. Therefore, the temperature tolerance range of the growth of this bacterium is 3-47℃, and it is expected to be used as a starter for low-temperature shrimp paste fermentation.

[0106] (2) Experiment on the ability of strains to produce protease

[0107] a) Comparison of proteolytic cycles

[0108] The strain was picked with a sterile toothpick and inoculated on skimmed milk powder medium, cultured at 37°C for 48 hours, and the diameter of the transparent zone and the diameter of the colony were recorded. The enzyme activity index of the hydrolytic protease of other halophilic bacteria from shrimp paste was compared. The enzyme activity index EI was defined as the ratio of the transparent zone diameter to the colony diameter.

[0109] After implementation, the hydrolysis zone of the strain on the skimmed milk powder plate is as follows Figure 6 The EI values ​​of different strains are different, as shown in Table 1. Compared with other strains from shrimp paste, the EI value of Bacillus halodenitrificans XJ-229 is much greater than that of other strains, which indicates that the strain has a strong proteolytic ability.

[0110] In summary, the salt-denitrifying Cladosporium XJ-229 isolated and obtained in Example 1 has good salt tolerance, the salt concentration range is suitable for the salt concentration requirement of the low-salt shrimp paste fermentation system, and has the ability to produce high protease.

[0111] Table 1 EI values ​​of different strains from shrimp paste

[0112]

[0113] b) Comparison of protease production capacity

[0114] Use an inoculation loop to pick a loop of strains and inoculate them into 100 mL of liquid TSB medium containing 3% (m / V) NaCl, and culture them at 37°C, 150 rpm for 18 h. Then, inoculate them into TSB medium containing 6% (m / V) NaCl at a 3% inoculum amount and culture them at 37°C, 150 rpm for 48 h, and obtain a viable count of 1.3×10 9CFU / mL of fermentation broth. The obtained fermentation broth was centrifuged at 8000×g, 4℃ for 10min, and the supernatant was collected as the crude enzyme solution for the determination of enzyme activity. Casein was used as the substrate, and the protease activity in the fermentation broth was determined according to the national standard "SB / T 10317-1999 Protease Activity Determination Method". The enzyme activity unit is defined as 1mL of enzyme solution, at 40℃, pH=7.5, hydrolyzing casein per minute to produce 1μg of tyrosine is 1 enzyme activity unit (U).

[0115] After implementation, the protease activity of each strain was Figure 7 Compared with other strains from shrimp paste, the protease activity of Bacillus halodenitrificans XJ-229 was significantly higher than that of other strains, with a protease activity of 15.38±1.34U / mL, indicating that the strain had a strong proteolytic ability.

[0116] Example 3 Application of salt-denitrifying Cladosporium XJ-229 in low-salt shrimp paste fermentation

[0117] (1) Preparation and fermentation of shrimp paste

[0118] The 2-4 cm small white shrimp purchased from a farmer's market in Jiangsu were washed, 16% (m / m) sea salt was added, drained and crushed into shrimp paste using a grinder, and then dispensed into 500 mL glass bottles, 350 g per bottle. The salt-denitrifying Cladosporium XJ-229 strain was cultured twice in TSB medium containing 16% NaCl, and the bacterial solution was adjusted to 1×10 7 CFU / mL, the bacterial solution was inoculated at a 2% (V / m) inoculation rate as the fermentation medium for shrimp paste, and then cultured at a constant temperature of 37°C for 30 days. The shrimp paste fermented without inoculation of the strain was used as the control to compare the changes in the indicators of the shrimp paste fermented with and without inoculation of the strain.

[0119] (2) Analysis of Hazards in Shrimp Paste

[0120] The total volatile basic nitrogen content in shrimp paste was determined according to the Chinese National Food Safety Standard GB5009.228-2016. The malondialdehyde content in shrimp paste was determined by spectrophotometry according to the Chinese National Food Safety Standard GB5009.181-2016. The eight main biogenic amines in shrimp paste were determined by high performance liquid chromatography, including tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine.

[0121] After implementation, the contents of amino acid nitrogen and several harmful substances in the samples are shown in Table 2. The amino acid nitrogen content in shrimp paste inoculated with the salt-denitrifying Cladosporium XJ-229 strain was 1.32±0.04g / 100g, the volatile basic nitrogen content was 118.04±6.31mg / 100g, the malondialdehyde content was 3.90±0.24mg / kg, and the total amount of biogenic amines was 326.14±5.85mg / kg, which were 12.65%, 16.63% and 24.56% lower than those of the control group without strain inoculation, respectively. The amino acid nitrogen content in shrimp paste inoculated with the salt-denitrifying Cladosporium XJ-229 strain is much higher than the 0.6g / 100g amino acid nitrogen content in shrimp paste specified in the "Aquatic Products Industry Standard of the People's Republic of China for Shrimp Paste SC / T3602-2016" and the 1.1g / 100g amino acid nitrogen content in shrimp paste specified in the "Domestic Trade Industry Standard of the People's Republic of China SB / T 10525-2009". The volatile basic nitrogen content is much lower than the 450mg / 100g volatile basic nitrogen content in shrimp paste specified in the "Domestic Trade Industry Standard of the People's Republic of China SB / T 10525-2009". In addition, Figure 8 As shown, compared with the control shrimp paste, the salt-denitrifying Cladosporium XJ-229 has a better degradation effect on tyrosine and histamine in biogenic amines, with a degradation rate of 32.7% for tyramine and 55.1% for histamine. The implementation results of this strain show that the strain can effectively reduce the content of harmful substances in shrimp paste, improve the safety of low-salt shrimp paste fermentation quality, and meet the requirements of shrimp paste industry standards.

[0122] Table 2 Amino acid nitrogen and several harmful substances in shrimp paste

[0123]

[0124] (3) Determination of free amino acid content in shrimp paste

[0125] The free amino acids in shrimp paste were determined by high performance liquid chromatography (HPLC), using an Agilent Hypersil ODS column with gradient elution, a mobile phase flow rate of 1.0 mL / min, a column temperature of 40 °C, proline detection at 262 nm, and other amino acids at 338 nm.

[0126] After implementation, the content of free amino acids in the samples is shown in Table 3. The higher amino acids in shrimp paste are mainly glutamic acid, glycine, alanine, lysine, leucine and aspartic acid, among which glutamic acid and aspartic acid are recognized as umami amino acids, alanine is a sweet amino acid, lysine has a sweet and umami taste, and leucine has a bitter taste, which provides an important and unique flavor for shrimp paste. The free amino acid content in shrimp paste inoculated with salt-denitrifying Bacillus subtilis XJ-229 is 8506.71±13.16mg / 100g, which is 211.06mg / 100g higher than that in shrimp paste without inoculation. Compared with the control group, aspartic acid, glutamic acid, glycine, alanine, tyrosine, cysteine, valine, methionine, phenylalanine, lysine, and methionine in shrimp paste inoculated with denitrifying Bacillus subtilis XJ-229 increased significantly, while histidine, threonine, isoleucine and leucine decreased. The increase of aspartic acid, glutamic acid and lysine indicates that the inoculation of salt-denitrifying Bacillus XJ-229 is beneficial to the increase of the umami taste of shrimp paste. The increase of glycine, alanine, lysine, cysteine ​​and methionine indicates that the inoculation of salt-denitrifying Bacillus XJ-229 is beneficial to the increase of the sweetness of shrimp paste. The decrease of bitter histidine, isoleucine and leucine indicates that the inoculation of salt-denitrifying Bacillus XJ-229 is beneficial to the reduction of the bitterness of shrimp paste. In short, compared with shrimp paste not inoculated with this strain, the inoculation of salt-denitrifying Bacillus XJ-229 can promote the increase of the umami taste of shrimp paste, while reducing the bitterness of shrimp paste, which is beneficial to the improvement of the quality and flavor of shrimp paste.

[0127] Table 3 Free amino acid content in shrimp paste

[0128]

[0129]

[0130] (4) Sensory evaluation of shrimp paste

[0131] Ten participants, 5 men and 5 women, who had experience in olfactory testing and had no bad habits such as alcoholism and smoking, were recruited, and the candidates were trained to describe and identify the odor quality of shrimp paste. 5g of sample was placed in a 15mL tasting cup, covered with a lid and placed at room temperature for 30 minutes. Each member was asked to perform the tasting independently in the tasting area and rinse their mouths with water between tasting samples. The shrimp paste samples were scored on a scale of 0 to 10 for shrimp flavor, fishy smell, fermented smell, ammonia smell, umami taste, salty taste, sweetness and bitterness according to sensory preferences.

[0132] After implementation, the sensory scores of shrimp paste are as follows Fig. 9 As shown. Compared with the control group, the shrimp paste inoculated with salt-denitrifying Cladosporium XJ-229 had reduced shrimp flavor, fishy smell and ammonia flavor, and increased umami and sweetness flavor. Shrimp paste inoculated with salt-denitrifying Cladosporium XJ-229 is more acceptable to people.

[0133] (5) SPME-GC-MS determination of flavor compounds

[0134] The volatile compounds in shrimp paste were determined using the SPME-GC-MS method. The volatile flavor compounds were analyzed using a Trace 1300ISO series gas chromatograph-mass spectrometer (Thermo Fisher Scientific Inc.), equipped with a TG-WAXMS (30m×0.25μm×0.25mm) capillary column, helium (99.9%) as the carrier gas, and a flow rate of 1mL / min. The oven temperature was set to: 32°C, 5min, 4°C / min to 60°C, 8°C / min to 100°C, and 12°C / min to 240°C. The temperatures of the transmission line and ion source were 250°C and 230°C, respectively. The mass spectrum from 35 to 300 m / z was obtained by electron impact ionization at 70eV. The obtained mass spectrum was compared with the NIST2.0 (Agilent Technologies Inc.) database. The relative percentage content of each compound in the shrimp paste sample was obtained by using an internal standard and peak area normalization method for relative quantitative calculation.

[0135] Table 4 shows the types and relative contents of volatile compounds in the shrimp paste of the control group and the shrimp paste inoculated with salt-denitrifying Cladosporium XJ-229 detected by GC-MS. A total of 32 volatile compounds were detected in the shrimp paste of the control group, including 7 alcohols, 2 acids, 1 lipid, 2 aldehydes, 4 ketones, 4 ethers, 1 amine, 7 pyrazines, 1 indole and 3 other compounds. A total of 40 volatile compounds were detected in the shrimp paste inoculated with salt-denitrifying Cladosporium XJ-229, including 12 alcohols, 2 acids, 3 lipids, 4 aldehydes, 4 ketones, 4 ethers, 2 amines, 4 pyrazines, 1 indole and 4 other compounds. The volatile compounds in the shrimp paste inoculated with salt-denitrifying Cladosporium XJ-229 increased compared with the types of volatile compounds in the shrimp paste without inoculation. Compared with the control group, the types and contents of alcohol compounds in shrimp paste inoculated with salt-denitrifying Bacillus XJ-229 were higher, which may be the reason why the fermentation taste score of shrimp paste fermented with salt-denitrifying Bacillus XJ-229 was higher. For example, the content of 3-methyl-1-butanol with fermentation and fruity aroma was higher in shrimp paste fermented with salt-denitrifying Bacillus XJ-229. Compared with the control group, the trimethylamine with fishy smell and indole with odor in shrimp paste fermented with salt-denitrifying Bacillus XJ-229 were reduced, which may be one of the reasons for its lower fishy smell and ammonia smell. These results show that inoculation with salt-denitrifying Bacillus XJ-229 can improve the flavor quality of low-salt shrimp paste.

[0136] Table 4 Volatile compounds in samples detected by SPME-GC-MS

[0137]

[0138]

[0139]

[0140] All the results showed that inoculation of salt-denitrifying Cladosporium XJ-229 could reduce the harmful components in low-salt shrimp paste, improve the nutritional quality of shrimp paste and improve the flavor quality of low-salt shrimp paste, making the flavor quality of low-salt shrimp paste more acceptable to people.

[0141] Example 4 Application of salt-denitrifying Cladosporium XJ-229 in low-temperature and low-salt shrimp paste fermentation

[0142] Specific implementation method Referring to Example 3, the difference is that the fermentation temperature is adjusted to 10°C and cultured for 30 days. The shrimp paste without inoculation of strains is used as a control to compare the changes in the contents of amino acid nitrogen, volatile basic nitrogen, biogenic amines, malondialdehyde and other components in the shrimp paste.

[0143] After implementation, the results were consistent with the conclusions drawn under high-temperature fermentation, that is, compared with the control group, the amino acid nitrogen content of the inoculated salt-denitrifying Bacillus XJ-229 increased, the volatile basic nitrogen content decreased, and the content of harmful substances such as biogenic amines and malondialdehyde decreased.

[0144] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A protease-producing strain of Virgibacillus halodenitrificans XJ-229, characterized in that: The salt-denitrifying Cladosporium XJ-229 has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 20241629.

2. A microbial preparation containing the salt-denitrifying Cladosporium XJ-229 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The microbial preparation comprises one or more of the living cells, inactivated cells, fermentation broth, lysate or extract of the salt-denitrifying Cladosporium XJ-229.

4. A product containing the salt-denitrifying Cladosporium XJ-229 according to claim 1, or the microbial preparation according to claim 2 or 3.

5. The product according to claim 4, characterized in that The products include food, medicine or health products.

6. The product according to claim 5, characterized in that The food includes fermented food; the fermented food includes fruit and vegetable products, dairy products, bean products, and meat products; optionally, the fermented food includes shrimp paste.

7. The product according to claim 5, characterized in that The dosage form of the medicine includes powder, granule, capsule, tablet, pill or oral solution.

8. A meat sauce starter, characterized in that: The meat sauce starter contains the salt-denitrifying Cladosporium XJ-229 according to claim 1.

9. The meat sauce starter as claimed in claim 8, characterized in that In the meat sauce starter, the number of the salt-denitrifying Cladosporium XJ-229 is ≥ 1×10 6 CFU / mL or 1×10 6 CFU / g.

10. A method for preparing low-salt shrimp paste, characterized in that: The salt-denitrifying Cladosporium XJ-229 described in claim 1 or the microbial preparation described in claim 2 or 3 is added to the shrimp product for fermentation.

11. The method according to claim 10, characterized in that The low salt refers to low NaCl content; the salt content in the low salt shrimp paste is 10-18% w / w.

12. The method according to claim 10, characterized in that The shrimp product comprises one or more of shrimp meat paste, shrimp meat chunks, and shrimp meat extract.

13. The method according to claim 10, characterized in that The fermentation conditions are: 10-40° C., and fermentation for at least 15 days.

14. Use of the salt-denitrifying Cladosporium XJ-229 of claim 1 or the microbial preparation of claim 2 or 3 in the preparation of shrimp paste or products containing shrimp paste.

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