A strain of halophilic tetragenococcus and its application

Abstract

The present application relates to the field of microbial technology, and specifically discloses a strain of Tetragenococcus halophilus and its application. The present application isolates and obtains Tetragenococcus halophilus ZF628 with high salt tolerance and high L-aspartate-β-decarboxylase activity from natural environments such as soy sauce and soy mash. The Tetragenococcus halophilus ZF628 is added to the fermentation process of fermented sauce and soy sauce for fermentation. In addition to significantly increasing the L-alanine content of the fermented sauce and soy sauce products, improving the taste and flavor, it can also increase lactic acid and has higher added value.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a strain of halophilic Tetragenococcus and its application. Background Art

[0002] L-alanine is a non-essential amino acid that can give food a gentle freshness and has the effect of harmonizing the saltiness, sourness, bitterness, and astringency of food. The sweetness of L-alanine is 1.2 times that of sugar, and it can also increase the persistence of taste. Compared with 0.5% sugar and 0.2% salt, the critical value of 0.06% alanine makes the taste of alanine last longer, making food not bland like water, but delicious food with a mature taste. Oyster sauce, yeast extract, soy sauce, sauce, etc. made from oysters are rich in alanine, which is also an important reason why these foods have a more harmonious umami taste than MSG.

[0003] The main methods for preparing L-alanine are chemical synthesis and biosynthesis (enzyme conversion and microbial conversion). The enzymatic conversion method uses L-aspartate-β-decarboxylase (ASD, EC 4.1.1.12) to directly catalyze L-aspartate to prepare L-alanine. L-aspartate-β-decarboxylase can specifically catalyze the decarboxylation of L-aspartate to produce L-alanine and CO2, and most L-aspartate-β-decarboxylases catalyze in the pH range of 4.0 to 5.5, and the catalytic process continuously consumes H + , resulting in a continuous increase in pH during the reaction. When pH>5.5, the enzyme activity decreases significantly. Microbial conversion is the main method for preparing L-alanine, that is, using a single strain such as Escherichia coli, Proteus, Bacillus subtilis, or multi-bacteria fermentation to prepare L-alanine, and then purifying and refining to obtain a high-purity L-alanine product, which has high requirements for equipment and technology.

[0004] In the condiment industry, L-alanine can not only enhance the sweetness and harmony of soy sauce, fermented sauce, etc., but also enhance the richness of fish sauce, etc., and enhance the continuity of taste. It helps to significantly improve the taste and flavor of condiments and enhance the competitiveness of condiments. Therefore, increasing the content of L-alanine in condiments has a development trend.

[0005] At present, most of the microorganisms commonly used in the industry to ferment and prepare L-alanine are non-food grade safe strains and have weak salt tolerance. Combining the fermentation process of traditional condiments such as soy sauce and paste, screening out food grade safe strains that exist naturally in nature, produce high L-alanine and tolerate high salt production environments has an irreplaceable advantage in improving the taste and flavor of fermented condiments. Summary of the invention

[0006] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide a halophilic tetragenococcus and its application. In order to increase the content of L-alanine in the fermentation process of condiments such as soy sauce and sauce, the present application provides a halophilic tetragenococcus with L-aspartic acid decarboxylation activity, which is applied to the fermentation process of fermented sauce and soy sauce, which can significantly increase the content of L-alanine in fermented sauce and soy sauce products.

[0007] To achieve the above purpose, the technical solution adopted by this application is:

[0008] In the first aspect, the present application provides a strain of Tetragenococcus halophilus ZF628, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 24, 2024, with a preservation number of GDMCC No: 65198, a classification number of Tetragenococcushalophilus, and a preservation address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The halophilic Tetragenococcus halophilus ZF628 provided in the present application is isolated from soy sauce mash, and has the characteristics of high salt tolerance, high L-aspartate-β-decarboxylase activity, and high L-alanine synthesis. The strain is applied to the fermentation process of fermented sauce and soy sauce, which can not only significantly increase the L-alanine content in the fermented sauce and soy sauce products, but also overcome the problem of weak salt tolerance of most fermentation strains for preparing L-alanine, and has high application value.

[0010] The halophilic Tetragenococcus halophilus ZF628 of the present application is suitable for growth in MRS medium supplemented with 5%-10% salt. Colonies appear after anaerobic culture in MRS agar medium supplemented with 5% salt for about 3 days. The colonies are round in shape, smooth in surface, milky white, and opaque.

[0011] The present application also provides a screening step for the above-mentioned Tetragenococcus halophilus ZF628, comprising:

[0012] Take crude oil or mash with high L-alanine content, dilute it with physiological saline and spread it on a salt-containing MRS screening solid plate, culture it in a strict anaerobic environment for a period of time, select different single colonies from the grown colonies, and purify the strains of different single colonies by streaking the plates 2-3 times to obtain pure strains.

[0013] The bacteria were activated using a salt-containing MRS activation medium, and the activated bacterial liquid was added to a salt-containing MRS screening medium at a ratio of 1% for continued fermentation. A strain with high L-alanine production was screened out through amino acid composition analysis, and the strain was subjected to 16S rRNA sequencing analysis, strain comparison, and salt tolerance test.

[0014] Preferably, the MRS screening solid plate is: MRS solid culture medium (pH 5.5) supplemented with 0.5% calcium carbonate and 10% sodium chloride.

[0015] MRS activation medium: MRS medium (pH 5.5) supplemented with 10% sodium chloride.

[0016] The MRS screening medium is: MRS medium (pH 5.5) supplemented with 1% sodium L-aspartate and 10% sodium chloride.

[0017] In a second aspect, the present application provides the use of the above-mentioned Tetragenococcus halophilus ZF628 in the preparation of condiment fermentation.

[0018] The present application isolates and obtains a food-grade safe strain with high salt tolerance, high L-aspartate-β-decarboxylase activity, and high L-alanine synthesis from natural environments such as soy sauce and mash, namely, Tetragenococcus halophilus ZF628, which overcomes the problem that most strains used for fermentation to prepare L-alanine have weak salt tolerance; the strain is applied to the fermentation process of condiments (fermented sauce and soy sauce), which can significantly increase the L-alanine content in fermented sauce and soy sauce products, and improve the taste and flavor.

[0019] As a preferred embodiment of the application described in the present application, the condiment includes soy sauce or sauce.

[0020] In a third aspect, the present application provides the use of the above-mentioned Tetragenococcus halophilus ZF628 in increasing the L-alanine content in soy sauce or paste.

[0021] In a fourth aspect, the present application provides a method for increasing the L-alanine content in a condiment, comprising adding the Tetragenococcus halophilus ZF628 before or during fermentation of the mash.

[0022] Compared with the traditional industrial preparation of L-alanine, adding a food-grade safe strain (Tetragenococcus halophilus ZF628) to the fermentation process of fermented paste and soy sauce to ferment and synthesize L-alanine fully utilizes the existing raw materials and does not require the separation and purification of L-alanine, saving process, manpower and time costs.

[0023] As a preferred embodiment of the method described in the present application, the condiment includes soy sauce or paste.

[0024] In a fifth aspect, the present application provides a method for preparing soy sauce or paste, comprising adding the Tetragenococcus halophilus ZF628 before or during the fermentation of the sauce mash.

[0025] As a preferred embodiment of the preparation method described in the present application, the Tetragenococcus halophilus ZF628 is inoculated into the koji material for soy sauce or the soy sauce mash in the early stage of fermentation for fermentation;

[0026] Alternatively, the Tetragenococcus halophilus ZF628 is inoculated into the koji for fermenting soy sauce or the mash in the early stage of fermentation for fermentation.

[0027] As a preferred embodiment of the preparation method described in the present application, the inoculation amount of Tetragenococcus halophilus ZF628 is 10 5 CFU / kg~10 6 CFU / kg.

[0028] Preferably, the inoculation amount of Tetragenococcus halophilus ZF628 inoculated into the soy sauce koji or the soy sauce mash in the early stage of fermentation is 10 6 CFU / kg;

[0029] The inoculation amount of Tetragenococcus halophilus ZF628 inoculated into the koji material for fermenting soy sauce or the soy sauce mash in the early stage of fermentation is 10 5 CFU / kg.

[0030] In a sixth aspect, the present application provides a soy sauce or paste, which is prepared using the above-mentioned preparation method.

[0031] Verified by the crude oil system, the alanine production rate of Tetragenococcus halophilus ZF628 in the crude oil system was 84%, and the L-alanine increase was about 0.58g / 100mL, which performed well. Tetragenococcus halophilus ZF628 can tolerate 24% sodium chloride and grow well under 16% sodium chloride conditions, proving that it can grow in the high-salt environment of fermented sauce and soy sauce fermentation.

[0032] Moreover, the sweetness and sourness of the crude oil fermented with the halophilic Tetragenococcus halophilus ZF628 of the present application are significantly improved, and the overall taste is significantly improved; compared with no addition of strains, the sweetness and sourness of the soybean paste fermented with the halophilic Tetragenococcus halophilus ZF628 are significantly improved, and the overall taste is significantly improved.

[0033] Compared with the prior art, this application has the following beneficial effects:

[0034] The present application provides a halophilic Tetragenococcus and its application. The present application isolates and obtains a halophilic Tetragenococcus (Tetragenococcushalophilus) ZF628 with high salt tolerance and high L-aspartate-β-decarboxylase activity from natural environments such as soy sauce and soy mash. During the fermentation process of fermented sauce and soy sauce, halophilic Tetragenococcus (Tetragenococcushalophilus) ZF628 is added for fermentation. In addition to significantly increasing the L-alanine content of the fermented sauce and soy sauce products, improving the taste and flavor, it can also bring about an increase in lactic acid, which has higher added value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a morphological diagram of Tetragenococcus halophilus ZF628;

[0036] Figure 2 This is a diagram showing the sensory evaluation results of the fermented crude oil in Example 5;

[0037] Figure 3 This is a diagram showing the sensory evaluation results of the fermented original sauce in Example 6. DETAILED DESCRIPTION

[0038] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0039] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0040] In the following examples:

[0041] The MRS screening solid plate is: MRS solid medium (pH 5.5) supplemented with 0.5% calcium carbonate and 10% sodium chloride.

[0042] MRS activation medium: MRS medium (pH 5.5) supplemented with 10% sodium chloride.

[0043] The MRS screening medium is: MRS medium (pH 5.5) supplemented with 1% sodium L-aspartate and 10% sodium chloride.

[0044] Example 1: Screening of lactic acid bacteria strains that produce high levels of L-aspartate-β-decarboxylase

[0045] This embodiment provides a method for screening a lactic acid bacteria strain that produces high L-aspartate-β-decarboxylase, comprising the following steps:

[0046] Gradient dilution of mash (10 -2 , 10 -3 , 10 -4 , 10 -5 ) sample, take 100 μL of diluted sample and spread it on MRS screening solid plate, place it in anaerobic bag at 30℃ and culture it for 5 days. Select strains with different morphologies from the plate and streak them on MRS screening solid plate for separation and purification 2-3 times, pick single clones and culture them in MRS activation medium at 30℃ for 3 days, inoculate them with 1% inoculation into MRS screening medium with Duchenne catheter, and observe the gas production of strains.

[0047] If the strain produces a large amount of gas, it is preliminarily proved that the strain can produce L-aspartate-β-decarboxylase and use L-aspartate to produce L-alanine and CO2. The fermentation liquid of the gas-producing strain is centrifuged at 8000rpm for 5 minutes, and the supernatant is taken. The amino acid content is determined by an amino acid analyzer to screen the strain with high L-alanine enhancement.

[0048] The results are shown in Table 1, and 3 strains with L-alanine production rates of more than 70% were screened out.

[0049] Table 1 L-alanine enhancement and production rate of lactic acid bacteria strains

[0050]

[0051]

[0052] Example 2: Crude oil system verification of lactic acid bacteria strains with high production of L-aspartate-β-decarboxylase

[0053] Crude oil system: 100% crude oil, sodium chloride concentration is about 17%, pH 5.2.

[0054] The three strains screened were verified in the crude oil system. They were inoculated into MRS activation medium and activated and cultured at 30°C for 3 days. They were inoculated into the crude oil system with a 1% inoculation amount and cultured for 10 days. The supernatant was taken after centrifugation at 8000rpm for 5 minutes, and the amino acid content was determined using an amino acid analyzer to screen bacteria with high L-alanine enhancement.

[0055] The results are shown in Table 2. The alanine production rate of the ZF628 strain in the crude oil system was 84%, and the L-alanine increase was about 0.58 g / 100 mL, which performed well.

[0056] Table 2 L-alanine enhancement and conversion rate of lactic acid bacteria strains in crude oil system

[0057] Strain number L-Alanine Boost Alanine production rate ZF625 0.50g / 100mL 72％ ZF628 0.58g / 100mL 84％ ZF630 0.45g / 100mL 66％

[0058] Example 3: Study on the properties of high-yield L-alanine strains

[0059] Strain activation: The single ZF628 colony obtained above was inoculated into MRS activation medium and activated for 5 days until the bacterial count reached 10 8 CFU / mL and above.

[0060] Using MRS screening medium as the basic medium, the tolerance of ZF628 to sodium chloride was investigated under the fermentation conditions of sodium chloride content of 12%, 16%, 20%, 24% and 26%, inoculation size of 1%, fermentation temperature of 30°C and fermentation time of 5d. The results are shown in Table 3. ZF628 can tolerate 24% sodium chloride and grow well under 16% sodium chloride conditions, proving that it can grow in the high-salt environment of fermented sauce and soy sauce fermentation.

[0061] Table 3 Tolerance of ZF628 to different NaCl contents

[0062] NaCl content ZF628 growth 12％ +++ 16％ +++ 20％ ++ 24％ w 26％ -

[0063] Example 4, Identification of ZF628 strain

[0064] The ZF628 strain obtained above was isolated and purified in MRS screening medium. The colony morphology was milky white, small, round, smooth and moist on the surface, and neat on the edges. Figure 1 As shown, facultative anaerobic.

[0065] 16S rRNA molecular biological identification: After the above strains were activated, DNA was extracted using a bacterial genomic DNA extraction kit, and PCR amplification was performed using universal primers 24F (5'-AAGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3').

[0066] The PCR reaction system was as follows: 12.5 μL of high-fidelity Taq enzyme premix and 1.25 μL of 10 pmol of each primer were added to each reaction solution, and the reaction conditions were: denaturation at 98°C for 30 s, annealing at 60°C for 20 s, extension at 72°C for 30 s, and 30 cycles.

[0067] The PCR products were sequenced after verification by agarose gel electrophoresis, and the sequencing results were compared with the NCBI database, and the bacterial species was identified as Tetragenococcus halophilus.

[0068] The above strain was preserved and named Tetragenococcus halophilus ZF628. The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 24, 2024, with the preservation number GDMCC No: 65198, the classification number Tetragenococcus halophilus, and the preservation address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0069] Example 5: Application of Tetragenococcus halophilus ZF628 in soy sauce pilot fermentation

[0070] Soybeans were washed and soaked, sterilized at 121°C for 25 minutes, and then mixed with roasted wheat in a ratio of 7:3. When the temperature dropped to 28°C, Aspergillus oryzae was inoculated at a ratio of 0.1% to make koji on a plate; the koji making temperature was 33-30°C and the koji making time was 40 hours. After the koji making was completed, the prepared koji material was mixed with 20°Bé salt water in a weight ratio of 1:2. After mixing, the ZF628 strain finally screened was added according to 10 6 The inoculum amount of CFU / kg mash was inoculated into the fermentation process. Tetragenococcus halophilus ZF628 was not added to the control group. After the fermentation was completed, the L-alanine, L-aspartic acid, amino acid nitrogen and lactic acid contents of the fermented crude oil were detected and sensory evaluation was performed.

[0071] The sensory evaluation was conducted by 15 people with sensory evaluation experience. The evaluation score ranged from 0 to 5 points, and the results were expressed as an average score, with a higher score representing a stronger flavor.

[0072] Table 4 Index test results of fermented crude oil

[0073]

[0074]

[0075] The evaluation results are as follows Figure 2 As shown in Table 4, compared with the control, the sweetness and sourness of the crude oil fermented with Tetragenococcus halophilus ZF628 were significantly improved, and the overall taste was significantly improved.

[0076] Example 6: Use of Tetragenococcus halophilus ZF628 in small-scale fermentation of fermented sauce

[0077] Soybeans were washed and soaked, and then steamed at 100°C for 10 minutes, and then mixed with wheat flour in a ratio of 7:3. When the temperature dropped to 28°C, Aspergillus oryzae was added at a ratio of 0.1% to make koji on a koji plate; the koji making temperature was 33-30°C, and the koji making time was 40 hours. After the koji making was completed, the prepared koji material was mixed with 20°Bé salt water at a weight ratio of 1:1.5. After mixing, the finally screened Tetragenococcus halophilus ZF628 was added at a weight ratio of 10 5 The inoculum amount of CFU / kg of sauce mash was inoculated into the fermentation process. The ZF628 strain was not added to the control group. After the fermentation was completed, the L-alanine, L-aspartic acid, amino acid nitrogen and lactic acid contents of the fermented crude oil were detected, and sensory evaluation was performed.

[0078] The sensory evaluation was conducted by 15 people with sensory evaluation experience. The evaluation score ranged from 0 to 5 points, and the results were expressed as an average score, with a higher score representing a stronger flavor.

[0079] Table 5 Test results of the indicators of fermented original sauce

[0080]

[0081] The evaluation results are as follows Figure 3 As shown in Table 5, compared with the control group, the sweetness and sourness of the soybean paste fermented with ZF628 were significantly improved, and the overall taste was significantly improved.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A strain of Tetragenococcus halophilus ZF628, characterized in that: The Tetragenococcus halophilus ZF628 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 24, 2024, with the collection number GDMCC No: 65198, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of Tetragenococcus halophilus ZF628 as claimed in claim 1 in the preparation of condiment fermentation.

3. The use according to claim 2, characterized in that The condiments include soy sauce or paste.

4. Use of the Tetragenococcus halophilus ZF628 as claimed in claim 1 in increasing the L-alanine content in soy sauce or paste.

5. A method for increasing the content of L-alanine in a condiment, characterized in that: The method comprises adding the Tetragenococcus halophilus ZF628 as claimed in claim 1 before or during fermentation of moromi.

6. The method according to claim 5, characterized in that The condiments include soy sauce or paste.

7. A method for preparing soy sauce or paste, characterized in that: The method comprises adding the Tetragenococcus halophilus ZF628 as claimed in claim 1 before or during fermentation of moromi.

8. The preparation method according to claim 7, characterized in that: Inoculating the halophilic Tetragenococcus ZF628 as claimed in claim 1 into the koji material for soy sauce or the soy sauce mash in the early stage of fermentation for fermentation; Alternatively, the Tetragenococcus halophilus ZF628 according to claim 1 is inoculated into koji for fermented soy sauce or moromi in the early stage of fermentation to carry out fermentation.

9. The preparation method according to claim 8, characterized in that: The inoculation amount of the halophilic Tetragenococcus halophilus ZF628 was 10 5 CFU / kg~10 6 CFU / kg.

10. A soy sauce or paste, characterized in that: The soy sauce or paste is prepared by the preparation method according to any one of claims 7 to 9.

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