A strain of Priesteria gigantea GD0038 and its application in improving the flavor of weed crab sauce

Abstract

The invention discloses a Priesteria gigantea GD0038 and an application thereof in improving the flavor of weed crab sauce. The strain is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242548 and a deposit date of November 13, 2024. The strain is applied to the preparation of weed crab sauce, can significantly improve the flavor of the weed crab sauce, increase the content of volatile substances in the fermented weed crab sauce, and increase the content of characteristic flavor substances such as 1-penten-3-ol, 1-nonanol, isovaleraldehyde, n-hexanol, n-octanol, 3-n-decanol, 2-heptanone, and other alcohols, pyrazines, and ketones, while reducing the content of unpleasant flavor substances such as butylamine, trimethylamine, and n-hexanal. The invention has very good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Priesteria gigantea GD0038 strain and application thereof in improving the flavor of weed crab sauce. Background Art

[0002] The weed crab, scientifically known as Sesamus haematops, also known as grinding crabs or crab crabs, belongs to the class Crustacea and family Squamaidae. It is a small freshwater crab. Its cephalothorax is square, with parallel lateral margins. Its forehead is broad, equal to or exceeding half the width of the cephalothorax. Its cheeks are covered with granules and hairs arranged in an alternating network. There are many species, the most common being the red-clawed Sesamus haematops (S. haema-tocheir). Its cephalothorax is 2.9 cm long. The legs of medium-sized Sesamus haematops are hairless, while the red walking legs are hairy.

[0003] Weed crab sauce is a traditional delicacy unique to Fuzhou, Fujian. Industrious farmers in the Minjiang River basin catch weed crabs during the autumn and winter off-seasons along the clean riverbanks. They clean the crabs in clean water, remove their tails, and add ingredients like rice wine, distiller's grains, monosodium glutamate, sugar, and glutinous rice. This sauce is then ground into a paste using a stone mill and seasoned with salt to create a delicious condiment. In Fuzhou, weed crab sauce is a must-have at wedding banquets, often used as a dip for jellyfish heads. Its delicious flavor is unforgettable. However, the flavor of naturally fermented weed crab sauce cannot be guaranteed; therefore, how to preserve and improve its flavor is crucial to its preparation. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a strain of Priesteria gigantea ( 巨大Priestia菌 ) GD0038 can be used in the preparation of weed crab sauce to enhance the flavor of the sauce.

[0005] In order to solve the above technical problems, the present invention provides a strain of Priesteria gigantea ( Priestia菌属 巨大 ) GD0038, deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 20242548, and the deposit date being November 13, 2024.

[0006] Based on the above, the present invention also provides a Priesteria gigantea ( 巨大Priestia菌 ) The screening method of GD0038 comprises the following steps:

[0007] (1) Initial screening of strains on plates: dilute the naturally fermented weed crab sauce 10-fold using sterile water, spread 100 μL on MRS agar solid medium, and culture at 30°C under oxygen consumption for 24 hours;

[0008] Single colonies with different morphologies were selected and made into bacterial suspensions with sterile water. The suspensions were spread on MRS agar solid medium and cultured at 30°C under oxygen consumption for 24 hours. This plate culture was repeated 4 times.

[0009] (2) Strain shake flask rescreening: The strains screened above were inoculated into MRS liquid culture medium and cultured at 37°C for 48 hours.

[0010] Furthermore, the preparation method of the naturally fermented weed crab sauce in step (1) is as follows: clean the weed crabs, remove the gills and crush them, and seal and ferment them at 21° C. for 40 days to obtain the naturally fermented weed crab sauce;

[0011] The MRS agar solid culture medium comprises the following components: 10 g / L beef protein powder; 10 g / L fish juice; 5 g / L yeast extract powder; 20 g / L glucose; 5 g / L sodium acetate; 2 g / L diammonium citrate; 0.58 g / L magnesium sulfate; 0.28 g / L manganese sulfate; 15.0 g / L agar powder; 0.1 g / L Tween 80; and a pH value of 6.2-6.4.

[0012] The MRS liquid culture medium comprises the following components: 10 g / L peptone; 10 g / L beef extract; 5 g / L yeast extract; 2 g / L dipotassium hydrogen phosphate; 2 g / L diammonium citrate; 5 g / L sodium acetate; 20 g / L glucose; 0.5 g / L magnesium sulfate; 0.25 g / L manganese sulfate; 0.1 g / L Tween 80; and a pH value of 6.2-6.4.

[0013] Based on the above, the present invention also provides a method for preparing weed crab sauce, comprising: taking weed crabs, crushing them, and inoculating the above-mentioned Priesteria gigantea ( 巨大Priestia菌 ) GD0038, fermented to make crab sauce.

[0014] Furthermore, the preparation method of the weed crab sauce comprises the following steps:

[0015] S1: Activated Priesteria gigantea ( 巨大Priestia菌 ) Inoculate GD0038 into the culture medium and incubate at 36-38°C for 20-30 hours to obtain the culture solution;

[0016] S2: Crush weed crabs, inoculate culture fluid or bacteria obtained by centrifuging the culture fluid, and culture at 36-38° C. for 40-50 days to obtain weed crab sauce.

[0017] Furthermore, the culture medium in step S1 is MRS liquid culture medium.

[0018] Furthermore, the culture conditions in step S1 are: culture at 37°C for 24 hours;

[0019] The culture conditions in step S2 are: culture at 37° C. for 40 days.

[0020] Furthermore, in step S2, the inoculation amount of the culture solution or bacteria is 3% to 10%.

[0021] Furthermore, in step S2, the wedelia are sterilized first and then crushed; the wedelia sterilization method is: soaking the wedelia in a sodium chloride solution.

[0022] The above-mentioned Priesteria gigantea ( 巨大Priestia菌 ) GD0038 can be used in the preparation of weed crab sauce.

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides Priesteria gigantea ( 巨大Priestia菌 ) GD0038 was screened from traditional fermented weed crab sauce. The raw material has strong adaptability, good physiological activity and growth characteristics, excellent salt tolerance, acid resistance, NaNO2 resistance, antibacterial properties, hydrophobicity, adhesion and automatic aggregation, etc. It can be used as an inoculation fermentation agent for weed crab sauce, which can solve the quality differences and safety problems caused by different batches of natural weed crab sauce.

[0025] Secondly, using Priesteria gigantea ( 巨大Priestia菌 ) The main aroma compounds of the weed crab sauce prepared by GD0038 fermentation are consistent with those of natural fermentation, and have a directional flavor enhancement effect, realizing the directional flavor improvement of natural fermentation.

[0026] In addition, using Priesteria gigantea ( 巨大Priestia菌 ) The weed crab sauce prepared by GD0038 fermentation also has the advantages of high unsaturated fatty acid content, strong antioxidant capacity, and excellent digestion characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a comparison chart of the number of volatile compound types in the two fermentation methods of Example 3 and Example 4. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0029] The present invention provides Priesteria gigantea ( 巨大Priestia菌GD0038 is a Gram-positive bacterium of the genus Bacillus, deposited with the China Center for Type Culture Collection under the accession number CCTCC NO: M 20242548, dated November 13, 2024, at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. This strain can be used to prepare weed crab sauce, exhibiting a targeted flavor-enhancing effect, achieving targeted flavor improvement through natural fermentation.

[0030] Example 1 Priesteria gigantea ( 巨大Priestia菌 ) Screening of GD0038

[0031] Priesteria gigantea ( 巨大Priestia菌 ) The screening method of GD0038 comprises the following steps:

[0032] (1) Initial screening of strains on plates: Use sterile water to make a 10-fold gradient dilution of the naturally fermented weed crab sauce, take 100 μL and spread it on MRS agar solid culture medium, and culture it at 30°C in an oxygen-consuming environment for 24 hours.

[0033] Single colonies with distinct morphologies were selected and suspended in sterile water. The suspension was then plated onto MRS agar and incubated at 30°C under aerobic conditions for 24 hours. This plate culture was repeated four times, resulting in a total of 40 strains.

[0034] Among them, the naturally fermented weed crab sauce is prepared by the following method: take weed crabs (caught in the waters of Zhanjiang, Guangdong Province in May 2024), clean them, remove the gills and crush them, seal and ferment them at 21°C for 40 days to obtain naturally fermented weed crab sauce.

[0035] The components of the MRS agar solid medium are as follows: 10 g / L beef protein powder; 10 g / L fish juice; 5 g / L yeast extract powder; 20 g / L glucose; 5 g / L sodium acetate; 2 g / L diammonium citrate; 0.58 g / L magnesium sulfate; 0.28 g / L manganese sulfate; 15.0 g / L agar powder; 0.1 g / L Tween 80; and a pH value of 6.2-6.4.

[0036] (2) Strain shake flask rescreening: The strains screened above were inoculated into MRS liquid culture medium and cultured at 37°C for 48 hours.

[0037] The components of MRS liquid culture medium are as follows: peptone 10 g / L; beef extract 10 g / L; yeast extract 5 g / L; dipotassium hydrogen phosphate 2 g / L; diammonium citrate 2 g / L; sodium acetate 5 g / L; glucose 20 g / L; magnesium sulfate 0.5 g / L; manganese sulfate 0.25 g / L; Tween 80 0.1 g / L; pH 6.2-6.4.

[0038] Example 2 Identification of bacterial species

[0039] (1) Identification of Priesteria gigantea GD0038: 16S rDNA sequence identification was performed using bacterial universal primers 27F / 1492R. 巨大Priestia菌 .

[0040] (2) Preservation method of Priesteria gigantea GD0038: Select a well-grown single colony from an MRS agar plate and inoculate it into 5 mL of MRS broth. Incubate at 30°C under aerobic conditions for 24 hours. Take 1 mL of the culture medium and add it to a cryovial containing 1 mL of 30% glycerol. Store in a -80°C freezer.

[0041] Example 3 Preparation of Weed Crab Sauce

[0042] The preparation method of weed crab sauce is as follows:

[0043] (1) Activation of bacterial strains: The strain Priesteria gigantea GD0038 was inoculated on a solid slant culture medium (the composition of which was the same as that of the MRS agar solid culture medium in Example 1), and cultured at 30°C under oxygen consumption for 48 hours to obtain an activated bacterial strain cultured on a slant solid culture for later use.

[0044] (2) Expansion culture: Take the activated bacterial strains mentioned above, add sterile water to make a bacterial suspension (add 5 mL of sterile water to one test tube), inoculate the bacterial suspension into the expansion medium (the composition is the same as that of the MRS liquid medium in Example 1), the inoculation amount is 4% (volume ratio), the culture temperature is 30°C, the culture time is 24 hours, and the culture solution is obtained. Adjust the bacterial concentration of the culture solution to 1×10 9 CFU / mL, centrifuged at 10000r / min for 10min, and obtained bacteria, which were used as a starter for the fermentation of weed crab sauce.

[0045] (3) Surface sterilization of weed crabs: Take weed crabs, remove the gills and crush them; place them in 20g / L NaCl solution and soak for 10 minutes to sterilize them, and repeat the soaking three times.

[0046] (4) Inoculation of strain: The prepared starter culture was inoculated into the crushed weeds (inoculation amount was 6% by volume of the bacterial solution, i.e., 6 mL of culture solution was inoculated into every 100 mL of crushed weeds) and fermented at 25°C for 40 days to obtain weed fermentation sauce with a solid content of 89.88% and a pH of 7.45.

[0047] Example 4 Preparation of Weed Crab Sauce by Natural Fermentation

[0048] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to Example 3, the difference is that the strain Priesteria megaterium GD0038 is not added in the fermentation step, and natural fermentation is carried out.

[0049] Example 5 Determination of Flavoring Substances in Weed Crab Sauce

[0050] The types and contents of volatile flavor substances in fermented weed crab sauce were detected by SPME-GC-MS, and the compounds were semi-quantitatively analyzed using the peak area of ​​the internal standard 2-methyl-3-heptanone. The test results are shown in Table 1 below.

[0051] SPME-GC-MS analysis of volatile flavor components

[0052] (1) Flavor extraction

[0053] Weigh 5.00 g ± 0.01 g of sample per vial and place it into a 25 mL headspace vial. After equilibration at room temperature for 10 minutes, insert an SPME needle into the silicone rubber liner of the vial and extend a 65 μm PDMS / DVB tip. Adsorb the sample in a 50°C water bath for 30 minutes. After adsorption is complete, remove the vial and insert it into the GC-MS inlet. Desorb the sample at 250°C for 2 minutes, followed by thermal desorption and GC-MS analysis.

[0054] (2) Chromatographic conditions

[0055] A DB-WAX elastic capillary column (60m×0.32mm×0.25μm) was used, with an injection port temperature of 250°C, splitless mode, a flow rate of 1.0mL / min, He as the carrier gas, an initial column temperature of 40°C, maintained for 3min, increased to 120°C at 5°C / min without retention, increased to 200°C at 10°C / min, maintained for 5min, increased to 240°C at 8°C / min, and retained for 5min.

[0056] (3) Mass spectrometry conditions

[0057] Electron impact (EI) ion source; electron energy 70 eV, filament emission current 200 μA, source temperature 200°C, detector voltage 350 V; full scan, 35–500 m / z.

[0058] Table 1 Volatile compounds in weed crab sauce detected by GC-MS

[0059]

[0060] The volatile compounds and their relative contents of weed crab sauce at different fermentation stages are shown in Table 1. Figure 1As can be seen, volatile flavor compounds extracted from weed crab sauce using SPME were analyzed by GC-MS, identifying a total of 70 volatile components (including 35 alcohols, 4 aldehydes, 3 amines, 4 pyrazines, 2 acids, 9 ketones, and 5 other compounds). Of these, 53 components were detected in the naturally fermented sample (Example 4), and 69 components were detected in the sample fermented with a starter culture (Example 3). This indicates that fermentation with Priesteria gigantea GD0038 gradually increases the abundance and content of volatile flavor components in weed crab sauce.

[0061] Comparing the data of Example 3 and Example 4, it can be seen that under the same fermentation conditions, inoculation of Priesteria gigantea GD0038 can significantly increase the content of the main volatile flavor alcohols, pyrazines, acids, and ketones in weed crab sauce, and significantly increase the content of volatile compounds such as characteristic flavor substances 1-penten-3-ol, 1-nonanol, isovaleraldehyde, n-hexanol, n-heptanol, and citronellol to 0.13%, 6.19%, 0.06%, 1.68%, 8.59%, and 3.47%, while reducing the content of undesirable flavors such as butylamine, trimethylamine, and n-hexanal to 0.28%, 0.52%, and 0.11%, respectively. Example 3 significantly improves the flavor of fermented weed crab sauce compared to Comparative Example 4, and has very good application prospects.

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A strain of Priesteria gigantea ( Priestia megaterium ), characterized in that, The Priesteria gigantea is Priesteria gigantea GD0038, and its deposit number is CCTCC NO: M 20242548.

2. Use of Priesteria gigantea as claimed in claim 1 in preparing weed crab sauce.

3. A method for preparing weed crab sauce, characterized in that: Weed crabs are mashed and inoculated with the Priesteria gigantea described in claim 1, and fermented to prepare weed crab sauce.

4. The method for preparing weed crab sauce according to claim 3, wherein The following steps are involved: S1: Inoculate activated Priesteria gigantea GD0038 into the culture medium and culture at 36-38°C for 20-30 hours to obtain a culture solution; S2: Crush weed crabs, inoculate the culture medium with the obtained bacteria by centrifugation, and culture at 25° C. for 40-50 days to obtain weed crab sauce.

5. The method for preparing weed crab sauce according to claim 4, wherein: The culture medium in step S1 is MRS liquid culture medium.

6. The method for preparing weed crab sauce according to claim 4, wherein: The culture conditions in step S1 are: culture at 37°C for 24 hours; The culture conditions in step S2 are: culture at 25° C. for 40 days.

7. The method for preparing weed crab sauce according to claim 4, wherein: The inoculation amount of the bacteria in step S2 is 3% to 10% by volume of the bacterial solution, that is, 3 to 10 mL of culture solution obtained by centrifugation is inoculated into every 100 mL of crushed wedeli.

8. The method for preparing weed crab sauce according to claim 4, wherein: In step S2, the wedeli are first sterilized and then crushed; the wedeli sterilization method is: soaking the wedeli in a sodium chloride solution.

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