P.megatherium GD0038 and application of P.megatherium GD0038 in improving flavor of wedelia chinensis

Abstract

The invention discloses a strain of P.megatherium GD0038 and application thereof in improving the flavor of wedelia chinensis, the strain is preserved in China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20242548, the preservation date is November 13, 2024, and when the strain is applied to preparation of the wedelia chinensis, the flavor of the wedelia chinensis can be obviously improved, the content of volatile substances in the fermented wedelia chinensis can be increased, and the wedelia chinensis can be used for preparing the wedelia chinensis. The content of special flavor substances such as 1-pentene-3-ol, 1-nonyl alcohol, isovaleraldehyde, n-hexanol, n-caprylic alcohol, 3-n-decanol, 2-heptanone and other alcohols, pyrazines and ketones is increased, meanwhile, the content of putidine, trimethylamine and n-hexanal with bad flavor is reduced, and the method has a very good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Priestia megaterium strain GD0038 and its application in improving the flavor of Sesarma dehaani paste. Background Art

[0002] Sesarma dehaani, also known as Sesarma messa and Sesarma parvirostris, belongs to the class Malacostraca and the family Grapsidae. It is a small freshwater crab. The carapace of Sesarma dehaani is square, and the left and right lateral margins are parallel. The frontal width is equal to or exceeds half of the carapace width. There are granules and hairs in the buccal region, arranged alternately in a reticular pattern. There are many species, and the common one is Sesarma haematocheir. The carapace length is 2.9 cm. The chelipeds of the medium-sized Sesarma dehaani are hairless, and the red walking legs are hairy.

[0003] Sesarma dehaani paste is a traditional specialty in Fuzhou, Fujian. During the slack season of autumn and winter, hardworking farmers in the Minjiang River Basin of Fuzhou catch Sesarma dehaani on the clean river bank, wash them with clean water, remove the tails, add auxiliary materials such as yellow rice wine, distiller's grains, monosodium glutamate, white sugar, and glutinous rice, grind them into paste with a stone mill, and add an appropriate amount of table salt to make a delicious condiment. In the Fuzhou area, Sesarma dehaani paste is an essential food for wedding banquets, usually used to dip jellyfish heads, and its delicious taste is unforgettable. However, the flavor of naturally fermented Sesarma dehaani paste cannot be guaranteed; therefore, how to guarantee and improve its flavor is of great significance for the preparation of Sesarma dehaani paste. Summary of the Invention

[0004] In view of the above problems, the present invention provides a Priestia megaterium strain ( Priestia megaterium ) GD0038, which can be applied to the preparation of Sesarma dehaani paste to improve the flavor of Sesarma dehaani paste.

[0005] To solve the above technical problems, the present invention provides a Priestia megaterium strain ( Priestia megaterium ) GD0038, which is deposited in the China Center for Type Culture Collection, with the deposit number of CCTCC NO: M 20242548, and the deposit date: November 13, 2024.

[0006] Based on the above, the present invention also provides a screening method for a Priestia megaterium strain ( Priestia megaterium ) GD0038, comprising the following steps: (1) Primary screening of strains on plates: Dilute the naturally fermented Sesarma dehaani paste by 10-fold gradient with sterile water, take 100 μL and spread it on MRS agar solid medium, and culture it statically for 24 hours at 30°C under aerobic conditions; Select single colony strains with different morphologies, make them into a bacterial suspension with sterile water, spread it on MRS agar solid medium, and culture it statically for 24 hours at 30°C under aerobic conditions. Repeat the plate culture 4 times in this way; (2) Shake flask rescreening of the strain: Inoculate the strain screened above into MRS liquid medium and culture it at 37 °C for 48 hours.

[0007] Furthermore, the preparation method of the naturally fermented Sesarma dehaani paste described in step (1) is as follows: Take Sesarma dehaani, wash it clean, remove the gills and break it up, and perform self-fermentation in a sealed manner at 21 °C for 40 days to obtain the naturally fermented Sesarma dehaani paste; The composition of the MRS agar solid medium is as follows: Beef protein powder 10 g / L; Fish juice 10 g / L; Yeast extract powder 5 g / L; Glucose 20 g / L; Sodium acetate 5 g / L; Diammonium citrate 2 g / L; Magnesium sulfate 0.58 g / L; Manganese sulfate 0.28 g / L; Agar powder 15.0 g / L; Tween 80 0.1 g / L; pH value is 6.2 - 6.4; The composition of the MRS liquid medium is 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 value 6.2 - 6.4.

[0008] Based on the above, the present invention also provides a preparation method of Sesarma dehaani paste, including: Take Sesarma dehaani, mash it, and inoculate the above-mentioned Priestia megaterium ( Priestia megaterium ) GD0038, and ferment to obtain Sesarma dehaani paste.

[0009] Furthermore, the preparation method of the Sesarma dehaani paste includes the following steps: S1: Inoculate the activated Priestia megaterium ( Priestia megaterium ) GD0038 strain into the medium and culture it at 36 - 38 °C for 20 - 30 hours to obtain a culture solution; S2: Take Sesarma dehaani, mash it, inoculate the culture solution or the cells obtained by centrifuging the culture solution, and culture it at 36 - 38 °C for 40 - 50 days to obtain Sesarma dehaani paste.

[0010] Furthermore, the medium selected in step S1 is MRS liquid medium.

[0011] Furthermore, the culture condition in step S1 is: Culture at 37 °C for 24 hours; The culture condition in step S2 is: Culture at 37 °C for 40 days.

[0012] Furthermore, the inoculation amount of the culture solution or the cells in step S2 is 3% - 10%.

[0013] Further, in step S2, the crabs are sterilized first and then mashed; the sterilization method for the crabs is: soaking the crabs in a sodium chloride solution.

[0014] The above-mentioned Priestia megaterium ( Priestia megaterium ) GD0038 can be applied to the preparation of crab paste.

[0015] The beneficial effects of the present invention are as follows: The Priestia megaterium ( Priestia megaterium ) GD0038 provided by the present invention is screened from traditionally fermented crab paste. It has strong raw material adaptability, good physiological activity and growth characteristics, and excellent salt tolerance, acid tolerance, resistance to NaNO 2 3, antibacterial property, hydrophobicity, adhesiveness, autoaggregation property, etc. It can be used as an inoculation fermenting agent for crab paste, and can solve the quality difference and safety problems caused by inconsistent batches of natural crab paste.

[0016] Secondly, the crab paste fermented with Priestia megaterium ( Priestia megaterium ) GD0038 has the same main aroma compounds as natural fermentation, has a directional flavor enhancement effect, and realizes the directional flavor improvement of natural fermentation.

[0017] In addition, the crab paste fermented with Priestia megaterium ( Priestia megaterium ) GD0038 also has the advantages of high unsaturated fatty acid content, strong antioxidant ability, and excellent digestion characteristics. Description of the Drawings

[0018] Figure 1 It is a comparison chart of the number of volatile compound species in two fermentation methods of Example 3 and Example 4. Detailed Embodiments

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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 part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0020] The Priestia megaterium ( Priestia megateriumGD0038 belongs to the genus Bacillus, a Gram-positive bacterium, which is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 20242548. The preservation date is November 13, 2024, and the preservation address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. This strain can be applied to the preparation of Sesarma dehaani H. Milne-Edwards sauce, with a directional flavor enhancement effect, achieving the directional flavor improvement of natural fermentation.

[0021] Example 1 Screening of Priestia megaterium ( Priestia megaterium ) GD0038 The screening method of Priestia megaterium ( Priestia megaterium ) GD0038 includes the following steps: (1) Primary screening of strains on plates: Use sterile water to perform 10-fold serial dilutions of naturally fermented Sesarma dehaani H. Milne-Edwards sauce. Take 100 μL and spread it on MRS agar solid medium, and incubate at 30 °C, aerobic, and static for 24 hours.

[0022] Select single colony strains with different morphologies, make them into bacterial suspensions with sterile water, spread them on MRS agar solid medium, and incubate at 30 °C, aerobic, and static for 24 hours. Repeat the plate culture 4 times in this way, and finally select 40 strains.

[0023] Among them, the naturally fermented Sesarma dehaani H. Milne-Edwards sauce is prepared by the following method: Take Sesarma dehaani H. Milne-Edwards (caught in the Zhanjiang Sea area of Guangdong Province in May 2024), wash it clean, remove the gills and break it up, and perform self-fermentation in a sealed manner at 21 °C for 40 days to obtain the naturally fermented Sesarma dehaani H. Milne-Edwards sauce.

[0024] The composition of the above MRS agar solid medium is as follows: beef protein powder 10 g / L; fish broth 10 g / L; yeast extract powder 5 g / L; glucose 20 g / L; sodium acetate 5 g / L; diammonium citrate 2 g / L; magnesium sulfate 0.58 g / L; manganese sulfate 0.28 g / L; agar powder 15.0 g / L; Tween 80 0.1 g / L; pH value 6.2 - 6.4.

[0025] (2) Re-screening of strains in shake flasks: Inoculate the strains screened above into MRS liquid medium and culture at 37 °C for 48 hours.

[0026] Among them, the composition of the MRS liquid medium is 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 value 6.2 - 6.4.

[0027] Example 2 Identification of the strain (1)Identification of Priestia megaterium GD0038: 16S rDNA sequence identification was performed using the universal bacterial primers 27F / 1492R, and it was Priestia megaterium .

[0028] (2)Preservation method of Priestia megaterium GD0038: Select well-grown single colonies from the MRS agar medium plate and inoculate them into 5 mL of MRS broth medium. Incubate statically under aerobic conditions at 30 °C for 24 h, and take 1 mL of the culture solution. Add it to a cryopreservation tube containing 1 mL of 30% glycerol and preserve it in a -80 °C refrigerator.

[0029] Example 3 Preparation of Sesarma dehaani paste The preparation method of Sesarma dehaani paste is as follows: (1)Strain activation: Inoculate the strain Priestia megaterium GD0038 on a solid slant medium (the component composition is the same as the MRS agar solid medium in Example 1), and incubate statically under aerobic conditions at 30 °C for 48 hours to obtain an activated strain in slant solid culture for standby.

[0030] (2)Subculture: Take the above-activated strain, add sterile water to make a bacterial suspension (add 5 mL of sterile water to one test tube), inoculate the bacterial suspension into the subculture medium (the component composition is the same as the MRS liquid medium in Example 1), the inoculation amount is 4% (volume ratio), the culture temperature is 30 °C, and the culture time is 24 hours to obtain a culture solution. Adjust the bacterial concentration of the culture solution to 1×10 9 CFU / mL, centrifuge at 10000 r / min for 10 min to obtain bacterial cells, which are used as a starter for the fermentation of Sesarma dehaani paste.

[0031] (3)Surface sterilization of Sesarma dehaani: Take Sesarma dehaani, remove the gills and then crush it; place it in a 20 g / L NaCl solution and soak for 10 min for sterilization, and repeat the soaking 3 times.

[0032] (4)Strain inoculation: Inoculate the above-prepared starter into the mashed Sesarma dehaani (the inoculation amount is 6%, based on the volume of the bacterial solution, that is: inoculate the bacterial cells obtained by centrifuging 6 mL of the culture solution into every 100 mL of mashed Sesarma dehaani) and ferment at 25 °C for 40 days to obtain Sesarma dehaani fermented paste, with a solid content of 89.88% and a pH of 7.45.

[0033] Example 4 Preparation of Sesarma dehaani paste by natural fermentation The specific implementation method refers to Example 3, and the difference is that in the fermentation step, the strain - Priestia megaterium GD0038 is not added, and natural fermentation is carried out.

[0034] Example 5 Determination of flavor substances in Sesarma dehaani paste The types and contents of volatile flavor compounds in fermented mud crab paste were detected by SPME-GC-MS. The peak area of the internal standard 2-methyl-3-heptanone was used for semi-quantitative analysis of the compounds, and the detection results are shown in Table 1 below.

[0035] SPME-GC-MS Analysis of Volatile Flavor Components (1)Flavor Substance Extraction Weigh 5.00 g ± 0.01 g of each sample and place it in a 25 mL headspace vial. After equilibrating the headspace vial containing the sample at room temperature for 10 min, insert the SPME syringe needle through the silicone rubber septum of the headspace vial, extend the 65 μm PDMS / DVB extraction head, and adsorb it in a 50 °C water bath for 30 min. After adsorption is complete, take it out and insert it into the GC-MS injection port for 2 min of desorption at 250 °C, and perform GC-MS detection by thermal desorption.

[0036] (2)Chromatographic Conditions DB-WAX elastic capillary column (60 m × 0.32 mm × 0.25 μm), injection port temperature 250 °C, splitless mode, flow rate 1.0 mL / min, carrier gas is He, initial column temperature 40 °C, hold for 3 min, increase to 120 °C at 5 °C / min, no retention, increase to 200 °C at 10 °C / min, hold for 5 min, increase to 240 °C at 8 °C / min, and retain for 5 min.

[0037] (3)Mass Spectrometry Conditions Electron impact (EI) ion source; electron energy 70 eV, filament emission current 200 μA, ion source temperature 200 °C, detector voltage 350 V; full scan, 35 - 500 m / z.

[0038] Table 1 Volatile Compounds in Mud Crab Paste Detected by GC-MS

[0039] The volatile compounds and their relative contents in mud crab paste at each fermentation stage are shown in Table 1. From Table 1 and the appendix Figure 1It can be seen that 70 volatile components were identified by GC-MS analysis of the volatile flavor substances extracted from Sesarma dehaani H. Milne-Edwards paste by SPME method (including 35 alcohols, 4 aldehydes, 3 amines, 4 pyrazines, 2 acids, 9 ketones and 5 other compounds). Among them, 53 components were detected in the naturally fermented sample (Example 4), and 69 components were detected in the sample fermented with the inoculated starter (Example 3). It shows that inoculating Priestia megaterium GD0038 for fermentation gradually increases the volatile flavor components and their abundance in Sesarma dehaani H. Milne-Edwards paste.

[0040] By comparing the data of Example 3 and Example 4, it can be seen that under the same fermentation conditions, inoculating Priestia megaterium GD0038 can significantly increase the contents of the main volatile flavor alcohols, pyrazines, acids and ketones in Sesarma dehaani H. Milne-Edwards paste. The contents of volatile compounds such as 1-penten-3-ol, 1-nonanol, isovaleraldehyde, n-hexanol, n-heptanol and citronellol, the characteristic flavor substances, are significantly increased to 0.13%, 6.19%, 0.06%, 1.68%, 8.59% and 3.47% respectively. At the same time, the contents of the off-flavor compounds putrescine, trimethylamine and n-hexanal are reduced to 0.28%, 0.52% and 0.11%. Example 3 significantly improves the flavor of the fermented Sesarma dehaani H. Milne-Edwards paste compared with Comparative Example 4 and has very good application prospects.

[0041] The above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the present invention in each example.

Claims

1. A strain of Priesteria gigantea, characterized in that The Priesteria gigantea is Priesteria gigantea ( Priestia megaterium ) GD0038, the 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 screening Priesteria gigantea as claimed in claim 1, characterized in that: The steps include: (1) Initial screening of strains on plates: Use sterile water to dilute the naturally fermented weed crab sauce 10 times in a gradient, take 100 μL and spread it on MRS agar solid medium, and culture it at 30°C, oxygen consumption, and static for 24 hours; Single colony strains with different morphologies were selected, and bacterial suspensions were prepared with sterile water, spread on MRS agar solid medium, and cultured at 30°C, oxygen consumption, and static for 24 hours. This plate culture was repeated 4 times; (2) Strain rescreening in shake flasks: The strains screened above were inoculated into MRS liquid culture medium and cultured at 37°C for 48 hours.

4. The method for screening Priesteria gigantea according to claim 3, characterized in that: The method for preparing the naturally fermented weed crab sauce in step (1) is as follows: taking weed crabs, cleaning them, removing their gills and crushing them, and sealing and self-fermenting them at 21° C. for 40 days to obtain the naturally fermented weed crab sauce; The components of the MRS agar solid culture medium are: 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; pH value is 6.2-6.4; The components of the MRS liquid culture medium are: 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.

5. A method for preparing weed crab sauce, characterized in that: Take the weed crab and crush it and inoculate the huge Priesteria bacteria ( Priestia megaterium )GD0038, fermented to make crab sauce.

6. The method for preparing weed crab sauce according to claim 5, characterized in that: The following steps are involved: S1: Activated Priesteria gigantea ( Priestia megaterium ) Inoculate GD0038 bacteria into the culture medium and culture at 36-38°C for 20-30 hours to obtain culture fluid; S2: Take weed crabs, crush them, inoculate the culture solution or the bacteria obtained by centrifuging the culture solution, and culture them at 36-38° C. for 40-50 days to obtain weed crab sauce.

7. The method for preparing weed crab sauce according to claim 6, characterized in that: The culture medium in step S1 is MRS liquid culture medium.

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

9. The method for preparing weed crab sauce according to claim 6, characterized in that: In step S2, the inoculation amount of the culture solution or the bacterial cells is 3% to 10%.

10. The method for preparing weed crab sauce according to claim 6, characterized in that: In step S2, the weeds are first sterilized and then crushed; the weed sterilization method is: soaking the weeds in a sodium chloride solution.

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