Enterococcus thailandicus and application thereof

Abstract

The application belongs to the field of biology and particularly relates to a high-yield caprylic acid Enterococcus thailandicus and application thereof. The Enterococcus thailandicus with the preservation number of CGMCC No. 25135 is screened in high-quality pit mud and is suitable for the environment of liquor brewing. The yield of caprylic acid of the Enterococcus thailandicus is 5.45 g / L when the Enterococcus thailandicus is fermented at 37 DEG C for 72 h, which is higher than the yield of caprylic acid of existing strains, and thus the Enterococcus thailandicus can be applied to liquor brewing as a strengthening agent.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biology, and particularly relates to a high-yield caprylic acid Enterococcus thailandicus and application thereof. BACKGROUND

[0002] In the process of liquor brewing, acids are precursor substances for forming esters, and can also constitute other flavor substances. Appropriate amount of organic acids can make the liquor body full and mellow, and the aftertaste long. Caprylic acid presents fruit aroma at low concentration, and is a precursor substance for forming ethyl caprylate.

[0003] Li Zhilin et al. found (Li Zhilin, Li Jing. Content and flavor contribution analysis of ethyl caprylate in Luzhou-flavor liquor [J]. Liquor-making Science and Technology, 2013 (4): 3.) that ethyl caprylate is an important substance in Luzhou-flavor liquor, and its aroma intensity value (OAV) is second only to ethyl hexanoate among esters. High-quality pit mud is one of the basic conditions for producing high-quality Luzhou-flavor liquor. The traditional high-quality pit mud is naturally matured by domesticating beneficial microorganisms through continuous production for many years, but this process needs a long time. The artificial pit mud cultivation technology by using compound microbial culture and old pit mud can shorten the maturation time of pit mud.

[0004] However, it is necessary to screen and mine high-quality related strains, which is the key to determine the effect. At present, the strain selection of pit mud cultivation technology mainly focuses on strains producing high hexanoic acid, and often ignores the ability of producing caprylic acid in the compound microbial system. In the existing literature, the content of caprylic acid produced by the strain is 0.066-4.277 g / L, and it is of great significance to screen and mine strains that can adapt to the brewing environment of liquor, do not destroy the balance of brewing microecology, and can produce high caprylic acid, for promoting the development of liquor-making technology. SUMMARY

[0005] The Enterococcus thailandicus strain provided by the present application is screened from high-quality old pit mud for Luzhou-flavor liquor, and has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 21, 2022, with the preservation number of CGMCC No. 25135 and the name of Enterococcus thailandicus zqw21.

[0006] The present application also provides an application of the Enterococcus thailandicus with high yield of caprylic acid. The Enterococcus thailandicus is used as a strengthening agent in the field of liquor brewing, such as artificial pit mud cultivation or intensified Daqu preparation, to increase the content of caprylic acid and provide precursor substances for the formation of ethyl caprylate. The Enterococcus thailandicus is used as a strengthening agent for biological strengthening of brewing microorganisms to develop new liquor products.

[0007] Preferably, the form of the enhancing bacteria agent includes liquid, lyophilized preparation or powder.

[0008] Preferably, the enhancing bacteria agent includes excipients. The excipients include culture medium of bacteria; the culture medium of bacteria includes but is not limited to yeast extract, glucose, peptone, water.

[0009] The present application also provides an enhancing agent for liquor brewing, which is prepared from the Enterococcus thailandicus zqw21. Specifically, the form of the enhancing bacteria agent is selected from liquid, lyophilized preparation or powder prepared after the Enterococcus thailandicus zqw21 is cultured. Optionally, the enhancing bacteria agent further includes excipients, and the excipients include culture medium of bacteria; the culture medium of bacteria includes but is not limited to yeast extract, glucose, peptone, water.

[0010] The Enterococcus thailandicus provided by the present application is screened in high-quality pit mud and is suitable for the environment of liquor brewing. The strain of the present application has an octanoic acid yield of 5.45 g / L after 72 h of fermentation at 37℃, which is higher than the octanoic acid yield of the strains in the existing literature. For example, CN201310587108. discloses a yeast strain capable of producing octanoic acid and decanoic acid and their corresponding ethyl esters, and the octanoic acid yield of the yeast strain is 4277.56 mg / L. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 GC-MS chart of octanoic acid produced by Enterococcus thailandicus zqw21.

[0012] Figure 2 GC-MS molecular fragment mass spectrum chart of octanoic acid produced by Enterococcus thailandicus zqw21.

[0013] Figure 3 GC-MS molecular fragment mass spectrum chart of octanoic acid standard.

[0014] Strain preservation information:

[0015] The Enterococcus thailandicus zqw21 of the present application was preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 21, 2022. The address of the preservation unit is No. 1, Beichen West Road, Beijing, China. The preservation number of the strain is CGMCC No. 25135, and the classification name of the strain is Enterococcus thailandicus. DETAILED DESCRIPTION

[0016] The present application will be further described below through specific examples, so as to better understand the present application, but the specific examples do not constitute a limitation on the present application.

[0017] The medium formula involved in the examples is as follows:

[0018] MRS liquid medium: glucose 20 g / L, peptone 10 g / L, yeast powder 4 g / L, beef infusion powder 5 g / L, Tween 80 1 g / L, triammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 1 g / L, pH 7.0, high-pressure steam sterilization at 115°C for 20 minutes.

[0019] MRS solid medium: 15 g / L agar was added to the MRS liquid medium, and high-pressure steam sterilization was performed at 115°C for 20 minutes.

[0020] Example 1: Isolation and identification of Enterococcus thailandicus

[0021] 1) Isolation method: 20 g of Luzhou Laojiao pit mud sample was placed in a conical flask containing 180 mL of sterile distilled water, and the sample was fully dispersed and mixed by constant temperature shaking for 10 min. 1 ml of sample suspension was taken, and dilution to 10 -2 ~10 -7 100 μL was taken from each gradient dilution, evenly coated on MRS solid medium plates containing 1% CaCO3, two plates were prepared in parallel, inverted, and incubated at 37°C under anaerobic conditions for 36-48 h, and observed in time.

[0022] 2) Streak purification: the plate with growing colonies was taken out, and single colonies with different colony morphology and obvious transparent circles were picked for secondary streaking until all single colonies were purified.

[0023] 3) Strain preservation: the single colonies of each strain after purification were picked into 5 mL of MRS liquid medium, incubated at 37°C under anaerobic conditions for 20-24 h, 1 mL of bacterial solution was taken into a bacteria preservation tube, 0.5 mL of 60% sterile glycerol solution was added, resuspended, and stored at -80°C.

[0024] 4) 16S rDNA sequence amplification: 1 mL of bacterial solution was centrifuged at 10,000 rpm for 5 min, the supernatant was removed, and bacterial slurry was obtained. CTAB solution and phenol-chloroform-isoamyl alcohol (25:24:1) were added to extract nucleic acid DNA. The upper clear liquid was obtained by centrifugation at 12,500 rpm for 5 min. An equal volume of chloroform-isoamyl alcohol (24:1) was added, and the mixture was mixed and centrifuged at 12,000 rpm for 5 min. The upper clear liquid was obtained by ethanol solution rinsing twice, and the supernatant was removed by centrifugation at 12,000 rpm for 5 min. The precipitate was blown dry, 50 μL of sterile water was added to resuspend it, and the bacterial strain DNA template was obtained. The primers were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. PCR amplification was performed, and the gene sequence of the 16S rDNA fragment was obtained by sequencing. The strain species information was determined by BLAST comparison of NCBI.

[0025] Finally, 4 strains of acid-producing strains were screened, which were identified as Enterococcus thailandicus zqw21, Enterococcus italicus rsj24, Weissella cibaria rsj26, and Enterococcus thailandicus rsj28. The 16S rDNA fragment nucleotide sequence of Enterococcus thailandicus zqw21 is shown as SEQ ID NO: 1:

[0026] tagggacggcctggctccaaaaaggttacctcaccgacttcgggtgttacaaactttcgtggtgtgacgg

[0027] gcggtgtgtacaaggcccgggaacgtattcaccgcggcgtgctgatccgcgattactagcgattccggct

[0028] tcatgtaggcgagttgcagcctacaatccgaactgagagaagctttaagagattagcttagcctcgcgac

[0029] ttcgcgactcgttgtacttcccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgac

[0030] gtcatccccaccttcctccggtttgtcaccggcagtcttgctagagtgcccaactaaatgatggcaacta

[0031] acaataagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacaaccatgca

[0032] ccacctgtcactttgcccccgaaggggaagctctatctctagagtggtcaaaggatgtcaagacctggta

[0033] aggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcctttg

[0034] agtttcaaccttgcggtcgtactccccaggcggagtgcttaatgcgttagctgcagcactgaagggcgga

[0035] aaccctccaacacttagcactcatcgtttacggcgtggactaccagggtatctaatcctgtttgctcccc

[0036] acgctttcgagcctcagcgtcagttacagaccagagagtcgccttcgccactggtgttcctccatatatc

[0037] tacgcatttcaccgctacacatggaattccactctcctcttctgcactcaagtctcccagtttccaatga

[0038] ccctccccggttgagccgggggctttcacatcagacttaagaaaccgcctgcgctcgctttacgcccaat

[0039] aaatccggacaacgcttgccacctacgtattaccgcggctgctggcacgtagttagccgtggctttctgg

[0040] ttagataccgtcaagggatgaacagttactctcatccttgttcttctctaacaacagagttttacgatcc

[0041] GAAAACCTTCTTCACGCAGCGGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCT

[0042] GCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCC AATGTGGCCGATCAC CCTCTCAGGTCGGCTATGCA

[0043] TCGTGGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCACC GC GGTCCATCCATCAGCGACACCC

[0044] GAAAGCGCCTTTCAAATCAAAACCATGCGGTTTCGATTGTTATACGGTATTAGCACCTGTTTCCAAGTGT

[0045] TATCCCCTTCTGATGGGCAGGTTACCCACGTGTTACTCACCCGTTCGCCACTCCTTTTCTTTCGGTGGAGCAAGCTCCG GTGAAAGAAAAAGCGTACGACTTGCATTTTTTAAGGGC.

[0046] Example 2: Related properties of Enterococcus thailandicus zqw21

[0047] Experiments show that the culture characteristics of Enterococcus thailandicus zqw21 are as follows: the optimum growth temperature is 37°C, the pH value is 7, it is facultative anaerobic, and it can grow under aerobic, anaerobic or micro-aerobic conditions. In terms of temperature tolerance, the bacteria grow well at 42°C, grow slowly at 45°C, and cannot grow at 48°C.

[0048] The biochemical properties of Enterococcus thailandicus zqw21 are as follows:

[0049] 1. The bacteria can produce acid by using 20 kinds of single carbon sources respectively, such as D-galactose, D-glucose, D-fructose, D-mannose, D-ribose, methyl-αD-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellotriose, D-maltose, D-gentiobiose, mannitol, esculin iron citrate, D-lactose, D-trehalose, potassium gluconate, glycerol (glycerin), potassium 5-ketoglucuronate, etc. under anaerobic conditions at 37°C for 48 hours. The utilization ability from strong to weak is as follows: D-galactose, D-glucose, D-fructose, D-mannose, D-ribose, methyl-αD-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellotriose, D-maltose, D-gentiobiose, stronger than mannitol, esculin iron citrate, D-lactose, D-trehalose, potassium gluconate, stronger than glycerol (glycerin), potassium 5-ketoglucuronate.

[0050] 2. The bacteria can utilize MRS medium to produce lactic acid 11.7 g / L and a variety of 41 volatile metabolites under micro-aerobic conditions at 37°C for 48 hours of static culture, including 33.6 mg / L of octanoic acid, 13.09 mg / L of acetic acid, 7.27 mg / L of nonanoic acid, 2.29 mg / L of hexanoic acid, 1.33 mg / L of n-heptanol, 1.33 mg / L of 1-hexadecene, 1.19 mg / L of 2,3-dihydro-2,2,6-trimethylbenzaldehyde, 1.07 mg / L of n-decanoic acid, 1.06 mg / L of phenylacetaldehyde, 1.06 mg / L of triisobutyl phosphate, 0.71 mg / L of phenylethanol, 0.62 mg / L of 2,6-dimethylpyrazine, 0.56 mg / L of heptanoic acid, 0.54 mg / L of iso-octanoic acid, 0.5 mg / L of benzaldehyde; and other trace amounts less than 0.5 mg / L of isovaleric acid 0.49 mg / L, vinyl acetate 0.48 mg / L, butyric acid 0.42 mg / L, geranylacetone 0.39 mg / L, ethyl linoleate 0.36 mg / L, ethyl palmitate 0.35 mg / L, 2-ethylhexanol 0.33 mg / L, diethylene glycol ether 0.31 mg / L, 3,4-dimethylbenzaldehyde 0.3 mg / L, undecane 0.28 mg / L, n-butanol 0.27 mg / L, 1-nonanol 0.27 mg / L, pentadecanol 0.27 mg / L, 2-ethylpyrazine 0.26 mg / L, 2,5-dimethylpyrazine 0.24 mg / L, ethyl oleate 0.22 mg / L, n-octanol 0.12 mg / L, benzothiazole 0.11 mg / L, acetophenone 0.1 mg / L, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate 0.1 mg / L, phenol 0.1 mg / L; and trace amounts less than 0.1 mg / L of 3-hydroxy-2,2,4-trimethylpentyl isobutyrate 0.09 mg / L, 2-methylpyrazine 0.04 mg / L, tributyl phosphate 0.03 mg / L, 2-methyl-6-vinylpyrazine 0.02 mg / L, diisobutyl phthalate 0.02 mg / L.

[0051] Example 3: Detection of the octanoic acid production ability of Enterococcus thailandicus zqw21

[0052] 1) Preparation of detection samples: The four strains with obvious acid production ability screened, i.e. Enterococcus thailandicus zqw21, Enterococcus italicus rsj24, Weissella cibaria rsj26, and Enterococcus thailandicus rsj28, were respectively dissolved to inoculate 10 mL of MRS liquid medium, and cultured at 37°C for 12-20 h, then 2% (volume ratio) of the inoculation amount was inoculated into 400 mL of MRS liquid medium for culture for 72 h. After fermentation, the fermented bacterial liquid was obtained, and the supernatant was taken after centrifugation at 10000 rpm for 5 min.

[0053] 2) Methods for determining volatile acids:

[0054] Headspace solid-phase microextraction (HSP) was employed: the supernatant was collected and added to a headspace vial, along with saturated NaCl solution. 2-Octanol (0.822 mg / ml) was used as an internal standard. The prepared sample was incubated at 60°C for 5 min to equilibrate, then extracted at 60°C for 50 min using a 50 / 30 μm DVB / CAR / PDMS extraction head. After extraction, desorption was performed at 250°C for 5 min at the GC inlet. Compound search results were matched with the NIST standard spectral library; compounds with a similarity of 80% or higher were confirmed as target compounds. A culture broth without added bacteria was used as a blank control group, and the content of each volatile substance was calculated.

[0055] GC-MS detection chromatographic conditions:

[0056] Gas chromatography conditions: HP-INNOWAX column (60m × 0.25mm × 0.25μm); Temperature program: initial temperature 40℃, hold for 5 min, increase to 100℃ at 4℃ / min, then increase to 230℃ at 6℃ / min.

[0057] Hold for 10 min with high-purity helium as the carrier gas (1.0 mL / min); injector temperature 250℃, no splitting.

[0058] Mass spectrometry conditions: electron ionization source, electron energy 70 eV; electron multiplier voltage 350 V; ion source temperature 230 °C; transfer line temperature 250 °C; mass range 40–450 m / z.

[0059] The volatile compounds in the fermentation products of the four strains screened in this invention were analyzed by HS-SPME / GC-MS. The results are shown in Table 1. The GC-MS results for the octanoic acid component are as follows: Figure 1 , Figure 2 , Figure 3 As shown, where Figure 1 GC-MS image of caprylic acid produced by Enterococcus tumefaciens zqw21 in Thailand; Figure 2 GC-MS molecular fragment mass spectrum of caprylic acid produced by Enterococcus thuringiensis zqw21 in Thailand; Figure 3 This is a GC-MS molecular fragment mass spectrum of octanoic acid standard.

[0060] Table 1. Results of volatile acid detection in fermentation broths of different bacterial strains

[0061]

[0062] From the above table, the Enterococcus thailandicus zqw21 produces 5.45 g / L of caprylic acid after 72 h of fermentation, and the difference in caprylic acid production is significant (P<0.01) compared with the other three strains. The Enterococcus thailandicus zqw21 is preserved, and the preservation number is CGMCC NO: 25135.

Claims

1. A type of Thai enterococcus ( Enterococcus thailandicus zqw21, its collection number is CGMCCNo.25135.

2. The application of Enterococcus tumefaciens zqw21 as described in claim 1 in the brewing of Baijiu (Chinese liquor).

3. The application as described in claim 2, characterized in that, The aforementioned Enterococcus tumefaciens zqw21 was used as an enhancing agent in the brewing of baijiu (Chinese liquor).

4. The application as described in claim 3, characterized in that, The aforementioned Enterococcus tumefaciens zqw21 was applied to the cultivation of artificial pit mud or the preparation of Daqu (a type of starter culture) to increase the octanoic acid content and provide precursor substances for the formation of ethyl octanoate.

5. The application as described in claim 3, characterized in that, The aforementioned Enterococcus tumefaciens zqw21 was used as an enhancing agent to biofortify brewing microorganisms for the development of baijiu products.

6. The application as described in any one of claims 3 to 5, characterized in that, The form of the enhancing agent is selected from liquid, lyophilized preparation or powder.

7. The application as described in claim 6, characterized in that, The enhanced microbial agent also includes excipients, which include culture medium substances for the bacteria; the culture medium substances for the bacteria include yeast extract, glucose, peptone and water.

8. A microbial agent for use in the brewing of baijiu (Chinese liquor), prepared from Enterococcus thamniosum zqw21 as described in claim 1.

9. The microbial enhancer as described in claim 8, characterized in that, The form of the enhancing agent is selected from the liquid, lyophilized preparation or powder prepared after culturing the Thai Enterococcus zqw21.

10. The microbial enhancer as described in claim 9, characterized in that, The enhanced microbial agent also includes excipients, which include culture medium substances for the bacteria; the culture medium substances for the bacteria include yeast extract, glucose, peptone and water.

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