Pediococcus pentosaceus and application thereof

Abstract

The application discloses a Pediococcus pentosaceus strain derived from high-temperature Daqu and application thereof, the strain is named Pediococcus pentosaceus zqw75, and the preservation number is CGMCC NO.25146.The strain can produce aroma substances such as lauryl acetate, n-heptanol, butyl dodecalactone, benzene acetaldehyde, methyl heptenone and pyrazine, and when the strain is used in combination with commercial sweet wine starter, the relative content of phenylethanol is increased from 8.562% to 10.455%, the relative content of ethyl butyrate is increased from 0.460% to 1.473%, the relative content of ethyl octanoate is increased from 0.754% to 1.672%, the relative content of ethyl n-hexanoate is increased from 0.832% to 3.446%, and the relative content of isoamyl acetate is increased from 0.832% to 2.155%; and the strain can produce aroma substances that are not produced by the commercial sweet wine starter, such as hexanoic acid, butyric acid, octanoic acid, ethyl hexanoate and lauryl acetate.The strain can be applied in the field of liquor making, including pit mud making and liquor flavor substance improvement, and has great industrial application value.

Description

Technical Field

[0001] This invention relates to the field of microbiology, particularly to the field of microbial fermentation technology, especially to the field of brewing, and specifically to a strain of Pediococcus pentosaceus and its application. Background Technology

[0002] Baijiu (Chinese white liquor) brewing involves natural inoculation and multi-species fermentation, with microorganisms being the main force in the process. The microorganisms involved in brewing are primarily yeasts, bacteria, and molds, which play a crucial role in the flavor, quality, and yield of baijiu. Broadly speaking, baijiu microorganisms are widely distributed and originate from a wide range of sources. They can be found in raw materials, water, air, yeast, fermented mash, and lees, as well as in the production site, tools, equipment, fermentation pits, and even human hands and shoes. However, the most important sources of microorganisms in brewing are primarily the spaces within the three production sites: yeast, fermentation pit mud, and lees. The most influential microorganisms in these areas are the molds, yeasts, and bacteria mentioned above.

[0003] Pediococcus pentosaceus is a Gram-positive bacterium belonging to the family Streptococceae and the genus Pediococcus. In recent years, the probiotic function of Pediococcus pentosaceus and its applications in agriculture, animal husbandry, food industry, and clinical practice have received considerable attention.

[0004] VIDHYASAGAR V et al. (VIDHYASAGAR V, JEEVARATNAM K. Evaluation of Pediococcus pentosaceus strains isolated from Idly batter for probiotic properties invitro[J]. Journal of functional foods, 2013, 5(1): 235-243.) found that Pediococcus pentosaceus can exert antibacterial effects on a variety of bacteria, such as Listeria, Salmonella typhi, Streptococcus salivarius, and Staphylococcus aureus, by producing bacteriocins or bacteriocinoids.

[0005] Chinese invention patent CN107099482B provides a strain of Pediococcus pentosaceus with accession number CGMCC No.14218. This strain is used to improve the flavor of fermented milk and increase the content of 2,3-butanedione and 3-hydroxy-2-butanone in fermented milk.

[0006] Chinese invention patent CN109971689B provides a strain of Pediococcus pentosaceus with accession number GDMCC No.60517. This strain is used for rice vinegar fermentation, resulting in rice vinegar containing amyl acetate, which has a banana-like fruit aroma.

[0007] Chinese invention patent CN109504637B provides a strain of Pediococcus pentosaceus with accession number CGMCC No.15957. This strain and the fermented yogurt prepared using this strain can relieve constipation and have a laxative effect. In addition, this strain has a significant inhibitory effect on the growth of pathogenic Staphylococcus aureus.

[0008] Chinese invention patent CN108060103B provides a strain of Pediococcus pentosaceus with accession number CGMCC No.15074. This strain has a fast growth rate and high acid production efficiency, and can be used as a forage silage additive to improve the quality of forage silage.

[0009] In the existing technology, there has never been any report of Pediococcus pentosaceus used in the brewing of baijiu, especially in enhancing the flavor of baijiu and producing aroma substances such as lauryl acetate, n-heptanol, butyl dodecyl lactone, phenylacetaldehyde, and methyl heptenone, or in combination with sweet wine koji to produce the above aroma substances, or in improving the production of the above aroma substances by sweet wine koji.

[0010] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0011] To address the shortcomings of existing technologies, this invention provides a strain of Pediococcus pentosaceus that can be used in conjunction with sweet wine koji in brewing fermentation agents to increase the yield of various aroma substances during fermentation, including acids, alcohols, esters, aldehydes, ketones, and pyrazines, thereby enhancing the variety and quantity of fermentation flavor substances.

[0012] According to a first aspect of the present invention, the present invention provides a strain of Pediococcus pentosaceus, named Pediococcus pentosaceus zqw75, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25146, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on June 21, 2022.

[0013] The *Pediococcus pentosacchari* zqw75 strain was obtained by screening from high-temperature Daqu (a type of starter culture), enriching it multiple times in a glucose-containing medium, and then screening and purifying it into pure cultures. BLAST alignment analysis of its 16S rRNA gene sequence (as shown in SEQ ID NO. 1) confirmed that the strain belonged to the genus *Pediococcus pentosacchari* and was named *Pediococcus pentosacchari* zqw75.

[0014] According to a second aspect of the present invention, the present invention also provides a microbial inoculum containing the aforementioned Pediococcus pentosaceus strain zqw75.

[0015] In one embodiment of the present invention, the microbial agent is one or more liquid or solid agents containing live cells of Pediococcus pentosacchari zqw75, freeze-dried Pediococcus pentosacchari zqw75 cells, or immobilized Pediococcus pentosacchari zqw75 cells, or the microbial agent is a Pediococcus pentosacchari zqw75 preparation existing in any other form.

[0016] According to a third aspect of the present invention, there is provided an application of *Pediococcus pentosaceus* according to the first aspect of the present invention in the production of aroma substances, wherein the aroma substances are selected from the group consisting of hexanoic acid, butyric acid, octanoic acid, lauryl acetate, n-heptanol, butyl dodecyl lactone, phenylacetaldehyde, methyl heptenone, isoamyl alcohol, n-octanol, 1-nonanol, phenethyl alcohol, 1-hexadecyl alcohol, geraniol, 3,4-dimethylbenzaldehyde, geraniol acetone, 2,6-dimethylpyrazine, and 2-ethylpyrazine.

[0017] According to a fourth aspect of the present invention, a brewing fermentation agent is provided, the brewing fermentation agent containing Pediococcus pentosaceus as described in the first aspect of the present invention.

[0018] In one embodiment of the present invention, the brewing fermentation agent further contains fermentation-active substances and auxiliary materials.

[0019] In one embodiment of the present invention, the fermentation active substance is one or more of brewing yeast, Rhizopus, and Aspergillus.

[0020] In one embodiment of the present invention, the excipient is one or more of the following: culture medium for bacteria, wheat bran, wheat, and barley.

[0021] According to a fifth aspect of the present invention, the application of Pediococcus pentosaceus according to the first aspect of the present invention, or the microbial agent according to the second aspect of the present invention, or the brewing fermentation agent according to the fourth aspect of the present invention, in brewing is provided.

[0022] In one embodiment of the present invention, the Pediococcus pentosaceus described in the first aspect of the present invention, or the microbial agent described in the second aspect of the present invention, or the brewing fermentation agent described in the fourth aspect of the present invention, is mixed with the substance to be fermented and then fermented.

[0023] Beneficial effects

[0024] The *Pediococcus pentosaceus* strain screened in this invention can produce aroma compounds such as hexanoic acid, butyric acid, octanoic acid, lauryl acetate, n-heptanol, butyl dodecyl lactone, phenylacetaldehyde, and methyl heptenone, as determined by volatile matter analysis. When used in combination with commercial sweet wine yeast, it can increase the yield of flavor compounds, with the relative content of phenylethanol increasing from 8.562% to 10.455%, ethyl butyrate from 0.460% to 1.473%, ethyl octanoate from 0.754% to 1.672%, ethyl hexanoate from 0.832% to 3.446%, and isoamyl acetate from 0.832% to 2.155%. Furthermore, it can produce aroma compounds not generated by commercial sweet wine yeast, such as hexanoic acid, butyric acid, octanoic acid, ethyl hexanoate, and lauryl acetate.

[0025] Preservation of biological materials

[0026] A strain of Pediococcus pentosaceus, named Pediococcus pentosaceus zqw75, was screened at medium and high temperature using Daqu (a type of Chinese liquor) and deposited on June 21, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.25146. Attached Figure Description

[0027] Figure 1 This is a microscopic image of Pediococcus pentosaceus zqw75 provided by the present invention;

[0028] Figure 2 This is the GC-MS spectrum of volatile aroma compounds produced by Pediococcus pentosaceus zqw75. Detailed Implementation

[0029] The present invention will now be described in detail with reference to specific embodiments.

[0030] The culture media involved in the following examples are as follows:

[0031] MRS liquid culture medium: glucose 20 g / L, peptone 10 g / L, yeast extract 4 g / L, beef extract 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, autoclaved at 115°C for 20 minutes.

[0032] MRS solid medium: Add 15 g / L agar to the MRS liquid medium and autoclave at 115°C for 20 minutes.

[0033] Example 1: Isolation, purification, and preservation of Pediococcus pentosaceus zqw75

[0034] 1) Separation method: Take 20g of Luzhou Laojiao medium-temperature Daqu sample and place it in an Erlenmeyer flask containing 180mL of sterile distilled water. Shake on a constant temperature shaker for 10min to thoroughly disperse and mix the sample. Take 1ml of the sample suspension and dilute it to 10 using the serial dilution method. -2 ~10 -7 Take 100 μL from each dilution and spread it evenly on an MRS solid medium plate. Prepare two parallel plates, invert them, and incubate them anaerobically at 37°C for 36–48 h, observing them regularly.

[0035] 2) Streak purification: Remove the plate from which colonies have grown, pick single colonies with different colony morphologies, and streak them a second time until all single colonies are purified.

[0036] 3) Preservation of bacterial strains: After purification, single colonies of each strain were picked into 5 mL of MRS liquid medium and incubated anaerobically at 37 °C for 20–24 h. 1 mL of bacterial solution was then transferred to a preservation tube, 0.5 mL of 60% sterile glycerol solution was added, the culture was resuspended, and the culture was stored at -80 °C.

[0037] Example 2: Determination of Volatile Aroma Compounds

[0038] (1) Prepare the bacterial suspension to be tested:

[0039] Six different bacterial strains were screened, and their glycerol preservation tubes were dissolved and inoculated into MRS liquid medium. The cultures were then anaerobic at 37℃ for 24 h and activated for three generations. After activation, the cultures were inoculated into 50 mL of MRS liquid medium at a 2% (v / v) inoculation rate and cultured for another 24 h. After fermentation, the bacterial culture was obtained, centrifuged at 10000 rpm for 5 min, and the supernatant was collected.

[0040] (2) GC-MS detection method:

[0041] Headspace solid-phase microextraction (HSP) was employed: the supernatant was collected and added to a headspace vial, along with saturated NaCl solution. Using 0.822 mg / ml 2-octanol as an internal standard, the prepared sample was equilibrated at 60 °C for 5 min, followed by extraction at 60 °C for 40 min using a 50 / 30 μm DVB / CAR / PDMS extraction head. After extraction, desorption was performed at 250 °C for 5 min via 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. The original culture medium was used as a blank control, and the content of each volatile substance was calculated.

[0042] GC-MS detection chromatographic conditions:

[0043] 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.

[0044] Hold for 10 min with high-purity helium as the carrier gas (1.0 mL / min); injector temperature 250℃, no splitting.

[0045] 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.

[0046] The test results are shown in Table 1. Among the screened strains, Rsj1 has the most outstanding aroma-producing ability, and the types and total content of aroma components are the highest among the six strains.

[0047] Table 1. Results of volatile aroma compound determination for 6 strains

[0048]

[0049]

[0050] Note: "——" indicates that the substance was not detected.

[0051] Example 3: Identification of the strain

[0052] 16S rDNA sequence amplification: Centrifuge 1 mL of Rsj1 bacterial culture at 10000 rpm for 5 min, discard the supernatant to obtain bacterial sludge, add CTAB solution and phenol-chloroform-isoamyl alcohol (25:24:1) to extract nucleic acid DNA, centrifuge at 12500 rpm for 5 min, collect the supernatant, add an equal volume of chloroform-isoamyl alcohol (24:1), mix well, centrifuge at 12000 rpm for 5 min, collect the supernatant, wash twice with ethanol solution, centrifuge at 12000 rpm for 5 min, discard the supernatant, blow the precipitate dry, and resuspend in 50 μL of sterile water to obtain the bacterial DNA template. Primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′ were used for PCR amplification.

[0053] The gene sequence of the 16S rDNA fragment was obtained by sequencing. The species information of the strain was determined by NCBI BLAST comparison of its 16S rDNA gene sequence (as shown in SEQ ID NO.1), and it was identified as Pediococcus pentosaceus and named Pediococcus pentosaceus zqw75.

[0054] Example 4: Enhancement of Fermentation Flavor by Pediococcus pentosaceus

[0055] (1) Weigh 100g of glutinous rice, steam the rice, rinse it with cold water, and then spread it out to cool. Next, put the cooled glutinous rice into a wide-mouthed bottle and add rice wine fermentation agent. Control group: Add commercially available sweet wine yeast (4g yeast / kg glutinous rice); Experimental group: Add commercially available sweet wine yeast (4g yeast / kg glutinous rice) and purified Pediococcus pentosaceus (addition amount 1×10⁻⁶). 7 (CFU / g yeast). Mix the starter culture with the cooled glutinous rice, build a nest, and then ferment the rice wine. Set the fermentation temperature to 30℃ and the time to 72 hours.

[0056] (2) Mix the fermented rice wine well, centrifuge at 8000 rpm for 10 min, and take the supernatant for volatile substance detection. The detection method is the same as in Example 2 above.

[0057] The test results are shown in Table 2. After adding Pediococcus pentosaceus, the flavor compounds were improved. The relative content of phenylethanol increased from 8.562% to 10.455%, the relative content of ethyl butyrate increased from 0.460% to 1.473%, the relative content of ethyl octanoate increased from 0.754% to 1.672%, the relative content of ethyl hexanoate increased from 0.832% to 3.446%, and the relative content of isoamyl acetate increased from 0.832% to 2.155%. It can also produce aroma compounds that are not produced by commercial sweet wine yeast, such as hexanoic acid, butyric acid, octanoic acid, ethyl hexanoate, and lauryl acetate.

[0058] Table 2. Results of aroma component analysis

[0059]

[0060]

[0061] Note: "——" indicates that the substance was not detected.

[0062] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. A strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ), characterized in that, The Pediococcus pentosaceus ( Pediococcus pentosaceus It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 25146 and deposit date of June 21, 2022. The Pediococcus pentosus can produce lauryl acetate, dodecyl butyl lactone, methyl heptenone and 2,6-dimethylpyrazine.

2. A microbial inoculant, characterized in that, The microbial agent contains Pediococcus pentosaceus as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is an agent containing live cells of Pediococcus pentosaceus or dried Pediococcus pentosaceus cells obtained by freeze-drying.

4. The microbial agent according to claim 2, characterized in that, The microbial agent is a liquid or solid agent containing immobilized Pediococcus pentosaceus cells.

5. The application of *Pediococcus pentosaceus* according to claim 1 in the production of aroma substances after fermentation, wherein, The aroma compounds are selected from the group consisting of hexanoic acid, butyric acid, octanoic acid, lauryl acetate, n-heptanol, butyl dodecyl lactone, phenylacetaldehyde, methyl heptenone, isoamyl alcohol, n-octanol, 1-nonanol, phenethyl alcohol, 1-hexadecyl alcohol, geraniol, 3,4-dimethylbenzaldehyde, geraniol acetone, 2,6-dimethylpyrazine, and 2-ethylpyrazine.

6. A brewing fermentation agent, characterized in that, The brewing fermentation agent contains Pediococcus pentosaceus as described in claim 1.

7. The brewing fermentation agent according to claim 6, characterized in that, The brewing fermentation agent also contains fermentation-active substances and auxiliary materials.

8. The brewing fermentation agent according to claim 7, characterized in that, The fermentation active substance is one or more of the following: brewer's yeast, Rhizopus, and Aspergillus.

9. The brewing fermentation agent according to claim 7, characterized in that, The excipient is a culture medium for bacteria.

10. The brewing fermentation agent according to claim 7, characterized in that, The auxiliary material is wheat bran.

11. The brewing fermentation agent according to claim 7, characterized in that, The auxiliary material is wheat or barley.

12. The application of Pediococcus pentosaceus as described in claim 1, or the microbial agent as described in any one of claims 2 to 4, or the brewing fermentation agent as described in any one of claims 6 to 11 in brewing.

13. The application according to claim 12, characterized in that, Fermentation is carried out by mixing the Pediococcus pentosaceus of claim 1, or the microbial agent of any one of claims 2 to 4, or the brewing fermentation agent of any one of claims 6 to 11 with the substance to be fermented.

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