Lactobacillus diolivorans strain capable of simultaneously producing 2-nonanol and ethyl oleate and application thereof

Abstract

The application belongs to the field of biology and particularly relates to a propandiol-feeding Lactobacillus lentus capable of simultaneously producing 2-nonanol and ethyl oleate and application thereof. The preservation number of the propandiol-feeding Lactobacillus lentus is CGMCC No.25141. The strain can produce 2-nonanol and ethyl oleate with contents of 0.330 mg / L and 0.213 mg / L respectively, and thus 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 lentilactobacillus diolivorans capable of simultaneously producing 2-nonanol and ethyl oleate and application thereof. BACKGROUND

[0002] 2-nonanol is also known as 2-nonyl alcohol, heptyl methyl carbinol and methyl heptyl carbinol. It has rich wax, green, butter and fruit fragrance. It is mainly used as a solvent, an organic synthesis intermediate and a flavor.

[0003] Ethyl oleate is also known as cis-9-octadecenoic acid ethyl ester, 9-octadecenoic acid ethyl ester, a colorless oily liquid, easily soluble in organic solvents. It is mainly used in the preparation of lubricants, water-resistant agents, resin toughening agents, surfactants, pharmaceutical excipients, plasticizers and ointment bases and other organic chemicals, and can also be used in the formulation of daily chemical fragrances and food fragrances, such as baked foods, dairy products, beverages and the like, and has high practical value in the chemical, pharmaceutical and food industries.

[0004] In the process of liquor brewing, acids are precursor substances for the formation of esters, and can also constitute other flavor substances. Appropriate amount of organic acids can make the liquor body full, mellow and long-lasting. It has been found that 2-nonanol and ethyl oleate have certain influence on the flavor of liquor body. As a characteristic sensory attribute of Maotai-flavor liquor, sauce aroma presents a significant positive correlation with ethyl isobutyrate, ethyl 2-methylbutyrate, ethyl isovalerate, 3-methylbutyric acid, benzaldehyde, 2-heptanone, ethyl acetate, ethyl phenylacetate, 2,5-dimethyl-3-n-pentylpyrazine, 3-octanone, dimethyl trisulfide, 2-nonanol, 2-phenylethanol, hexanal and (2E)-nonenal (Yu S. Identification of aroma components in different Chinese liquors [D]. Shanghai Institute of Technology, 2015.). Meanwhile, the chemical components of the empty cup aroma of Maotai-flavor liquor were determined by GC / MS, and 13 kinds of compounds were identified, among which ethyl palmitate, ethyl oleate and ethyl linoleate were the main components (Wu H. Q., Zhang G. Y., He S. M. et al. Study on the aroma components of Maotai-flavor liquor: 2. GC / MS analysis of the empty cup aroma of Zhen liquor and Maotai liquor [J]. Journal of Analysis and Testing, 1996, 15(4): 4.).

[0005] Lactic acid bacteria, as one of the main microorganisms in liquor brewing, have both flavor characteristics and microecological functions, and few lactic acid bacteria capable of producing 2-nonanol and ethyl oleate are reported in the existing research. SUMMARY

[0006] The present invention provides a strain of lactic acid bacteria that can simultaneously produce 2-nonanol and ethyl oleate, which can be obtained from the mash in the process of brewing baijiu. This strain can be applied to the brewing of alcoholic beverages.

[0007] Therefore, this invention provides a lactic acid bacterium capable of simultaneously producing 2-nonanol and ethyl oleate, which was deposited on June 21, 2022, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. CGMCC NO. 25141, and its classification name is Lentilactobacillus diolivorans.

[0008] The present invention also provides the application of the propylene glycol-treated Lactobacillus in relation to ethyl palmitate. Preferably, the ethyl palmitate is 2-nonanol and / or ethyl oleate.

[0009] Specifically, the application involves fermenting *Lactobacillus propylene glycol* to produce ethyl palmitate. More specifically, the fermentation is carried out by static incubation at 37°C for 2 days at a pH of 7.0, with an inoculum size of 2% (×10⁷ cfu / mL) of *Lactobacillus propylene glycol*.

[0010] More preferably, the fermentation medium is composed of 16g ​​glucose, 10g tryptone, 2.5g yeast powder, 5g beef extract, 3.85g bovine brain, 4.9g bovine heart, 0.5g Tween 80, 1g ammonium citrate, 2.5g sodium chloride, 2.5g anhydrous sodium acetate, 0.05g magnesium sulfate, 0.025g manganese sulfate, 1.25g disodium hydrogen phosphate, and 1g dipotassium hydrogen phosphate per liter.

[0011] In a specific experiment, using the above-mentioned fermentation process, the strain produced 0.330 mg / L of 2-nonanol and 0.213 mg / L of ethyl oleate.

[0012] This invention also provides an application of *Lactobacillus propylene glycol* that can simultaneously produce 2-nonanol and ethyl oleate. This *Lactobacillus propylene glycol* is used as an enhancing agent in the field of baijiu brewing, such as in baijiu brewing starter culture and fermentation in cellars.

[0013] Preferably, the enhancing agent is in the form of a liquid, a lyophilized formulation, or a powder. Preferably, the enhancing agent includes excipients. The excipients include culture medium for the bacteria; the culture medium includes, but is not limited to, yeast extract, glucose, peptone, and water.

[0014] This invention also provides an enhancer for baijiu (Chinese liquor) brewing, prepared from *Lactobacillus propylene glycol-modified*, which can simultaneously produce 2-nonanol and ethyl oleate. Specifically, the enhancer is selected from liquid, freeze-dried, or powder forms prepared from *Lactobacillus propylene glycol-modified* after cultivation. Optionally, the enhancer further includes excipients, including culture medium for the bacteria; the culture medium includes, but is not limited to, yeast extract, glucose, peptone, and water.

[0015] This invention provides a propylene glycol-based Lactobacillus that can simultaneously produce 2-nonanol and ethyl oleate, which can provide strain resources for optimizing the brewing process of baijiu (Chinese liquor) and fermentation in cellars. It can add aroma components to the fermentation production of baijiu, improve the taste of the liquor, and play other functional roles, and has a broad market application prospect. Attached Figure Description

[0016] Figure 1 This is a GC-MS molecular fragment mass spectrum of 2-nonanol produced by the strain in Example 1.

[0017] Figure 2 This is the GC-MS molecular fragment mass spectrum of the 2-nonanol standard in Example 1.

[0018] Figure 3 This is a GC-MS molecular fragment mass spectrum of ethyl oleate produced by the strain in Example 1.

[0019] Figure 4 This is a GC-MS molecular fragment mass spectrum of the ethyl oleate standard in Example 1.

[0020] Strain preservation information:

[0021] The *Lentilactobacillus diolivorans* strain of this invention was deposited on June 21, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. The accession number for this strain is CGMCC No. 25141, and its taxonomic name is *Lentilactobacillus diolivorans*. Detailed Implementation

[0022] The present invention will be further illustrated below with specific embodiments in order to better understand the present invention, but these embodiments do not constitute a limitation thereof.

[0023] The culture medium formulations involved in the examples are as follows:

[0024] 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 6.0, autoclaved at 115°C for 20 minutes.

[0025] MRS solid medium: Add 15 g / L agar to the MRS liquid medium and autoclave at 115°C for 20 minutes.

[0026] Fermentation medium: glucose 16g / L, tryptone 10g / L, yeast extract 2.5g / L, beef extract 5g / L, bovine brain 3.85g / L, bovine heart 4.9g / L, Tween 80 0.5g / L, ammonium citrate 1g / L, sodium chloride 2.5g / L, anhydrous sodium acetate 2.5g / L, magnesium sulfate 0.05g / L, manganese sulfate 0.025g / L, disodium hydrogen phosphate 1.25g / L, dipotassium hydrogen phosphate 1g / L, pH 6.0, autoclaved at 115℃ for 20 minutes.

[0027] Example 1: Isolation and screening of strains

[0028] Separation method: Take 20g of Luzhou Laojiao mash 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 of 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.

[0029] 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.

[0030] Strain preservation: Pick a single colony of each strain after purification into 5 mL of LMR broth, incubate at 37°C for 20-24 h, transfer 1 mL of the bacterial culture to a preservation tube, add 0.5 mL of 60% sterile glycerol solution, resuspend, and store at -80°C.

[0031] Secondary screening: The bacterial strain was inoculated into MRS medium with glycerol and incubated at 37°C for 2-3 days. The volatile products were then qualitatively determined using headspace solid phase microextraction / gas chromatography-mass spectrometry (HS-SPME / GC-MS) to screen for target strains.

[0032] The specific methods for HS-SPME / GC-MS are as follows:

[0033] ①HS-SPME extraction conditions

[0034] Insert the extraction head into the headspace of the sample vial and adsorb at 60℃ for 60 min. After adsorption, remove the extraction head and insert it into the gas chromatograph injection port for desorption at 230℃ for 10 min.

[0035] ②GC analysis conditions

[0036] Chromatographic column: Rtx-Wax capillary column, 60m in length, 0.25mm in inner diameter, and 0.25μm in film thickness; Carrier gas: He; Flow rate: 1.0ml / min, split ratio: 20:1; Column temperature: Injector temperature maintained at 230℃, initial gas chromatographic column temperature maintained at 35℃ for 5min, then increased to 230℃ at 5℃ / min and held for 15min.

[0037] ④MS analysis conditions

[0038] Ion source temperature and interface temperature: 230℃; Ionization method: EI + Electron energy: 70 eV; Scan quality range: 35–500 m / z.

[0039] Four strains of lactic acid bacteria were screened from this batch of fermented mash samples, as shown in Table 1. Based on the HS-SPME / GC-MS results, one strain, numbered zqw42, was found to be capable of simultaneously producing 2-nonanol and ethyl oleate. Further research is needed on this strain.

[0040] Table 1. HS-SPME / GC-MS results of the strains

[0041] Strains zqw40 zqw41 zqw42 zqw66 2-nonanol + + + + ethyl oleate -- -- + --

[0042] Note: "+" indicates the presence of this substance; "--" indicates the absence of this substance.

[0043] Example 2: Molecular identification of bacteria

[0044] The target strains obtained through purification and screening were collected by centrifugation of fresh bacterial culture in the logarithmic growth phase. Genomic DNA was extracted using a bacterial genome extraction kit. The full-length 16S rDNA sequence was amplified using universal primers 27F / 1541R for lactic acid bacteria, as follows:

[0045] 27F(5′-AGAGTTTGATCCTGGCTCAG-3′)

[0046] 1541R(5′-AAGGAGGTGATCCAGCC-3′)

[0047] ①Reaction system (50 μl)

[0048]

[0049] ②Reaction Procedure

[0050]

[0051] PCR products were separated and tested by 1.0% agarose gel electrophoresis at a voltage of approximately 11 V / cm for 20 min.

[0052] The PCR products were purified according to the instructions of the small-volume gel recovery PCR product purification kit from Shanghai Sangon Biotech Co., Ltd., and the sequencing was performed by Shanghai Sangon Biotech Co., Ltd.

[0053] The obtained 16S rDNA sequence is shown in SEQ ID NO:1:

[0054] GTGCAGGCGGCATGCTATACATGCAAGTCGAACGCGTCTTGGTCAATGATTTTAGGTGCTTGCACTTGACTGATTTGACATTAAGACGAGTGGCGAACTGGTGAGTAACACGTGGGTAACCTGCCCTTGAAGTAGAGGATAACACTTGGAAACAGGTGCTAATACTGCATAACAACGAAAACCGCCTGGTTTTCGTTTGAAAGATGGCTTCGGCTATCGCTTTAGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGATGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTGGAGAAGAACGGATGTCAGAGTAACTGTTGACATCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACCGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACT.

[0055] The gene sequence of the 16S rDNA fragment obtained by sequencing was compared with NCBI's BLAST to determine the species information of the strain. It was identified as Lentilactobacillus diolivorans and named Lentilactobacillus diolivorans zqw42.

[0056] Example 3: Morphological characteristics and physicochemical properties of propylene glycol-treated slow-growing latex

[0057] 1. Experiments show that the culture characteristics of *Lactobacillus propylene glycol* zqw42 are as follows: the optimal growth temperature is 37℃, the optimal pH is 7, it is facultative anaerobic, and it can grow under aerobic, anaerobic, or microaerobic conditions. Regarding temperature tolerance: the bacterium grows well at 37℃, grows slowly and has a low bacterial concentration at 42℃, and cannot grow at 45℃ and 48℃.

[0058] 2. Under anaerobic conditions at 37℃ for 48 hours, this bacterium, as identified by the API 50CH kit, can produce acid using 30 single carbon sources, including L-arabinose, D-ribose, D-xylose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, D-cellobiose, D-maltose, D-lactose, D-micobiose, D-sucrose, D-trehalose, D-melatotriose, D-raffinose, D-thulose; methyl-β-D-xylanopyranoside, D-galactose, methyl-α-D-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin; glycerol, ferric citrate of esculenta, D-gentiobiose, D-tagatose, D-arabinol, and potassium gluconate. Their utilization ability, from strongest to weakest, is as follows: L-arabinose, D-ribose, D-xylose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, D-cellobiose, D-maltose, D-lactose, D-micobiose, D-sucrose, D-trehalose, D-melatotriose, D-raffinose, D-thulene; methyl-βD-xylanopyranoside, D-galactose, methyl-αD-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin; glycerol, ferric citrate of aesculin, D-gentiobiose, D-tagatose, D-arabinol, potassium gluconate.

[0059] 3. Under microaerobic conditions at 37℃, this bacterium was cultured statically in MRS liquid medium for 48 hours. The non-volatile metabolites were detected by HPLC. It was found that the bacterium could produce lactic acid 6.8 g / L, acetic acid 0.92 g / L, and ethanol 3.9 g / L.

[0060] Example 4: Fermentation experiment of Lactobacillus zqw42 (propylene glycol-treated)

[0061] After dissolving the obtained glycerol preservation tube of *Lactobacillus propylene glycol-treated* zqw42, it was inoculated into 10 mL of MRS liquid medium at a 5% inoculation rate. After static incubation at 37°C for 24 h, it was inoculated into fermentation medium at a 2% inoculation rate and cultured continuously for 2 days. The detection method was the same as that in Example 1. After 2 days of continuous fermentation, the yields of 2-nonanol and ethyl oleate produced by *Lactobacillus propylene glycol-treated* zqw42 were 0.330 mg / L and 0.213 mg / L, respectively.

Claims

1. A type of propylene glycol-producing *Lactobacillus* that simultaneously produces 2-nonanol and ethyl oleate (… Lentilactobacillus diolivorans zqw42, its collection number is CGMCC No.25141.

2. The application of Lactobacillus propylene glycol ZQW42 as an enhancing agent in Baijiu brewing as described in claim 1, for increasing the content of 2-nonanol and ethyl oleate.

3. The application as described in claim 2, characterized in that, The propylene glycol-containing Lactobacillus zqw42 was applied to the starter culture for Baijiu brewing or as an additive in fermentation pits to increase the content of 2-nonanol and ethyl oleate.

4. The application as described in claim 3, characterized in that, The additive is selected from liquid, lyophilized formulation or powder.

5. The application as described in claim 4, characterized in that, The additive also includes excipients, which include culture medium for bacteria; the culture medium for the propylene glycol-treated Lactobacillus zqw42 includes yeast extract, glucose, peptone, and water.

6. The application of Lactobacillus propylene glycol-producing strain ZQW42 as described in claim 1 in the production of 2-nonanol and ethyl oleate.

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