CN116218743A - Propylene glycol slow-producing lactobacillus capable of simultaneously producing 2-nonanol and ethyl oleate and application thereof - Google Patents

Abstract

The invention belongs to the field of biology, and particularly relates to propylene glycol slow-producing lactobacillus capable of simultaneously producing 2-nonanol and ethyl oleate and application thereof. The enterococcus Thailand accession number: CGMCC No.25135, which is screened in high quality pit mud and is suitable for the environment of brewing white spirit, experiments show that when the strain is fermented for 72 hours at 37 ℃, the octanoic acid yield reaches 5.45g/L, which is higher than the octanoic acid yield of the existing strain, so the strain can be used as an enhanced microbial inoculum for brewing white spirit.

Description

Propylene glycol slow-producing lactobacillus capable of simultaneously producing 2-nonanol and ethyl oleate and application thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to a propylene glycol slow-growing lactobacillus (Lentilactobacillus diolivorans) capable of simultaneously producing 2-nonanol and ethyl oleate and application thereof.

Background

2-nonanol is also known as 2-nonylalcohol, heptyl methylmethanol, methylheptyl methanol. Has rich wax fragrance, green fragrance, cream fragrance and fruit fragrance. Mainly used as solvent, organic synthesis intermediate and perfume.

Ethyl oleate, also known as cis-9-octadecenoic acid ethyl ester (cis-9-Octadecenoic acid, ethyl ester), 9-octadecenoic acid ethyl ester (9-Octadecenoic acid ethyl ester), is a colorless oily liquid, and is readily soluble in organic solvents. The preparation method is mainly used for preparing lubricants, water-repellent agents, resin toughening agents, surfactants, pharmaceutical excipients, plasticizers, ointment matrixes and other organic chemicals, can also be used in daily chemical essence and food essence formulas, such as baked foods, dairy products, beverages and the like, and has higher practical value in the industries of chemical industry, medicines, foods and the like.

During the brewing process of white spirit, acids are precursors to esters and may also constitute other flavour substances. Proper amount of organic acid can make wine body plump, mellow and long in aftertaste. The research shows that the 2-nonanol and the ethyl oleate have a certain influence on the flavor of the wine body. Maotai-flavor is taken as the characteristic sensory attribute of the Maotai-flavor liquor, and has obvious positive correlation with ethyl isobutyrate, ethyl 2-methylbutyrate, ethyl isovalerate, 3-methylbutyrate, benzaldehyde, 2-heptanone, amyl acetate, ethyl phenylacetate, 2, 5-dimethyl-3-n-amyl pyrazine, 3-octanone, dimethyl trisulfide, 2-nonanol, 2-phenethyl alcohol, hexanal, (2E) -nonenal (in the identification research [ D ] of aroma substances of liquor with different flavors in China, shanghai application technical college, 2015.). Meanwhile, the chemical components of the Maotai-flavor type wine empty cupped flavor are determined by adopting a chromatography-mass spectrometry (GC/MS) method, and 13 compounds are identified, wherein ethyl palmitate, ethyl oleate, ethyl linoleate and the like are used as main components (Wu Huiqin, zhang Guiying, he Shouming and the like: maotai-flavor type wine aroma component research: 2: GC/MS analysis [ J ] analysis and test report of Maotai-flavor wine empty cupped flavor components, 1996,15 (4): 4).

Lactic acid bacteria are used as one of main microorganisms in white spirit brewing, have flavor characteristic function and microecological function, and are less in the prior researches, and can produce 2-nonanol and ethyl oleate by fermentation.

Disclosure of Invention

The lactobacillus which can simultaneously produce 2-nonanol and ethyl oleate is obtained by screening from fermented grains in the process of brewing white spirit, and can be applied to brewing wine.

Therefore, the invention provides a lactobacillus capable of simultaneously producing 2-nonanol and ethyl oleate, which is preserved in China general microbiological culture Collection center (CGMCC) for culture Collection of microorganisms in the year 2022, month 6 and 21, and has the preservation number: CGMCC No.25141, the classification name is propylene glycol-eating Lactobacillus chrous (Lentilactobacillus diolivorans).

The invention also provides application of the propylene glycol-eating lactobacillus chromenensis in ethyl palmitate. Preferably, the ethyl palmitate is 2-nonanol and/or ethyl oleate.

Specifically, the application is to ferment propylene glycol-fed lactobacillus chromenensis to produce ethyl palmitate. More specifically, the fermentation was stationary cultured at 37℃for 2 days at a pH of 7.0, and the inoculum size of the propylene glycol-feeding Lactobacillus chrous was 2% (. Times.107 cfu/mL).

More preferably, the fermentation medium has a composition comprising 16g glucose, 10g tryptone, 2.5g yeast powder, 5g beef extract, 3.85g beef brain, 4.9g beef 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, 1g dipotassium hydrogen phosphate per liter of medium.

In a specific experiment, the fermentation process is adopted, and the content of 2-nonanol and ethyl oleate produced by the strain is 0.330mg/L and 0.213mg/L respectively.

The invention also provides an application of the propylene glycol slow-producing lactobacillus capable of simultaneously producing the 2-nonanol and the ethyl oleate, and the propylene glycol slow-producing lactobacillus capable of simultaneously producing the 2-nonanol and the ethyl oleate is used as an enhanced microbial inoculum in the field of white wine brewing, such as in white wine brewing yeast and pit fermentation.

Preferably, the form of the enhanced bacterial agent comprises a liquid, a lyophilized preparation or a powder. Preferably, the enhanced microbial agent comprises an auxiliary material. The auxiliary materials comprise culture medium substances of bacteria; culture materials for the bacteria include, but are not limited to: yeast extract, glucose, peptone and water.

The invention also provides an enhancer for brewing white spirit, which is prepared from the propylene glycol slow-producing lactobacillus capable of simultaneously producing 2-nonanol and ethyl oleate. Specifically, the form of the enhanced bacterial agent is selected from liquid, freeze-dried preparation or powder prepared by culturing the propylene glycol slow-growing lactobacillus. Optionally, the enhanced bacterial agent further comprises an auxiliary material, wherein the auxiliary material comprises a bacterial culture medium substance; culture materials for the bacteria include, but are not limited to: yeast extract, glucose, peptone and water.

The invention provides the propylene glycol slow-growing lactobacillus capable of simultaneously producing 2-nonanol and ethyl oleate, which can provide strain resources for optimization of liquor brewing yeast and pit fermentation processes, can add aroma components for fermentation production of liquor, improve the taste of the liquor, and exert other functional effects, and has wider market application prospects.

Drawings

FIG. 1 is a mass spectrum of 2-nonanol GC-MS molecular fragments produced by the strain of example 1.

FIG. 2 is a mass spectrum of GC-MS molecular fragments of the 2-nonanol standard of example 1.

FIG. 3 is a mass spectrum of ethyl oleate GC-MS molecular fragments produced by the strain of example 1.

FIG. 4 is a GC-MS molecular fragment mass spectrum of the ethyl oleate standard of example 1.

Strain preservation information:

the propylene glycol slow-growing lactobacillus is preserved in the China general microbiological culture Collection center, called CGMCC for short, in 2022, 6 and 21 days, and the preservation unit address is North Chenxi Lu No. 1 and No. 3 in the Korean region of Beijing city. The preservation number of the strain is CGMCC No.25141, and the strain is classified and named as propylene glycol-eating Lactobacillus chrous Lentilactobacillus diolivorans.

Detailed Description

The invention will be further illustrated by the following specific examples in order to provide a better understanding of the invention, but without limiting the invention thereto.

The medium formulation referred to in the examples:

MRS liquid medium: glucose 20g/L, peptone 10g/L, yeast powder 4g/L, beef extract powder 5g/L, tween 80 1g/L, triammonium citrate 2g/L, sodium acetate 5g/L, magnesium sulfate 0.2g/L, manganese sulfate 0.05g/L, dipotassium hydrogen phosphate 1g/L, pH6.0, and high pressure steam sterilization at 115 ℃ for 20 minutes.

MRS solid medium: 15g/L agar was added to the MRS broth and autoclaved at 115℃for 20 minutes.

Fermentation medium: 16g/L glucose, 10g/L tryptone, 2.5g/L yeast powder, 5g/L beef extract, 3.85g/L beef brain, 4.9g/L beef heart, 0.5g/L tween 80, 1g/L ammonium citrate, 2.5g/L sodium chloride, 2.5g/L anhydrous sodium acetate, 0.05g/L magnesium sulfate, 0.025g/L manganese sulfate, 1.25g/L disodium hydrogen phosphate, 1g/L dipotassium hydrogen phosphate and sterilizing by high-pressure steam at the pH of 6.0,115 ℃ for 20 minutes.

Example 1: isolation and screening of strains

The separation method comprises the following steps: taking 20g of a fermented grains sample of the Luzhou Laojiao, putting the sample into a conical flask filled with 180mL of sterile distilled water, and oscillating for 10min by a constant-temperature shaking table to fully scatter and mix the sample uniformly. Taking 1mL of sample suspension, diluting to 10 by adopting a double ratio dilution method ^-2 ～10 ^-7 100 mu L of each gradient of diluent is sucked and uniformly coated on an MRS solid culture medium plate, two plates are prepared in parallel, inverted, placed under anaerobic condition at 37 ℃ for culture for 36-48h, and observed in time.

And (3) scribing and purifying: and taking out the plate with the colonies, picking single colonies with different colony morphologies, and carrying out secondary streaking until all the single colonies are purified.

And (3) strain preservation: single colony of each strain after purification is picked into 5mLMRS liquid culture medium, placed at 37 ℃ for static culture for 20-24 hours, 1mL of bacterial liquid is sucked into a bacteria-preserving tube, 0.5mL of 60% sterile glycerol solution is added, resuspended and placed at-80 ℃ for preservation.

And (3) re-screening: inoculating strain glycerol pipe into MRS culture medium, standing at 37deg.C for 2-3 days, and qualitatively determining volatile product by headspace solid phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS) to screen target strain.

The HS-SPME/GC-MS method is as follows:

(1) HS-SPME extraction conditions

Inserting the extraction head into the head space of the sample bottle, adsorbing at 60deg.C for 60min, taking out the adsorbed extraction head, inserting into gas chromatography sample inlet, and desorbing at 230deg.C for 10min.

(2) GC analysis conditions

Chromatographic column: rtx-Wax capillary chromatographic column, column length 60m, inner diameter 0.25mm, liquid film thickness 0.25 μm; carrier gas: he; flow rate: 1.0ml/min, split ratio: 20:1, a step of; column temperature: the temperature of the sample inlet is kept at 230 ℃, the temperature of the initial gas chromatographic column is kept at 35 ℃ for 5min, and the temperature is raised to 230 ℃ at 5 ℃/min and kept for 15min.

(4) MS analysis conditions

The ion source temperature and the interface temperature are 230 ℃; ionization mode: EI (electronic equipment) ⁺ The method comprises the steps of carrying out a first treatment on the surface of the Electron energy: 70ev; scanning quality range: 35-500 m/z.

4 strains of lactic acid bacteria are screened out in the fermented grain samples in the batch, 1 strain with the capacity of simultaneously producing 2-nonanol and ethyl oleate is obtained according to the HS-SPME/GC-MS result and numbered zqw42, and further research is carried out on the strains.

TABLE 1 results of strains HS-SPME/GC-MS

Strain	zqw40	zqw41	zqw42	zqw66
					2-nonanol	+	+	+	+
Oleic acid ethyl ester	--	--	+	--

Note that: "+" is the inclusion of this material; "-" does not contain this material

Example 2: molecular characterization of bacteria

Purifying and screening the obtained target strain, taking fresh bacterial liquid in logarithmic growth phase, centrifugally collecting bacterial cells, and extracting genome DNA by using a bacterial genome extraction kit. The full-length sequence of the 16S rDNA is amplified by adopting a lactobacillus universal primer 27F/1541R, and the method is concretely as follows:

27F(5′-AGAGTTTGATCCTGGCTCAG-3′)

1541R(5′-AAGGAGGTGATCCAGCC-3′)

(1) reaction system (50 μl)

(2) Reaction procedure

The PCR products were checked by electrophoresis on a 1.0% agarose gel at a voltage of about 11V/cm for 20min.

The purification of PCR products was performed as described in the Shanghai Biotechnology Co small amount of gel recovery PCR product purification kit, and sequencing was performed by Shanghai Biotechnology Co.

The sequence of the 16S rDNA is shown as SEQ ID NO. 1:

GTGCAGGCGGCATGCTATACATGCAAGTCGAACGCGTCTTGGTCAATGATTTTAGGTGCTTGCACTTGACTGATTTGACATTAAGACGAGTGGCGAACTGGTGAGTAACACGTGGGTAACCTGCCCTTGAAGTAGAGGATAACACTTGGAAACAGGTGCTAATACTGCATAACAACGAAAACCGCCTGGTTTTCGTTTGAAAGATGGCTTCGGCTATCGCTTTAGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGATGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTGGAGAAGAACGGATGTCAGAGTAACTGTTGACATCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACCGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACT GACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAACCTAAGAGATTAGGCGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCAGCATTTAGTTGGGCACTCTAGCAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAAACCGCGAGGTCAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGGTAACCTTCGGGGGCCAGCCGTCTAAGGTGGACAGAGATTGTG。

sequencing the gene sequences of the obtained 16S rDNA fragments were aligned by BLAST at NCBI, strain species information was determined, identified as Lactobacillus paracasei (Lentilactobacillus diolivorans), and designated as Lactobacillus paracasei zqw (Lentilactobacillus diolivorans).

Example 3: morphology and physicochemical characteristics of propylene glycol-based slow-growing masts

1. Experiments show that the culture characteristics of the propylene glycol-fed Lactobacillus chrous zqw: the optimal growth temperature is 37 ℃, the pH value is 7, and the growth can be realized under the conditions of facultative anaerobism, aerobics, anaerobic or micro-aerobics. In terms of temperature resistance: the strain grows well at 37 ℃, grows slowly at 42 ℃ and has low concentration of bacterial liquid, and cannot grow at 45 ℃ and 48 ℃.

2. The strain is cultured for 48 hours under the anaerobic condition at 37 ℃, and is identified by an API50CH kit, and L-arabinose, D-ribose, D-xylose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-sucrose, D-trehalose, D-melezitose, D-raffinose and D-turbinase can be respectively utilized; methyl-beta D-xylopyranoside, D-galactose, methyl-alpha D-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin; 30 single carbon sources such as glycerol, esculin ferric citrate, D-gentiobiose, D-tagatose, D-arabitol, potassium gluconate and the like produce acid. Its utility capability is from strong to weak: l-arabinose, D-ribose, D-xylose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-sucrose, D-trehalose, D-melezitose, D-raffinose, D-turinose; methyl-beta D-xylopyranoside, D-galactose, methyl-alpha D-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin; glycerol, escin ferric citrate, D-gentiobiose, D-tagatose, D-arabitol and potassium gluconate.

3. The bacterium is subjected to static culture for 48 hours by utilizing an MRS liquid culture medium under the microaerophilic condition at 37 ℃, and the non-volatile metabolites of the bacterium are detected by HPLC high performance liquid chromatography, so that the bacterium can be metabolized to produce 6.8g/L of lactic acid, 0.92g/L of acetic acid and 3.9g/L of ethanol.

Example 4: fermentation experiment of propylene glycol-based Lactobacillus chrous zqw42

After the obtained propylene glycol-based Lactobacillus chrous zqw glycerol storage tube was dissolved, the tube was inoculated into 10mL of a liquid-filled MRS medium at an inoculum size of 5%, and after 24 hours of stationary culture at 37℃the tube was inoculated into a fermentation medium at an inoculum size of 2%, and the culture was continued for 2 days, and the detection method was the same as that of example 1. After continuous fermentation for 2 days, the produced 2-nonanol and ethyl oleate yield of propylene glycol-based Lactobacillus chrous zqw42 was 0.330mg/L and 0.213mg/L, respectively.

Claims (9)

1. Can simultaneouslyPropylene glycol slow-producing lactobacillus for producing 2-nonanol and ethyl oleateLentilactobacillus diolivorans) The preservation number is CGMCC No.25141.

2. The use of the propylene glycol-based Lactobacillus chrous according to claim 1 in brewing white spirit.

3. The use according to claim 2, wherein the propylene glycol-based lactobacillus chromen is used as an enhancing agent in brewing white spirit.

4. The use according to claim 3, wherein the propylene glycol-based lactobacillus chromen is used in a starter for brewing white spirit or as an additive in pit fermentation to increase the content of 2-nonanol or ethyl oleate.

5. The use according to any one of claims 3 to 4, wherein the additive is in a form selected from the group consisting of a liquid, a lyophilized formulation or a powder.

6. The use according to claim 5, wherein the additive further comprises an adjuvant comprising a culture medium substance of a fungus; culture materials for the bacteria include, but are not limited to: yeast extract, glucose, peptone and water.

7. A starter for brewing white spirit or an additive in fermentation is prepared from the propylene glycol-based Lactobacillus chrous.

8. The additive of claim 7, wherein the additive is in a form selected from the group consisting of a liquid, lyophilized preparation, and a powder prepared from the propylene glycol-based Lactobacillus chrous after culturing.

9. The use of claim 8, wherein the enhanced bacterial agent further comprises an adjunct comprising a bacterial culture medium material; culture materials for the bacteria include, but are not limited to: yeast extract, glucose, peptone and water.

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