Lactobacillus buchneri strain and its use

Abstract

The present invention belongs to the biological field, and specifically relates to a strain of Lentilactobacillus buchneri that ferments and produces a variety of floral and fruity flavor substances and its use. In view of the fact that there are fewer species of Lentilactobacillus buchneri in the existing liquor brewing process, the research on the flavor substances, especially aroma substances, produced by the fermentation of Lentilactobacillus buchneri is blank, and the present invention provides a strain of Lentilactobacillus buchneri and its use. It can simultaneously produce floral and fruity alcohol substances, including ethyl elaidate, n-octanol, n-hexanol, 1-nonanol, phenylethyl alcohol, methyl heptenone, and geranyl acetone. Therefore, the strain can be used to prepare natural floral and fruity flavor substances, used for the optimization of fermented grains microorganisms, and strengthen the abundance of aroma-producing microorganisms in the liquor brewing process, and has good industrial application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biology, and in particular relates to a Lactobacillus buchneri strain capable of fermenting and producing a variety of floral and fruity flavor substances and a use thereof. Background Art

[0002] There are many different flavoring substances. With the exception of a few, most are non-nutritive and present in very small amounts in food, yet they have significant sensory effects. Based on their chemical structure (functional groups), they can be divided into volatile substances such as esters, alcohols, aldehydes, acids, phenols, ketones, hydrocarbons, furans, pyrazines, and sulfur-containing and heterocyclic compounds. Based on their sensory effects, they can be categorized as fruity, green, spicy, woody, and aldehyde-based. For example, esters and alcohol-aldehydes have pleasant fruity and floral aromas, pyrazines have a toasted aroma, sulfides have a gluey or rubbery odor, short-chain fatty acids such as butyric acid have sweaty and sour odors, phenols have an ink-like or cellar-mud odor, and guaiacol has a fruity aroma. The concentration and olfactory threshold of these flavoring substances influence the perception of food flavor.

[0003] Lactobacillus brevis is a Gram-positive anaerobic bacterium. Current studies have found that it plays an important role in feed fermentation and food fermentation. For example, Yang H et al. (Yang H, Wang B, Zhang Q, et al. Improvement of Fermentation Quality in the Fermented Total Mixed Ration with Oat Silage. 2021.) studied the effects of adding oat silage to fermented total mixed diets on fermentation, chemical composition and bacterial communities. After adding silage, bacterial diversity increased during the fermentation process, and heterologous fermentative lactic acid bacteria such as Lactobacillus brevis, Lentilactobacillus brevis, and Companilactobacillus versmoldensis appeared. Koichi Tanabe et al. (Koichi Tanabe, Masaki Monguchi, Ryoga Inoue, et al. Lentilactobacillus buchneri domination during the fermentation of Japanese traditional fermented fish (funazushi). 2022.) isolated and identified strains of traditional Japanese fermented fish, and the results showed that Lentilactobacillus buchneri was the dominant strain in the fermentation. However, Lentilactobacillus buchneri rarely appears in existing studies on the microorganisms in the koji, cellar mud, and mash during the baijiu brewing process, and no research has been conducted on the flavor substances, especially aroma substances, produced by Lentilactobacillus buchneri fermentation. Summary of the Invention

[0004] In view of the fact that few species of Lentilactobacillus buchneri appear in the existing liquor brewing process and the lack of research on flavor substances, especially aroma substances, produced by fermentation of Lentilactobacillus buchneri, the present invention provides a Lentilactobacillus buchneri strain and its use.

[0005] The Lactobacillus buchneri of the present invention has a deposit number of CGMCC NO. 25139. The deposit date is June 21, 2022, and the depositary is the General Microbiology Center of the China Culture Collection Administration (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0006] The 16S rDNA sequence of the above-mentioned Lactobacillus buchneri is shown in SEQ ID NO: 1.

[0007] Among them, the Lactobacillus buchneri can simultaneously produce ethyl elaidic acid ester, n-octanol, n-hexanol, 1-nonanol, phenylethyl alcohol, methyl heptenone, and geranyl acetone.

[0008] Among them, the contents of ethyl elaidate, n-octanol, n-hexanol, 1-nonanol, phenylethyl alcohol, methyl heptenone, and geranyl acetone are 0.842, 2.019, 0.433, 0.708, 1.856, 0.485, and 0.255 mg / L, respectively.

[0009] The biological characteristics of the Lactobacillus brunneri are as follows: on the MRS agar medium, the colonies are raised, white, round, and have neat edges.

[0010] The growth conditions of the Lactobacillus buchneri are as follows: growth temperature of 30°C-40°C, optimum temperature of 37°C, pH value of 5.0-7.0, and growth period of 20-48h.

[0011] The present invention also provides use of the above-mentioned Lactobacillus buchneri in liquor brewing.

[0012] The present invention also provides the use of Lactobacillus buchneri in fermenting and producing a variety of floral and fruity flavor substances.

[0013] Among them, in the above-mentioned use, the floral and fruity flavor substances are floral and fruity alcohol substances, including ethyl elaidate, n-octanol, n-hexanol, 1-nonanol, phenylethyl alcohol, methyl heptenone, and geranyl acetone.

[0014] Beneficial effects: The Lactobacillus buchneri provided by the present invention is a lactic acid bacteria isolated from a sample of Luzhou Laojiao fermented mash, which has the ability to produce a variety of floral and fruity flavor substances and is named zqw40. The strain has a short culture time, simple and easy-to-obtain raw materials, and strong adaptability. It can simultaneously produce ethyl oleate, n-octanol, n-hexanol, 1-nonanol, phenylethyl alcohol, methyl heptenone, and geranylacetone, with contents of 0.842, 2.019, 0.433, 0.708, 1.856, 0.485, and 0.255 mg / L, respectively. Therefore, the strain can be used to prepare natural floral and fruity flavor substances, be used for fermented mash microbial optimization, and enhance the abundance of aroma-producing microorganisms in the liquor brewing process, and has good industrial application prospects.

[0015] The present invention discloses a strain of Lentilactobacillus buchneri, with the deposit number CGMCC No. 25139. The deposit date is June 21, 2022, and the depositor is the General Microbiology Center of the China Culture Collection Administration (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The strain is classified as Lentilactobacillus buchneri. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a microscopic examination image of Lactobacillus brunneri in Example 1 of the present invention;

[0017] Figure 2 This is the GC-MS detection spectrum of volatile substances of the strain in Example 1 of the present invention. DETAILED DESCRIPTION

[0018] The Lactobacillus buchneri of the present invention has a deposit number of CGMCC NO. 25139. The deposit date is June 21, 2022, and the depositary is the General Microbiology Center of the China Culture Collection Administration (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0019] The 16S rDNA sequence of the above-mentioned Lactobacillus buchneri is shown in SEQ ID NO: 1.

[0020] SEQ ID NO: 1 16S rDNA sequence of Lactobacillus buchneri

[0021]

[0022] The biological characteristics of the Lactobacillus brunneri are as follows: on the MRS agar medium, the colonies are raised, white, round, and have neat edges.

[0023] The growth conditions of the Lactobacillus buchneri are as follows: growth temperature of 30°C-40°C, optimum temperature of 37°C, pH value of 5.0-7.0, and growth period of 20-48h.

[0024] The slow-growing lactobacillus buchneri of the present invention is separated from a Luzhou Laojiao fermented grains sample and can simultaneously produce ethyl elaidate, n-octanol, n-hexanol, 1-nonanol, phenylethyl alcohol, methyl heptenone and geranylacetone, with the contents of these being 0.842, 2.019, 0.433, 0.708, 1.856, 0.485 and 0.255 mg / L, respectively.

[0025] The present invention also provides use of the above-mentioned Lactobacillus buchneri in liquor brewing.

[0026] The present invention also provides the use of Lactobacillus buchneri in fermenting and producing a variety of floral and fruity flavor substances.

[0027] Among them, in the above-mentioned use, the floral and fruity flavor substances are floral and fruity alcohol substances, including ethyl elaidate, n-octanol, n-hexanol, 1-nonanol, phenylethyl alcohol, methyl heptenone, and geranyl acetone.

[0028] The specific method of the present invention for producing floral and fruity alcohol substances by fermenting Lactobacillus brunneri is as follows: inoculating Lactobacillus brunneri into a fermentation medium, culturing at 37°C for 24 hours, and obtaining a pH value of 6.0; the inoculation amount of Lactobacillus brunneri is 2% (×10 7 cfu / mL).

[0029] More specifically, the fermentation medium is composed of 16 g glucose, 10 g tryptone, 2.5 g yeast powder, 5 g beef extract, 3.85 g bovine brain, 4.9 g bovine heart, 0.5 g Tween 80, 1 g ammonium citrate, 2.5 g sodium chloride, 2.5 g anhydrous sodium acetate, 0.05 g magnesium sulfate, 0.025 g manganese sulfate, 1.25 g disodium hydrogen phosphate, and 1 g dipotassium hydrogen phosphate per liter of culture medium.

[0030] Using the above fermentation process, Lactobacillus buchneri zqw40 can produce a variety of floral and fruity flavor substances, including ethyl elaidate, n-octanol, n-hexanol, 1-nonanol, phenylethyl alcohol, methyl heptenone, and geranyl acetone, with contents of 0.842, 2.019, 0.433, 0.708, 1.856, 0.485, and 0.255 mg / L, respectively.

[0031] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0032] The culture medium formula involved in the embodiment is:

[0033] MRS liquid medium: glucose 20 g / L, peptone 10 g / L, yeast powder 4 g / L, beef extract powder 5 g / L, Tween 80 1 g / L, ammonium citrate tribasic 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, potassium hydrogen phosphate 1 g / L, pH 6.0, high-pressure steam sterilization at 115 ° C for 20 min.

[0034] MRS solid medium: Add 15 g / L agar to MRS liquid medium and sterilize by high-pressure steam at 115°C for 20 minutes.

[0035] Fermentation medium: glucose 16 g / L, tryptone 10 g / L, yeast powder 2.5 g / L, beef extract 5 g / L, bovine brain 3.85 g / L, bovine heart 4.9 g / L, Tween 80 0.5 g / L, ammonium citrate 1 g / L, sodium chloride 2.5 g / L, anhydrous sodium acetate 2.5 g / L, magnesium sulfate 0.05 g / L, manganese sulfate 0.025 g / L, disodium hydrogen phosphate 1.25 g / L, dipotassium hydrogen phosphate 1 g / L, pH 6.0, autoclave at 115°C for 20 min.

[0036] Example 1: Isolation and purification of strains

[0037] 1) Separation method: Take 20g Luzhou Laojiao fermented grains sample, put it into a conical flask filled with 180mL sterile distilled water, shake it on a constant temperature shaker for 10min to fully break up and mix the sample. Take 1mL of sample suspension and dilute it to 10% by the doubling dilution method. -2 ~10 -7 , aspirate 100 μL from each gradient dilution and evenly spread it on the MRS solid culture medium plate, prepare two plates in parallel, invert them, place them at 37℃ for 36-48h, and observe them in time.

[0038] 2) Streak purification: Remove the plate with colonies, pick single colonies with different colony morphologies, and perform secondary streaking until all single colonies are purified.

[0039] 3) Strain Preservation: After purification, single colonies of each strain were placed into 5 mL of MRS liquid culture medium and incubated at 37°C for 20-24 h. 1 mL of the culture medium was transferred to a preservation tube, and 0.5 mL of 60% sterile glycerol solution was added, resuspended, and stored at -80°C.

[0040] 4) GC-MS detection:

[0041] Headspace solid-phase microextraction (SPME) was used: the bacterial suspension was centrifuged, the supernatant was collected and added to a headspace vial. Saturated NaCl solution was then added. The prepared sample was equilibrated at 60°C for 5 minutes, then extracted using a 50 / 30μm DVB / CAR / PDMS tip at 60°C for 40 minutes. Desorption was then performed at 250°C for 5 minutes in the GC inlet. Compound search results were matched against the NIST standard library, and compounds with a similarity of at least 80% were confirmed as the target compounds.

[0042] GC-MS detection chromatographic conditions:

[0043] Gas chromatography conditions: HP-INNOWAX column (60 m × 0.25 mm × 0.25 μm); temperature program: initial temperature 40 °C, hold for 5 min, increase to 100 °C at 4 °C / min, then increase to 230 °C at 6 °C / min,

[0044] Maintain for 10 min, the carrier gas is high-purity helium (1.0 mL / min); the injection port temperature is 250°C, and there is no split.

[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] Through GC-MS detection combined with sensory scoring, a lactic acid bacteria strain with a strong floral and fruity aroma in the fermentation liquid was obtained among the isolated strains, and this strain was selected for further research.

[0047] Example 2: Identification of strains

[0048] 16S rDNA sequence amplification: 1 mL of target strain culture was centrifuged at 10,000 rpm for 5 minutes. The supernatant was removed and the DNA was extracted with CTAB solution and phenol-chloroform-isoamyl alcohol (25:24:1). The supernatant was removed by centrifugation at 12,500 rpm for 5 minutes. An equal volume of chloroform-isoamyl alcohol (24:1) was added, mixed, and centrifuged at 12,000 rpm for 5 minutes. The supernatant was removed and rinsed twice with ethanol. The supernatant was removed by centrifugation at 12,000 rpm for 5 minutes. The pellet was air-dried and resuspended in 50 μL of sterile water to obtain the strain DNA template. PCR amplification was performed using primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO: 2) and 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO: 3).

[0049] The gene sequence of the 16S rDNA fragment was obtained by sequencing, and the species information of the strain was determined by NCBI BLAST comparison. It was identified as Lentilactobacillus buchneri and named Lentilactobacillus buchneri zqw40.

[0050] Example 3: Detection of the ability to produce floral and fruity flavor substances

[0051] After dissolving a glycerol tube of Lactobacillus brucei zqw40, the cells were inoculated into MRS liquid medium and incubated at 37°C for 24 h. After activation for three generations, the cells were inoculated into 50 mL of fermentation medium at a 2% (volume ratio) inoculum and incubated for 24 h. After fermentation, the bacterial solution was centrifuged at 10,000 rpm for 5 min and the supernatant was collected. The detection method was the same as that in Example 1, using 0.822 mg / mL of 2-octanol as the internal standard and the original culture medium as a blank control to calculate the content of each volatile substance.

[0052] The detection results of floral and fruity flavor substances are shown in Table 1. Lactobacillus buchneri zqw40 can produce ethyl elaidate, n-octanol, n-hexanol, 1-nonanol, phenylethyl alcohol, methyl heptenone, and geranyl acetone, with contents of 0.842, 2.019, 0.433, 0.708, 1.856, 0.485, and 0.255 mg / L, respectively.

[0053] Table 1 Detection results of floral and fruity flavor substances in fermentation broth of Lactobacillus brucei zqw40

[0054] Floral and fruity flavor substances Content mg / L Sensory characteristics Ethyl elaidate 0.842 Floral, fruity and oily notes n-octanol 2.019 Strong oily and citrus smell n-Hexanol 0.433 Aroma of young branches and leaves, with a hint of wine, fruit and fat 1-nonanol 0.708 Pleasant aroma of rose and orange Phenylethyl alcohol 1.856 Soft, pleasant and long-lasting rose scent Methyl heptenone 0.485 Fruity and fresh aroma Geranyl acetone 0.255 Magnolia scent

[0055] It should be noted that the specific features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in any one or more embodiments. In addition, those skilled in the art may combine and combine the different embodiments and features of the different embodiments described in this specification without any contradiction.

Claims

1. Lactobacillus buchneri ( Lentilactobacillus buchneri ), characterized in that: The deposit number is: CGMCCNO. 25139.

2. Use of the Lactobacillus buchneri described in claim 1 in liquor brewing.

3. The use of the Lactobacillus buchneri of claim 1 in fermenting and producing a variety of floral and fruity flavor substances, characterized in that: The floral and fruity flavor substances are ethyl elaidate, n-octanol, n-hexanol, 1-nonanol, phenylethyl alcohol, methyl heptenone and geranyl acetone.

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