A novel JP1 strain of strictly anaerobic *Lactobacillus* and its applications

Abstract

This invention provides a novel *Lactobacillus* strain JP1, a strictly anaerobic bacterium, and its applications, belonging to the field of bioengineering technology. This strain JP1 was isolated from the mash of strong-aroma baijiu (Chinese liquor) from Songhe Town. It belongs to the genus *Lactobacillus* (…). Sporolactobacillus This is a potential new species of *Lactobacillus*, and no other species in the genus *Lactobacillus* have been reported to ferment and produce butyric acid, ethyl butyrate, hexanoic acid, and ethyl hexanoate. The strain JP1 described in this application possesses various enzymatic activities and can ferment hexanoic acid, ethyl hexanoate, butyric acid, and ethyl butyrate at different time points using saccharified wine mash as a substrate, with a relatively high yield of butyric acid. It has significant application potential in the fermentation industry.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a novel strictly anaerobic strain JP1 with good organic acid and ester production and multiple enzyme activity characteristics, and its application in food. Background Technology

[0002] The main components of strong-aroma baijiu are ethanol and water, accounting for about 98%, while the remaining 2% of trace components are key to its unique aroma and flavor [Liu H, Sun B. Effect of Fermentation Processing on the Flavor of Baijiu[J]. J. Agric. Food Chem, 2018, 66(22): 5425~5432]. Although trace components account for a small proportion of the liquor, they play a crucial role in the quality and unique flavor formation of the liquor [Zhao Dongrui. Study on the antioxidant and anti-inflammatory properties of flavor substances and phenolic flavor substances in Gujinggong liquor[D]. [Doctoral Dissertation]. Guangdong: South China University of Technology, 2019. The concentration and ratio of four esters, namely ethyl acetate, ethyl lactate, ethyl butyrate and ethyl hexanoate, have an important influence on the flavor of Chinese liquor [Wei Y, Zou W, Shen C, et al. Basic flavor types and component characteristics of Chinese traditional liquors: a review [J]. Journal of Food Science, 2020, 85(12): 4096~4107]. In addition to the four main esters mentioned above, other esters that significantly contribute to aroma include ethyl octanoate, ethyl valerate, ethyl 2-methylbutyrate, ethyl 3-methylbutyrate, ethyl 2-methylbutyrate, ethyl 2-methylbutyrate, and ethyl 3-phenylpropionate. These esters produce unique aromas, giving strong-aroma baijiu a rich cellar fragrance and a sweet, mellow, and full-bodied taste [Xu Y, Zhao J, Liu X, et al. Flavor mystery of Chinese traditional fermented baijiu: The great contribution of ester compounds[J]. Food Chemistry, 2022, 369: 130920].

[0003] Acids are important flavor compounds in alcoholic beverages, mainly including acetic acid, lactic acid, hexanoic acid, butyric acid, formic acid, propionic acid, valeric acid, isovaleric acid, and phenylacetic acid. Acids are important precursors for the formation of esters, and they can eliminate some of the bitterness in baijiu (Chinese white liquor), making it more mellow and harmonious. Butyric acid imparts a unique creamy flavor to baijiu (Chinese liquor). The esters or aromatic compounds it produces have fruity notes [Liu S, Bischoff KM, Leathers TD, et al. Butyric acid from anaerobic fermentation of lignocellulosic biomass hydrolysates by Clostridium tyrobutyricum strain RPT-4213[J]. Bioresource Technology, 2013, 143: 322-329]. It is mainly produced by Clostridium species, including Clostridium tyrobutyricum, Clostridium butyricum, Clostridium beijerinckii, Clostridium populeti, and Clostridium thermobutyricum. Thermobutyricum, Clostridium pasteurianum, etc. [Tang Wan. Exploratory Study on the Metabolic Mechanism and Application of Clostridium butyricum [D]. [Master's Thesis]. Zhejiang: Zhejiang University of Technology, 2018].

[0004] The microorganisms in the fermented grains mainly come from the brewing starter culture and pit mud, with a smaller portion originating from the raw materials themselves and the production environment. The microbial community is complex, primarily containing yeast, mold, and bacteria [Qian W, Lu ZM, Chai LJ, et al. Cooperation within the microbial consortia of fermented grains and pit mud drives organic acid synthesis in strong-flavor Baijiu production[J]. Food Research International, 2021, 147: 110449]. Yeasts mainly produce esters and aroma compounds; molds mainly provide saccharification power, converting starches into sugars that can be directly used by microorganisms; and bacteria mainly produce organic acids for Baijiu production [Qian W, Lu ZM, Chai LJ, et al. Cooperation within the microbial consortia of fermented grains and pit mud drives organic acid synthesis in strong-flavor Baijiu production[J]. Food Research International, 2021, 147: 110449].

[0005] The genus *Sporolactobacillus* is a bacterial group, with *Sporolactobacillus inulinus* as its type species, established in 1963. This genus currently contains 12 officially published species and 2 unnamed species [Parte, AC, SardàCarbasse, J., Meier-Kolthoff, JP, Reimer, LCand]. M. (2020). List of Prokaryotic names with Standing in Nomenclature (LPSN) moves to the DSMZ. International Journal of Systematic and Evolutionary Microbiology, 70, 5607-5612; DOI: 10.1099 / ijsem.0.004332. Most *Sporolactobacillus* species are facultative anaerobic and exist in various environments, such as vineyard soils, wine fermentation agents, and chicken feed [Yanagida, F., & Suzuki, KI (2015). *Sporolactobacillus*. Bergey's Manual of Systematics of Archaea and Bacteria, 1-8.]. It has been reported that *Sporolactobacillus* can ferment to produce D-lactic acid [Klotz, S., Kuenz, A., & Prüβe, U. (2017). Nutritional requirements and the impact of yeast extract on the d-lactic acid production by *Sporolactobacillus inulinus*. *Green Chemistry*, 19(19), 4633-4641.], and has certain probiotic effects [Sanders, ME, Morelli, L., & Tompkins, TA (2003). Sporeformers as human probiotics: Bacillus, *Sporolactobacillus*, and *Brevibacillus*. *Comprehensive reviews in food science and food safety*, 2(3), 101-110.]. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a novel spore-forming Lactobacillus strain JP1 that is strictly anaerobic and its applications. This strain possesses various enzymatic activity characteristics and can ferment saccharified liquor as a substrate at different time periods to produce hexanoic acid, ethyl hexanoate, butyric acid, and ethyl butyrate, among which butyric acid production is relatively high.

[0007] To achieve the above objectives, the specific solution adopted by the present invention is as follows:

[0008] A novel, strictly anaerobic strain, JP1, belonging to the genus *Sporolactobacillus*, has been deposited with accession number CGMCC NO.27858 at the China General Microbiological Culture Collection Center, Beijing, China, on July 7, 2023.

[0009] A microbial inoculant comprising the aforementioned novel strain JP1.

[0010] The application of the aforementioned novel strain JP1 or microbial inoculant in food production. Further, the food is a fermented food. Even further, the food is an alcoholic beverage.

[0011] A method for brewing strong-aroma baijiu involves using the aforementioned new strain JP1 as the fermentation strain.

[0012] [Beneficial Effects]

[0013] Strain JP1 was isolated from samples of strong-aroma baijiu mash fermented in Songhe Town at 1 and 3 days of fermentation. The strain is strictly anaerobic, rod-shaped, with peritrichous flagella; it is Gram-positive. The strain can grow on both R2A and RCM media. The growth temperature range is 20-40℃, with an optimum temperature of 30-35℃; the pH range is 4-7.5, with an optimum pH of 5.0-5.5; the ethanol tolerance range is 0-5%; and the NaCl tolerance range is 0-6%. The strain is not sensitive to amoxicillin, ampicillin, bacitracin, kanamycin, neomycin, neomycin, piperacillin, and rifampin, but is sensitive to chloramphenicol, erythromycin, gentamicin, vancomycin, ofloxacin, and tetracycline. Strain JP1 can utilize glycerol, L-arabinose, ribose, galactose, glucose, fructose, mannose, sorbitol, α-methyl-D-glucoside, N-acetylglucosamine, arbutin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, starch, D-minobiose, and D-tagatose as carbon sources to grow and produce acid. The following carbon sources cannot be used to produce acids: erythritol, D-arabinose, D-xylose, L-xylose, agonol, β-methyl-D-xylose, sorbitol, rhamnose, eugenol, inositol, mannitol, α-methyl-D-mannose, amygdalin, aesculin, salicylic acid, inulin, pinealose, raffinose, glycogen, xylitol, gentiobiose, D-misugar, D-rockose, L-rockose, D-arabinitol, L-arabinitol, gluconate, 2-keto-gluconate, 5-keto-gluconate.

[0014] The strain showed positive protease and cellulase activity, and possessed intracellular esterase activity; however, it was negative for amylase, oxidase, catalase, indole production, urease, and β-glucosidase. Strain JP1 showed positive reactions for alkaline phosphatase, leucine aromatic aminoaminase, valine aromatic aminoaminase, cystine aromatic aminoaminase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, and β-uronic acid aglycone. It was negative for esterase (C4), lipolipase (C8), lipolipase (C14), trypsin, chymotrypsin, α-glucosidase, β-glucosidase, N-acetyl-glucosamine, α-mannose, and β-fucoside.

[0015] After 3 days of anaerobic fermentation in the saccharification broth, GC-MS analysis of strain JP1 revealed the following volatile substances: isobutanol, ethanol, n-hexanol, n-propanol, isoamyl alcohol, n-pentanol; acetic acid; ethyl hexanoate, ethyl acetate, etc. The relative content of ethanol reached 117.5368 μg / mL, and the relative content of ethyl hexanoate was 0.1461 μg / mL. With extended fermentation time, strain JP1 mainly produced butyric acid and ethyl butyrate. The strain produced the highest butyric acid content, reaching 18.912 μg / mL, at 30℃. During the fermentation of the mash by strain JP1, the esterification process primarily involved the production of ethyl butyrate with prolonged fermentation time.

[0016] When strain JP1 uses ethanol as a substrate, the esters are ethyl acetate and ethyl butyrate, which increase with fermentation time; the acids are acetic acid and butyrate, with butyrate being the predominant component, reaching its maximum content of 14.583 μg / mL after 14 days of fermentation. When strain JP1 uses ethyl acetate as a substrate, the number of esters increases, with seven types present, including butyl formate, ethyl acetate, methyl acetate, butyl acetate, propyl acetate, and ethyl propionate. When strain JP1 uses hexanoic acid as a substrate, the esters are ethyl hexanoate, methyl hexanoate, and ethyl acetate; the acid is acetic acid. Strain JP1 possesses esterase activity, promoting the synthesis of ethyl hexanoate from hexanoic acid and ethanol.

[0017] Strain JP1 is a potential new species in the genus *Sporolactobacillus*. No other strains in this genus have been reported to ferment and produce butyric acid, ethyl butyrate, hexanoic acid, and ethyl hexanoate. Furthermore, most other strains are aerobic or facultative aerobic, but our isolated strain JP1 is strictly anaerobic, possesses diverse enzymatic activities, and can ferment saccharified mash at different time points to produce hexanoic acid, ethyl hexanoate, butyric acid, and ethyl butyrate, with a relatively high yield of butyric acid. It has significant application potential in the fermentation industry.

[0018] Preservation Notes: A novel, strictly anaerobic strain JP1, belonging to *Sporolactobacillus*, has been preserved with accession number CGMCC NO.27858. The depository institution is the China General Microbiological Culture Collection Center, located in Beijing, China. The preservation date is July 7, 2023. Attached Figure Description

[0019] Figure 1 Plate and electron micrograph of strain JP1.

[0020] Figure 2 Liquid fermentation morphology of strain JP1 in R2A medium.

[0021] Figure 3 Phylogenetic NJ tree of strain JP1 16S rRNA gene sequence.

[0022] Figure 4 Antibiotic experiment of strain JP1; In the figure: A, amoxicillin; B, ampicillin; C, bacitracin; D, chloramphenicol; E, erythromycin; F, gentamicin; G, vancomycin; H, ofloxacin; I, kanamycin; J, neomycin; K, neomycin; L, tetracycline; M, piperacillin; N, rifampin.

[0023] Figure 5 Detection of protease and cellulase in strain JP1.

[0024] Figure 6 Analysis of fermentation materials over 7 days at different temperatures.

[0025] Figure 7 Analysis of fermentation materials after 14 days at different temperatures.

[0026] Figure 8 Analysis of fermentation materials at different temperatures over 21 days.

[0027] Figure 9 Analysis of fermentation materials at different temperatures over 28 days. Detailed Implementation

[0028] This study uses the mash of strong-aroma baijiu from different periods in Songhe Town as the research object to explore new groups of anaerobic microorganisms, study the physiological and biochemical characteristics and fermentation characteristics of potential new strains, and investigate their ability to produce acid and esters during fermentation, laying the foundation for further exploration and utilization of potential new microbial resources in industrial production.

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments.

[0030] 1. Materials and Methods

[0031] 1.1 Materials and Reagents

[0032] 1.1.1 Sample Collection

[0033] Samples of fermented mash were collected from the fermentation cellars of Songhe Winery Co., Ltd. in Songhe Town, Luyi County, Henan Province. According to the fermentation cycle, samples from the upper, middle, and lower sections of the fermentation cellar at 1, 3, 6, 9, 13, 21, 27, 33, 48, and 60 days of fermentation were collected and mixed separately, resulting in a total of 10 samples. These samples were placed in sterilized centrifuge tubes. Samples intended for pure culture were stored at 4°C, while samples intended for culture-free storage were stored at -80°C. Specific sample information is shown in Table 1.

[0034] Table 1 Information on fermented mash samples

[0035] 1.1.2 Types and Composition of Culture Media

[0036] 1.1.2.1 Culture medium for strain isolation

[0037] R2A medium: 0.5g yeast extract, 0.5g peptone, 0.5g casein hydrolysate, 0.5g glucose, 0.5g soluble starch, 0.3g K2HPO4, 0.024g MgSO4·7H2O, 0.3g sodium pyruvate, 15.0g agar, 1L distilled water, pH 7.0, sterilized at 121℃ for 20min.

[0038] Fortified Clostridium tumefaciens medium (RCM): 10.0g peptone, 10.0g beef meal, 3.0g yeast powder, 5.0g glucose, 1.0g soluble starch, 5.0g sodium chloride, 3.0g sodium acetate, 0.5g L-cysteine ​​hydrochloride, 15g agar, pH 6.8±0.1.

[0039] 1.1.2.2 Physiological and biochemical assays and enzyme activity screening culture medium

[0040] The culture media used for the temperature growth range, antibiotic sensitivity, ethanol tolerance, salinity tolerance, and pH growth experiments were selected from the optimal growth media in Section 1.1.2.1.

[0041] Fermentation medium: saccharification liquid of mash raw material; culture medium: mixture of sorghum and wheat.

[0042] Preparation method: Mix and crush the raw materials for brewing Songhe wine into powder, add 4 times the volume of water and high temperature amylase (1000U / kg), cook and gelatinize at 92℃ for 1 hour, quickly cool to below 60℃, add saccharifying enzyme (3000U / kg), saccharify at 60℃ for 2 hours, filter with gauze, and adjust the sugar content of the supernatant to 13-14°Bx with distilled water.

[0043] Enzyme activity detection medium:

[0044] (1) Amylase activity assay medium

[0045] Gao's medium: KNO3 1g, MgSO4·7H2O 0.5g, soluble starch 20g, K2HPO4 0.5g, agar 20g, water 1000mL, autoclave at 121℃ for 30min.

[0046] (2) Cellulase activity detection medium

[0047] 20g CMA-Na, 2.5g peptone, 2.5g Na2HPO4, 1.5g KH2PO4, 15g agar, 1000mL distilled water, pH 7.0-7.5, autoclaved at 121℃ for 15min.

[0048] (3) Protease activity detection medium

[0049] Solution l: K2HPO4 0.4g, MgSO4·7H2O 0.2g, mannitol 10g, yeast extract powder 3g, agar 15g, water 500mL, pH 7.0-7.5, autoclaved at 115℃ for 15min.

[0050] Solution 2: 5% skim milk powder, 50g; water, 500mL, autoclave at 110℃ for 15min. Mix solutions 1 and 2 thoroughly and pour into plates.

[0051] (4) Esterase activity assay medium

[0052] 100 mL of polyvinyl alcohol (PVA) emulsion, 0.5 g glucose, 0.5 g sodium chloride, 1 g potassium nitrate, 1 mL trace salt, 15 g agar powder, 1000 mL distilled water, natural pH. PVA emulsion: Add 10 mL of glyceryl tributyrate to 30 mL of 3% polyvinyl alcohol, place in a refrigerator (5-10℃) and let stand for 1-2 hours. Then, treat with an ultrasonic homogenizer (100W) for 30 minutes until the solution changes from separate layers to a uniform milky white color and no longer separates. Sterilize the emulsion and culture medium separately (autoclave at 115℃ for 15 minutes). Add 1 mL of emulsion to every 100 mL of culture medium and pour into plates (prepare the PVA emulsion fresh each time).

[0053] 1.2 Instruments and Equipment

[0054] LD2X-50KBS Vertical High-Temperature Sterilizer, Shanghai Shenan Medical Instrument Factory; SW-CJ-ZG Single-Person Clean Workbench, Suzhou Zhong Cleaning Company; BPS-150 Biochemical Incubator, Shanghai Xunbo Experimental Instrument Company; HZQ-B Digital Display Constant Temperature Shaker, Suweier Experimental Instrument Company; Centrifuge 5430R High-Speed ​​Refrigerated Centrifuge, Merck AG, Germany; SMART-N Water Purification System, Likang Biomedical Holding Co., Ltd.; A35 Anaerobic Workstation, DWS, UK; 7890A-5975C Gas Chromatography-Mass Spectrometry System, Agilent Technologies, USA; JSM-7900F Scanning Electron Microscope, NEC Japan; JEOL JEM-2100PLUS Transmission Electron Microscope, NEC Japan.

[0055] 1.3 Methods

[0056] 1.3.1 Isolation and Purification of Anaerobic Bacteria by Pure Culture

[0057] The following operation was performed in the anaerobic workstation: the mash sample was diluted to 10 using a gradient dilution method. -3 Spread 100 μL of bacterial suspension onto an isolation plate and incubate at 28℃ for 3-7 days. Observe the growth and morphology of the bacterial strains on the isolation plate. Purify different morphological microorganisms on R2A medium. Preserve the purified strains in 5 mL anaerobic culture flasks with inner stoppers using glycerol tubes (30% glycerol).

[0058] 1.3.2 Observation of strain morphology

[0059] The inoculated strain was cultured in R2A and RCM media at 28℃ for 3-4 days, and the cell color, morphology, and pigment production were observed. Alternatively, the inoculated strain was cultured in R2A liquid medium at 28℃ in a shaker for 3-4 days. The cells were collected by centrifugation at 4000 r / min, and washed 2-3 times with physiological saline to remove culture medium components, finally preparing a physiological saline bacterial suspension. The microscopic morphology of the strain was observed using scanning electron microscopy and transmission electron microscopy, respectively.

[0060] 1.3.3 Cloning and Phylogenetic Analysis of the 16S rRNA Gene of the Strains

[0061] The genome of the anaerobic strain was extracted using a kit (Sangon Biotech). Polymerase chain reaction (PCR) amplification was performed using primers for the 16S rRNA gene (27F: 5'-AGTTTGATCMTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The amplification system consisted of 10 μL of 2×Taq PCR StarMix (Kangrun Biotechnology), 1 μL of each primer, 1 μL of template DNA, and 7 μL of deionized water. The reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for 32 cycles; followed by a final extension at 72℃ for 10 min. The 16S rRNA gene was cloned and sequenced at Sangon Biotech Shanghai. The 16S rRNA gene sequence was compared using EZBioCloud to analyze the similarity of the strain's 16S rRNA gene and preliminarily determine the strain's taxonomic position. A phylogenetic tree of strain NJ with 16S rRNA gene was constructed using MEGA 7.0.

[0062] 1.3.4 Identification of Physiological and Biochemical Indicators

[0063] 1.3.4.1 Growth experiments at different temperatures

[0064] The strain was inoculated onto R2A solid medium and incubated in constant temperature incubators at 15, 20, 25, 30, 35, 40, and 45℃ for 3-5 days. Its growth was observed to determine the temperature range and optimal growth temperature of the strain. Three replicates were set up for the experiment.

[0065] 1.3.4.2 Antibiotic susceptibility test

[0066] A bacterial suspension was prepared using 0.85% physiological saline and spread onto R2A solid medium. Antibiotic susceptibility testing was performed using the disk diffusion method. Disks containing the following antibiotics were amoxicillin (10 μg / disc), ampicillin (10 μg / disc), bacitracin (0.04 U / disc), chloramphenicol (30 μg / disc), erythromycin (15 μg / disc), gentamicin (10 μg / disc), vancomycin (30 μg / disc), ofloxacin (10 μg / disc), kanamycin (30 μg / disc), neomycin (30 μg / disc), tetracycline (30 μg / disc), piperacillin (10 μg / disc), and rifampin (5 μg / disc). The cultures were incubated at 28°C for 4 days to observe the antibiotic resistance characteristics of the strain. The experiment was performed in triplicate, and two or more consistent results were recorded as positive experimental data.

[0067] 1.3.4.3 Enzyme Activity Detection

[0068] Using the spot inoculation method, representative strains were inoculated onto amylase, cellulase, protease, and esterase activity detection plates, respectively. The plates were incubated at 35℃ for 3-4 days, and the presence and size of the clear zone around the colonies were observed to determine the characteristics of protease, pectinase, and esterase production. Iodine staining was used to observe the presence and size of the clear zone around the strains to determine amylase production. Staining with 0.2% Congo red for 30 minutes, followed by rinsing with 2% NaCl until the eluent was colorless, and the presence and size of the clear zone around the strains were observed to determine the characteristics of cellulase production.

[0069] 1.3.4.4 Growth experiments at different pH levels

[0070] Using RCM medium as the base medium, different buffering substances were added:

[0071] pH 1.0, 25 mL KCl (0.025 mol / L) / 67 mL HCl (0.025 mol / L); pH 2.0, 72.5 mL Phosphoric acid (0.1 mol / L) / 27.5 mL HCl (0.1 mol / L); pH 3.0, 93 mL Citric acid (0.1 mol / L) / 7 mL HCl (0.1 mol / L); pH 4.0, 65.5 mL Citric acid (0.1 mol / L) / 34.5 mL HCl (0.1 mol / L); pH 5.0, 41 mL Citric acid (0.1 mol / L) / 59 mL HCl (0.1 mol / L); pH 6.0, 88 mL KH₂PO₄ (1 / 15 mol) / 12 mL Na₂HPO₄ (1 / 15 mol); pH 7.0, 39 mL KH₂PO₄ (1 / 15 mol) / 6 mL Na2HPO4(1 / 15mol); pH 8.0, 5mL KH2PO4(1 / 15mol) / 95mL Na2HPO4(1 / 15mol); pH 9.0, 10mL Na2CO3(0.1mol) / 90mL NaHCO3(0.1mol); pH10.0, 60mL Na2CO3(0.1mol) / 40mL NaHCO3(0.1mol).

[0072] 1.3.4.5 API reagent strip detection

[0073] According to the API operation manual, API ZYM, API 50CH, API20NE, and API anaerobic reagent strips (bioMérieux) were used to detect the enzymatic characteristics, carbon source acid production, carbon source utilization, and other physiological and biochemical characteristics of the strains. The strains were incubated at 35°C.

[0074] 1.3.4.6 Ethanol Tolerance Test

[0075] During the fermentation of distiller's grains, microorganisms produce a certain amount of ethanol, and the volume fraction increases continuously with the extension of fermentation time. A certain volume fraction of ethanol will have varying degrees of inhibitory effect on microbial growth. Different volumes of ethanol (final volume fractions: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10%) were added to 25 mL screw-top anaerobic flasks containing 10 mL of sterile RCM liquid medium, and 200 μL of bacterial suspension (OD) was added. 600 nm ≈1.0), cultured in a shaker at 37℃ and 140 r / min for 3–5 days, and then the OD was measured. 600 nm The value was used to determine the ethanol tolerance range. The experiment was set up with three replicates, and the average of the three values ​​was recorded as the experimental data.

[0076] 1.3.4.7 Gram Test

[0077] Gram staining assay was performed on the strain using a Gram staining kit.

[0078] 1.3.4.8 Anaerobic Experiment

[0079] The growth of the strain was tested under aerobic, facultative, and strictly anaerobic conditions to determine its aerobic characteristics.

[0080] 1.3.5 Detection and Analysis of Volatile Substances in Fermentation Broth of Strains under Anaerobic Conditions

[0081] Fermentation was conducted on the strain at different temperatures (25, 30, and 35°C), and the optimal fermentation temperature was determined based on the types and relative contents of volatile substances. Ethanol, ethyl acetate, and hexanoic acid were used as substrates at the optimal temperature. The volatile components in the fermentation broth were detected by GC-MS. After fermentation, 20 mL of the fermentation broth was centrifuged at 5000 rpm for 5 min, and 8 mL of the supernatant was used for GC-MS. 8 mL of the sample was added to a 20 mL headspace vial containing 3 g of sodium chloride and 10 μL of 4-octanol. The sample was processed using a headspace sampler at the following temperatures: sample processing temperature 60°C, quantitative loop / valve temperature 100°C, transfer line temperature 110°C, sample processing time 45 min, pressure equilibration time 0.25 min, and injection time 1 min.

[0082] Gas chromatography (GC) conditions: Injector temperature 240℃, carrier gas He, flow rate 1mL / min, split ratio 10:1, column oven temperature program: first, start at an initial temperature of 50℃, hold for 2min, then increase to 80℃ at a rate of 3℃ / min, then increase to 230℃ at a rate of 5℃ / min, and hold for 10min.

[0083] Mass spectrometry (MS) conditions: EI+ ion source, quadrupole temperature 150℃, auxiliary heating temperature 250℃, ion source temperature set to 230℃, filament flow rate 0.20mA, electron energy 70eV, detector voltage 350V. Signal acquisition was performed in full scan mode, with a scan range of 29–350 m / z.

[0084] Qualitative and semi-quantitative analysis: Volatile compounds with a matching degree greater than 75 were retrieved from the standard spectral library for qualitative analysis. The content of volatile flavor substances was calculated based on the ratio of the peak area of ​​the internal standard 4-octanol to that of acids, esters, alcohols and other flavor substances.

[0085] 2 Results and Analysis

[0086] 2.1 Strain Isolation

[0087] One facultative anaerobic bacterium was purified and appeared white on R2A solid medium; the strain number was recorded as JP1. Morphological characteristics were compared between different culture media. Analysis of the pure culture results showed that this strain was isolated from samples taken at 1 and 3 days of fermentation, but not from other samples.

[0088] 2.2 Strain morphology

[0089] 2.2.1 Morphological characteristics of strain JP1

[0090] Strain JP1 is a Gram-positive strain. Its plate morphology after 3 days of growth on R2A solid medium is as follows: Figure 1 As shown in (a), the colonies are white bacteria with surface secretions, no substrate hyphae, and are easily picked up. Scanning electron microscopy (b) shows that the strain is rod-shaped, 0.5-0.6 μm in diameter, with flocculent secretions around the cells. Transmission electron microscopy (c) shows that the strain is rod-shaped, with flagella and pili on the cells, and a large amount of secretions around the cells. The fermentation broth morphology is as follows. Figure 2 As shown, the cells are rod-shaped, secrete a large amount of extracellular polymers, and have peritrichous flagella.

[0091] 2.3 Molecular identification and phylogenetic analysis of the 16S rRNA gene of strain JP1

[0092] The 16S rRNA gene alignment results showed that strain JP1 was most similar to *Sporolactobacillus shoreae*, with a similarity of 92.41%, followed by *S. putidus* (92.14%), *S. vineae* (91.94%), and *S. shoreicorticis* (91.64%). The phylogenetic NJ tree of strain JP1's 16S rRNA gene is shown below. Figure 3As shown, strain JP1 clusters within a branch of the genus *Sporolactobacillaceae*. Preliminary assessment identifies the strain as a potential new species within the genus *Sporolactobacillaceae*, with the taxonomic position as: Bacteria, Firmicutes, Bacilli, Bacillales, Sporolactobacillaceae, *Sporolactobacillaceae*.

[0093] The 16S rRNA gene clone sequence of strain JP1 is shown in SEQ ID NO: 1.

[0094] 2.4 Physiological and Biochemical Experiments

[0095] 2.4.1 Temperature tolerance test of strain JP1

[0096] As shown in Table 2, the growth range of strain JP1 is 20-40℃, with the optimal growth temperature being 30-35℃. Growth is slower at 20 and 25℃ than at 30 and 37℃; the growth trend is best at 30-35℃; growth begins to weaken at 40℃, and growth is almost stagnant at 45℃ due to high temperature inhibition.

[0097] Table 2 Temperature and growth of strain JP1

[0098] 2.4.2 Antibiotic Experiment with Strain JP1

[0099] like Figure 4 The results showed that chloramphenicol, erythromycin, gentamicin, vancomycin, ofloxacin, and tetracycline were six antibiotics that had a significant inhibitory effect on strain JP1; while amoxicillin, ampicillin, bacitracin, kanamycin, neomycin, neomycin, piperacillin, and rifampin were eight antibiotics that had no inhibitory effect on strain JP1.

[0100] 2.4.3 Enzyme activity characteristics of the strain

[0101] Under various enzyme activity solid culture conditions, strain JP1 was positive for protease and cellulase, weakly positive for amylase, and negative for esterase, oxidase, and catalase.

[0102] 2.4.4 Identification of strain JP1 using the API system

[0103] (1) Detection of strain JP1 API 20NE

[0104] API 20NE results showed that strain JP1 was positive for nitrate reduction and also positive for hydrolysis of glucose, aesculin, gelatin, p-nitro-D-methylgalactose, and mannose. It was negative for hydrolysis of tryptophan, arginine, and urea, and negative for utilization of glucose, arabinose, mannitol, N-acetylglucosamine, maltose, gluconate, capric acid, adipic acid, malic acid, citric acid, and phenylacetic acid.

[0105] (2) Detection of strain JP1 API 50CH

[0106] API 50CH results showed that strain JP1 can utilize glycerol, L-arabinose, ribose, galactose, glucose, fructose, mannose, sorbitol, α-methyl-D-glucoside, N-acetylglucosamine, arbutin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, starch, D-minobiose, and D-tagatose as carbon sources for growth and acid production. The following carbon sources cannot be used to produce acids: erythritol, D-arabinose, D-xylose, L-xylose, agonol, β-methyl-D-xylose, sorbitol, rhamnose, eugenol, inositol, mannitol, α-methyl-D-mannose, amygdalin, aesculin, salicylic acid, inulin, pinealose, raffinose, glycogen, xylitol, gentiobiose, D-misugar, D-rockose, L-rockose, D-arabinitol, L-arabinitol, gluconate, 2-keto-gluconate, 5-keto-gluconate.

[0107] (3) Detection of strain JP1 API ZYM

[0108] API ZYM results showed that strain JP1 was positive for alkaline phosphatase, leucine aromatic aminoaminase, valine aromatic aminoaminase, cystine aromatic aminoaminase, acid phosphatase, naphthol-AS-BI-phosphatase, α-galactosidase, β-galactosidase, and β-uronic acid aglycone. It was negative for esterase (C4), lipolipase (C8), lipolipase (C14), trypsin, chymotrypsin, α-glucosidase, β-glucosidase, N-acetyl-glucosamine, α-mannose, and β-fucoside.

[0109] (4) Detection of strain JP1 API 20A

[0110] API 20A results showed that strain JP1 was negative for indole formation, urease, and β-glucosidase reactions, but positive for protease. JP1 can utilize glucose, lactose, sucrose, maltose, salicylate, xylose, arabinose, glycerol, cellobiose, mannose, raffinose, and trehalose as carbon sources for growth. It cannot utilize mannitol, pinosanose, sorbitol, or rhamnose as carbon sources for acid production.

[0111] 2.4.5 Ethanol tolerance test of strain JP1

[0112] As shown in Table 3, the control group was selected from the freshly added bacterial culture before fermentation, and its OD was measured. 600 nm The value was 0.030. Analysis showed that strain JP1 exhibited some growth at ethanol concentrations ranging from 0% to 6% (v / v), but its growth was inhibited with further increases in ethanol concentration. This indicates that higher concentrations of ethanol have an inhibitory effect on the strain. Its ethanol tolerance range is 0-6%.

[0113] Table 3 Ethanol tolerance test OD of strain JP1 600 nm value

[0114] 2.4.6 Salt tolerance test of strain JP1

[0115] As shown in Table 4, the unfermented culture that had just been added to the culture was used as a control, and its OD was measured. 600 nm The value was 0.030. Analysis showed that strain JP1 exhibited some growth at salt concentrations ranging from 0% to 6%, but its growth was inhibited with further increases in salt concentration. The NaCl tolerance range for strain JP1 was 0-6%.

[0116] Table 4 Salt tolerance growth of the strains

[0117] 2.4.7 pH growth range detection of strain JP1

[0118] Analysis shows that strain JP1 can grow in the pH range of 4-7.5, with the optimal pH being 5-5.5. The specific growth conditions are shown in Table 5.

[0119] Table 5 Growth of strains at different pH values

[0120] 2.5 Analysis of volatile substances in the fermentation broth of strain JP1

[0121] (1) Detection of volatile substances during fermentation in different culture media

[0122] After 3 days of anaerobic fermentation in the mash saccharification liquid and R2A medium, the volatile substances of strain JP1 were detected by GC-MS, as shown in Table 6. In the saccharification liquid, a variety of volatile components were detected, including: isobutanol, ethanol, n-hexanol, n-propanol, isoamyl alcohol, n-pentanol; acetic acid; ethyl hexanoate, ethyl acetate, etc. The relative content of ethanol reached 117.5368 μg / mL; ethyl hexanoate was detected, with a relative content of 0.1461 μg / mL. In the R2A medium, ethanol, acetic acid, and 2,3-butanediol were detected, with the relative content of ethanol being 30.2195 μg / mL. In the mash saccharification liquid, after 3 days of fermentation, strain JP1 was able to produce ethyl hexanoate, an important flavor compound in strong-aroma baijiu, and also produced ethyl acetate. Esters in fermented mash are mainly synthesized by microorganisms with esterification capabilities using acids and alcohols as substrates. The detection of ester formation in the fermented mash saccharification liquid indicates that strain JP1 has a certain esterification function. However, the strain's physiological and biochemical esterase tests were negative. This may be because the esterase in strain JP1 is an inducible enzyme, and the substrates used in the physiological and biochemical experiments cannot induce the relevant esterase, while the fermented mash contains the corresponding substrates, thus inducing its production. Furthermore, fermentation experiments demonstrated that strain JP1 can utilize the fermented mash saccharification liquid to produce ethanol and acetic acid.

[0123] Table 6. Results of volatile substance detection for strain JP1 in different culture media.

[0124] (2) Detection of volatile substances in saccharified liquor at different fermentation times

[0125] Temperature experiments showed that JP1 grew best at 25, 30, and 35℃. Therefore, fermentation was conducted at these three temperatures, and the types and contents of volatile substances produced were analyzed. The results... Figure 6-9 As shown in Table 6, the strain produced the highest butyric acid content (18.912 μg / mL) at 30℃. Fermentation was carried out at 30℃ using ethanol, ethyl acetate, and hexanoic acid as substrates. The volatile compounds were analyzed after fermentation at different times, as shown in Table 6. With prolonged fermentation time, strain JP1 mainly produced butyric acid, ethyl butyrate, and ethanol. During the fermentation of the mash by strain JP1, the esterification process primarily involved the production of ethyl butyrate with increasing fermentation time.

[0126] During the fermentation of baijiu mash, brewer's yeast mainly produces ethanol; lactic acid bacteria, lactobacilli, and Bacillus mainly produce acetic acid, which can be produced as ethyl acetate in the presence of microorganisms with esterification properties. Clostridium hexanoate in the mash can produce hexanoate, which, under microbial esterification, produces ethyl hexanoate, an important flavor compound in strong-aroma baijiu. Strain JP1, fermented with saccharified baijiu mash, showed the presence of hexanoate and ethyl hexanoate. Therefore, fermentation was carried out at different temperatures with extended fermentation times using ethanol, ethyl acetate, and hexanoate as substrates.

[0127] When ethanol is used as a substrate, the esters produced by strain JP1 are ethyl acetate and ethyl butyrate, which increase with prolonged fermentation time. The acids produced are acetic acid and butyrate, with butyrate being the predominant acid. The butyrate content reaches its maximum at 14 days of fermentation, at 14.583 μg / mL. It does not change much with the extension of time to 21 days and decreases slightly at 28 days. Considering the gradual increase in ethyl butyrate content, it is speculated that the butyrate yield of strain JP1 increases with prolonged fermentation time. Under the action of strain JP1, butyrate undergoes esterification with ethanol to be converted into ethyl butyrate.

[0128] When strain JP1 uses ethyl acetate as a substrate, the number of esters increases, with seven types present, including butyl formate, ethyl acetate, methyl acetate, butyl acetate, propyl acetate, and ethyl propionate. The relative content of ethyl propionate reaches 3.158 μg / mL, and the relative content of butyl acetate reaches 1.411 μg / mL. The main acids are butyric acid and acetic acid, with butyric acid reaching 8.431 μg / mL.

[0129] When hexanoic acid is used as a substrate, strain JP1 produces ethyl hexanoate, methyl hexanoate, and ethyl acetate as esters, and acetic acid as the acid. After the addition of hexanoic acid, compared with the blank control, strain JP1 can produce ethyl hexanoate, with the highest relative content reaching 38.517 μg / mL at 14 days of fermentation, significantly higher than that at 3 days of fermentation (0.1461 μg / mL). This indicates that strain JP1 possesses esterase activity and can promote the synthesis of ethyl hexanoate from hexanoic acid and ethanol.

[0130] Table 6. Results of volatile substance detection for strain JP1 under different substrates and fermentation times.

[0131] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

1. A new strictly anaerobic strain JP1, belonging to the genus *Lactobacillus* (…). Sporolactobacillus The sample has been deposited with accession number CGMCC NO.27858, deposited at the China General Microbiological Culture Collection Center in Beijing, China, on July 7, 2023.

2. A microbial inoculant comprising the novel strain JP1 of claim 1.

3. Use of the novel strain JP1 of claim 1 or the microbial inoculant of claim 2 in food production.

4. Use according to claim 3, characterized in that: The food is a fermented food.

5. Use according to claim 4, characterized in that: The food is an alcoholic product.

6. A method for brewing Luzhou-flavor liquor, characterized in that: Fermentation brewing is performed using the novel strain JP1 of claim 1 as a fermentation starter.

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