Ivory c. propinquum strain for producing flavoring substances and application thereof

Abstract

This invention discloses a strain of *Kroppenstedtia eburnea* producing flavor compounds and its applications, belonging to the field of microbial technology. This invention provides a strain of *Kroppenstedtia eburnea*, KE1613, derived from the mash used in the brewing of Maotai-flavor baijiu. This strain was deposited on January 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 29828. *Kroppenstedtia eburnea* KE1613 can produce a variety of volatile aroma compounds, including 2,5-dimethylpyrazine, an important flavor compound in Maotai-flavor baijiu, and simultaneously produces phenylethanol and fatty acids. This enhances the Maotai-flavor of the mash during fermentation and also provides floral and fruity aromas, which is of significant practical importance for improving the flavor of baijiu, especially Maotai-flavor baijiu, during production.

Description

Technical Field

[0001] This invention relates to a strain of *Cropensia ivoryensis* that produces flavor compounds and its applications, belonging to the field of microbial technology. Background Technology

[0002] According to aroma differences, baijiu (Chinese liquor) can be divided into 12 major aroma types, including strong aroma, sauce aroma, and light aroma, among which sauce aroma, strong aroma, light aroma, and rice aroma are the four basic aroma types. Sauce aroma baijiu is renowned for its elegant and delicate aroma and mellow, full-bodied flavor. Its production process is characterized by "four highs and two longs, one large and one many," namely, high-temperature stacking, high-temperature distillation, high-temperature fermentation, and high-temperature koji making; long production cycle and long storage time; large amount of koji used, and multiple rounds of distillation. Sauce aroma baijiu possesses an elegant style characterized by being "colorless (or slightly yellow) and transparent, with a prominent, elegant, and delicate sauce aroma, a mellow and harmonious flavor, a long aftertaste, and a lingering fragrance even in an empty glass," making it the most popular of the three major aroma types of baijiu today (sauce, strong, and light).

[0003] The rich aroma and flavor compounds in baijiu, including esters, alcohols, aldehydes, acids, and pyrazines, determine its quality and style, and are also important components of its flavor. Among all types of baijiu, sauce-aroma baijiu has the most diverse and highest content of pyrazine compounds (headspace solid-phase microextraction-gas chromatography for the detection of pyrazine compounds in baijiu, Huang Jialing et al., China Brewing, 2016, 35(8): 163-168). Pyrazines are a class of six-membered heterocyclic compounds containing nitrogen atoms at positions 1 and 4, and have important flavor value. Pyrazines are mostly caramel and roasted, and are important flavor compounds in sauce-aroma baijiu. On the one hand, due to their low flavor threshold, they can make an important contribution to the typical aroma composition of baijiu, and at the same time have a synergistic effect, enriching the aroma of baijiu; on the other hand, they also give baijiu health benefits and play an important role in the prevention of cardiovascular and cerebrovascular diseases.

[0004] Floral and fruity aromas are also typical of soy sauce-flavored baijiu. The main substances contributing to floral and fruity aromas are esters, such as ethyl isovalerate, ethyl acetate, and ethyl hexanoate. Acids are indispensable precursors for the synthesis of esters. Some alcohols can also contribute to floral and fruity aromas, such as phenylethanol and 2-butanol.

[0005] *Kroppenstedtia* is a dominant bacterial microorganism in the production of Daqu (a type of starter culture) and the fermentation process of high-temperature Daqu mash in soy sauce-flavored liquor, with a relative abundance of approximately 3.49%-41.73% (Spatial heterogeneity of the microbiome and metabolome profiles of high-temperature Daqu in the same workshop. Food Research International, 2022, 156:111298). *Kroppenstedtia* belongs to the Thermoactinomycetaceae family of the Bacilli class. It is an aerobic, thermostable microorganism with an optimal growth temperature of 45–50℃. Temperature is a crucial factor affecting the quality of fermentation in soy sauce-flavored liquor, reflecting whether the fermentation process is normal. During the fermentation process, the temperature of the mash can reach up to 50℃. Current research based on metatranscriptomics and metagenomics has revealed that *Cropensis* is a functional microorganism involved in fatty acid biosynthesis during the brewing process of soy sauce-flavored liquor (Daqu and environmental microbiota regulate fatty acid biosynthesis via driving the core microbiotain soy sauce aroma type liquor fermentation. International Journal of Food Microbiology, 2024, 408:110423). Functional annotation of genes encoding enzymes from six rounds of Daqu fermentation has shown that *Cropensis ivoryensis* encodes α-amylase, which participates in carbohydrate metabolism during Daqu fermentation. Furthermore, Spearman correlation analysis shows a significant positive correlation between *Cropensis* and flavor compounds such as amino acids and acetic acid during Daqu fermentation.

[0006] Fermentation of distiller's grains involves high temperatures, and the heat tolerance of the bacterial strains, their metabolic activity at these temperatures, and the production of flavor compounds are key factors influencing strain selection. Therefore, to ensure smooth fermentation, it is necessary to comprehensively consider the quality of the strains, ensuring they can adapt to high-temperature environments, maintain high metabolic activity, and produce ideal flavor compounds under these conditions. This is crucial for ensuring fermentation efficiency and the quality of the final product. Although *Cropensella* is a dominant bacterial microorganism in the fermentation process of distiller's grains, few strains have been isolated and cultured in pure form, leaving the metabolic characteristics of *Cropensella* at the strain level and its contribution to flavor during the fermentation process of soy sauce-flavored liquor unclear.

[0007] Therefore, isolating and purifying Croppenstein microorganisms from fermented mash piles and analyzing their physiological and metabolic characteristics has important theoretical and applied value for exploring and analyzing high-quality fermented mash resources and developing new baijiu brewing and regulation technologies. It also has important practical significance for improving the flavor of baijiu in production. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a strain of *Kroppenstedtia eburnea*, KE1613, derived from the mash used in the brewing of Maotai-flavor liquor. This strain was deposited on January 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 29828. Strain KE1613 can adapt to relatively high fermentation temperatures (45℃, 50℃) and can ferment to produce flavor compounds such as 2,5-dimethylpyrazine, phenylethanol, and fatty acids.

[0009] The first objective of this invention is to provide an ivory clopenster bacteria strain KE1613, which has the accession number CGMCC No. 29828.

[0010] The functions of the strain *Cropensia ivoryensis* KE1613 of this invention are as follows:

[0011] (1) In liquid fermentation, the volatile aroma compounds produced by the ivory cropensis strain KE1613 were 1.88 times higher than those of the blank control group, with a yield of 22.19 mg / L. Pyrazines accounted for the largest proportion in the fermentation broth, reaching 70.35%. Compared with the control, the pyrazine content in KE1613 was 9.07 mg / L higher, with the yield of 2,5-dimethylpyrazine reaching 5.26 mg / L, which was 1.5 times that of the control group. The yield of phenylethanol reached 0.42 mg / L, and the yield of total acid reached 0.22 mg / L.

[0012] (2) In solid-state fermented sorghum, at 45℃, the ivory cropensis strain KE1613 produced 0.84 μg / g of 2,5-dimethylpyrazine, 13.13 μg / g of phenylethanol, and 12.08 μg / g of total acid, with the highest yield of isovaleric acid at 6.53 μg / g; at 50℃, the production of 2,5-dimethylpyrazine reached 0.80 μg / g, 12.95 μg / g of phenylethanol, and 16.55 μg / g of total acid, with the highest yield of isovaleric acid at 6.88 μg / g.

[0013] (3) Croppensteine ​​Ivory KE1613 can produce a variety of volatile aroma compounds in both TSB liquid culture medium and solid fermentation sorghum culture medium. It can produce 2,5-dimethylpyrazine, an important flavor compound of soy sauce-flavored liquor, and simultaneously produce phenylethanol and fatty acids, making the soy sauce-flavor of the fermented mash more prominent and providing floral and fruity aromas. This is of great practical significance for improving the flavor of liquor, especially soy sauce-flavored liquor, in production.

[0014] Combining the above effects, the ivory clopinster KE1613 of the present invention can produce flavor substances well in liquid and solid fermentation at relatively high temperatures (45°C, 50°C), and has the potential to be applied to wine production and improve the flavor of wine products.

[0015] In one embodiment, *Croponectinus ivoryensis* KE1613 is capable of metabolizing to produce 2,5-dimethylpyrazine, and simultaneously producing phenylethanol and fatty acids.

[0016] In one embodiment, during liquid fermentation, the volatile aroma compounds produced by the *Cropensia ivoryensis* KE1613 strain were 1.88 times higher than those of the blank control group, with a yield of 22.19 mg / L. Among them, the yield of 2,5-dimethylpyrazine reached 5.26 mg / L, the yield of phenylethanol reached 0.42 mg / L, and the yield of total acid reached 0.22 mg / L.

[0017] In one embodiment, when the above-mentioned strain is fermented in solid state at 45°C, the yield of 2,5-dimethylpyrazine reaches 0.84 μg / g, the yield of phenylethanol reaches 13.13 μg / g, and the yield of total acid reaches 12.08 μg / g, of which the yield of isovaleric acid is the highest at 6.53 μg / g.

[0018] In one embodiment, when the above-mentioned strain is fermented in solid state at 50°C, the yield of 2,5-dimethylpyrazine reaches 0.80 μg / g, the yield of phenylethanol reaches 12.95 μg / g, and the yield of total acid reaches 16.55 μg / g, of which the yield of isovaleric acid is the highest at 6.88 μg / g.

[0019] In one embodiment, the biological characteristics of the aforementioned strains are as follows: the colonies are ivory-colored, flat, irregular in shape, with wavy edges, and a dull surface. In the later stages of growth, the colonies become radially wrinkled and have an earthy odor. Cold field scanning electron microscopy reveals that the hyphae of strain KE1613 are long and curved, with a single endospore developing at the tip of the unbranched hyphae. The mycelial size is approximately (0.3-0.5) μm to (4-6) μm.

[0020] In one embodiment, the growth conditions for the above-mentioned strain are: aerobic conditions, cultured at 45-55°C for 3-4 days.

[0021] A second objective of this invention is to provide a microbial agent containing the aforementioned *Cropsporium ivoryense* KE1613.

[0022] In one embodiment, the viable count of Croppenstein KE1613 per milliliter or per gram of bacterial agent reaches 1 × 10⁻⁶. 8 CFU.

[0023] The third objective of this invention is to provide a method for increasing the content of volatile aroma compounds in the fermentation of baijiu (Chinese liquor), the method comprising: adding the above-mentioned *Cropspermum ivoryense* KE1613 or the above-mentioned microbial inoculant to the baijiu fermentation process.

[0024] In one embodiment, the method includes: inoculating *Cropensella ivoryensis* KE1613 into a liquid fermentation medium for fermentation, with an inoculation amount of 1–8% (v / v).

[0025] In one embodiment, the fermentation temperature is 45–55°C.

[0026] In one implementation, the fermentation time is 72–96 hours.

[0027] In one embodiment, the method includes inoculating *Cropensella ivoryensis* KE1613 into a solid-state fermented sorghum culture medium at an inoculation amount of 20–25% v / w.

[0028] In one embodiment, the fermentation temperature is 45–55°C and the fermentation time is 12–15 days.

[0029] In one embodiment, a method for producing 2,5-dimethylpyrazine, phenylethanol, and fatty acids by liquid fermentation of *Croponectinus ivoryensis* strain includes the following steps: after sterilization of the liquid fermentation medium, the above-mentioned *Croponectinus ivoryensis* strain is inoculated, and the medium is cultured under aerobic conditions with shaking.

[0030] In one embodiment, the liquid fermentation medium (TSB medium) is composed of: 17.0 g / L tryptone, 5.0 g / L sodium chloride, 3.0 g / L soybean peptone, 2.5 g / L dipotassium hydrogen phosphate, and 2.5 g / L glucose.

[0031] In one embodiment, the solid-state fermented sorghum culture medium is prepared by adding 100g of red tassel sorghum and 80mL of deionized water to a 250mL Erlenmeyer flask.

[0032] A fourth objective of this invention is to provide the application of the aforementioned *Cropspermia ivoryensis* KE1613 or the aforementioned microbial inoculant in brewing.

[0033] In one embodiment, the wine is a distilled spirit, a fermented spirit, or a blended spirit.

[0034] In one embodiment, the alcoholic beverage includes common alcoholic products such as baijiu, huangjiu, wine, and beer.

[0035] The present invention also provides the application of the above-mentioned Croppensteine ​​KE1613 or the above-mentioned microbial agent in stack fermentation.

[0036] Beneficial effects

[0037] This invention relates to *Kroppenstedtia eburnean*, isolated and screened from *Kroppenstedtia* microorganisms in fermented mash during stacking fermentation. The strain, with accession number CGMCC No. 29828 and named KE1613, can adapt to relatively high fermentation temperatures and produces a variety of volatile aroma compounds in both TSB liquid medium and solid-state fermentation sorghum medium. It produces 2,5-dimethylpyrazine, an important flavor compound in soy sauce-flavored baijiu, and simultaneously produces phenylethanol and fatty acids, thus enhancing the soy sauce-flavor of the fermented mash and providing floral and fruity aromas. This has significant practical implications for improving the flavor of baijiu, especially soy sauce-flavored baijiu, during production.

[0038] Specifically:

[0039] (1) In liquid fermentation, the volatile aroma compounds produced by the ivory cropensis strain KE1613 were 1.88 times higher than those of the blank control group, with a yield of 22.19 mg / L. Pyrazines accounted for the largest proportion in the fermentation broth, reaching 70.35%. Compared with the control, the pyrazine content in KE1613 was 9.07 mg / L higher, with the yield of 2,5-dimethylpyrazine reaching 5.26 mg / L, which was 1.5 times that of the control group. The yield of phenylethanol reached 0.42 mg / L, and the yield of total acid reached 0.22 mg / L.

[0040] (2) In solid-state fermentation, at 45℃, the ivory cropensis strain KE1613 produced 0.84 μg / g of 2,5-dimethylpyrazine, 13.13 μg / g of phenylethanol, and 12.08 μg / g of total acid, with the highest yield of isovaleric acid at 6.53 μg / g; at 50℃, the production of 2,5-dimethylpyrazine reached 0.80 μg / g, the production of phenylethanol reached 12.95 μg / g, and the total acid reached 16.55 μg / g, with the highest yield of isovaleric acid at 6.88 μg / g.

[0041] (3) In liquid fermentation and solid fermentation at 50℃, strain KE1613 was superior to strain 6E22, which also belongs to Clopentomeus, and had a higher yield of flavor compounds. In liquid fermentation, strain KE1613 had 71% more volatile aroma compounds than strain 6E22. In solid fermentation at 50℃, strain KE1613 had 24.0% more volatile aroma compounds than strain 6E22.

[0042] Biological Preservation Materials

[0043] The ivory Kroppenstedtia eburnea strain KE1613 of this invention was deposited on January 31, 2024 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo.29828, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0044] Figure 1 It is the ivory clopenster strain KE1613, in which Figure 1 A is a diagram of colony morphology; Figure 1 B is a cold field scanning electron microscope image. Detailed Implementation

[0045] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0046] This invention isolates and screens a strain of Kroppenstedtia eburnea, numbered KE1613, from the accumulated mash of soy sauce liquor. This strain is capable of metabolizing and producing 2,5-dimethylpyrazine, as well as phenylethanol and fatty acids.

[0047] The *Kroppenstedtia eburnean* strain of this invention, taxonomically named, was deposited on January 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 29828, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0048] The 16S rRNA gene sequence of this strain was compared with the EzBioCloud database to determine its species. The full-length 16S rRNA gene sequence is 1526 bp. It has the highest homology with the type strain Kroppenstedtia eburnea DSM45196. TWith a similarity of 99.16% and a difference of 11 bases, the 16S rRNA gene sequence of the *Cropensia ivoryensis* strain of the present invention is shown in SEQ ID NO: 1.

[0049] The *Croplastrus ivoryensis* strain provided by this invention produces 2,5-dimethylpyrazine and simultaneously produces phenylethanol and fatty acids using TSB liquid fermentation medium. The fermentation medium is formulated with 17.0 g / L tryptone, 5.0 g / L sodium chloride, 3.0 g / L soybean peptone, 2.5 g / L dipotassium hydrogen phosphate, and 2.5 g / L glucose.

[0050] To better obtain the yield of volatile aroma compounds, the *Croponstrom ivoryensis* strain provided in this invention, when KE1613 is cultured in TSB liquid fermentation medium at a fermentation temperature of 45°C, 100 mL of fermentation broth is placed in a 250 mL Erlenmeyer flask and cultured on a constant temperature shaker at a speed of 180 rpm for 72–96 hours, can yield a fermentation broth containing 2,5-dimethylpyrazine, phenylethanol, and fatty acids.

[0051] Using the above-mentioned liquid fermentation process, the volatile aroma compounds produced by the *Croponema ivoryense* strain KE1613 were 1.88 times higher than those of the blank control group, with a yield of 22.19 mg / L. Among them, the yield of 2,5-dimethylpyrazine reached 5.26 mg / L, the yield of phenylethanol reached 0.42 mg / L, and the yield of total acid reached 0.22 mg / L.

[0052] This invention simulates the ability of *Croponema ivoryense* strain to produce volatile aroma compounds during stacking fermentation. KE1613 was cultured on solid sorghum medium, and two solid-state fermentation groups were set up and placed in constant temperature incubators at fermentation temperatures of 45℃ and 50℃, respectively, for 15 days.

[0053] The solid-state fermentation sorghum culture medium formula is to add 100g of red tassel sorghum and 80mL of deionized water to a 250mL Erlenmeyer flask.

[0054] Using the above-mentioned solid-state fermentation process, the *Cropensida ivoryensis* strain KE1613 produced 0.84 μg / g of 2,5-dimethylpyrazine, 13.13 μg / g of phenethyl alcohol, and 12.08 μg / g of total acid under a culture temperature of 45°C, with isovaleric acid being the highest yield at 6.53 μg / g. Under a culture temperature of 50°C, the yield of 2,5-dimethylpyrazine reached 0.80 μg / g, the yield of phenethyl alcohol reached 12.95 μg / g, and the yield of total acid reached 16.55 μg / g, with isovaleric acid being the highest yield at 6.88 μg / g.

[0055] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0056] Raw materials used in the examples:

[0057] The mash samples were taken from a representative distillery of Maotai-flavor liquor, specifically from the mash on days 1, 4, and 9 of the third fermentation stage. Each sample was taken from three points: 50cm below the ground and 20cm deep. Approximately 50g of mash was collected from each point, mixed thoroughly, placed in a sterile resealable bag, and stored at 4℃ with ice packs.

[0058] Tryptic soy peptone (TSB) medium (g / L): tryptic 17-18, sodium chloride 4.5-5.5, soy peptone 2.5-3.5, dipotassium hydrogen phosphate 2-3, glucose 2-3.

[0059] TSB solid medium: Based on TSB liquid medium, with 2% w / v agar added.

[0060] Solid-state fermentation sorghum culture medium: Add 100g of red tassel sorghum and 80mL of deionized water to a 250mL Erlenmeyer flask.

[0061] Example 1: Screening of Croppenstein strain KE1613 in ivory

[0062] (1) Sampling and preparation: The mash samples were taken from a representative distillery of Maotai-flavor liquor, specifically from the mash on days 1, 4, and 9 of the third fermentation stage. Each mash sample was taken from 50cm below the ground, at a depth of 20cm, with three sampling points. Approximately 50g of mash was collected from each point, mixed thoroughly, and placed in a sterile self-sealing bag. The samples were stored at 4℃ with ice packs and then sent back to the laboratory for subsequent microbial isolation.

[0063] (2) Screening for bacteria: Weigh 10.0g of the fermented mash sample and add it to a 250mL Erlenmeyer flask containing 100mL of sterile physiological saline and an appropriate amount of glass beads. Place the flask in a constant temperature shaker and shake at 45℃ and 180r / min for 30min. Let it stand for 10min until the mash settles to the bottom of the flask to obtain a bacterial suspension. Take the supernatant and perform serial dilutions (10⁻¹-10⁻⁷). Take 100μL of the bacterial suspension and spread it on TSB solid medium. Place the flask in a 45℃ incubator and incubate for 2-3 days. After the colonies grow, select single colonies with different colony morphologies at regular intervals, isolate and purify them three times, and then inoculate them into TSB liquid medium. Incubate the liquid medium on a constant temperature shaker at 45℃ and 180r / min for 2 days.

[0064] The strain was screened and named KE1613.

[0065] Example 2: Molecular identification of strain KE1613

[0066] The strain KE1613 obtained from the screening in Example 1 was subjected to molecular identification.

[0067] Take 2 mL of bacterial culture and extract bacterial DNA for species identification according to the instructions of the bacterial DNA rapid extraction kit (Nanjing Novizan Biotechnology Co., Ltd.). Use universal primers (27F, 1492R) for bacterial 16S rRNA gene to perform PCR amplification on the extracted microbial DNA. Send the PCR product to Wuxi Tianlin Biotechnology Co., Ltd. for sequencing.

[0068] Primers used:

[0069] 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO: 2);

[0070] 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO: 3);

[0071] PCR amplification reaction 20μL system: 27F 1μL, 1492R 1μL, 2×Taq Master Mix (Vazyame) 12.5μL, sterile ultrapure water 4.5μL, DNA template 1μL.

[0072] The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 7 min; denaturation at 95℃ for 25 s, annealing at 56℃ for 30 s, extension at 72℃ for 20 s, for 35 cycles; 72℃ for 7 min.

[0073] Purification: Based on the PCR results, the taxonomic position of the strain was determined, and the culture medium of the target strain was spread on TSB solid medium to obtain single colonies.

[0074] The 16S rRNA gene sequence of the strain was obtained, and its species was determined by homology comparison with the type strains in the EzBioCloud database. The full length of the 16S rRNA gene sequence is 1526 bp, and its similarity with the type strain with the highest homology, Kroppenstedtia eburnea DSM 45196T, is 99.16%, differing by 11 bases. The 16S rRNA gene sequence of the Kroppenstedtia eburnea strain of the present invention is shown in SEQ ID NO: 1.

[0075] Following the above method, the high-performance *Cropensia ivoryensis* strain KE1613 was obtained and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29828. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. The deposit date is January 31, 2024.

[0076] Example 3: Physiological and biochemical characteristics of *Cropensia ivoryensis* KE1613 and preparation of inoculum.

[0077] (1) Cell morphology

[0078] Croppenstein KE1613 was inoculated onto TSB solid medium and incubated at 45°C for 48 hours. The colony morphology on the solid medium was observed. The colonies were ivory-colored, flat, irregular in shape, with wavy edges and a dull surface. In the later stages of growth, the colonies became radially wrinkled and had an earthy smell.

[0079] The bacterial culture, after three subcultures and growth to the logarithmic growth phase, was spread onto agar plates, allowing the bacteria to cover the entire plate. Bacterial cells were picked, washed three times with PBS buffer, and fixed twice with 2.5% v / v glutaraldehyde solution. The microscopic morphology of the bacterial cells was observed using a cold field scanning electron microscope. Under cold field scanning electron microscopy, the hyphae of strain KE1613 were long and curved, with a single endospore developing at the tip of the unbranched hyphae. The hyphae were approximately (0.3-0.5) μm × (4-6) μm in size.

[0080] (2) Growth curve of Clopenella ivory strain KE1613

[0081] Croppensteine ​​ivory strain KE1613 was streaked on solid TSB medium, and single colonies were selected and cultured in liquid TSB medium to obtain seed culture with good growth viability. The seed culture was then transferred at a 1% v / v inoculum to 250 mL Erlenmeyer flasks containing 100 mL of liquid TSB medium with an appropriate amount of glass beads. Fermentation broth was collected every 4 hours for growth analysis. Uninoculated medium served as a control, and each group was divided into three replicates.

[0082] Growth curve analysis showed that the strain grew slowly from 0 to 16 hours, indicating a lag phase; it grew rapidly after 18 hours, entering the logarithmic growth phase; and it entered a plateau phase between 22 and 28 hours, with OD... 600 The pH ranged from 2.8 to 3.2. The pH of strain K.1613 showed an increasing trend throughout its growth, changing from an initial pH of 7.46 to 8.46.

[0083] (3) Preparation of microbial agents

[0084] The method for preparing the inoculum from Croppenstein KE1613 is as follows:

[0085] The obtained Kroppenstedtia eburnean KE1613 was inoculated into TSB medium and cultured at 45°C for 24 hours to obtain primary seed culture. Then, the culture was expanded stepwise to three generations at an inoculation ratio of 10% per generation to obtain tertiary seed culture.

[0086] Take the third-stage seed culture from the above steps and inoculate it into 100 mL of TSB liquid medium at an inoculation ratio of 10% (v / v). Ferment and culture at 45°C for 24 h. Then, collect the bacterial cells by centrifugation at 8000 rpm for 10 min at 4°C. Add a freeze-drying protectant (skim milk) according to the bacterial cell content in a clean bench, mix well, and then freeze-dry under vacuum. Finally, seal the freeze-dried microbial agent.

[0087] The specific preparation method of the bacterial agent is as follows: Kroppenstedtia eburnean KE1613 cells are mixed with skim milk at a ratio of 5:1 (v / v), that is, 500 mL of fermentation broth corresponds to 100 mL of skim milk. The mixture is dispensed into glass petri dishes, with each dish containing 100 mL. The dishes are sealed with plastic wrap and pre-frozen at -80℃ for 2 hours. After pre-freezing, the mixture is freeze-dried in a vacuum freeze dryer at a vacuum degree of 66.66 Pa for 24 hours to obtain a solid bacterial agent containing Kroppenstedtia eburnean KE1613.

[0088] The viable count of Croppenstein KE1613 per milliliter or per gram of bacterial agent reaches 1×10⁻⁶. 8 CFU.

[0089] Example 4: Liquid fermentation of *Cropensia ivoryensis* KE1613 to produce high yields of volatile aroma compounds

[0090] The selected *Cropensia ivoryensis* strain KE1613 was streaked on solid TSB medium. Single colonies were selected and cultured in liquid TSB medium to obtain seed culture with good growth viability. The inoculum was transferred at a 1% v / v inoculation rate to 250 mL Erlenmeyer flasks containing 100 mL of liquid TSB medium with an appropriate amount of glass beads. The flasks were incubated at 180 rpm for 72–96 hours. Fermentation broth was collected every 4 hours for metabolic characterization analysis. Uninoculated medium served as a control. Each group was divided into three replicates. The content of volatile aroma compounds in the fermentation broth was determined using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

[0091] Another strain of *Croplastrum ivoryense*, strain number 6E22, was also detected during the screening process. The screening and identification methods were the same as in Examples 1 and 2. Group 6E22 was set up using the same method as described above, and its aroma production was compared with that of KE1613.

[0092] The specific method for HS-SPME-GC-MS determination is as follows:

[0093] (1) Preprocessing methods

[0094] Take 30 mL of the culture that has reached the stationary phase and transfer it to a 50 mL centrifuge tube. Centrifuge at 8000 rpm for 10 min at 4 °C. Take 5 mL of the supernatant and transfer it to a 20 mL headspace vial. Add 2.3 g of sodium chloride and 10 μL of chromatographic grade tert-amyl alcohol internal standard solution.

[0095] (2) Analysis conditions

[0096] Instrument: Gas Chromatography-Mass Spectrometry System (GC-MS) from Agilent Technologies, USA.

[0097] Detection conditions: Chromatographic column: DB-Wax capillary column, 30m long, 0.25mm inner diameter, 0.25μm film thickness; Carrier gas: He; Flow rate: 1.0mL / min, injection port temperature: 250℃, desorption for 5min, splitless injection; Gas phase temperature program: initial temperature 40℃, hold for 3min, increase to 60℃ at 5℃ / min, then increase to 230℃ at 10℃ / min, hold for 8min; Mass spectrometry conditions: ion source temperature 260℃, interface temperature 200℃, electron energy 70eV, scan mass range 25–350 amu. Qualitative analysis was performed using the NIST17 spectral library, and the relative content of various substances was calculated using the internal standard method.

[0098] The results are shown in Table 1. The volatile aroma compounds produced by the ivory cropensis strain KE1613 were 1.88 times higher than those of the blank control group, with a yield of 22.19 mg / L. Pyrazines accounted for the largest proportion in the fermentation broth, reaching 70.35%. Compared with the blank control group, the pyrazine content in KE1613 was 9.24 mg / L higher. Among them, the yield of 2,5-dimethylpyrazine reached 5.26 mg / L, which was 1.5 times that of the control group, the yield of phenylethanol reached 0.42 mg / L, and the yield of total acid reached 0.22 mg / L.

[0099] Table 1. Content of volatile aroma compounds in two strains of Clopentomeus ivory during liquid fermentation.

[0100]

[0101]

[0102] Based on this, the total amount of volatile aroma compounds in the fermentation broth of strain KE1613 was significantly higher than that of the control group and strain 6E22. Among them, important flavor substances such as pyrazines, acids, and alcohols were generally significantly higher than those of strain 6E22, and the total amount of volatile aroma compounds was 71% higher than that of strain 6E22.

[0103] Example 5: High-yield volatile aroma compounds from solid-state sorghum fermentation using *Croponectinella ivory* KE1613.

[0104] The purified KE1613 strain was inoculated into TSB liquid medium at a 1% v / v inoculum and cultured until the plateau phase. The cells were collected by centrifugation at 8000 rpm for 10 min at 4°C, washed twice with PBS buffer, and resuspended. Strain 6E22 was prepared using the same method. 20 mL of the strain resuspension was added to each solid-state fermented sorghum medium, mixed well, and then incubated at 45°C and 50°C respectively. A blank control of 20 mL of sterile water was used. Each treatment was performed in triplicate.

[0105] The volatile compound composition of sorghum fermented by the strain under different temperature conditions was detected by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). 5.0 g of sample was placed in a 50 mL centrifuge tube, 20 mL of ultrapure water was added, and the mixture was thoroughly vortexed and sonicated at 4 °C for 30 min, followed by centrifugation at 8000 rpm for 10 min. 5 mL of the supernatant was transferred to a 20 mL headspace vial, and 2.3 g of sodium chloride and 10 μL of 8.05 g / L chromatographic grade tert-amyl alcohol internal standard solution were added. The HS-SPME-GC-MS procedure and analytical method were the same as in Example 4.

[0106] The groups are as follows:

[0107] Control group: 20 mL of sterile water was added to the solid-state fermented sorghum culture medium.

[0108] Groups KE1613 and 6E22: Each strain was inoculated into TSB liquid medium at an inoculum of 1% v / v and cultured until the plateau phase. The cells were collected by centrifugation at 8000 rpm for 10 min at 4°C, washed twice with PBS buffer, and resuspended. 20 mL of the strain resuspension was added to each solid-state fermented sorghum medium, with an inoculum of 20% v / w, resulting in a viable cell count of approximately 2 × 10⁻⁶. 8 CFU / mL.

[0109] The results showed that when strain KE1613 fermented in solid-state at 45℃, the yield of 2,5-dimethylpyrazine reached 0.84 μg / g, the yield of phenethyl alcohol reached 13.13 μg / g, and the yield of total acid reached 12.08 μg / g, with isovaleric acid being the highest yield at 6.53 μg / g. When fermented in solid-state at 50℃, the yield of 2,5-dimethylpyrazine reached 0.80 μg / g, the yield of phenethyl alcohol reached 12.95 μg / g, and the yield of total acid reached 16.55 μg / g, with isovaleric acid being the highest yield at 6.88 μg / g.

[0110] The contents of volatile aroma compounds in solid-state fermented sorghum were compared between strains KE1613 and 6E22 at different temperatures (45℃ and 50℃), and the results are shown in Tables 2 and 3.

[0111] Table 2. Content of volatile aroma compounds of various strains in solid-state fermented sorghum at 45℃

[0112]

[0113] Table 2 shows that strains 6E22 and KE1613 exhibited different aroma-producing properties at 45℃. Strain 6E22 had a higher total amount of volatile aroma compounds and pyrazine content than strain KE1613, while strain KE1613 had higher phenylethanol and total acid content. At this temperature, KE1613 provided a more intense floral and fruity aroma, while strain 6E22 provided an excessive amount of nutty aroma.

[0114] Table 3. Content of volatile aroma compounds of various strains in solid-state fermented sorghum at 50℃

[0115]

[0116] Table 3 shows that at 50℃, strain KE1613 exhibits significantly better aroma production performance than strain 6E22. The total amount of volatile aroma compounds in strain KE1613 is 24.0% higher than that in strain 6E22. Except for the pyrazine content, which is similar between the two strains, the contents of other volatile aroma compounds are higher in strain KE1613.

[0117] Based on Tables 2 and 3, and considering the higher temperatures involved in solid-state fermentation, strain KE1613 is more suitable for alcoholic beverage production. In conclusion, strain KE1613 can effectively produce flavor compounds during both liquid and solid-state fermentation at higher temperatures (45℃ and 50℃), demonstrating its potential for application in alcoholic beverage production and improving the flavor of alcoholic products.

[0118] The nucleotide sequence of the 16S rRNA of the *Croplastrus ivoryensis* strain SEQ ID NO:1:

[0119]

[0120] It should be noted that the specific features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in one or more embodiments. Furthermore, those skilled in the art can combine and integrate the different embodiments described in this specification and the features of those embodiments without contradiction.

Claims

1. A strain of Kroppenstedtia eburnea KE1613, characterized in that, The ivory-colored *Cropensterella* KE1613 has the accession number CGMCC No. 29828.

2. A microbial inoculant, characterized in that, The microbial agent contains the ivory-colored Croppenstein bacterium KE1613 as described in claim 1.

3. A method for increasing the content of volatile aroma compounds in the fermentation of white liquor, characterized in that, The method includes adding the Ivory-colored Clopenstemon KE1613 of claim 1 or the microbial agent of claim 2 to the fermentation process of baijiu.

4. The method of claim 3, wherein, The method includes: inoculating ivory-colored Croppensteine ​​KE1613 into a liquid fermentation medium for fermentation, with an inoculation amount of 1~8% v / v.

5. The method of claim 4, wherein, The fermentation temperature is 45~55℃.

6. The method of claim 4, wherein, Fermentation time is 72~96 hours.

7. The method according to claim 3, characterized in that, The method includes inoculating ivory-colored Croppensteine ​​KE1613 into a solid-state fermented sorghum culture medium at an inoculation amount of 20-25% v / w.

8. The method of claim 7, wherein, The fermentation temperature is 45~55℃, and the fermentation time is 12~15 days.

9. The application of the ivory-colored Croppensteine ​​KE1613 of claim 1 or the microbial agent of claim 2 in liquid fermentation brewing.

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