New crocodylomorphs and applications thereof
By screening and identifying the heat-resistant Croppensteine strain MZ35, the problem of insufficient tetramethylpyrazine production in high-temperature solid-state fermentation environment was solved, significantly improving the flavor quality of Maotai-flavor liquor.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SICHUAN UNIV
- Filing Date
- 2026-06-17
- Publication Date
- 2026-07-24
Smart Images
Figure CN122445541A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a strain of *Cropensida ivoryensis* that produces pyrazine compounds, especially tetramethylpyrazine. Kroppenstedtia eburnea New strains and their applications. Background Technology
[0002] Maotai-flavor baijiu is characterized by high-temperature stacking, high-temperature fermentation, and multiple rounds of fermentation. Its flavor formation process is complex, with the microbial community playing a crucial role. High-temperature koji (a type of starter culture) serves as an important saccharifying and fermenting agent and aroma-generating agent in the brewing process of Maotai-flavor baijiu. It not only provides a source of microorganisms for fermentation but also participates in the formation of various flavor compounds and their precursors. Since both the high-temperature koji and the fermented mash are kept in a high-temperature environment for extended periods, heat-resistant microorganisms capable of adapting to high temperatures and participating in flavor formation are a vital factor influencing the quality and style of Maotai-flavor baijiu.
[0003] Pyrazine compounds are important nitrogen-containing heterocyclic flavor compounds in Maotai-flavor baijiu, typically exhibiting aroma characteristics such as roasted, nutty, caramel, and cooked grain aromas. They are a crucial material basis for the typical flavor of Maotai-flavor baijiu. Among them, tetramethylpyrazine is one of the representative pyrazine compounds in Maotai-flavor baijiu, and its content and accumulation level are closely related to the roasted, caramel, and Maotai-flavor characteristics in the liquor. Therefore, improving the formation capacity of tetramethylpyrazine in the fermentation system is of great significance for improving the flavor quality of Maotai-flavor baijiu and enhancing its typical style characteristics.
[0004] Existing research indicates that various heat-resistant bacteria exist in high-temperature daqu (fermentation starter) and sauce-flavored baijiu mash, among which *Croplastrum* spp. (… Kroppenstedtia *Croplastridium* is a common thermotolerant microbial group in high-temperature daqu and fermentation systems. These microorganisms can adapt to high-temperature environments and may participate in the formation of flavor compounds during baijiu fermentation. However, current research on *Croplastridium* mainly focuses on its community distribution, relative abundance changes in high-temperature daqu or fermentation mash, and its correlation with flavor compounds. Research and application on the flavor metabolism capabilities of specific pure culture strains, particularly their targeted promotion of tetramethylpyrazine formation, remains limited.
[0005] While existing technologies disclose some applications of *Croponsetia ivoryensis* in baijiu fermentation or flavor enhancement, their effects are mostly focused on increasing the total amount of volatile aroma compounds or promoting the production of substances such as 2,5-dimethylpyrazine, phenylethanol, and fatty acids (Patent CN117987317A). For *Croponsetia ivoryensis* strains that can generate tetramethylpyrazine as their core function in solid-state fermentation systems, existing technologies still lack targeted strain resources and application methods.
[0006] Therefore, it is necessary to screen out more *Croponstrom ivoryensis* strains that can adapt to the high-temperature solid-state fermentation environment and have the ability to produce tetramethylpyrazine, so as to achieve the directional accumulation of tetramethylpyrazine in the fermentation system and provide new functional strain resources and technical means to improve the roasted aroma, nutty aroma, caramel aroma and soy sauce aroma characteristics of soy sauce-flavored liquor. Summary of the Invention
[0007] To provide more heat-resistant, pyrazine-producing, especially tetramethylpyrazine-producing, brewing microbial resources, this invention screened, isolated, and identified a heat-resistant, high-tetramethylpyrazine-producing, and multi-flavor-producing *Croplastrus ivoryensis* strain from soy sauce-flavored daqu (a type of starter culture). Kroppenstedtia eburnea New strain MZ35.
[0008] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, the present invention provides an ivory-colored Clopenstegia ( Kroppenstedtia eburnea MZ35, with accession number CCTCC NO: M 20252598.
[0009] The ivory-colored Croppensteine MZ35 forms milky white to ivory-colored colonies on TSB solid medium. The colony edges are relatively neat and have the characteristic of radial growth from the center outwards. The cells are irregular short rods or ellipsoids.
[0010] The 16S rRNA gene sequence of the ivory-colored Croppensteine MZ35 is shown in SEQ ID NO: 1.
[0011] Among them, the ivory-colored Croppensteine MZ35 is an aerobic and thermostable bacterium that grows well at 37–45 °C and can maintain growth at 55 °C, with a growth pH of 6.0–8.0.
[0012] The *Cropensator ivory* MZ35, when used in a solid-state fermentation system, can generate tetramethylpyrazine. Preferably, the tetramethylpyrazine content is not less than 2.0 mg / 100 g; more preferably not less than 3.0 mg / 100 g; and even more preferably not less than 3.6 mg / 100 g.
[0013] Secondly, the present invention provides a microbial inoculant containing the aforementioned *Cropensida ivoryensis* MZ35.
[0014] The viable count of *Cropensella ivory* MZ35 in the microbial agent is not less than 1 × 10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g, preferably not less than 1×10 8 CFU / mL or 1×108 CFU / g.
[0015] The microbial agent is a liquid agent, a solid agent, or a freeze-dried powder.
[0016] Preferably, the solid bacterial agent is prepared by adding a freeze-drying protectant to a bacterial suspension, followed by pre-freezing and vacuum freeze-drying to obtain solid bacterial powder.
[0017] More preferably, the freeze-drying protectant is one or any combination of skim milk powder, trehalose, sucrose, and glycerin.
[0018] Thirdly, the present invention provides the application of the above-mentioned ivory-colored Clopenstemon MZ35 or microbial inoculants in the fermentation of baijiu, the flavor regulation of mash, or the preparation of starter culture.
[0019] The liquor mentioned is at least one of the following: soy sauce aroma type, light aroma type, or strong aroma type.
[0020] Preferably, the application refers to the use of the above-mentioned *Croponthes ivory* MZ35 or microbial inoculants in the fermentation of baijiu, flavor regulation of mash, or preparation of starter culture to produce tetramethylpyrazine, acetoin, and / or trimethylpyrazine.
[0021] Fourthly, the present invention provides the application of the above-mentioned ivory-colored Croppensteine MZ35 or microbial inoculants in the production of flavor substances in the preparation of fermented foods.
[0022] The flavoring substance is selected from at least one of tetramethylpyrazine, acetoin, trimethylpyrazine, 2,3-butanedione, 2,3-butanediol, 2,5-dimethylpyrazine, phenethyl alcohol, 2,3-dihydrobenzofuran, 4-ethyl-2-methoxyphenol, 2,3,5-trimethyl-6-ethylpyrazine, methyl hexadecanoate, or 2,6-dimethylpyrazine.
[0023] Fifthly, the present invention provides a method for increasing the tetramethylpyrazine content in a solid-state fermentation system for baijiu, comprising the following steps: inoculating the above-mentioned ivory-colored Croppensteine MZ35 or microbial inoculant into the solid-state fermentation system, and then fermenting.
[0024] The fermentation temperature is 40–45 °C, and the fermentation time is 3–14 days.
[0025] The solid-state fermentation system is either a sorghum solid-state fermentation system or a sauce-flavored baijiu mash enhanced fermentation system.
[0026] The inoculation amount of the ivory-colored Clopensteg MZ35 or microbial agent is 10-30 v / w.
[0027] Among them, inoculation is carried out in the form of bacterial suspension, and the bacterial suspension OD 600The OD value is 1.0 to 5.0. Optimal OD is preferred. 600 It is version 3.0.
[0028] Beneficial Effects: This invention isolated, screened, and identified a heat-resistant strain of *Cropstomium ivory* MZ35, which is highly productive of pyrazine compounds, especially tetramethylpyrazine, from a soy sauce-flavored baijiu (Chinese liquor) starter culture. The strain has the preservation number CCTCC NO: M20252598. This strain can adapt to high-temperature solid-state fermentation and the stacking fermentation environment of soy sauce-flavored baijiu mash, and promotes the formation of tetramethylpyrazine. Inoculating MZ35 into sorghum solid-state fermentation or baijiu mash enhanced fermentation systems can increase the tetramethylpyrazine content; under optimized conditions, it can reach 3.74 mg / 100 g, and in baijiu mash enhanced fermentation systems, it can increase the content by 66.88%. Furthermore, this strain can also promote the accumulation of soy sauce-flavored substances or precursors such as acetoin and trimethylpyrazine. The strain and inoculum of this invention can be used for baijiu stacking fermentation, flavor regulation of baijiu mash, and preparation of solid-state fermentation products enriched with tetramethylpyrazine, and has application value in enhancing roasted aroma, nutty aroma, and soy sauce aroma characteristics. Attached Figure Description
[0029] Figure 1 The image shows the morphology of *Cropensida ivoryensis* MZ35 obtained by screening in Example 1 of this invention under a scanning electron microscope.
[0030] Figure 2 The effect of different temperatures, pH values, and dissolved oxygen conditions on the growth of *Cropensida oryzae* MZ35 in Example 2 of this invention is illustrated in A, where A represents temperature, B represents pH, and C represents dissolved oxygen conditions.
[0031] Preservation information of *Cropensida ivoryensis* MZ35 of this invention *Cropenstra ivory-colored* MZ35, with accession number CCTCC NO: M 20252598, was deposited on November 20, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. It is classified and named as follows: Kroppenstedtia eburnea MZ35. Detailed Implementation
[0032] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.
[0033] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).
[0034] In this document, “and / or” means and includes any and all possible combinations of one or more of the associated listed items. For example, “the composition contains A and / or B” can be interpreted as the composition contains A, the composition contains B, or the composition contains both A and B.
[0035] In one embodiment of the present invention, a heat-resistant strain MZ35 was isolated, screened, and identified from a soy sauce-flavored koji (a type of starter culture). This strain forms milky-white to ivory-colored colonies on TSB solid medium, with relatively neat colony edges and a radial growth pattern from the center outwards. Its 16S rRNA gene sequence is shown in SEQ ID NO: 1. Based on the morphological and molecular biological characteristics of the microorganism, strain MZ35 was thus identified as *Croppedia ivoryensis*. Kroppenstedtia eburnea It was deposited in CCTCC on November 20, 2025, with accession number CCTCC NO: M 20252598.
[0036] In one embodiment of the present invention, the screened *Cropensida ivoryensis* MZ35 is subjected to solid-state fermentation in a sorghum solid-state fermentation system or a Maotai-flavor liquor mash enhanced fermentation system. By optimizing the fermentation culture conditions, high yields of pyrazine compounds, especially tetramethylpyrazine, can be achieved. In some examples, the fermentation temperature is 40–45 °C, and the fermentation time is 3–14 days. In some examples, the inoculum size is 10–30 v / w%. In some examples, inoculation is performed in the form of bacterial suspension, and the OD of the bacterial suspension is [not specified]. 600 The OD value is 1.0 to 5.0. Optimal OD is preferred. 600 It is version 3.0.
[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. The advantages and features of this invention will become clearer with this description. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions were performed under conventional conditions in the art or as recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following embodiments are commercially available.
[0038] The culture media used in the following examples are as follows: TSB liquid medium: tryptone 17.0 g / L, NaCl 5.0 g / L, soybean peptone 3.0 g / L, K2HPO4 2.5 g / L, glucose 2.5 g / L; sterilized at 121 ℃ for 20 min.
[0039] TSB solid medium: Add 20.0 g / L agar to TSB liquid medium and sterilize at 121 ℃ for 20 min.
[0040] Solid-state fermentation sorghum culture medium: Weigh 100 g of sorghum, add 80 mL of deionized water, place in a 250 mL Erlenmeyer flask, sterilize at 121 ℃ for 30 min, and cool before use.
[0041] The mash for Maotai-flavor liquor: mash from the third round of fermentation at a Maotai-flavor liquor distillery in the Chishui River basin.
[0042] Example 1: The Ivory-colored Clopenstegia of the present invention ( Kroppenstedtia eburnea Isolation, screening and identification of MZ35 1. Isolation and screening of *Cropensida ivoryensis* MZ35 Weigh 10 g of the soy sauce-flavored Daqu sample into a sterilized 250 mL Erlenmeyer flask, add 90 mL of sterile physiological saline, and add sterile glass beads. Shake in a shaker at 50 ℃ and 200 r / min for 30 min, then let stand for 20 min and take the supernatant for later use.
[0043] The supernatant was serially diluted with sterile physiological saline to prepare 10^-1, 10^-3, 10^-5, 10^-6, and 10^-7 dilutions. 100 μL of each dilution was evenly spread onto TSB solid medium and incubated upside down in a 45 ℃ incubator for 1–2 days.
[0044] A large number of single colonies with different colony morphologies were streaked onto TSB solid medium. The isolation and purification were repeated multiple times until a single strain with consistent colony morphology was obtained. One of the strains with a typical radial colony morphology was named MZ35.
[0045] The above process was used to isolate and purify thermostable microorganisms from soy sauce-flavored Daqu samples, and to screen for single colonies with different morphologies. MZ35 formed milky white to ivory-colored colonies on TSB solid medium, with relatively neat colony edges and a radial growth pattern from the center outwards. Scanning electron microscopy (SEM) showed that the bacterial cells were irregularly short rod-shaped or ellipsoidal. Figure 1 The cell surface is relatively rough, and some cells show slight wrinkled structures. The length of a single cell is about 5-10 μm.
[0046] 2. Molecular identification of 16S rRNA from *Cropensida ivoryensis* MZ35 Genomic DNA of strain MZ35 was extracted, isolated, and purified using a bacterial genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd., catalog number: B518255-0050), and the 16S rRNA gene sequence was amplified using universal bacterial primers.
[0047] The PCR amplification primers are as follows: Forward primer 27F, SEQ ID NO: 2: 5'-AGAGTTTGATCCTGGCTCAG-3'; Reverse primer 1492R, SEQ ID NO: 3: 5'-GGTTACCTTGTTACGACTT-3'.
[0048] The PCR amplification conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 56 °C annealing for 30 s, 72 °C extension for 90 s, for a total of 35 cycles; and a final extension at 72 °C for 7 min.
[0049] The 16S rRNA gene sequence (SEQ ID NO: 1) was compared with the NCBI database using BLAST and homology analysis. It was found that strain MZ35 was similar to *Cropensida ivoryensis*. Kroppenstedtia eburnea The type strain is highly homologous. Therefore, based on the morphological and molecular biological characteristics of the microorganism, strain MZ35 was identified as *Croplastrus ivoryensis*. Kroppenstedtia eburnea It was deposited on November 20, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The accession number is CCTCC NO: M 20252598.
[0050] SEQ ID NO: 1: 16S rRNA gene sequence of *Cropensida ivoryensis* MZ35:
[0051] Example 2: Analysis of the growth characteristics of *Cropensida ivoryensis* MZ35 Croppensteine ivory-colored bacteria MZ35 was inoculated into 50 mL of TSB liquid medium at a 3% (v / v) inoculum and cultured at 30 ℃, 37 ℃, 45 ℃ and 55 ℃ for 36 h, respectively. The OD of the culture medium was measured. 600 The TSB liquid medium without bacterial culture was used as a blank control.
[0052] The results are as follows Figure 2 As shown in Figure A, the OD of *Cropensida ivoryensis* MZ35 after incubation at 45 °C for 24 h is... 600 The value was approximately 3.07, OD at 36 h. 600 The value was approximately 3.078; the OD value after incubation at 55 °C for 36 h was... 600 The value is approximately 2.699, indicating that *Cropensida ivoryensis* MZ35 can maintain growth under relatively high temperatures, demonstrating a certain degree of heat resistance. Considering the growth performance at different temperatures, the optimal growth temperature for *Cropensida ivoryensis* MZ35 is 45 ℃, and it can maintain growth at 55 ℃.
[0053] Ivory-colored Croppensteine MZ35 was inoculated into TSB liquid medium at pH 6.0, 7.0, 8.0, and 9.0 at a 3% (v / v) inoculation rate and cultured at 45 °C for 36 h. The OD of the culture medium was then measured. 600 value.
[0054] The results are as follows Figure 2 As shown in B, MZ35 grows well in the pH range of 6.0-8.0, with better growth at pH 6.0 and pH 7.0. At pH 9.0, the early growth of the strain was somewhat affected, but it recovered later. These results indicate that MZ35 is suitable for growth in neutral to slightly alkaline environments.
[0055] The effect of dissolved oxygen conditions on the growth of *Cropensida dentata* MZ35 was further investigated. MZ35 was cultured for 36 h under both static and shaking incubation conditions. Figure 2 As shown in Figure C, the results indicate that the biomass under shaking culture conditions was significantly higher than that under static culture conditions, suggesting that *Cropensida ivoryensis* MZ35 is an aerobic bacterium that thrives in conditions with sufficient oxygen supply.
[0056] The values 50 / 250 mL, 100 / 250 mL, and 150 / 250 mL represent the dissolved oxygen environment: 50 mL of TSB liquid culture medium is added to a 250 mL Erlenmeyer flask. The first number is the volume of culture medium, and the second number is the nominal capacity of the culture flask, which is "volume / volume of the shake flask". This setting is to change the oxygen supply conditions of the culture system. Generally, at the same shaking speed, the smaller the volume of culture medium, the more thorough the contact and mixing with air, and the stronger the oxygen supply capacity; the larger the volume of culture medium, the weaker the oxygen supply capacity.
[0057] Example 3: Analysis of the ability of *Cropensida ivoryensis* MZ35 to produce tetramethylpyrazine in solid-state fermentation of sorghum. 1. Testing Methods Ivory-colored Croppensteine MZ35 was inoculated into TSB liquid medium and cultured at 45 °C until the logarithmic growth phase or stationary phase. The bacterial cells were collected by centrifugation and resuspended in sterile water to prepare OD. 600 Bacterial suspension with a value of 3.0.
[0058] 20 mL of bacterial suspension was inoculated into solid-state fermented sorghum culture medium at an inoculation rate of 20 v / w%. After mixing, the mixture was placed in a 45°C incubator and incubated statically for 14 days. A blank control was prepared by adding 20 mL of sterile water. Each treatment was performed in triplicate. After fermentation, samples were taken and stored at -80°C for later use.
[0059] Headspace solid-phase microextraction-gas chromatography-mass spectrometry was used to detect volatile compounds in fermentation samples.
[0060] The headspace solid-phase microextraction (SPE) method described above is as follows: 1 g of fermentation sample is weighed and placed in a 20 mL headspace vial. Internal standard solution is added, and the sample is equilibrated at 60 °C for 5 min. Subsequently, the SPE fiber is inserted into the headspace vial, and extraction is performed at the same temperature for 50 min. After extraction, the SPE fiber is inserted into the injection port of a gas chromatograph-mass spectrometer (GC-MS), and desorption is performed at 250 °C for 5 min.
[0061] GC conditions were as follows: DB-WAX capillary column was used; injection port temperature was 250 ℃; temperature program was as follows: initial temperature 40 ℃ held for 3-4 min, temperature increased to 120 ℃ at 5 ℃ / min, then increased to 230 ℃ at 10-15 ℃ / min and held for 4-8 min; carrier gas was high-purity helium.
[0062] MS conditions were: electron impact ion source, electron energy 70 eV, ion source temperature 250 ℃, mass scan range 30-500 m / z. The NIST spectral library was used for qualitative analysis, and the content of target volatile substances was calculated using either the internal standard method or the external standard method.
[0063] 2. Effects of *Cropensida ivoryensis* MZ35 on tetramethylpyrazine formation in solid-state fermented sorghum. Compared with the blank control group that was not inoculated with MZ35, the content of pyrazine compounds in solid-state fermented sorghum with added MZ35 was significantly increased, especially the content of tetramethylpyrazine was significantly increased (Table 1).
[0064] Under static fermentation conditions of 45 ℃ for 14 days, the tetramethylpyrazine content in the fermentation system reached 3.74 mg / 100 g. Simultaneously, *Cropensida ivory* MZ35 also promoted the accumulation of substances such as acetoin and trimethylpyrazine, with acetoin content reaching 0.97 mg / 100 g and trimethylpyrazine content reaching 0.57 mg / 100 g. Acetoin can serve as an important precursor in the formation of tetramethylpyrazine, while trimethylpyrazine belongs to the pyrazine flavor compounds associated with soy sauce aroma. These results indicate that MZ35 not only promotes the accumulation of tetramethylpyrazine but also improves the composition of pyrazine flavor compounds in the solid-state fermentation system. Furthermore, it can produce small amounts of 2,3-butanediol, phenylethanol, and 2,3-butanedione, which contribute to enriching the flavor profile of baijiu.
[0065] Table 1. Effects of MZ35 on tetramethylpyrazine and related substances in the solid-state fermentation system at the end of 14 days of fermentation.
[0066] The above results indicate that MZ35 is suitable for the targeted enrichment of tetramethylpyrazine in sorghum solid-state fermentation systems and can be used to prepare solid-state fermentation products rich in tetramethylpyrazine, contributing more flavor compounds in baijiu brewing.
[0067] Example 4: Analysis of the tetramethylpyrazine production capacity of *Cropensida ivoryensis* MZ35-enhanced Maotai-flavor Baijiu mash 1. Test Methods Take 100 g of the fermented mash of a sauce-flavored baijiu that was just put into the cellar, and add 20 mL of OD. 600 MZ35 bacterial suspension with a pH of approximately 3.0 was thoroughly mixed and then placed in constant temperature incubators at 40 ℃, 45 ℃, and 50 ℃ for static fermentation for 4 days. A mash with 20 mL of sterile water was used as a blank control, and each treatment was performed in triplicate.
[0068] After fermentation, samples were taken, and the content of volatile flavor compounds in the mash was detected using the headspace solid-phase microextraction-gas chromatography-mass spectrometry method described in Example 3, with a focus on analyzing the changes in tetramethylpyrazine, acetoin, and trimethylpyrazine. The volatile flavor composition of the experimental group and the control group is shown in Table 2.
[0069] Table 2. Effects of Cromendum ivory-colored fortification MZ35 on flavor compounds at different stacking temperatures.
[0070] Table 2 (Continued) Effects of Croppensteine MZ35 fortification on flavor compounds at different stacking temperatures
[0071] Table 2 shows that the types and contents of flavor compounds produced by *Cropensida ivoryensis* MZ35 varied significantly under different stacking temperatures. However, compared to the control group, the levels of esters and alcohols increased at different temperatures, while the levels of pyrazines also increased significantly.
[0072] 2. Effects of *Cropensida ivoryensis* MZ35 on tetramethylpyrazine formation in fermented mash The results are shown in Table 3. The addition of MZ35 significantly altered the tetramethylpyrazine content in the mash of Maotai-flavor liquor. Specifically, 40 ℃ was more conducive to the accumulation of tetramethylpyrazine, and the tetramethylpyrazine content in the MZ35-treated group was significantly higher than that in the blank control group. 45 ℃ was more conducive to the formation of trimethylpyrazine.
[0073] Table 3. Effects of Clopentomeus ivory-colored MZ35 reinforcement on tetramethylpyrazine and related substances during stacking.
[0074] The results indicate that fortification with *Cropensida ivoryensis* MZ35 can increase the content of tetramethylpyrazine, acetoin, and trimethylpyrazine during the fermentation of Maotai-flavor liquor mash, especially at 40℃, where the tetramethylpyrazine content increased by 66.88%.
[0075] In summary, *Croponectinus ivoryensis* MZ35 can be used as a functional strain for enhancing the fermentation of soy sauce-flavored baijiu mash, thereby increasing the tetramethylpyrazine content in the mash and improving the roasted, nutty, caramel, and soy sauce-related flavor characteristics.
[0076] Example 5: Preparation and application of *Cropensida ivoryensis* MZ35 microbial inoculant. 1. Preparation of microbial inoculants MZ35 was inoculated into TSB liquid medium and cultured at 45 ℃ and 180-200 r / min for 24-36 h to obtain seed culture. The seed culture was then transferred to fresh TSB liquid medium at an inoculation rate of 5-10% (v / v) for further expansion culture until OD reached. 600 The value is 3.0.
[0077] Collect bacterial cells by centrifugation of the culture medium, wash with sterile water or PBS buffer, and resuspend to obtain a liquid microbial inoculum, wherein the viable count of the microbial inoculum is 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.
[0078] A lyophilization protectant is added to the bacterial suspension, followed by pre-freezing and vacuum freeze-drying to obtain solid bacterial powder. The lyophilization protectant can be any one or a mixture of skim milk powder, trehalose, sucrose, and glycerol, all of which are suitable strains for this invention.
[0079] 2. Application of microbial inoculants Referring to Examples 3 or 4, the prepared microbial inoculant can be used to replace the seed liquid of *Croponecten bacterium* MZ35 in sorghum solid-state fermentation, fermentation of soy sauce-flavored baijiu mash, or for food fermentation that requires enrichment of pyrazines, especially tetramethylpyrazines.
[0080] The above results indicate that the *Croponectinus ivoryensis* MZ35 provided by this invention can adapt to the solid-state fermentation environment and promote the formation of tetramethylpyrazine in sorghum solid-state fermentation and intensified fermentation systems of Maotai-flavor liquor mash. It can also generate a variety of other flavor substances that are beneficial to enriching the flavor and taste of liquor, such as 2,3-butanediol, phenylethanol, and 2,3-butanedione. Therefore, the *Croponectinus ivoryensis* MZ35 of this invention is suitable for increasing the tetramethylpyrazine content in the fermentation system and improving the relevant flavor characteristics of Maotai-flavor liquor.
[0081] The above description is merely an exemplary embodiment of the present invention. It should be noted that those skilled in the art can make improvements to the present invention without departing from the inventive concept, and all such improvements fall within the scope of protection of the present invention.
Claims
1. Ivory-colored Clopenstead ( Kroppenstedtia eburnea MZ35, characterized in that, The accession number is CCTCC NO: M 20252598.
2. A microbial inoculant, characterized in that, Contains the ivory-colored Croppensteine MZ35 as described in claim 1.
3. The application of the ivory-colored Clopenstemon MZ35 as described in claim 1 or the microbial agent as described in claim 2 in the fermentation of baijiu, the flavor regulation of mash, or the preparation of starter culture.
4. The application according to claim 3, characterized in that, The liquor is at least one of the following: soy sauce aroma type, light aroma type, or strong aroma type.
5. The application of the ivory-colored Clopenstemon MZ35 of claim 1 or the microbial agent of claim 2 in the production of tetramethylpyrazine, acetoin and / or trimethylpyrazine in the fermentation of baijiu, the flavor regulation of mash or the preparation of starter culture.
6. The application of the *Cropensia ivoryensis* MZ35 of claim 1 or the microbial agent of claim 2 in the production of flavor substances in the preparation of fermented foods, characterized in that... The flavoring substance is selected from at least one of tetramethylpyrazine, acetoin, trimethylpyrazine, 2,3-butanedione, 2,3-butanediol, 2,5-dimethylpyrazine, phenethyl alcohol, 2,3-dihydrobenzofuran, 4-ethyl-2-methoxyphenol, 2,3,5-trimethyl-6-ethylpyrazine, methyl hexadecanoate, or 2,6-dimethylpyrazine.
7. A method for increasing the tetramethylpyrazine content in a solid-state fermentation system for baijiu (Chinese liquor), characterized in that, The process includes the following steps: inoculating the ivory-colored Croppensteine MZ35 of claim 1 or the microbial agent of claim 2 into a solid-state fermentation system, and then fermenting.
8. The method according to claim 7, characterized in that, The fermentation temperature is 40–45 °C.
9. The method according to claim 7, characterized in that, The solid-state fermentation system is either a sorghum solid-state fermentation system or a sauce-flavored baijiu mash enhanced fermentation system.
10. The method according to claim 7, characterized in that, The inoculum amount of *Cropensida ivoryensis* MZ35 or microbial agent is 10–30 v / w.