Acetobacter xylinum with low fusel yield and application of acetobacter xylinum

Abstract

The invention discloses acetobacter Indonesii with low fusel oil yield and an application of the acetobacter Indonesii. The strain of the Acetobacter indicum X3 is preserved in the China Center for Type Culture Collection (CCTCC), the preservation date is July 15, 2025, and the preservation number is CCTCC NO.M20251613. The strain of the Acetobacter indicum X3 has the advantages that the strain of the Acetobacter indicum X3 is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO.M20251613; the acetobacter strain is obtained by separating and purifying mash of the rice-flavor liquor, fusel oil in the rice-flavor liquor can be reduced by 14.2%, the acetobacter seed solution and distiller's yeast of the rice-flavor liquor are prepared into mixed yeast to be applied to brewing of rice-flavor raw liquor, the total acid content in a produced liquor body reaches 0.35 g / L and is increased by 66.7% compared with that of a production control group, and the production cost is reduced. The total ester content reaches 0.35 g / L and is improved by 47.8% compared with that of a control group, and the flavor purity and the comprehensive score of a wine body are optimal through sensory evaluation.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a strain of Acetobacter acetobacter with low fusel oil production and its application in reducing the fusel oil content of rice-flavored baijiu. Background Technology

[0002] Rice-aroma baijiu, one of the four major traditional Chinese baijiu aroma types, is unique for its elegant and mellow sweet flavor. Representative products such as Guilin Sanhua Jiu and Xiangshan Jiu embody the millennia-old brewing wisdom of the Lingnan region. Its brewing process uses rice as raw material and small-grain yeast as the saccharification and fermentation agent, undergoing semi-solid saccharification and short-term liquid fermentation to create a unique style characterized by "elegant honey aroma and pure rice aroma." However, this aroma type has long faced a technical bottleneck of "high alcohol and low ester content"—the content of higher alcohols (fusel oils) in the liquor is significantly higher than in other aroma types, especially isoamyl alcohol and isobutanol, leading to adverse reactions such as headaches and hangovers after consumption, severely restricting its market competitiveness and health benefits.

[0003] Fusel oils refer to monohydric alcohols containing three or more carbon atoms, primarily produced by yeast metabolism. Their formation pathways include both amino acid degradation metabolism and carbohydrate synthesis metabolism. In rice-aroma baijiu production, although the protein content of rice is lower than that of grains like sorghum, the semi-solid-state fermentation process results in a higher amino acid nitrogen content in the saccharified mash. Furthermore, the metabolic characteristics of the small-batch microbial community (such as Rhizopus and yeast) easily lead to excessive accumulation of fusel oils. In addition, traditional open fermentation processes pose a high risk of environmental microbial contamination, and the lack of proper temperature control (e.g., temperatures easily exceeding 40°C in summer) further exacerbates fusel oil formation.

[0004] To address the high fusel oil content in baijiu (Chinese liquor), the industry has explored solutions through multi-dimensional technological innovation in recent years. Currently, the main methods to reduce fusel oil content in baijiu are breeding low-fusel oil-producing yeasts or optimizing fermentation and distillation processes. However, existing research largely focuses on optimizing single yeast strains, with insufficient research on the synergistic mechanisms of molds and bacteria in the starter culture, and a lack of systematic research on fusel oil metabolism in rice-aroma baijiu. Therefore, while preserving the typical flavor of rice-aroma baijiu, selecting low-fusel oil-producing strains that inhibit high-fusel oil-producing yeasts from the perspective of microbial interactions has become a key means of controlling fusel oil content during the brewing process. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by focusing on the selection of strains that produce low fusel oil. A new strain of Acetobacter indonesiensis X3 is provided, which has a high ability to reduce fusel oil. This strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M20251613 and deposit date of July 15, 2025.

[0006] The *Acetobacter spp.* X3 strain provided by this invention was isolated and screened from rice-flavored baijiu mash and is preserved in a laboratory strain bank. The colony morphology of this strain after 18 hours of culture on MRS plates is: light yellow, round colonies with a smooth surface, a raised center, and regular edges. The microscopic morphology after 18 hours of culture on MRS liquid medium in a shaker is: cells are oval to short rod-shaped, indicating Gram-negative bacteria.

[0007] The low-fusel oil-producing Acetobacter indonesiensis X3 involved in this invention is obtained by the following method: using rice-aroma baijiu mash as the microbial screening source. First, rice juice is allowed to ferment statically for 7 days. After fermentation, the fusel oil content in the fermentation broth is measured, and strains that significantly reduce fusel oil production are screened. Then, this strain is mixed with rice-aroma baijiu starter in a certain proportion to form a mixed starter, with pure rice-aroma baijiu starter as the control starter. Rice fermentation is carried out, and the alcohol yield, fusel oil content, and other key quality indicators are compared after distillation.

[0008] As one of the objectives of this invention, it also provides the application of Acetobacter Indonesianis X3 in the brewing of rice-aroma baijiu to effectively reduce the fusel oil content of the baijiu. Specifically, the Acetobacter Indonesianis X3 is mixed with rice-aroma baijiu yeast and then fermented.

[0009] As one of the objectives of this invention, this invention also provides a microbial inoculant comprising *Acetobacter ingrainiensis* with low fusel oil production, whose preservation number is CCTCC NO.M20251613. The microbial inoculant may be a solid or liquid inoculant.

[0010] As one of the objectives of this invention, this invention also provides a rice-aroma type of baijiu, which is brewed from a microbial inoculum containing a low-yield fusel oil Indonesian Acetobacter spp. with preservation number CCTCC NO.M20251613.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The low-fusel oil-producing Acetobacter indonesiensis X3 of this invention is isolated from rice-aroma baijiu mash and can be used in baijiu production to reduce the fusel oil content in rice-aroma baijiu by 14.2%, significantly improving the quality of baijiu. Attached Figure Description

[0013] Figure 1 This is a macroscopic morphological diagram of the X3 bacterial colony;

[0014] Figure 2 This shows the growth of strain X3 at different temperatures;

[0015] Figure 3 This shows the growth of strain X3 at different ethanol concentrations;

[0016] Figure 4 This shows the growth of strain X3 at different glucose concentrations;

[0017] Figure 5 This shows the growth of strain X3 under different acetic acid concentrations;

[0018] Figure 6 This shows the growth of strain X3 at different pH levels. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0020] Example 1

[0021] This embodiment screens strains that produce low fusel oil using the following method:

[0022] 1.1 Materials:

[0023] A total of 39 strains were selected from the microbial strain bank of Tianlongquan Winery's rice-aroma baijiu mash and Jinpai Co., Ltd.'s microbial research laboratory, including 19 yeast strains, 15 lactic acid bacteria strains, and 5 acetic acid bacteria strains.

[0024] 1.2 Culture medium preparation:

[0025] ①YPD liquid culture medium: glucose 20g / L, peptone 20g / L, yeast extract 10g / L, pH natural.

[0026] ②YPD agar medium: glucose 20g / L, peptone 20g / L, yeast extract 10g / L, agar powder 20g / L, pH natural.

[0027] ③ MRS liquid culture medium: glucose 20g / L, casein digest 10g / L, beef extract 10g / L, yeast extract 4g / L, triammonium citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, dipotassium hydrogen phosphate 2g / L, Tween 80 1.08g / L, pH 5.7±0.2.

[0028] ④ MRS agar medium: glucose 20g / L, peptone 10g / L, beef extract 8g / L, yeast extract 4g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 80 1g / L, agar 14g / L, pH 6.5±0.2.

[0029] ⑤ Rice juice culture medium: Weigh 100.0g of crushed rice (particle size 0.45mm), add 800mL of tap water, cook for 2 hours, cool, add 1.35g of amylase (3700 activity units) and 0.05g of saccharifying enzyme (100,000 activity units) for saccharification and liquefaction, stir evenly and place in a 60℃ water bath for 2-3 hours. During this period, if the test paper does not turn blue when tested with dilute iodine solution, continue heating and boiling for 5 minutes. After cooling, filter, determine the sugar content, and freeze for later use.

[0030] 1.3 Strain isolation, screening and activation

[0031] To target microorganisms originating from the fermented mash, 10 mL of the mash solution was dissolved in an Erlenmeyer flask containing 90 mL of sterile physiological saline. The solution was thoroughly shaken. 1 mL of the solution was then transferred from the Erlenmeyer flask into a test tube containing 9 mL of sterile water. This mixture was then diluted sequentially to prepare 10 mL solutions. -5 10 -6 10 -7 The suspension was prepared, and 200 μL of each suspension was spread onto MRS agar medium and incubated at 37°C for 1-2 days. After colonies grew, the culture was purified by streaking on MRS agar medium until pure culture was obtained.

[0032] For lactic acid bacteria and acetic acid bacteria from the strain bank, 100 μL of bacterial culture from a glycerol storage tube was transferred into a 10 mL MRS liquid culture tube and incubated at 37°C for 18 h. The culture was then streaked onto an MRS agar plate using an inoculation loop and incubated at 37°C for 18 h. A single colony was then inoculated into MRS liquid culture medium and incubated at 37°C for 18 h to obtain a seed culture, which was then stored in a refrigerator for later use.

[0033] For yeast derived from the strain bank, 100 μL of bacterial culture from a glycerol preservation tube was transferred to a 10 mL YPD liquid tube and cultured at 30°C and 150 rpm / min in a shaker for 24 h. The culture was then streaked onto a YPD plate using an inoculation loop for isolation. After incubation at 30°C upside down for 48 h, a loopful of a single colony was inoculated into YPD liquid medium and cultured at 30°C and 150 rpm / min in a shaker for 24 h to obtain the seed culture, which was then stored in a refrigerator for later use.

[0034] 1.4 Screening of strains producing low fusel oil

[0035] Adjust the sugar content of rice juice culture medium to Bx8 with water, dispense 10 mL into test tubes, and sterilize at 121℃ for 20 min. Inoculate the activated seed culture medium at a 2% (v / v) inoculation rate. Yeast is incubated at 30℃ for 7 days, while lactic acid bacteria and acetic acid bacteria are incubated at 37℃ for 7 days. After fermentation, centrifuge the fermentation broth at 8000 rpm for 5 min, collect the supernatant, filter through a 0.45 μm filter membrane, and perform gas chromatography analysis on the filtered fermentation supernatant. Strains with low fusel oil content are selected for further solid-state fermentation validation.

[0036] Gas chromatography results showed that among the 39 strains selected from the microbial library, after initial screening for the production of fusel oil through fermentation, strains B943, B944, B958, X3, X7, X9, and X1, a total of 12 strains, produced fusel oil content lower than the control group (70.084 mg / L). In the yeast samples, all strains produced fusel oil content lower than the control group, and three strains had significantly lower fusel oil yields than the control group, namely Y128 (15.28 mg / L), Y242 (29.6 mg / L), and L18 (27.63 mg / L).

[0037] This embodiment uses the following method to verify the fermentation of strains that produce low fusel oil:

[0038] (1) Preparation with koji: The culture medium of the strain to be verified with low yield of fusel oil is mixed with rice aroma type baijiu koji in a specific ratio. Each sample requires 2g of rice aroma type baijiu koji and 1mL of culture medium of the strain to be verified, which is used to verify the semi-solid fermentation effect of rice aroma type baijiu.

[0039] (2) Semi-solid small-scale fermentation experiment

[0040] In the laboratory pilot test platform, 200g of rice was taken for each sample, and water was added at a grain-to-water ratio of 1:1 (g:mL). The rice was steamed at 121℃ for 10min, cooled to about 30℃, and the starter culture was added. The mixed starter culture prepared in step (1) was added at a rate of 1% of the rice mass. The starter culture and rice were thoroughly mixed and then put into a 2L fermentation bottle for saccharification at 30℃ for 24h (open saccharification, with a moist two-layer gauze covering the mouth of the fermentation bottle). After saccharification, 130% water (by weight of rice) was added, and fermentation was carried out for 14d. The mash was poured into a conical flask, heated with an electric furnace, and the wine was distilled. 25mL of the first wine was discarded, and 100mL of the main wine was added to obtain the rice-aroma type original wine.

[0041] (3) Raw wine testing and data comparison analysis

[0042] Accurately transfer 1 mL of the raw wine obtained in step (2) into a 2 mL sample bottle, add 10 μL of triple internal standard working solution (tert-amyl alcohol (IS1): 17028.1 mg / L, n-amyl acetate (IS2): 16864 mg / L, 2-ethylhexanol (IS3): 12104.2 mg / L), cap the bottle, shake well, and determine the content of each component in the raw wine by gas chromatography. Chromatographic conditions: Agilent 780A gas chromatograph, FID detector, CP-WAX capillary column (50 m * 0.2 μm * 0.25 mm). Temperature program: column temperature 40℃ held for 8 min, then increased to 150℃ at 5℃ / min. The injection port temperature was 250℃, the detector temperature was 260℃, the air-to-hydrogen flow rate ratio was 300:30, the carrier gas was nitrogen, the split ratio was 30:1, the column flow rate was 1.0 mL / min, and the injection method was an autosampler. The detection results are shown in Table 1.

[0043] Table 1. Gas chromatography results of low fusel oil-producing strains in small-scale trials of rice-aroma baijiu (unit: g / L)

[0044]

[0045] As shown in Table 1, strain X3 exhibited a 13% reduction in fusel oil content (1.38 g / L) compared to the control group, while its total acid and total ester content increased by 340% and 166.7%, respectively. These combined results indicate that strain X3 performed optimally in reducing fusel oil content and improving ester balance, thus identifying it as a low-fusel oil-producing strain.

[0046] Example 2

[0047] This example explores the effect of different inoculum amounts on the production of fusel oil by low-fusel oil-producing strains.

[0048] In solid-state pilot-scale experiments, it was preliminarily determined that strain X3 performed optimally in reducing fusel oils and improving ester balance in rice-aroma baijiu. To further explore the application value of strain X3, especially to achieve better results in the core objective of "impurity reduction," it is necessary to finely control its fermentation process parameters. The inoculum size is one of the most critical and controllable factors affecting the microbial fermentation process, metabolite profile, and final product quality. A systematic study of the influence mechanism of strain X3 on fusel oil synthesis in rice-aroma baijiu under different inoculum sizes will provide stronger technical support and better process schemes for subsequent pilot-scale and even large-scale applications. Specific operations are as follows:

[0049] The mixed yeast was prepared by adding 0.5 mL, 1 mL, 1.25 mL, 1.5 mL and 2 mL of seed liquid of strain X3 to step (1) of implementation case 1. Two rounds of semi-solid small-scale test were carried out according to step (2) of implementation case 1. The original wine samples were tested according to step (3) of implementation case 1. The test results are shown in Table 2.

[0050] Table 2. Gas chromatography results of small-scale tests on rice-aroma baijiu with different inoculum amounts for strain X3 (unit: g / L)

[0051]

[0052] As shown in Table 2, the content of fusel oil in all X3 inoculation groups was lower than that in the control group. The impurity reduction effect increased with the increase of inoculation amount. The effect was best when the inoculation amount was 2 mL, with a reduction of 22%, followed by an inoculation amount of 1.25 mL, with a reduction of 14.2%. In terms of total acid and total ester, all X3 groups were significantly higher than the control group. With the increase of inoculation amount, the content of total acid and total ester increased accordingly. When the inoculation amount was 2 mL, the content of total acid and total ester was the highest, with the increase of 123.8% for total acid and 108.7% for total ester. In terms of alcohol content, the alcohol content generally decreased after X3 inoculation. The alcohol content was the lowest and fluctuated the most when the X3 inoculation amount was 2 mL.

[0053] Example 3

[0054] This example demonstrates sensory evaluation of small-scale wine samples with different inoculation amounts of strain X3.

[0055] Because the total acidity of the wine sample increased significantly after the addition of strain X3, and the alcohol content also decreased to varying degrees, sensory evaluation was carried out to assess the impact of different inoculation amounts on the quality of the wine. Five company-level baijiu judges were organized to evaluate and score the wine samples. The results are shown in Table 3.

[0056] Table 3 Sensory evaluation scores of small-scale wine samples with different inoculation amounts for strain X3

[0057]

[0058] As shown in Table 3, the addition of strain X3 significantly improved the sensory quality of rice-aroma baijiu. The 0.5 mL inoculation group had the highest overall score (91.2 points), followed by the 1.25 mL group (91.16 points), both of which were better than the control group (90.2 points). Based on the gas chromatography detection index and sensory evaluation data of the small-scale test samples, without affecting the alcohol yield, the X3 inoculation amount of 1.25 mL showed the best performance in terms of flavor purity and overall score (pure honey aroma, no negative flavors). Furthermore, the gas chromatography detection index of the samples verified its good impurity reduction effect and ester balance. Therefore, the optimal inoculation amount of strain X3 for reducing fusel oil in rice-aroma baijiu was determined to be 1.25 mL.

[0059] Example 4

[0060] This example demonstrates the tolerance of strain X3, which produces low fusel oil.

[0061] The fermentation process of baijiu (Chinese liquor) is highly complex, and strains are affected by stress conditions such as high temperature, high sugar, high alcohol, and high osmotic pressure. Therefore, certain physiological characteristics of the strains are required in production, and obtaining strains and conducting research on their tolerance to the living environment is fundamental for industrial applications. We conducted a series of tolerance tests on the low-yield fusel oil strain X3, as detailed below:

[0062] (1) Temperature resistance test

[0063] The seed culture of strain X3 was inoculated into MRS liquid medium at a 2% inoculum size. After static incubation at 25℃, 30℃, 37℃, 40℃, 42℃, and 45℃ for 24 h, the absorbance was measured at 600 nm. The results are shown below. Figure 2 .

[0064] from Figure 2 It can be seen that within the temperature range of 25℃-37℃, the cell biomass of Acetobacter X3 increases and remains at a high level with increasing temperature. When the temperature reaches 42℃, the growth of the strain is somewhat inhibited, but its cell biomass still remains at a high level. When the temperature reaches 45℃, the OD... 600 The value decreased significantly, indicating that high temperature strongly inhibits the growth of Acetobacter X3 (and may even lead to the death of a large number of bacteria). In summary, Acetobacter X3 can tolerate a maximum temperature of 42℃.

[0065] (2) Ethanol resistance test

[0066] The ethanol concentration of MRS liquid medium was adjusted to 0%, 3%, 6%, 9%, 12%, 15%, and 18% (v / v) with anhydrous ethanol. Seed culture of strain X3 was inoculated at a 2% inoculum size. After static incubation at 37℃ for 24 h, the absorbance was measured at 600 nm. The results are shown below. Figure 3 .

[0067] The high concentration of ethanol produced during fermentation can be toxic to the metabolism of microbial cells, causing them to stop growing or die. Figure 3 It can be seen that the growth of the strain is inhibited with increasing ethanol content. Strain X3 can tolerate 9% ethanol content, but its biomass decreases significantly with further increases in ethanol content. Since the ethanol concentration in the mash does not exceed 6% in the later stages of fermentation, this strain can tolerate increasing ethanol content during fermentation.

[0068] (3) High sugar tolerance test

[0069] The glucose concentration of MRS liquid medium was adjusted to 20 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, 500 g / L, and 600 g / L. Seed culture of strain X3 was inoculated at a 2% inoculum size and incubated at 37℃ for 24 h. The absorbance was then measured at 600 nm. The results are shown below. Figure 4 .

[0070] from Figure 4 It can be seen that the cell biomass of strain X3 decreases with increasing glucose concentration. When the glucose concentration is 400 g / L, its cell biomass still reaches more than 40% of the initial glucose concentration (20 g / L), indicating that the strain can still grow in a high glucose environment of 40%, showing good tolerance.

[0071] (4) Acetic acid resistance test

[0072] The acetic acid content of MRS liquid medium was adjusted to 0%, 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, and 1.3% (v / v) with acetic acid. Seed culture of strain X3 was inoculated at a 2% inoculum and incubated at 37℃ for 24 h. The absorbance was then measured at 600 nm. The results are shown below. Figure 5 .

[0073] Depend on Figure 5 It can be seen that as the volume fraction of acetic acid increases, the OD of Acetobacter X3 increases. 600 The value showed a gradual decreasing trend, indicating that acetic acid inhibited its growth; however, even when the volume fraction of acetic acid reached 1.3%, the strain could still maintain a certain growth capacity (OD). 600 The cell biomass of the strain was still greater than 0.5%, indicating that it had not completely stopped growing and demonstrating good tolerance to acetic acid. Further observation showed that when the acetic acid volume fraction was 1.1%, the cell biomass of the strain could still reach more than 40% of the group without acetic acid, indicating that the maximum tolerable acetic acid volume fraction for this strain was 1.1%.

[0074] (5) pH resistance test

[0075] The pH of the MRS liquid medium was adjusted to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 6.5 using 0.1 mol / L HCl and 0.1 mol / L NaOH, respectively. The seed culture of strain X3 was inoculated at a rate of 2%, and after static incubation at 37℃ for 24 h, the absorbance was measured at 600 nm. The results are shown below. Figure 6 .

[0076] from Figure 6 OD of Acetobacter x3 600 The trend of OD value with pH shows that as pH decreases, the growth of the strain is gradually inhibited. When the pH is below 4.0, the OD value... 600 The pH value was significantly reduced, bacterial growth was significantly inhibited, and cell biomass was reduced to below 0.5, indicating that the minimum tolerable pH for Acetobacter X3 is 4.0.

[0077] The colony morphology of Acetobacter Indonesianis X3 cultured on MRS plates for 18 hours is as follows: pale yellow, round colonies with a smooth surface, a raised center, and regular edges. The microscopic morphology of cells cultured on MRS liquid medium in a shaker for 18 hours is as follows: cells are oval to short rod-shaped, and the bacteria are Gram-negative.

Claims

1. Acetobacter indonesiensis X3, characterized by low fusel oil production, It was deposited at the China Center for Type Culture Collection on July 15, 2025, with accession number CCTCCNO.M20251613.

2. The application of the low-fusel oil-producing Acetobacter Indonesianis X3 as described in claim 1 in reducing the fusel oil content in Baijiu brewing.

3. The application according to claim 2, characterized in that, The liquor in question is a rice-aroma type.

4. The application according to claim 2 or 3, characterized in that, The low-yield fusel oil Indonesian Acetobacter X3 was mixed with rice-flavored baijiu yeast and then fermented.

5. A microbial inoculant, characterized in that, It contains the low-yield fusel oil Indonesian Acetobacter X3 as described in claim 1.

6. The microbial agent according to claim 5, characterized in that, The microbial agent is either a solid agent or a liquid agent.

7. A rice-aroma type of baijiu, characterized in that, It is brewed from the microbial inoculant described in claim 5.

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