Screening and identification of pseudomonas sp. strain for producing feruloyl esterase at high yield and use of pseudomonas sp. strain as bioaugmentation agent in fermented foods

Abstract

Provided are the screening and identification of a Pseudomonas sp. strain for producing feruloyl esterase at high yield and a use of the Pseudomonas sp. strain as a bioaugmentation agent in fermented foods, relating to the technical field of brewing. By means of screening, a Pseudomonas sp. strain A-4H for producing feruloyl esterase at high yield is obtained. After cultivation for 36 h, the Pseudomonas sp. strain A-4H can produce ferulic acid at a concentration of 153.79±2.36 mg / L, and the activity of feruloyl esterase in a fermentation broth reaches 136.63±10.27 U / L. The addition of the Pseudomonas sp. A-4H during the brewing of Huangjiu can effectively increase the content of ferulic acid, the content of phenolic acid, and the content of organic acid in Huangjiu, providing an effective biological pathway for increasing the content of ferulic acid in fermented foods.

Description

Screening, identification, and application as a biofortifier of a high-yield ferulic acid esterase-producing Pseudomonas aeruginosa in fermented foods Technical Field

[0001] This invention relates to the screening, identification, and application as a biofortifier in fermented foods of a strain of Pseudomonas aeruginosa that produces high levels of ferulic acid esterase, and belongs to the field of brewing technology. Background Technology

[0002] Yellow wine is one of the oldest types of alcoholic beverages in my country and occupies an important position in Chinese brewing. It has a mellow taste, delicate aroma, and is rich in nutrients, possessing unique health benefits. Wheat koji, mainly divided into raw and cooked wheat koji, is a saccharifying and fermenting agent rich in various microorganisms and enzymes. Currently, 975 kinds of volatile flavor compounds have been detected in Chinese yellow wine, including phenols, alcohols, esters, aldehydes, ketones, acetals, etc. These important volatile compounds originate from various aspects such as raw materials, koji used, brewing water, and even the environment.

[0003] Ferulic acid (FA) is a widely used hydroxycinnamic acid. Various scientific studies have shown that FA has significant health protection potential, including antioxidant, antiviral, anti-inflammatory, and vascular function benefits. The FA content in traditionally fermented rice wine is approximately 0.57–3.98 mg / L. As a precursor, the FA content is related to the raw materials used in fermentation and the koji (fermentation starter). For example, polyphenols in glutinous rice interact with amylopectin and amylose in different ways, influencing their effects and being hydrolyzed by α-amylase. Ferulic acid is the main phenolic acid compound in wheat bran and husk. However, ferulic acid is cross-linked with cellulose, hemicellulose, and lignin through ether and ester bonds, making its release difficult. Therefore, selecting koji with high yields of ferulic acid esterase, cellulase, and xylanase is crucial for increasing the FA content in fermented rice wine. Furthermore, during rice wine fermentation, FA can be converted into compounds such as vanillin, 4-vinylguaiacol, caffeic acid, and vanillic acid by enzymes secreted by different microorganisms, enriching the aroma of rice wine.

[0004] In the existing technology, the ferulic acid content in rice wine is too low, making it difficult to exert the functional benefits of ferulic acid and enhance the aroma of rice wine. Increasing the ferulic acid content in rice wine plays an important role in improving the quality of rice wine. Summary of the Invention

[0005] Based on existing technical and process problems, this invention aims to provide a high-yield ferulic acid esterase strain, a screening method, and its applications.

[0006] A high-yield ferulic acid esterase-producing Pseudomonas sp. A-4H was deposited on January 2, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2025002, located at Wuhan University, Wuhan, China.

[0007] In one embodiment of the present invention, the Pseudomonas sp. A-4H was obtained from wheat koji through isolation, purification and screening verification.

[0008] In one embodiment of the present invention, the screening step is as follows:

[0009] Step 1: Pre-treatment of wheat koji

[0010] Accurately weigh 10g of wheat koji sample and place it in a sterilized Erlenmeyer flask. Add 90mL of sterile physiological saline and a few sterile glass beads. Incubate in a 37℃ constant temperature water bath with shaking for 4 hours for later use.

[0011] Step 2: Isolation and purification of the strain

[0012] Take the pretreated wheat koji suspension from step one and perform a 10-fold serial dilution four times. Take 10 3 10 4 10 5 10 6 200 μL of diluted bacterial suspension was spread onto ferulic acid esterase solid selection medium and incubated upside down in a 37°C incubator. Single colonies with different morphological appearances were selected and streaked onto LB solid medium for further isolation and purification.

[0013] Step 3: Screening and liquid fermentation verification of ferulic acid esterase-producing strains.

[0014] Each isolated strain was inoculated into a ferulic acid esterase liquid selection medium for liquid fermentation experiments to produce ferulic acid. The content of ferulic acid was determined by high performance liquid chromatography, and the strain with the strongest ferulic acid production capacity was named A-4H.

[0015] The present invention provides a microbial preparation containing the aforementioned Pseudomonas A-4H.

[0016] In one embodiment of the present invention, the number of Pseudomonas A-4H cells in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0017] The present invention provides a product containing the aforementioned Pseudomonas A-4H or the aforementioned microbial preparation.

[0018] The present invention provides a method for producing ferulic acid, wherein the Pseudomonas aeruginosa A-4H or the microbial preparation is added to an environment containing ferulic acid esters for fermentation.

[0019] In one embodiment of the present invention, the ferulic acid esters include methyl ferulic acid, ethyl ferulic acid, oligosaccharide ferulic acid esters, or polysaccharide ferulic acid esters.

[0020] This invention provides a method for preparing wheat koji, wherein the Pseudomonas aeruginosa A-4H and Penicillium oxalate M1816 are simultaneously inoculated into one or more of wheat, wheat bran, rice, millet, corn, and glutinous rice.

[0021] This invention provides a method for increasing the ferulic acid content in rice wine by adding the Pseudomonas A-4H or the microbial preparation to the rice wine raw materials for fermentation.

[0022] In one embodiment of the present invention, Penicillium oxalate M1816 is also added to the raw materials of rice wine.

[0023] In one embodiment of the present invention, the amount of *Pseudomonas aeruginosa* A-4H added to the rice wine raw materials is not less than 1 × 10⁻⁶. 6 CFU / mL.

[0024] In one embodiment of the present invention, the ratio of Pseudomonas aeruginosa A-4H and Penicillium oxalate M1816 added to the rice wine raw materials is (1-8):1.

[0025] In one embodiment of the present invention, fermentation is carried out at 15–28°C for at least 20 days.

[0026] This invention provides the application of the aforementioned Pseudomonas A-4H or the aforementioned microbial preparation in the preparation of fermented foods. Beneficial effects:

[0027] This invention screened and obtained a high-yielding Pseudomonas sp. strain A-4H of ferulic acid esterase. After 36 hours of cultivation, Pseudomonas A-4H produced ferulic acid at a content of 153.79 ± 2.36 mg / L, and the ferulic acid esterase activity in the fermentation broth reached 136.63 ± 10.27 U / L. Adding Pseudomonas A-4H to the brewing of rice wine can effectively increase the ferulic acid, phenolic acid, and organic acid content in the rice wine, providing an effective biological pathway for increasing the ferulic acid content in brewed foods.

[0028] Preservation of biological materials

[0029] A strain of Pseudomonas sp. A-4H, taxonomically named Pseudomonas sp. A-4H, was deposited on January 2, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025002, located at Wuhan University, Wuhan, China. Attached Figure Description

[0030] Figure 1: Ability of the screening strains to produce ferulic acid in liquid screening medium;

[0031] Figure 2: Ferulic acid esterase activity of the fermentation broth of the top 20 strains with high FA production.

[0032] Figure 3: Colony morphology of strain A-4H;

[0033] Figure 4: Phylogenetic tree of strain A-4H;

[0034] Figure 5: Tolerance evaluation of strain A-4H;

[0035] Figure 6: Block morphology (A) and temperature and humidity change curves during fermentation (B). Detailed Implementation

[0036] The structures, proportions, and sizes illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and to enable them to understand and read the invention. They are not intended to limit the conditions under which the invention can be implemented and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size, without affecting the effects and objectives of the invention, should still fall within the scope of the technical content disclosed in this invention.

[0037] Terminology Explanation:

[0038] The terms “comprising,” “including,” “having,” and “containing” as used herein mean “including but not limited to,” “including but not limited to,” “having but not limited to,” and “containing but not limited to,” and are used interchangeably with the corresponding phrases. Unless the context clearly indicates otherwise, the term “or” as used herein is used to mean “and / or” and is used interchangeably with it. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, the examples of suitable methods and materials set forth below are illustrative only and do not limit the invention in any way.

[0039] As used in this invention, the term "wheat starter" refers to a fermenting agent made from wheat or other grains (such as rice, corn, etc.) through a specific process. In some embodiments, it specifically refers to a solid fermenting agent obtained by inoculating grains with Pseudomonas aeruginosa A-4H or Pseudomonas aeruginosa A-4H and Penicillium oxalate M1816 and then fermenting them.

[0040] The term "enhanced wheat koji" as used in this invention refers to the block koji obtained by co-inoculating grains with Penicillium oxalate M1816 and Pseudomonas aeruginosa A-4H in a specific ratio.

[0041] The term "block koji" as used in this invention refers to a solid block fermenting agent obtained by pressing wheat koji into blocks. In some embodiments, this includes fortified wheat koji (wheat inoculated with Penicillium oxalate M1816 and Pseudomonas aeruginosa A-4H and pressed into koji blocks) and unfortified wheat koji (wheat inoculated with Penicillium oxalate M1816 and pressed into koji blocks).

[0042] The term "saccharification" as used in this invention refers to the biochemical process of converting complex carbohydrates such as starch into simple sugars (such as glucose). In some embodiments, saccharification power is defined as the number of milligrams of glucose produced by converting 1 g of oven-dried wheat koji into soluble starch in 1 hour at 35°C and pH = 4.6, denoted as U / g.

[0043] The term "liquefaction" as used in this invention refers to the process of converting polysaccharides such as starch into a liquid syrup. In some embodiments, liquefaction power is defined as the number of grams of starch that 1 g of oven-dried wheat koji can liquefy in 1 hour at 35°C and pH = 4.6, denoted as U / g.

[0044] The term "pre-fermentation" as used in this invention refers to a pretreatment or preliminary fermentation process of the raw materials before the main fermentation process. In some embodiments, it generally refers to the period from day 1 to day 5 after the raw materials are introduced, during which the starch rapidly saccharifies and the yeast in the mash multiplies rapidly, entering the alcoholic fermentation stage. Due to the fermentation action of the yeast, the temperature of the mash rises rapidly. When it reaches 35°C, the mash is aerated 2 to 3 times a day to replenish oxygen and increase yeast activity. "Post-fermentation" refers to the period from day 5 to day 20 of fermentation, during which the mash sinks and fermentation enters a slow phase. To prevent the rice wine from spoiling, it is moved to an environment of 15°C.

[0045] The analytical methods involved in the following embodiments are as follows:

[0046] Determination of physicochemical properties of wheat koji: The determination of saccharification and liquefaction power refers to the general analytical method of brewing koji in QB / T 4257-2011; the determination of cellulose and xylanase activity is performed using the DNS method.

[0047] Determination of ferulic acid esterase activity in wheat koji: Extraction of crude enzyme solution: Accurately weigh 5.00 g of wheat koji (convert to oven-dry weight, DW) to a value accurate to 0.001 g, place it in a 250 mL sterile Erlenmeyer flask, add 60 mL of acetate-sodium acetate buffer solution (pH = 4.6 100 mM), and extract in a 30 °C constant temperature shaker at 180 r / min for 1 h. Centrifuge at 12000 r / min at 4 °C for 15 min, and collect the supernatant as the crude enzyme solution to be tested. The ferulic acid (FA) content was determined by high performance liquid chromatography.

[0048] Determination of ferulic acid esterase activity in fermentation broth: 1 mL of fermentation broth was centrifuged, and 0.5 mL of the supernatant was collected. 1.5 mL of an acetate-sodium acetate buffer solution (pH = 4.6, 0.1 M) containing 200 mg / L methyl ferulic acid was added, and the mixture was reacted in a 30°C water bath for 10 min. After the reaction was complete, 2 mL of 10% glacial acetic acid was added to terminate the reaction, and the FA content generated was measured. Definition of ferulic acid esterase activity: Under conditions of 30°C and pH = 4.6, the amount of enzyme required to degrade methyl ferulic acid to produce 1 μmol of ferulic acid per minute is defined as 1 unit of enzyme activity (U). In the following examples, the enzyme activity corresponding to a unit mass of ferulic acid esterase was calculated, and the unit is U / g.

[0049] Physicochemical index testing of rice wine:

[0050] The physicochemical properties of alcohol content, total acid, and amino nitrogen were determined according to the methods in the national standard GB13662-2018, and the reducing sugar was determined by the 3,5-dinitrosalicylic acid method.

[0051] Determination of ferulic acid and other phenolic acid content in rice wine: Sample pretreatment: Take 0.5 mL of liquid screening culture medium and fermented rice wine samples respectively, add 1.5 mL of methanol, centrifuge at 10000 rpm for 10 min at 4℃, and after centrifugation, take the supernatant and filter it through a 0.22 μm organic filter membrane. Determine the ferulic acid content using high performance liquid chromatography (HPLC). HPLC conditions: Column: Athena C18-WP (5 μm, 250 × 4.6 mm); Flow rate: 1 mL / min; Column temperature: 32℃; Detection wavelength: 320 nm; Mobile phase A: 1% aqueous acetic acid solution; Mobile phase B: pure acetonitrile. Elution program: 0–8 min: A: 80%, B: 20%; 8–13 min: A: 72%, B: 28%; 13–19 min: A: 80%, B: 20%; 19–25 min: A: 80%, B: 20%.

[0052] Determination of Organic Acids in Shaoxing Rice Wine: The organic acid content in Shaoxing rice wine was determined by HPLC. Sample pretreatment: 1 mL of Shaoxing rice wine sample was added to a 5 mL EP tube, followed by 0.2 mL of 30% zinc sulfate and 0.2 mL of 10% potassium ferrocyanide. Water was added to a final volume of 4 mL. The mixture was centrifuged at 8000 g for 5 min, and 1 mL of the supernatant was filtered through a 0.22 μm aqueous filter membrane. Mobile phase: 0.02 mol / L potassium dihydrogen phosphate solution; detection wavelength: 210 nm; column temperature: 30℃, isocratic elution; flow rate: 0.8 mL / min; chromatographic column: Athena C18-WP (5 μm, 250 × 4.6 mm).

[0053] The culture medium components involved in the following examples are as follows:

[0054] Enrichment medium: NaCl 0.3g, (NH4)2SO4 1.3g, K2HPO4 0.3g, MgSO4·7H2O 0.3g and sterile water 90mL, sterilized at 115℃ for 20min;

[0055] Ferulic acid esterase solid screening medium: NaCl 0.3g, (NH4)2SO4 1.3g, K2HPO4 0.3g, MgSO4·7H2O 0.3g, 18g agar and 1000mL sterile water, sterilized at 115℃ for 20min, cooled and then added 15mL of ethyl ferulic acid ester (10% V / V dissolved in N,N-dimethylformamide), and shaken well;

[0056] Ferulic acid esterase liquid screening medium: Based on the above ferulic acid solid screening medium, without adding agar, and using methyl ferulic acid instead of ethyl ferulic acid;

[0057] LB solid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 20g agar, 1L ultrapure water, natural pH, sterilized at 121℃ for 20min.

[0058] The saccharifying enzymes used in the following examples were purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10017-250g, and the liquefying enzymes were purchased from Solarbio, catalog number #A8750-250g.

[0059] The Penicillium oxalicum M1816 involved in the following examples has been disclosed in the literature "Enzyme production potential of Penicillium oxalicum M1816 and its application in ferulic acid production".

[0060] The brewing yeast RWBLY1906 SHL involved in the following examples has been disclosed in the literature "Selection of brewing yeast with low fusel alcohol production and high acetate production and the formation mechanism of differences in rice wine fermentation".

[0061] Example 1: Physiological and biochemical identification of Pseudomonas sp. A-4H strain

[0062] 1. Screening of strains

[0063] Step 1: Pre-treatment of wheat koji

[0064] Accurately weigh 10g of wheat koji (from Shaoxing, Zhejiang) sample and place it in a sterilized Erlenmeyer flask. Add 90mL of sterile physiological saline and a few sterile glass beads. Incubate in a 37℃ constant temperature water bath with shaking for 4 hours for later use.

[0065] Step 2: Isolation and purification of the strain

[0066] Take the pretreated wheat koji suspension from step one and perform a 10-fold serial dilution four times. Take 10 3 10 4 10 5 10 6 200 μL of diluted bacterial suspension was spread onto ferulic acid esterase solid selection medium and incubated upside down in a 37°C incubator. Single colonies with different morphological appearances were selected and streaked onto LB solid medium for further isolation and purification.

[0067] Step 3: Screening and liquid fermentation verification of ferulic acid esterase-producing strains.

[0068] Fifty-six strains capable of producing a clear zone were screened from wheat koji. Since the ratio of the diameter of the clear zone to the colony diameter showed no significant difference (P>0.05), these 56 strains were inoculated into liquid selection medium to determine their ferulic acid (FA) production capacity. The top 20 strains with the highest FA production were selected to measure ferulic acid esterase activity. The culture methods and specific conditions were as follows: the 56 strains were inoculated into 96-well plates containing LB liquid medium at an inoculation rate of 4% v / v (initial inoculation concentration 1×10⁻⁶). 6 After incubation at 37°C for 36 hours (CFU / mL), the OD values ​​of 56 strains were analyzed. 600 Adjust the solution to 0.8 using sterile water, then inoculate it into liquid selection medium containing ferulic acid esterase at an inoculation rate of 4% v / v (initial inoculation concentration of 1×10⁻⁶). 6 Incubate at 600 rpm and 37°C for 36 h (CFU / mL).

[0069] The FA content of 56 strains was determined and is shown in Figure 1. Among them, strain A-4H produced FA of 153.79±2.36 mg / L after 36 h of culture. Strains A-10, Sb, A-6 and A-1H also produced high FA contents, which were 58.46±0.32 mg / L, 47.13±0.03 mg / L, 32.80±0.17 mg / L and 32.33±0.13 mg / L, respectively.

[0070] Figure 2 shows the ferulic acid esterase activity of the fermentation broth of the first 20 strains. Among them, the ferulic acid esterase activity of the fermentation broth of strain A-4H was 136.63±10.27 U / L.

[0071] 2. Physiological and biochemical identification of the strain

[0072] (1) Morphological characteristics of strains

[0073] When strain A-4H was inoculated into LB medium, the resulting colonies were dot-like, waxy, opaque, pale yellow, with raised surfaces and neat edges. Under a 1000x microscope, the A-4H cells were observed to be rod-shaped, appearing singly or in short chains (Figure 3).

[0074] (2) DNA extraction and bacterial identification

[0075] DNA was extracted from the strain using the CATB method, followed by PCR amplification and sequencing analysis. The 16S rRNA gene of the selected strain was amplified using universal primers 27F / 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'5'-TACGGTTACCTTGTTACGACTT-3'-3'), and a phylogenetic tree was generated using MEGA11 software. As shown in Figure 4, the results indicate that A-4H belongs to the genus *Pseudomonas*.

[0076] The Pseudomonas sp. A-4H strain was deposited on January 2, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025002, located at Wuhan University, Wuhan, China.

[0077] Example 2: Properties of Pseudomonas sp. A-4H

[0078] (1) Ethanol tolerance: LB liquid medium was dispensed into 150 mL Erlenmeyer flasks, sterilized at 115 °C for 20 min, cooled to room temperature, and then anhydrous ethanol was added to make the final concentration of ethanol in the LB liquid medium 0, 2, 4, 6 and 8% vol. Then, an inoculum of 1% (10 6The mycelial dry weight was measured after the experiment was completed. The mycelial dry weight was measured after the experiment was completed. (CFU / mL) of Pseudomonas bacteria were inoculated into the culture and cultured at 37℃ for 6 days. Each experiment was repeated 3 times.

[0079] (2) Reducing sugar tolerance: Reducing sugar was added to LB liquid medium at concentrations of 90, 70, 50, and 30 g / L, dispensed into 150 mL Erlenmeyer flasks, sterilized at 115 °C for 20 min, and after cooling to room temperature, the flasks were incubated with 1% (10 g / L) of reducing sugar. 6 Inoculate the Pseudomonas sylvestris culture at an inoculum size of CFU / mL and incubate at 37°C for 6 days. Each experiment was repeated in 3 replicates, and the dry weight of the mycelium was measured.

[0080] (3) Lactic acid tolerance: LB liquid medium was dispensed into 150 mL Erlenmeyer flasks, shaken well, sterilized at 115 °C for 20 min, cooled to room temperature, and then filtered sterilized lactic acid was added to achieve final lactic acid concentrations of 0, 2, 4, 6, and 8 g / L in the LB liquid medium. Then, 1% (1×10⁻⁶) was added. 6 Inoculate the Pseudomonas sylvestris culture at an inoculum size of CFU / mL and incubate at 37°C for 6 days. Each experiment was repeated 3 times. The dry weight of the mycelium was measured after the experiment.

[0081] (4) Temperature tolerance: Dispense LB liquid medium into 150 mL Erlenmeyer flasks, shake well, sterilize at 115 °C for 20 min, and when the temperature of the LB liquid medium has cooled to room temperature, then add 1% (1×10⁻⁶) of the medium to the flasks. 6 Inoculation with Pseudomonas aeruginosa at a concentration of CFU / mL was carried out, and cultured at 37, 45, 50 and 55 °C for 6 days. Each experiment was repeated 3 times. The dry weight of the mycelium was measured after the experiment.

[0082] The results showed that the Pseudomonas aeruginosa A-4H could grow under conditions of 0-8% vol alcohol, 30-90 g / L reducing sugar, 0-8 g / L lactic acid, and 37-55℃. Among these conditions, the growth was best under the following conditions: 0-2% vol alcohol, 0-2 g / L lactic acid, 50-70 g / L reducing sugar, and 37-45℃ (Figure 5).

[0083] Example 3: Pseudomonas sp. A-4H fortified malt koji

[0084] (1) Preparation of Pseudomonas sp. A-4H seed culture:

[0085] Pseudomonas A-4H was inoculated into LB liquid medium and cultured for 36–48 hours at 37°C and 180 rpm to obtain a primary seed culture. Then, 4% of this seed culture was inoculated into fresh medium and cultured for 36–48 hours to obtain a secondary seed culture. The concentration was then adjusted to 1 × 10⁻⁶ using a hemocytometer. 6 per mL.

[0086] (2) Pseudomonas sp.A-4H Enhanced Wheat Production

[0087] The process for making hand-fortified block koji involves selecting high-quality wheat from the current year, simply washing it, and then uniformly crushing it into 5-6 pieces. 2275g of the crushed wheat is mixed with 9.5% bran (w / w), and 750mL of water at 80℃ is added. When the temperature drops to 32-35℃, 6% v / m seed solution is added (with an inoculation live bacteria ratio of P. oxalicum M1816:Pseudomonas aeruginosa = 4:1, and the initial total live bacteria count is 1×10⁻⁶). 6 CFU / mL (total water content controlled at 36%), were boosted with Pseudomonas A-4H, Pseudomonas A-10, and Pseudomonas Sb, respectively. The unboosted group was inoculated with only 1×10⁻⁶ CFU / mL. 6 Wheat koji was prepared using *Penicillium oxalate* M1816 at CFU / mL. After rapid mixing, the inoculated wheat koji was pressed into shape using a mold (28.0cm × 18.0cm × 10.0cm). After the koji blocks formed, they were left to stand for 30 to 45 minutes, and then transferred to a room dedicated to solid-state fermentation. The fermentation parameters of the koji blocks are shown in Table 1. After fermentation, the enzyme activities of the koji blocks were measured, as shown in Table 2. The A-4H enhanced group showed better enzyme activity, with higher saccharification power, liquefaction power, xylan and cellulose enzyme activities, and ferulic acid ester activities.

[0088] Table 1 Fermentation parameters of block koji

[0089] Table 2 Basic Physicochemical Indicators of Wheat Qu

[0090] The inoculation ratio of Penicillium oxalate M1816 to Pseudomonas aeruginosa A-4H in the enhanced group was adjusted to 1:1 to 8:1. When the inoculation ratio was 8:1, the saccharification power of wheat koji was the highest. When the inoculation ratio was adjusted to 4:1, the saccharification power decreased slightly, while the liquefaction power and ferulic acid esterase activity were the highest. Therefore, a Penicillium oxalate M1816:Pseudomonas aeruginosa A-4H inoculation ratio of 4:1 was selected for subsequent experiments.

[0091] Example 4: Application of reinforced koji in the production of rice wine

[0092] Yeast activation: Saccharomyces cerevisiae RWBLY1906 SHL from glycerol tubes were streaked onto agar plates in an aseptic environment and incubated at 28°C for 48 hours. Single colonies of yeast from the plates were then inoculated into YPD medium in an aseptic environment and incubated at 30°C for 36–48 hours, maintaining a temperature between 28–30°C, not exceeding 30°C. This culture is then ready for use as YPD culture medium.

[0093] Preparation of yeast starter: Steamed rice was mixed with liquefying enzyme (700 U / g rice), saccharifying enzyme (5 U / g rice), and malt koji (0.1 U / g rice) to prepare a saccharification solution. The mixture was then incubated in a 60°C water bath for 6 hours. After saccharification, the solution was filtered, dispensed into Erlenmeyer flasks, and sterilized. The yeast starter was prepared according to a 10... 6 Yeast was inoculated at an initial inoculum of CFU / mL and cultured at a constant temperature for 36–48 h. The ingredient system for the rice wine is shown in Table 3. The total fermentation time was 20 days. The first 5 days were the pre-fermentation, with the temperature controlled at 28℃ and the yeast stirred twice a day. The subsequent 15 days were the post-fermentation, with the temperature controlled at 15℃.

[0094] Table 3 Ingredients for Yellow Rice Wine Brewing

[0095] Tests of physicochemical properties of rice wine:

[0096] There were no significant differences in alcohol content and total acid content between wheat koji fortified with strain A-4H and unfortified wheat koji. However, the total sugar content of the fortified wheat koji was significantly higher than that of the unfortified wheat koji (Table 4). The basic physicochemical indicators of rice wine brewed with fortified and unfortified wheat koji met the physicochemical requirements for rice wine in GB / T13662-2018.

[0097] Table 4 Basic Physicochemical Indicators of Shaoxing Wine

[0098] Tracking the changes in ferulic acid content during fermentation, it can be found that the ferulic acid content in the fortified group and the unfortified group showed the same trend during fermentation, with a rapid increase followed by a decrease and eventually leveling off. Among them, the ferulic acid content in the fortified group of rice wine was significantly higher than that in the unfortified group throughout the entire fermentation process (Table 5).

[0099] Table 5. Changes in ferulic acid content during fermentation.

[0100] The changes in total phenol content during the fermentation process are shown in Table 6. The changes in total phenol content during fermentation are consistent with the changes in ferulic acid content, showing a rapid increase followed by a decrease and eventually leveling off. Among them, the total phenol content of the rice wine brewed in the A-4H fortified group was consistently higher than that in the non-fortified group during fermentation. At the end of fermentation, the total phenol content of the A-4H fortified group was approximately 1.15 times that of the non-fortified group.

[0101] Table 6. Changes in total phenol content during fermentation.

[0102] The phenolic acid content of the rice wine in the A-4H fortified group and the unfortified group was measured after fermentation, as shown in Figure 7. The rice wine brewed in the A-4H fortified group had significantly higher contents of vanillic acid, syringic acid, protocatechuic acid, p-coumaric acid, chlorogenic acid, and 4-vinylguaiacol than the unfortified group. Among them, coumaric acid, protocatechuic acid, vanillic acid, and syringic acid may give rice wine its antihypertensive properties, effectively improving its efficacy.

[0103] Table 7. Content of other phenolic acids at the end of fermentation.

[0104] After fermentation, the A-4H-fortified group had higher levels of lactic acid, acetic acid, and citric acid than the unfortified group, while its oxalic acid content was lower (Table 8). Organic acids play a crucial role in the taste, color, and flavor of Shaoxing wine. For example, lactic acid has a mild sour taste and good acidity. Succinic acid is the most complex organic acid in terms of taste; its sourness is mild, but the flavor intensifies upon entry, initially salty and then bitter, and it can stimulate saliva production. Therefore, Shaoxing wine fermented with pseudomonocyte A-4H fortification also produces a superior taste.

[0105] Table 8 Organic acid content after fermentation

[0106] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Pseudomonas sp. A-4H, characterized in that, The Pseudomonas A-4H strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2025002 and deposit date of January 2, 2025.

2. A microbial preparation containing the Pseudomonas A-4H of claim 1.

3. The microbial preparation as described in claim 2, characterized in that, In the aforementioned microbial preparation, the number of Pseudomonas A-4H cells is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

4. A product containing the Pseudomonas A-4H of claim 1 or the microbial preparation of claim 2 or 3.

5. A method for producing ferulic acid, characterized in that, The Pseudomonas A-4H of claim 1, or the microbial preparation of claim 2 or 3, is added to an environment containing ferulic acid esters for fermentation.

6. The method as described in claim 5, characterized in that, The ferulic acid esters include methyl ferulic acid, ethyl ferulic acid, oligosaccharide ferulic acid esters, or polysaccharide ferulic acid esters.

7. A method for preparing wheat koji, characterized in that, The Pseudomonas A-4H of claim 1 and Penicillium oxalate M1816 were simultaneously inoculated into a wheat koji substrate, wherein the wheat koji substrate includes one or more of wheat, wheat bran, rice, millet, corn, and glutinous rice.

8. A method for increasing the ferulic acid content in rice wine, characterized in that, The Pseudomonas A-4H of claim 1, or the microbial preparation of claim 2 or 3, is added to the raw materials of rice wine for fermentation.

9. The method as described in claim 8, characterized in that, The raw materials for rice wine also include Penicillium oxalate M1816.

10. The method as described in claim 9, characterized in that, The amount of Pseudomonas A-4H added to the raw materials for rice wine is not less than 1×10⁻⁶. 6 CFU / mL.

11. The method as described in claim 10, characterized in that, The ratio of Pseudomonas aeruginosa A-4H to Penicillium oxalate M1816 in the raw materials for rice wine is (1-8):

1.

12. The method as described in claim 10, characterized in that, Ferment at 15–28°C for at least 20 days.

13. The use of the Pseudomonas A-4H of claim 1 or the microbial preparation of claim 2 or 3 in the preparation of fermented foods.

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