Screening and identification of pseudomonas capable of producing high-yield feruloyl esterase and application of pseudomonas as biological enhancer in fermented food

Abstract

The invention discloses screening and identification of pseudomonas capable of producing high-yield feruloyl esterase and application of the pseudomonas as a biological enhancer in fermented food, and belongs to the technical field of brewing. According to the invention, a strain of pseudomonas sp. A-4H with high yield of feruloyl esterase is obtained by screening. After the pseudomonas A-4H is cultured for 36 hours, the content of ferulic acid which is 153.79 + / -2.36 mg / L can be generated, and the enzyme activity of feruloyl esterase in fermentation liquor reaches 136.63 + / -10.27 U / L; when the pseudomonas A-4H is added in the brewing process of the yellow rice wine, the ferulic acid content, phenolic acid content and organic acid content in the yellow rice wine can be effectively increased, and an effective biological way is provided for increasing the ferulic acid content in brewed food.

Description

Technical Field

[0001] The invention relates to the screening and identification of a strain of Pseudomonas producing high-yield ferulic acid esterase and the application of the strain as a biological enhancer in fermented food, belonging to the technical field of brewing. Background Art

[0002] Huangjiu is one of the oldest wines in my country and occupies an important position in China's brewed wine. Huangjiu has a soft taste, elegant aroma, rich nutrition, and unique health care functions. Wheat koji, mainly divided into raw wheat koji and cooked wheat koji, is a saccharification and fermentation agent rich in various microorganisms and enzyme systems. At present, there are 975 kinds of volatile flavor substances detected in Huangjiu in my country, including phenols, alcohols, esters, aldehydes, ketones, acetals, etc. These important volatile compounds come from various aspects such as raw materials, koji, brewing water and even the environment.

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

[0004] In the prior art, 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 the existing technical problems and process problems, the present invention aims to provide a strain with high ferulic acid esterase production, a screening method and an application.

[0006] A strain of Pseudomonas sp. A-4H that produces high levels of ferulic acid esterase was deposited in the China Center for Type Culture Collection (CCTCC) on January 2, 2025, with the deposit number CCTCC NO:M 2025002. The address is: Wuhan University, Wuhan, China.

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

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

[0009] Step 1: Pretreatment of malt koji

[0010] Accurately weigh 10 g of malt koji sample and put it into a sterilized conical flask, add 90 mL of sterile saline and put in a few sterile glass beads, and shake and culture in a constant temperature water bath at 37°C for 4 hours.

[0011] Step 2: Isolation and purification of strains

[0012] Take the pre-treated malt koji suspension in step 1 and dilute it 10 times four times. 3 , 10 4 , 10 5 , 10 6 200 μL of the diluted bacterial suspension was spread on the ferulic acid esterase solid screening medium and placed in an incubator at 37°C for inversion culture. Single colonies with different colony morphology were selected and streaked on LB solid medium for further separation and purification.

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

[0014] The isolated strains were inoculated into ferulic acid esterase liquid screening medium for liquid fermentation to produce ferulic acid. The ferulic acid content was determined using high performance liquid chromatography, and the strain with the strongest ferulic acid production ability was named A-4H.

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

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

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

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

[0019] In one embodiment of the present invention, the ferulate ester includes methyl ferulate, ethyl ferulate, oligosaccharide ferulate or polysaccharide ferulate.

[0020] The invention provides a method for preparing wheat koji, wherein the pseudomonas A-4H and penicillium oxalicum M1816 are inoculated into one or more of wheat, bran, rice, millet, corn and glutinous rice at the same time.

[0021] The invention provides a method for increasing the content of ferulic acid in yellow wine. The method comprises adding the Pseudomonas A-4H or the microbial preparation into yellow wine raw materials for fermentation.

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

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

[0024] In one embodiment of the present invention, in the rice wine raw material, the cell ratio of Pseudomonas A-4H and Penicillium oxalicum M1816 added is (1-8):1.

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

[0026] The present invention provides application of the Pseudomonas A-4H or the microbial preparation in preparing fermented food.

[0027] Beneficial effects:

[0028] The present invention screens and obtains a strain of Pseudomonas sp. A-4H that produces high ferulic acid esterase. The Pseudomonas sp. A-4H can produce a ferulic acid content of 153.79±2.36 mg / L after culturing for 36 hours, and the ferulic acid esterase activity in the fermentation broth reaches 136.63±10.27 U / L; adding the Pseudomonas sp. A-4H in the brewing of yellow rice wine can effectively increase the ferulic acid content, phenolic acid content and organic acid content in the yellow rice wine, providing an effective biological approach for increasing the ferulic acid content in brewed food.

[0029] Biomaterial Deposit

[0030] A strain of Pseudomonas sp. A-4H, taxonomically named: Pseudomonas sp. A-4H, was deposited in the China Center for Type Culture Collection on January 2, 2025, with the deposit number CCTCC NO: M 2025002, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1: Screening strains for their ability to produce ferulic acid in liquid screening medium;

[0032] Figure 2 : Screening the ferulic acid esterase activity of the fermentation broth of the top 20 strains with the highest FA production;

[0033] Figure 3 : Colony morphology of strain A-4H;

[0034] Figure 4 : Phylogenetic tree of strain A-4H;

[0035] Figure 5 : Tolerance evaluation of strain A-4H;

[0036] Figure 6 : Koji block morphology (A) and temperature and humidity change curve during fermentation (B). DETAILED DESCRIPTION

[0037] The structures, proportions and sizes illustrated in the drawings in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0038] Terminology explanation:

[0039] The terms "comprising", "including", "having", and "containing" used in the present invention mean "including but not limited to", "including but not limited to", "having but not limited to", and "containing but not limited to", and can be used interchangeably with the corresponding phrases. Unless the context clearly indicates otherwise, the term "or" used in this article is used in this article to mean the term "and / or" and can be 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 the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the examples of suitable methods and materials described below are illustrative only and do not limit the present invention in any way.

[0040] The term "wheat koji" used in the present invention refers to a starter culture made from wheat or other cereals (such as rice, corn, etc.) through a specific process. In certain embodiments, it specifically refers to a solid starter culture obtained by fermenting cereals inoculated with Pseudomonas A-4H or Pseudomonas A-4H and Penicillium oxalicum M1816.

[0041] The term "fortified malt koji" used in the present invention refers to the block koji obtained by fermenting grains with Penicillium oxalicum M1816 and Pseudomonas aeruginosa A-4H inoculated into grains at a specific ratio.

[0042] The term "block koji" used in the present invention refers to a solid block starter obtained by briquetting wheat koji. In certain embodiments, it includes fortified wheat koji (wheat inoculated with Penicillium oxalicum M1816 and Pseudomonas A-4H and pressed into koji blocks) and unfortified wheat koji (wheat inoculated with Penicillium oxalicum M1816 and pressed into koji blocks).

[0043] The term "saccharification" as used in the present invention refers to the biochemical process of converting complex carbohydrates such as starch into simple sugars (such as glucose). In certain embodiments, the saccharification power refers to the number of milligrams of glucose produced by converting soluble starch into 1g of absolute dry malt koji in 1h at 35°C and pH=4.6, and the symbol is U / g.

[0044] The term "liquefaction" used in the present invention refers to the process of converting polysaccharides such as starch into liquid syrup. In certain embodiments, the liquefaction power refers to the number of grams of starch that can be liquefied by 1g of absolute dry malt koji in 1 hour at 35°C and pH=4.6, with the symbol U / g.

[0045] The term "pre-fermentation" used in the present invention refers to a pretreatment or preliminary fermentation process of the raw materials before the main fermentation process. In certain embodiments, it generally refers to the period from 1 to 5 days after the material is dropped, when the starch is rapidly saccharified and the yeast in the mash multiplies in large quantities, and the alcohol fermentation stage is entered. Due to the fermentation of the yeast, the temperature of the mash rises rapidly. When it rises to 35°C, it is ventilated 2 to 3 times a day to supplement oxygen and increase the activity of the yeast; "post-fermentation" refers to the period from 5 to 20 days after the fermentation, when the mash sinks and the fermentation enters a slow period. In order to prevent the rice wine from becoming sour, it is moved to a 15°C environment.

[0046] The analytical methods involved in the following embodiments are as follows:

[0047] Determination of physical and chemical indicators of malt koji: the determination of saccharification and liquefaction power refers to QB / T 4257-2011 General Analysis Method for Brewing Daqu; the cellulose and xylanase activities are determined by DNS method.

[0048] Determination of ferulic acid esterase activity in malt koji: Extraction of crude enzyme solution: Accurately weigh 5.00g malt koji (converted to absolute dry weight, DW when calculating), accurate to 0.001g, place in a 250mL sterilized conical flask, add 60mL of acetic acid-sodium acetate buffer solution (pH=4.6100mM), place in a 30℃ constant temperature shaker at 180r / min for 1h, 12000r / min, 4℃ centrifuge for 15min, and take the supernatant as the crude enzyme solution to be tested. Ferulic acid (FA) content is determined using high performance liquid chromatography.

[0049] Determination of ferulic acid esterase activity in fermentation broth: The ferulic acid esterase activity in fermentation broth is determined by taking 1 mL of fermentation broth, centrifuging and taking 0.5 mL of supernatant, adding 1.5 mL of acetic acid-sodium acetate buffer (pH=4.6, 0.1 M) containing 200 mg / L of methyl ferulate, and reacting in a water bath at 30°C for 10 min. After the reaction is completed, 2 mL of 10% glacial acetic acid is added to terminate the reaction, and the FA content generated by the reaction is determined. Definition of ferulic acid esterase activity: Under the conditions of 30°C and pH=4.6, the amount of enzyme required to degrade methyl ferulate to generate 1 μmol of ferulic acid per minute is 1 enzyme activity unit (U). In the following embodiments, the enzyme activity corresponding to the unit mass of ferulic acid esterase is calculated in U / g.

[0050] Physical and chemical index detection of rice wine:

[0051] The physical and chemical indicators 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.

[0052] Determination of ferulic acid and other phenolic acids in rice wine: Sample pretreatment: Take 0.5 mL of liquid screening culture medium and fermented rice wine sample respectively, add 1.5 mL of methanol, centrifuge at 4 ° C, 10000 rpm for 10 min, take the supernatant after centrifugation and pass it through a 0.22 μm organic filter membrane, and use high performance liquid chromatography to determine the content of ferulic acid. High performance liquid chromatography conditions: chromatographic column: Athena C18-WP (5 μm, 250×4.6 mm); flow rate: 1 mL / min; column temperature 32 ° C; detection wavelength: 320 nm; mobile phase A: 1% acetic acid aqueous 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%.

[0053] Determination of organic acids in rice wine: HPLC method was used to determine the content of organic acids in rice wine: Sample pretreatment method: Take 1mL of rice wine sample and add it to a 5mL EP tube, then add 0.2mL 30% zinc sulfate and 0.2mL 10% potassium ferrocyanide, add water to 4mL, centrifuge at 8000g for 5min, take 1mL of supernatant, and filter through a 0.22μm aqueous phase filter. Mobile phase: 0.02mol / L potassium dihydrogen phosphate solution; detection wavelength is 210nm; column temperature: 30℃, isocratic elution, flow rate: 0.8mL / min; chromatographic column: Athena C18-WP (5μm, 250×4.6mm).

[0054] The culture medium components involved in the following examples are as follows:

[0055] Enrichment medium: NaCl 0.3 g, (NH4)2SO4 1.3 g, K2HPO4 0.3 g, MgSO4·7H2O 0.3 g and sterile water 90 mL, sterilized at 115 °C for 20 min;

[0056] Ferulic acid esterase solid screening medium: NaCl 0.3g, (NH4)2SO41.3g, K2HPO40.3g, MgSO4·7H2O 0.3g, 18g agar and sterile water 1000mL, sterilize at 115℃ for 20min, add 15mL of ethyl ferulate (10% V / V dissolved in N,N-dimethylformamide) after cooling, and shake well;

[0057] Ferulic acid esterase liquid screening medium: based on the above-mentioned ferulic acid solid screening medium, agar is not added, and methyl ferulate is used instead of ethyl ferulate;

[0058] LB solid medium: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, agar 20 g, ultrapure water 1 L, natural pH, 121 ° C, sterilization for 20 min.

[0059] The saccharifying enzyme involved in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with the product number S10017-250g, and the liquefying enzyme was purchased from Solarbio with the product number #A8750-250g.

[0060] The Penicillium oxalicum (P. oxalicum) M1816 involved in the following examples has been disclosed in the document "Enzyme production potential of Penicillium oxalicum M1816 and its application in ferulic acid production".

[0061] The brewer's yeast RWBLY1906 SHL involved in the following examples has been disclosed in the document "Breeding of brewer's yeast with low fusel alcohol production and high acetate production and the mechanism of formation of rice wine fermentation differences".

[0062] Example 1: Physiological and biochemical identification of Pseudomonas sp. A-4H strain

[0063] 1. Screening of strains

[0064] Step 1: Pretreatment of malt koji

[0065] Accurately weigh 10 g of malt koji (from Shaoxing, Zhejiang) sample and put it into a sterilized conical flask, add 90 mL of sterile saline and put in a few sterile glass beads, and shake and culture in a constant temperature water bath at 37°C for 4 hours for use.

[0066] Step 2: Isolation and purification of strains

[0067] Take the pretreated malt koji suspension in step 1 and dilute it 10 times four times. 3 , 10 4 , 10 5 , 10 6 200 μL of the diluted bacterial suspension was spread on the ferulic acid esterase solid screening medium and placed in an incubator at 37°C for inversion culture. Single colonies with different colony morphology were selected and streaked on LB solid medium for further separation and purification.

[0068] Step 3: Screening of ferulic acid esterase producing strains and liquid fermentation verification

[0069] A total of 56 strains that can obviously produce transparent circles were screened out from wheat koji. Since there was no significant difference in the ratio of the diameter of the transparent circle to the diameter of the colony (P>0.05), the above 56 strains that can produce transparent circles were inoculated in liquid screening culture medium to determine the ability to produce FA, and the top 20 strains with higher FA content were selected to determine the ferulic acid esterase activity. The culture method and specific conditions are as follows: 56 strains were inoculated in 96 deep-well plates, the contents of the deep-well plates were LB liquid culture medium, and the inoculation amount was 4% v / v (the initial inoculation concentration was 1×10 6 CFU / mL), 600 rpm, temperature 37 ° C, after culturing for 36 h, the OD of 56 strains was 600 Sterile water was used to adjust the concentration to 0.8, and then inoculated into the liquid screening medium containing ferulic acid esterase, and the inoculation volume was 4% v / v (the initial inoculation concentration was 1×10 6 CFU / mL), 600 rpm, temperature 37°C, culture for 36 h.

[0070] Determination of FA content of 56 strains Figure 1 As shown, strain A-4H was able to produce 153.79±2.36 mg / L of FA after 36 h of culture, and strains A-10, Sb, A-6, and A-1H also produced relatively high levels of FA, 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.

[0071] The ferulic acid esterase activity of the fermentation broth of the first 20 strains was determined. Figure 2 The ferulic acid esterase activity of the fermentation broth of strain A-4H was 136.63±10.27U / L.

[0072] 2. Physiological and biochemical identification of strains

[0073] (1) Morphological characteristics of strains

[0074] The strain A-4H was inoculated in LB medium. The colonies formed were dot-shaped, waxy, opaque, light yellow, with raised surfaces and neat edges. Under a 1000x microscope, the A-4H bacteria were rod-shaped, appearing singly or in short chains ( Figure 3 ).

[0075] (2) DNA extraction and bacterial identification

[0076] The DNA of the strains was extracted using the CATB method, and PCR amplification and sequencing analysis were performed. The 16S rRNA gene of the selected strains was amplified using the bacterial universal primers 27F / 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'5'-TACGGTTACCTTGTTACGACTT-3'-3'), and the phylogenetic tree was generated using MEGA11 software. Figure 4 As shown, the results showed that A-4H belonged to the genus Pseudomonas.

[0077] The Pseudomonas sp. A-4H was deposited in the China Center for Type Culture Collection on January 2, 2025, with a deposit number of CCTCC NO: M 2025002, and the deposit address is Wuhan University, Wuhan, China.

[0078] Example 2: Characteristics of Pseudomonas sp. A-4H

[0079] (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 anhydrous ethanol was added to make the final ethanol concentration in the LB liquid medium 0, 2, 4, 6, and 8% vol. Then, the inoculum size was adjusted to 1% (10 6 CFU / mL) were inoculated with Pseudomonas bacterial liquid and cultured at 37°C for 6 days. Each test was repeated 3 times, and the dry weight of mycelium was measured after the experiment.

[0080] (2) Reducing sugar tolerance: Reducing sugar was added to LB liquid medium at 90, 70, 50 and 30 g / L, and the mixture was dispensed into 150 mL Erlenmeyer flasks. The mixture was sterilized at 115 °C for 20 min and cooled to room temperature. The mixture was then stirred at 1% (10 6 CFU / mL) was inoculated into Pseudomonas liquid and cultured at 37°C for 6 days. Each experiment was repeated 3 times and the dry weight of mycelium was measured.

[0081] (3) Lactic acid tolerance: LB liquid medium was dispensed into 150 mL Erlenmeyer flasks, shaken, sterilized at 115°C for 20 min, cooled to room temperature, and then filtered and sterilized lactic acid was added to make the final concentration of lactic acid in the LB liquid medium 0, 2, 4, 6, and 8 g / L. Then, 1% (1×10 6 CFU / mL) was inoculated into Pseudomonas liquid and cultured at 37°C for 6 days. Each test was repeated 3 times, and the dry weight of mycelium was measured after the experiment.

[0082] (4) Temperature tolerance: The LB liquid culture medium was dispensed into 150 mL Erlenmeyer flasks, shaken, and sterilized at 115 °C for 20 min. When the LB liquid culture medium cooled to room temperature, the medium was heated to 1% (1 × 10 6 The inoculum size of 1000 μg / mL was inoculated with Pseudomonas liquid and cultured at 37, 45, 50 and 55 °C for 6 days. Each test was repeated 3 times and the dry weight of mycelium was measured after the experiment.

[0083] The results showed that the Pseudomonas A-4H could grow under the conditions of 0-8% vol alcohol, 30-90 g / L reducing sugar, 0-8 g / L lactic acid, and 37-55°C, and the growth state was good under the conditions of 0-2% vol alcohol, 0-2 g / L lactic acid, 50-70 g / L reducing sugar, and 37-45°C. Figure 5 ).

[0084] Example 3: Pseudomonas sp. A-4H fortified malt

[0085] (1) Preparation of Pseudomonas sp. A-4H seed solution:

[0086] Pseudomonas A-4H was inoculated into LB liquid medium and cultured for 36 to 48 hours at 37°C and 180 rpm to obtain a primary seed solution. Then, it was inoculated into fresh medium at a 4% inoculum and cultured for 36 to 48 hours to obtain a secondary seed solution. The concentration was then adjusted to 1×10 6 Pieces / mL.

[0087] (2) Production of Pseudomonas sp. A-4H fortified malt

[0088] The production process of hand-fortified block koji is to select high-quality wheat of the year, wash it briefly, and then evenly grind it into 5-6 pieces. Mix 2275g of the grinded wheat with 9.5% bran (w / w), and add 750mL of 80℃ water. When the temperature drops to 32-35℃, add 6% v / m seed liquid (where the ratio of inoculated live bacteria count P.oxalicum M1816:Pseudomonas=4:1, and the initial total live bacteria count of inoculation is 1×106 CFU / mL, the total water content was controlled at 36%), and the cells were fortified with Pseudomonas A-4H, Pseudomonas A-10, and Pseudomonas Sb, respectively. The unfortified group was inoculated with only 1×10 6 CFU / mL of oxalicum Penicillium M1816 was used to make wheat koji. After rapid mixing, the inoculated wheat koji was pressed into shape using a mold (28.0cm×18.0cm×10.0cm). After the block koji was formed, it was placed for 30 to 45 minutes and then transferred to a room dedicated to solid fermentation. The fermentation parameters of the block koji are shown in Table 1. After the fermentation, the enzyme activity of the block koji was measured as shown in Table 2. The A-4H enhanced group had good enzyme activity, with high saccharification power, liquefaction power, xylan and cellulose, and ferulic acid esterase activity.

[0089] Table 1 Block koji fermentation parameters

[0090]

[0091] Table 2 Basic physical and chemical indicators of malt koji

[0092]

[0093]

[0094] The inoculation ratio of Penicillium oxalicum M1816 and Pseudomonas A-4H in the enhanced group was adjusted to 1:1-8:1. When the inoculation ratio was 8:1, the saccharification power of malt 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, the inoculation ratio of Penicillium oxalicum M1816: Pseudomonas A-4H was selected as 4:1 for subsequent experiments.

[0095] Example 4: Application of fortified block koji in the production of yellow rice wine

[0096] Yeast activation: streak the saccharomyces cerevisiae RWBLY1906 SHL in the glycerol tube on a plate in a sterile operating table and culture it in a 28°C incubator for 48 hours. Inoculate the single colony of yeast on the plate into the YPD medium in a sterile operating table and culture it at 30°C for 36 to 48 hours. The temperature is controlled at 28 to 30°C and does not exceed 30°C. It can be used as a YPD bacterial solution feed.

[0097] Preparation of yeast: Steamed rice was mixed with liquefaction enzyme (700U / g rice), saccharification enzyme (5U / g rice) and malt koji (0.1U / g rice) to prepare saccharification solution; then the mixture was placed in a 60℃ water bath for 6 hours. After saccharification, the mixture was filtered and sterilized in triangular bottles. 6The yeast was inoculated with an initial inoculum of 1000 CFU / mL and cultured at a constant temperature for 36-48 hours. The ingredient system of rice wine is shown in Table 3. The total fermentation time is 20 days. The first 5 days are the pre-fermentation, the temperature is controlled at 28℃, and the rake is opened twice a day. The post-fermentation lasts for 15 days, and the temperature is controlled at 15℃.

[0098] Table 3 Ingredients for brewing rice wine

[0099]

[0100] Determination of physical and chemical indicators of rice wine:

[0101] There was no significant difference in alcohol content and total acid content between the malt koji fortified with strain A-4H and the unfortified malt koji, and the total sugar content of the fortified malt koji was significantly higher than that of the unfortified malt koji (Table 4). The basic physical and chemical indicators of rice wine brewed with fortified and unfortified malt koji met the physical and chemical requirements of rice wine in GB / T13662-2018.

[0102] Table 4 Basic physical and chemical indicators of rice wine

[0103]

[0104] By tracking the changes in ferulic acid content during the fermentation process, it can be found that the ferulic acid content in the fortified group and the unfortified group has the same trend in the fermentation process, which is a rapid increase followed by a decrease and finally a flattening. The ferulic acid content in the fortified group of rice wine has been significantly higher than that in the unfortified group throughout the fermentation process (Table 5).

[0105] Table 5 Changes in ferulic acid content during fermentation

[0106]

[0107] The changes in total phenol content during the fermentation process are shown in Table 6. The changes in total phenol content during the fermentation process were consistent with the trend of ferulic acid content, which increased rapidly, then decreased and finally leveled off. The total phenol content of the rice wine brewed by the A-4H enhanced group was always higher than that of the unenhanced group during the fermentation process. At the end of fermentation, the total phenol content of the A-4H enhanced group was about 1.15 times that of the unenhanced group.

[0108] Table 6 Changes of total phenol content during fermentation

[0109]

[0110] At the end of fermentation, the phenolic acid content of rice wine in the A-4H enhanced group and the unenhanced group was measured as shown in Figure 7. The contents of vanillic acid, syringic acid, protocatechuic acid, p-coumaric acid, chlorogenic acid and 4-vinylguaiacol in the rice wine brewed by the A-4H enhanced group were significantly higher than those in the unenhanced group. Among them, coumaric acid, protocatechuic acid, vanillic acid and syringic acid may give rice wine anti-hypertensive properties and effectively improve the efficacy of rice wine.

[0111] Table 7 Content of other phenolic acids at the end of fermentation

[0112]

[0113]

[0114] After fermentation, the lactic acid, acetic acid and citric acid contents of the A-4H enhanced group were higher than those of the unenhanced group, and the oxalic acid content was lower than that of the unenhanced group (Table 8). Organic acids play an important role in determining the taste, color and flavor of rice wine. Lactic acid has a soft sour taste and good sour quality. Succinic acid is the most complex organic acid with a soft sour taste. It becomes stronger after entering the mouth, first salty and then bitter, and can induce saliva secretion. Therefore, rice wine fermented by Pseudomonas A-4H also produces a better effect in taste.

[0115] Table 8 Organic acid content after fermentation

[0116]

[0117] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

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

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

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

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

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

6. The method according to claim 5, characterized in that The ferulic acid ester includes methyl ferulate, ethyl ferulate, oligosaccharide ferulate or polysaccharide ferulate.

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

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

9. The method according to claim 8, characterized in that Penicillium oxalicum M1816 is also added to the rice wine raw materials.

10. The method according to claim 9, characterized in that In the yellow wine raw material, the amount of Pseudomonas A-4H added is not less than 1×10 6 CFU / mL.

11. The method according to claim 10, characterized in that: In the rice wine raw material, the cell ratio of the added Pseudomonas A-4H and Penicillium oxalicum M1816 is (1-8):

1.

12. The method according to claim 10, characterized in that: Ferment at 15-28°C for at least 20 days.

13. Use of the Pseudomonas A-4H according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of fermented food.

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