Red rice milk fermented rice koji based on biological fermentation and preparation method of red rice milk fermented rice koji

Abstract

The invention discloses red rice milk fermented rice koji based on biological fermentation and a preparation method of the red rice milk fermented rice koji. The red rice milk fermented rice koji is prepared by inoculating a culture base material with a composite strain containing Hahndrikia coagulans YTQ15, and fermenting. The hatherdrikia coagulans YTQ15 is an excellent strain with strong amylase production capacity and high temperature tolerance, and can form a composite strain with rhizopus oryzae, saccharomyces cerevisiae and monascus purpureus, and the composite strain and the rhizopus oryzae, the saccharomyces cerevisiae and the monascus purpureus jointly ferment a culture base material composed of rice bran, lotus seed hulls and rice flour to prepare the rice koji. The prepared rice koji has good liquefying and saccharifying effects on brown rice and other raw materials, and the alcoholization level is low; the rice koji is stable in quality and convenient to use, a rice milk product prepared from the rice koji has bright natural monascus red color and has both rice fragrance and fermentation fragrance, and the ethanol content is 0.5% or below.

Description

Technical Field

[0001] The invention belongs to the technical field of food microbial product production, and particularly relates to red rice milk fermented rice koji based on biological fermentation and a preparation method thereof. Background Art

[0002] With the improvement of modern living standards, people's demand for health and nutrition is growing; in addition, the introduction of the big food concept means that people's diets are more diverse, with a decrease in staple food intake and an increase in side dishes. Coarse grain products are increasingly attracting widespread attention from consumers because they are rich in dietary fiber, vitamins, minerals and trace elements, especially products made from brown rice, coix seeds, etc. Among them, rice milk is a product made by hydrolyzing macromolecules such as starch and protein in these raw materials into small molecular structures through modern process technology. It has the characteristics of delicate taste and easy digestion, and has rich nutritional value. It can supplement the nutritional deficiencies of milk powder, so it has broad market prospects.

[0003] At present, the processing technology of rice milk is divided into non-enzymatic method, enzymatic method and fermentation method, wherein the fermentation method is to utilize the effect of enzyme produced by microbial growth metabolism, and the macromolecular substances in the raw material are hydrolyzed, thereby giving rice milk rich nutrition and unique flavor, without adding auxiliary materials or additives such as sweetener, skim milk powder, sodium carboxymethyl cellulose, and having advantages that enzymatic method and non-enzymatic method cannot match. However, the microbial strains used at present are mostly limited to lactic acid bacteria or yeast, and the types are relatively single, and the early stage process still needs amylase and saccharifying enzyme to gelatinize, and the process is complicated. In addition, the addition of composite strains mostly needs to be activated and added respectively, and the process is complicated and the processing cycle is long. Summary of the invention

[0004] The invention aims to provide a red rice milk fermented rice koji based on biological fermentation and a preparation method thereof.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A red rice milk fermented rice koji based on biological fermentation is prepared by fermentation with glutinous rice flour, rice bran and lotus seed shell as base materials, inoculated with a composite strain consisting of Heyndrickxia coagulans YTQ15, Rhizopus oryzae 40469, Saccharomyces cerevisiae JH301 and Monascus purpurogenus FJMR36. The Heyndrickxia coagulans YTQ15, whose scientific name is Heyndrickxiacoagulans, has been deposited in the China Center for Type Culture Collection in Wuhan, with a deposit number of CCTCC NO: M20242065 and a deposit date of September 25, 2024.

[0007] The preparation method of the red rice milk fermented rice koji based on biological fermentation is as follows:

[0008] 1) Base material preparation

[0009] The components are weighed and mixed evenly according to the mass ratio of glutinous rice flour: rice bran: lotus seed shell: CaCO3 = 6:1:1:0.05 (on a dry basis), wherein the lotus seed shell needs to be crushed and passed through a 40-mesh sieve; RO water is added to the mixed base material, and the material is fully stirred to make the water content of the material 40%, and the material is allowed to stand for 0.5-1.0h, and sterilized at 105°C for 30min to obtain a culture medium.

[0010] 2) Preparation of bacterial strains

[0011] The coagulans Hendrixella YTQ15, Rhizopus oryzae 40469, Saccharomyces cerevisiae JH301 and Monascus purpurogenus FJMR36 were activated and expanded to prepare solid bacterial agents;

[0012] 3) Vaccination

[0013] Inoculum amount of bacterial strains: Rhizopus oryzae 40469 0.125%, Hyndrickella coagulans YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, Monascus purpurogenum FJMR36 0.1%;

[0014] Weigh each bacterial agent according to the inoculum amount, mix them evenly, and then stir them evenly into the sterilized culture medium to obtain the inoculum material.

[0015] 4) Fermentation

[0016] The inoculated material is evenly piled on the fermentation shelf (thickness 4-5cm), the temperature is controlled at 33-35℃, and the humidity is 90-95%; after 12-15 hours, the material is evenly spread (thickness 2.0-2.5cm), the temperature is controlled at 32±2℃, and the culture is continued for 70-72 hours, during which time it is turned over every 24 hours; then it is statically cultured for 94-96 hours.

[0017] 5) Drying and storage

[0018] The materials are collected, moved into an oven, and dried at 39-40° C. until the moisture content is below 10% to obtain rice koji; the materials are vacuum packed and stored at 4-6° C. in the dark.

[0019] The present invention adopts the above technical scheme, takes rice bran, lotus seed shell and sticky rice flour as culture medium, takes coagulans YTQ15, Rhizopus oryzae 40469, Saccharomyces cerevisiae JH301 and Monascus purpureus FJMR36 as strains, and ferments to prepare rice koji. The composite strain of fermented rice koji contains coagulans YTQ15, Monascus purpureus, Rhizopus oryzae and Saccharomyces cerevisiae, is a composite fermentation agent rich in multiple enzymes and probiotics, and is also the driving force for decomposing starch, protein and other components in raw materials to form the appearance, aroma and nutrients of rice milk. The rice koji prepared by the present invention has stable performance, convenient use and high bioconversion rate, has good liquefaction and saccharification effects on raw materials such as brown rice, and has a low alcoholization level. The ethanol content of rice milk prepared by the rice koji is less than 0.5%, and in addition to having rice fragrance, fermentation fragrance and rich nutrients, it also has bright natural red red color of Monascus purpureus, and can increase the physiological active components of the product Monacolin k and the like. Therefore, the rice koji of the present invention can be used to produce probiotic high-value nutritious rice milk. In addition, the rice koji uses rice bran, lotus seed shells, etc. as natural proliferation factors, which can effectively reduce production costs while maintaining the amount of live bacteria to the greatest extent, and realize the high-value utilization of by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the enzyme production ability of the isolated strain on the plate.

[0021] Figure 2 It is the enzyme production capacity of the isolated strain in liquid culture medium.

[0022] Figure 3 is the growth amount of the isolated strain at different temperatures.

[0023] Figure 4 This is the colony morphology of strain YTQ15.

[0024] Figure 5 This is the Gram staining result of strain YTQ15 (light microscope, ×1000).

[0025] Figure 6 This is the phylogenetic tree of strain YTQ15 based on 16S rDNA gene sequence analysis.

[0026] Figure 7 It refers to the saccharification and liquefaction power of rice koji under different bacterial strain ratios.

[0027] Figure 8 It is the saccharification power and liquefaction power of rice koji under different material ratios.

[0028] Fig. 9 It is the effect of different culture conditions on the saccharification and liquefaction power of rice koji.

[0029] Fig.10It is a schematic diagram of the process flow of rice koji of the present invention. DETAILED DESCRIPTION

[0030] Example 1 Isolation, screening, identification and preservation of strains

[0031] 1 Materials and methods

[0032] 1.1 Materials

[0033] 1.1.1 Isolation samples and sources

[0034] (1) Sweet wine koji: collected by laboratory staff from a winery in Fujian.

[0035] 1.1.2 Culture medium

[0036] (1) LB medium: finished product, produced by Qingdao Haibo Biotechnology Co., Ltd.

[0037] (2) Selective culture medium: LB as the basic culture medium, supplemented with 1% soluble starch and 2% agar.

[0038] (3) Fermentation medium: peptone 1%, NaCl 1%, soluble starch 1%, pH 7.0.

[0039] 1.2 Methods

[0040] 1.2.1 Strain isolation and purification

[0041] Aseptically homogenize the sweet wine koji sample, take 5g and put it in 45mL sterile water, shake it on a shaker at 150r / min for 1-2h, and then treat it in an 80℃ water bath for 30min. Use the 10-fold gradient dilution spreading method to spread the appropriate dilution gradient (so that the number of colonies on each plate is between 10 and 50) on the LB medium plate, culture it at 37℃ for 24h, observe the morphological characteristics of the colonies (such as color, size, integrity of the edge of the colony, etc.), pick the strains that resemble spore-forming bacteria, inoculate them in another blank LB medium plate and continue to culture them, repeat several times until the strains are completely purified; transfer them to the LB slant, culture them at 37℃ for 24h, and store them in a refrigerator at 4℃ for standby use.

[0042] 1.2.2 Screening of high-yield amylase strains

[0043] (1) Evaluation of amylase production capacity

[0044] The purified isolated strain was made into a bacterial suspension, which was appropriately diluted and spread on a selective culture medium so that 3-5 colonies grew on each plate. The plate was cultured at 37°C for 24 hours. Iodine solution was added to the plate, and the hydrolysis zone diameter (H) and colony diameter (C) around the colonies were measured. The ratio of the two, i.e., H / C, was used to preliminarily determine the enzyme production capacity of the strain.

[0045] On the basis of the above experiment, we selected strains with larger H / C, activated them and inoculated them in equal amounts into fermentation medium, and cultured them at 37°C and 150r / min for 48h; took 40mL of fermentation broth in a 50mL centrifuge tube, centrifuged it at 4°C and 9000r / min for 10min, took the supernatant, and used the kit method to determine the amylase activity, and analyzed the enzyme production capacity of the strain according to the enzyme activity. Among them, the definition of amylase activity is: the amylase in 1mL of enzyme solution liquefies 1mg of soluble starch in 1h at 40°C and pH 6.0, which is defined as 1 activity unit, expressed as U / mL.

[0046] (2) Temperature tolerance evaluation

[0047] The high-yield amylase strain was used as the starting bacteria, and the seed solution was obtained by activation with LB liquid medium. The seed solution of each strain was uniformly diluted to OD 600nm The concentration of the culture medium was 0.5, and then 10% was inoculated into fresh LB liquid culture medium. The temperature was set to 40, 45, 50, 55, and 60°C, the shaking speed was 180 r / min, and the culture was carried out for 24 hours. The amount of bacteria in each treatment was determined, and the adaptability of the strain to the temperature was analyzed.

[0048] 1.2.3 Strain identification

[0049] (1) Morphological observation

[0050] The colony morphology of the strain was observed with reference to the Bergey's Manual of Bacterial Identification (8th edition); the morphological characteristics of the bacterial cells were observed under a microscope after Gram staining.

[0051] (2) Molecular Biological Identification

[0052] The genomic DNA of the target strain was extracted using a bacterial gene DNA extraction kit, and the genomic DNA was used as a template. The primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-CTACGGCTAC CTTGTTACGA-3') were used for PCR amplification, and the PCR amplification products were detected by 1% agarose gel electrophoresis. The purified PCR amplification products were sent to Shangya Biotechnology (Fuzhou) Co., Ltd. for DNA sequencing. The obtained sequences were compared with BLAST in the GenBank database of NCBI. The 16S rDNA gene sequences of strains with higher homology were selected, and the neighbor-joining (NJ) method in MEGA4.0 software was used to construct a phylogenetic tree.

[0053] 2 Results and analysis

[0054] 2.1 Isolation and screening of strains with high amylase activity

[0055] A total of 16 pure strains of spore-like bacteria were isolated from the collected samples, numbered YTQ11-17, YTQ21-25, and YTQ31-34. The utilization of starch by each strain is shown in the following table. Figure 1 As shown. On the selective medium plate, the ratio of the decomposition circle to the colony diameter of each strain was significantly different (p < 0.05). The ratios of strains YTQ16, YTQ12 and YTQ15 were relatively high, and the differences were not large; the ratios of YTQ22 and YTQ31 were slightly lower, but both were above 2.90, indicating that these strains have strong enzyme production capabilities.

[0056] The enzyme production capacity of the five strains YTQ12, YTQ15, YTQ16, YTQ22 and YTQ31 in liquid culture medium was further analyzed. Figure 2 The results were slightly different from the above test results, but the activities of the three strains YTQ12, YTQ15, and YTQ16 were still relatively high, and significantly different from the other strains (p < 0.05); among them, YTQ12 had the highest amylase activity, followed by YTQ15 and YTQ16, with no significant difference between the two.

[0057] The temperature tolerance of YTQ12, YTQ15 and YTQ16 was evaluated. Figure 3 . At 40-45℃, the bacterial counts of the three strains were relatively close; when the temperature continued to rise, the bacterial counts of YTQ12 and YTQ16 decreased significantly faster than that of YTQ15, indicating that YTQ15 has stronger temperature tolerance. Therefore, YTQ15 is an excellent strain with strong amylase production ability and high temperature tolerance. Applying it to the preparation of rice milk is expected to improve the utilization rate of raw materials and save costs.

[0058] 2.2 Species identification of strain YTQ15

[0059] The colony morphology of strain YTQ15 after being cultured on LB plate at 37℃ for 24h is as follows Figure 4 YTQ15 colonies are round, light yellow, smooth and moist, with neat edges and a protrusion in the middle. The bacteria are blue-purple after Gram staining ( Figure 5 ), indicating that it is a Gram-positive bacterium; the bacteria are rod-shaped and arranged in pairs or chains.

[0060] The phylogenetic tree of strain YTQ15 was constructed based on the 16S rDNA gene sequence. Figure 6Strain YTQ15 and Heyndrickxia coagulans are clustered in one branch and have the closest relationship. Combined with the observation of colony morphology, strain YTQ15 can be identified as Heyndrickxia coagulans. According to literature reports, Heyndrickxia coagulans was originally named Bacillus coagulans and is a probiotic that can be used in food.

[0061] Heyndrickxia coagulans YTQ15 was deposited in the China Center for Type Culture Collection in Wuhan, with the deposit number CCTCC NO:M 20242065 and the deposit date of September 25, 2024.

[0062] Example 2 Study on the preparation process of rice koji

[0063] The determination or preparation method involved in this embodiment is as follows:

[0064] (1) Rice milk preparation method

[0065] The raw rice is soaked for 3-4 hours, drained, water (twice the amount of rice) is added, sterilized at 105° C. for 30 minutes, and after appropriate cooling, 1.0-1.5% of rice koji is added, and the mixture is water-bathed at 55-60° C. for 10-12 hours; the rice milk is obtained by squeezing and filtering, and the content of soluble solids (hereinafter referred to as solids) and other indicators are determined.

[0066] (2) Evaluation method of liquefaction and saccharification capacity

[0067] The saccharification power and liquefaction power are used to evaluate the fermentation capacity of rice koji. The saccharification power is carried out according to the iodine titration method in QB / T 5188-2017 "Brewing Red Koji", and the liquefaction power is carried out according to QB / T 4257-2011 "General Analysis Method for Brewing Red Koji".

[0068] (3) Determination of moisture content

[0069] The direct drying method in GB 5009.3-2016 “Determination of water content in food” was adopted.

[0070] (4) pH determination

[0071] The first method in GB 12456-2021 "Determination of total acid in food", i.e., the acid-base indicator titration method, is adopted.

[0072] (5) Bacterial count determination

[0073] The plate count method was used.

[0074] (6) Ethanol content

[0075] Determined according to the method specified in GB / T 12143.

[0076] (7) Sensory evaluation method

[0077] Referring to GB / T 10220 "General Theory of Sensory Analysis Methodology" and other methods, 8 trained sensory assessors were selected to describe the profile of brown rice milk according to the evaluation criteria shown in Table 1, and color, tissue state, flavor, texture, and overall acceptability were selected as sensory descriptors. The sum of the average score of each item and the product of the corresponding weight was the final sensory score of the brown rice milk, where 1 point indicated extremely poor, 5 points indicated moderate, and 9 points indicated very good; the full score was 9 points, and the higher the score, the better the sensory quality.

[0078] Table 1 Sensory evaluation standards for brown rice milk

[0079]

[0080]

[0081] 1 Materials

[0082] 1.1 Bacteria strains and sources

[0083] (1) Monascus purpureus FJMR36, a self-isolated strain, with a deposit number of CCTCCNO: M2017313, which has been disclosed in Chinese patent CN108034591B.

[0084] (2) Rhizopus oryzae 40469, purchased from China Industrial Microbiological Culture Collection Center.

[0085] (3) Saccharomyces cerevisiae JH301, a self-isolated strain, with a deposit number of CCTCCNO: M 2015226, which has been disclosed in Chinese patent CN105176854B.

[0086] (4) Heyndrickxia coagulans YTQ15, a self-isolated strain, was deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20242065.

[0087] 1.2 Culture medium

[0088] (1) Medium M1: 1 L 10° malt extract, 2% agar, pH 5.4, sterilized at 121°C for 15 min.

[0089] (2) Medium M2: 2% glucose, 2% trypsin, 1% yeast extract, sterilized at 121°C for 20 min.

[0090] (3) Medium M3: 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0, sterilized at 121°C for 15 min.

[0091] (4) Medium M4: rice bran, water content 40%, sterilized at 105°C for 30 min.

[0092] (5) Medium M5: Indica rice was soaked in 0.9% lactic acid overnight, rinsed and drained, and sterilized at 105°C for 30 min.

[0093] 1.3 Raw materials

[0094] Rice bran, glutinous rice flour, lotus seed shell: commercially available, the water content of each raw material is controlled below 10%, and they are sealed and stored.

[0095] 2 Test methods

[0096] 2.1 Preparation of bacterial strains

[0097] (1) Activation and expansion of Rhizopus oryzae: Rhizopus oryzae 40469 was inoculated on the slant of the medium M1 test tube and activated and cultured at 30±2°C for 4-5 days. The spores on the slant were washed off with an appropriate amount of sterile water to prepare 1.0×10 6 / mL spore suspension; inoculate the spore suspension into the medium M4 at 10% (v / m), 30±2°C, 95±2% humidity, and culture for 70-72h; collect the culture material, dry it at 40°C to a moisture content of less than 10%, and obtain the solid bacterial agent of Rhizopus oryzae 40469 (bacterial amount at 10 9 CFU / g), stored in -20℃ refrigerator for future use.

[0098] (2) Activation and expansion of purple Monascus: Inoculate purple Monascus FJMR36 on the slant of the medium M1 test tube and activate and culture at 30±2℃ for 6-7 days. Wash the slant spores with an appropriate amount of sterile water to prepare 1.0×10 6 / mL spore suspension; inoculate the spore suspension into the medium M5 at 10% (v / m), 30±2℃, 95±2% humidity, and culture for 164-168h; collect the culture material, dry it at 40℃ to a moisture content of less than 10%, crush it, pass it through a 60-mesh sieve, and obtain a purple Monascus FJMR36 solid powder (bacterial quantity is 10 8 CFU / g),

[0099] Store in -20℃ refrigerator for future use.

[0100] (3) Activation and expansion of cerevisiae yeast: Saccharomyces cerevisiae JH301 was inoculated into medium M2 and activated and cultured at 28±2°C for 22-24 h. Then, the inoculum was transferred to fresh medium M2 at a rate of 10% (v / v) and cultured at 28±2°C for 22-24 h. The cells were centrifuged and mixed with a protective agent, granulated, and dried to prepare cerevisiae JH301 inoculum granules (with a bacterial count of 10 8 CFU / g), stored in -20℃ refrigerator for future use.

[0101] (4) Activation and expansion of coagulant Hyndrickella YTQ15: Coagulant Hyndrickella YTQ15 was inoculated into medium M3 and activated and cultured at 37±2°C for 18-24 h. Then, the inoculum was transferred to fresh medium M3 at a 10% (v / v) inoculum volume and expanded and cultured at 37±2°C for 18-24 h. The cells were centrifuged and mixed with a protective agent, granulated, and freeze-dried to prepare coagulant Hyndrickella YTQ15 inoculum granules (the bacterial amount was 10 9 CFU / g), stored in -20℃ refrigerator for future use.

[0102] 2.2 Study on the proportion of compound bacteria added

[0103] On the basis of previous research, with improving raw material utilization as the main target, Rhizopus oryzae 40469, Monascus purpurogenus FJMR36, Saccharomyces cerevisiae JH301 and Hyndrickella cerevisiae YTQ15 were selected as composite strains, and the inoculation amounts of Monascus purpurogenus FJMR36 and Saccharomyces cerevisiae JH301 were kept unchanged at 0.1% (referring to 0.1g of Monascus purpurogenus FJMR36 inoculated in 100g of culture material, the same below) and 0.2%, respectively. The inoculation amounts of Rhizopus oryzae 40469 and Hyndrickella cerevisiae YTQ15 were adjusted, and the combined strains were inoculated into glutinous rice flour with a moisture content of 40%, and cultured at 30°C and 95% humidity for 3d to obtain rice koji. After the end, the saccharification and liquefaction abilities of the rice koji treated in each treatment were measured to determine the appropriate addition ratio of each strain.

[0104] The specific processing is as follows:

[0105] FH1: Rhizopus oryzae 40469 0.5%, Hyndrickella coagulans YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, Monascus purpurogenum FJMR36 0.1%;

[0106] FH2: Rhizopus oryzae 40469 0.25%, Hyndrickella coagulans YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, Monascus purpurogenum FJMR36 0.1%;

[0107] FH3: Rhizopus oryzae 40469 0.17%, Hyndrickella coagulans YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, Monascus purpurogenum FJMR36 0.1%;

[0108] FH4: Rhizopus oryzae 40469 0.125%, Hyndrickella coagulans YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, Monascus purpurogenum FJMR36 0.1%;

[0109] FH5: Rhizopus oryzae 40469 0.1%, Hyndrickella coagulans YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, Monascus purpurogenum FJMR36 0.1%.

[0110] The present invention hopes to promote the positive synergistic accumulation of amylase and saccharifying enzyme in rice koji by rationally matching different strains, thereby improving its liquefaction and saccharification capacity. Figure 7 . As the inoculation ratio of Rhizopus oryzae 40469 decreased, the saccharification and liquefaction capacity of rice koji first increased and then decreased. If the inoculation amount of Rhizopus oryzae was too large, it would produce competitive inhibition on other enzyme-producing strains; while if the inoculation amount was too small, it would affect the enzyme production capacity of the bacteria. Therefore, in this experiment, the appropriate inoculation amount of each bacteria was: Rhizopus oryzae 40469 0.125%, Hyndrix coagulans YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, and Monascus purpurogenum FJMR36 0.1%. Under this condition, the liquefaction power and saccharification power of rice koji reached 1.81g / g·h and 1242.6mg / g·h, respectively, which were significantly higher than those of other treatments.

[0111] 2.3 Study on culture medium ratio

[0112] Preliminary experiments have shown that rice bran, glutinous rice flour and lotus seed shells can provide essential nutrients or suitable conditions for the growth of strains. On this basis, the study clarified the appropriate ratio of each component of the culture medium to further improve the growth of bacteria and enzyme activity. The specific treatment is:

[0113] (1) MB1: glutinous rice flour: rice bran: lotus seed shell = 10:1:1;

[0114] (2) MB2: glutinous rice flour: rice bran: lotus seed shell = 8:1:1;

[0115] (3) MB3: glutinous rice flour: rice bran: lotus seed shell = 6:1:1;

[0116] (4) MB4: glutinous rice flour: rice bran: lotus seed shell = 4:1:1;

[0117] (5) MB5: glutinous rice flour: rice bran: lotus seed shell = 2:1:1;

[0118] The moisture content of all treatments was 40%, and the lotus seed shell was crushed and passed through a 40-mesh sieve before use. The appropriate bacterial species added in the above test were inoculated (Rhizopus oryzae 40469 0.125%, Hyndrickella coagulans YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, and Monascus purpurogenum FJMR36 0.1%), and cultured at 30°C and 95% humidity for 3 days to obtain rice koji; after the end, the saccharification and liquefaction capacity of the rice koji in each treatment was measured to determine the appropriate culture medium ratio.

[0119] The influence of the ratio of each material in the culture medium on the liquefaction and saccharification capacity of rice koji, such as Figure 8 As shown. The change in the ratio of rice bran, glutinous rice flour and lotus seed shell has little effect on saccharification power, but has a significant effect on liquefaction power; within a certain range, when the ratio of glutinous rice flour decreases, that is, when the ratio of rice bran and lotus seed shell increases relatively, the liquefaction power is significantly improved. The liquefaction and saccharification power of MB3 treatment are the highest, reaching 1.89g / g·h and 1265.8mg / g·h respectively, but there is no significant difference with MB4 treatment. It suggests that the three components play a certain role in the growth of strains. Among them, glutinous rice flour is the main nutrient source, while rice bran and lotus seed shell help to loosen the culture matrix and improve the growth conditions of strains while providing trace elements. Therefore, glutinous rice flour: rice bran: lotus seed shell = 6:1:1 or 4:1:1 can be selected as the culture medium in the future.

[0120] 2.4 Culture condition test

[0121] (1) Effects of temperature, humidity and culture time on rice koji performance

[0122] The culture medium is prepared according to the ratio of glutinous rice flour: rice bran: lotus seed shell = 6:1:1 and inoculated (Rhizopus oryzae 40469 0.125%, Hyndrickella condensa YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, Monascus purpureus FJMR36 0.1%), and different culture conditions are carried out, i.e. different temperatures (25°C, 28°C, 30°C, 32°C, 35°C), or different humidity (85%, 90%, 95%), or different culture time (2d, 3d, 4d). After the culture is completed, the saccharification and liquefaction capacity of the rice koji is measured to determine the appropriate culture parameters. Among them, the water content of the culture medium is 40%; during the temperature gradient test, the humidity is 95% and the time is 3d; during the humidity gradient test, the temperature is 32°C and the time is 3d; during the culture time test, the temperature is 32°C and the humidity is 95%.

[0123] Effects of temperature, humidity and incubation time on the properties of rice koji, such as Fig. 9AC. The liquefaction and saccharification power at 28℃ and 35℃ were both high and had no significant difference, indicating that slightly lower temperatures were conducive to the growth and enzyme production of filamentous fungi such as Rhizopus, and slightly higher temperatures were conducive to the growth and metabolism of spore-forming fungi. At 32℃, the growth of both types of strains was taken into account, so the accumulation of amylase and saccharifying enzyme was the highest. 95% humidity conditions were more conducive to strain growth, but there was no significant difference from 90% conditions; therefore, appropriate humidity parameters could be selected according to product performance requirements. The culture time had a significant effect on the fermentation performance, and the enzyme activity was the highest at 4d, so the culture time needed to be strictly controlled to achieve the maximum accumulation of amylase and saccharifying enzyme.

[0124] (2) Effect of culture medium pH on rice koji performance

[0125] Based on glutinous rice flour: rice bran: lotus seed shell = 6:1:1, different proportions of CaCO3 were added to adjust the pH value of the material, and the moisture content of the culture medium was maintained at 40%. After inoculation, the culture was cultured at 32°C and 95% humidity for 4 days. The saccharification and liquefaction abilities of the rice koji were measured to clarify the effect of pH value on the growth and enzyme production of the strain, as well as the appropriate amount of CaCO3 added.

[0126] The details are as follows:

[0127] a) Glutinous rice flour: rice bran: lotus seed shell: CaCO3 = 6:1:1:0

[0128] b) Glutinous rice flour: rice bran: lotus seed shell: CaCO3 = 6:1:1:0.03

[0129] c) glutinous rice flour: rice bran: lotus seed shell: CaCO3 = 6:1:1:0.05

[0130] d) Glutinous rice flour: rice bran: lotus seed shell: CaCO3 = 6:1:1:0.07

[0131] like Fig. 9 D. The pH value of the natural material combination is acidic. With the increase of CaCO3 addition, the pH value of the culture medium gradually increases. When the pH value is close to neutral, the liquefaction and saccharification ability of rice koji is the highest. At this time, the amount of CaCO3 added satisfies the glutinous rice flour: rice bran: lotus seed shell: CaCO3 = 6:1:1:0.05. When the pH value further rises to alkaline, its liquefaction and saccharification power are lower than that under acidic conditions. This shows that the acidic environment is more conducive to the metabolic accumulation of related enzymes.

[0132] Example 3

[0133] A kind of red rice milk fermented rice koji based on biological fermentation and preparation method thereof

[0134] According to the results of Example 2, the best rice koji preparation process is as follows: 1. Preparation of base material

[0135] Weigh and evenly mix the base material components according to the mass ratio of glutinous rice flour: rice bran: lotus seed shell: CaCO3 = 6:1:1:0.05 (on a dry basis), wherein the lotus seed shell needs to be crushed and passed through a 40-mesh sieve; add RO water to the mixed base material, stir well to make the water content of the material 40%, let it stand for 0.5-1.0h, and sterilize at 105°C for 30min to obtain a culture medium.

[0136] 2. Preparation of bacterial strains

[0137] 2.1 Medium formulation

[0138] (1) Medium M1: 1 L 10° malt extract, 2% agar, pH 5.4, sterilized at 121°C for 15 min.

[0139] (2) Medium M2: 2% glucose, 2% trypsin, 1% yeast extract, sterilized at 121°C for 20 min.

[0140] (3) Medium M3: 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0, sterilized at 121°C for 15 min.

[0141] (4) Medium M4: rice bran, water content 40%, sterilized at 105°C for 30 min.

[0142] (5) Medium M5: Indica rice was soaked in 0.9% lactic acid overnight, rinsed and drained, and sterilized at 105°C for 30 min.

[0143] 2.2 Activation and expansion of bacterial strains

[0144] (1) Activation and expansion of Rhizopus oryzae: Rhizopus oryzae 40469 was inoculated on the slant of the medium M1 test tube and activated and cultured at 30±2°C for 4-5 days. The spores on the slant were washed off with an appropriate amount of sterile water to prepare 1.0×10 6 / mL spore suspension; inoculate the spore suspension into the culture medium M4 at 10% (v / m), 30±2°C, 95±2% humidity, and culture for 70-72h; collect the culture material, dry it at 40°C to a moisture content of less than 10%, obtain the solid bacterial agent of Rhizopus oryzae 40469, and store it in a refrigerator at -20°C for standby use.

[0145] (2) Activation and expansion of purple Monascus: Inoculate purple Monascus FJMR36 on the slant of the medium M1 test tube and activate and culture at 30±2℃ for 6-7 days. Wash the slant spores with an appropriate amount of sterile water to prepare 1.0×10 6 / mL spore suspension; inoculating the spore suspension into culture medium 5 at 10% (v / m), 30±2°C, 95±2% humidity, and culturing for 164-168h; collecting the culture material, drying it at 40°C to a moisture content of less than 10%, crushing it, passing it through a 60-mesh sieve, obtaining a purple Monascus FJMR36 solid powder, and storing it in a refrigerator at -20°C for standby use.

[0146] (3) Activation and expansion of brewer's yeast: Saccharomyces cerevisiae JH301 was inoculated into medium M2 and activated and cultured at 28±2°C for 22-24 h. Then, the inoculum was transferred to fresh medium M2 at a 10% (v / v) inoculation rate and expanded and cultured at 28±2°C for 22-24 h. The cells were collected by centrifugation, mixed with a protective agent, granulated, and dried to prepare Saccharomyces cerevisiae JH301 inoculum granules. The granules were stored in a refrigerator at -20°C for future use.

[0147] (4) Activation and expansion of Bacillus coagulans: Bacillus coagulans YTQ15 was inoculated into medium M3, activated and cultured at 37±2°C for 18-24 h, and then transferred to fresh medium M3 at a 10% (v / v) inoculum amount, expanded and cultured at 37±2°C for 18-24 h, and the cells were collected by centrifugation, mixed with a protective agent, granulated, and vacuum-freeze-dried to prepare Bacillus coagulans YTQ15 inoculum granules, which were stored in a refrigerator at -20°C for later use.

[0148] 3. Vaccination

[0149] The inoculum amount of bacterial strains is: Rhizopus oryzae 40469 0.125%, Hyndrickella coagulans YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, and Monascus purpurogenum FJMR36 0.1%. Each bacterial agent is weighed separately, mixed evenly, and then evenly mixed into the sterilized culture medium to obtain an inoculum material.

[0150] 4. Fermentation

[0151] The inoculated material is evenly piled on the fermentation shelf (thickness 4-5cm), the temperature is controlled at 33-35℃, and the humidity is 90-95%; after 12-15 hours, the material is evenly spread (thickness 2.0-2.5cm), the temperature is controlled at 32±2℃, and the culture is continued for 70-72 hours, during which time it is turned over every 24 hours; then it is statically cultured for 94-96 hours.

[0152] 5. Drying and storage

[0153] The materials are collected, moved into an oven, and dried at 39-40° C. until the moisture content is below 10% to obtain rice koji; the materials are vacuum packed and stored at 4-6° C. in the dark.

[0154] Example 4: Pilot test of rice koji and study on its application effect

[0155] The rice koji prepared in Example 3 was used for pilot testing; a commercially available starter was used as a control to prepare brown rice milk, and its utilization rate and saccharification capacity of different raw materials were detected, and sensory evaluation was performed to analyze the utilization effect of rice koji on raw materials. Wherein, raw material utilization rate (%) = (raw material dry weight - fermentation residue dry weight) / raw material dry weight × 100%.

[0156] The method for preparing brown rice milk is as follows:

[0157] Soak the brown rice for 3-4 hours, drain the water, add water (twice the weight of the brown rice), sterilize at 105° C. for 30 minutes, add 1.0-1.5% of the weight of the brown rice of the rice koji prepared in Example 3 or a commercially available starter after appropriate cooling, and water bath at 55-60° C. for 10-12 hours; obtain brown rice milk by squeezing and filtration.

[0158] After testing, the amount of composite Aspergillus oryzae prepared by the present invention reached 2.1×10 9 cfu / g, liquefaction power is 2.26g / g·h, and saccharification power is 1550.2mg / g·h. The rice koji was applied to brown rice fermentation, and the effect is shown in Table 2. Taking the commercial fermentation agent as the control, it can be seen that the raw material utilization rate and solidification of the homemade rice koji are significantly higher than those of the control group (p < 0.05); it can be seen that the saccharification and liquefaction capabilities of the rice koji prepared by this process are relatively high, which can significantly improve the utilization rate of raw materials and save costs. In addition, the rice milk prepared using the rice koji not only has rice fragrance and fermentation fragrance, but also has a bright red color, that is, it has a higher appearance value; at the same time, the ethanol content in the rice milk product is very low, which is 0.35g / 100g.

[0159] Table 2 Utilization effect of rice koji on brown rice

[0160]

[0161] Note: “ / ” means the red price is too small and not detected.

Claims

1. A red rice milk fermented rice koji based on biological fermentation, characterized in that: It is obtained by inoculating a composite bacterial strain including Heyndrickxia coagulans YTQ15 into a culture medium and fermenting it. The Heyndrickxia coagulans YTQ15, whose scientific name is Heyndrickxia coagulans, has been deposited in the China Center for Type Culture Collection in Wuhan, with a deposit number of CCTCC NO:M 20242065 and a deposit date of September 25, 2024.

2. A red rice milk fermented rice koji based on biological fermentation according to claim 1, characterized in that, The culture medium comprises glutinous rice flour, rice bran and lotus seed shell.

3. A red rice milk fermented rice koji based on biological fermentation according to claim 1, characterized in that, The composite bacterial strain consists of Hyndrickella coagulans YTQ15, Rhizopus oryzae 40469, Saccharomyces cerevisiae JH301 and Monascus purpurogenum FJMR36.

4. A method for preparing red rice milk fermented rice koji based on biological fermentation as claimed in claim 1, characterized in that, The following steps are involved: 1) Base material preparation The glutinous rice flour, rice bran, lotus seed shell and CaCO3 are uniformly mixed to obtain a mixed base material, water is added to the mixed base material, and the mixture is fully stirred to make the water content of the material 40%, and the mixture is allowed to stand for 0.5-1.0h and sterilized to obtain a culture medium. 2) Preparation of bacterial strains The coagulans Hendrixella YTQ15, Rhizopus oryzae 40469, Saccharomyces cerevisiae JH301 and Monascus purpurogenus FJMR36 were activated and expanded to prepare solid bacterial agents; 3) Vaccination Inoculum amount of bacterial strains: Rhizopus oryzae 40469 0.125%, Hyndrickella coagulans YTQ15 0.5%, Saccharomyces cerevisiae JH301 0.2%, Monascus purpurogenum FJMR36 0.1%; Weigh each bacterial agent according to the above inoculation amount, mix them evenly, and then evenly mix them into the sterilized culture medium to obtain an inoculation material; 4) Fermentation The inoculated materials are evenly piled on the fermentation shelf, and the temperature is controlled at 33-35℃ and the humidity is 90-95%; after 12-15 hours, the materials are evenly spread, the temperature is controlled at 32±2℃, and the culture is continued for 70-72 hours, during which the materials are turned over every 24 hours; then the materials are statically cultured for 94-96 hours; 5) Drying The materials are collected, moved into an oven, and dried to obtain rice koji.

5. A method for preparing red rice milk fermented rice koji based on biological fermentation according to claim 3, characterized in that, In step 1), the raw material mass ratio is as follows: glutinous rice flour: rice bran: lotus seed shell: CaCO3 = 6:1:1:0.

05.

6. A method for preparing red rice milk fermented rice koji based on biological fermentation according to claim 3, characterized in that, In step 1), the sterilization is performed at 105° C. for 30 minutes.

7. A method for preparing red rice milk fermented rice koji based on biological fermentation according to claim 3, characterized in that, In step 2), the lotus seed shells are crushed to a particle size of no more than 40 meshes.

8. A method for preparing red rice milk fermented rice koji based on biological fermentation according to claim 3, characterized in that, In step 4), the material is evenly piled on the fermentation rack to a thickness of 4-5 cm; after 12-15 hours, the material is evenly spread to a thickness of 2.0-2.5 cm.

9. A method for preparing red rice milk fermented rice koji based on biological fermentation according to claim 3, characterized in that, In step 5), the temperature of the drying process is 39-40° C., and the drying is performed until the moisture content is below 10%.

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