Flower fragrance type direct vat set rice milk fermented rice koji and preparation method thereof

Abstract

The invention discloses flower fragrance type direct vat set rice milk fermented rice koji and a preparation method thereof. The floral direct vat set rice milk fermented rice koji is prepared by inoculating a compound strain containing rhizopus arrhizus CMR21 into a culture base material and fermenting. Rhizopus arrhizus CMR21 is liquefied, has high saccharifying power and produces flower fragrance, and can be subjected to synergistic co-fermentation with aspergillus oryzae, Ferbienberg's yeast and lactobacillus plantarum to prepare a culture base material consisting of rice flour, bran and tofu skin, so that the flower fragrance type direct vat set rice milk fermented rice koji is prepared. The prepared rice koji is stable in quality, convenient to use, high in raw material utilization rate and low in alcoholization level; the rice milk prepared from the rice koji has unique flower fragrance, has rice fragrance and fermentation fragrance, and has nutritional and probiotic effects.

Description

Technical Field

[0001] The invention belongs to the technical field of food microbial product production, and particularly relates to a flower-scented direct-throw rice milk fermented rice koji and a preparation method thereof. Background Art

[0002] Rice milk is a pure natural plant protein beverage that is rich in nutrition and has certain health functions. The raw materials used to prepare rice milk include rice, brown rice and coix seed. The rice milk prepared from them is not only rich in nutrition and rice flavor, but also does not contain lactose, so it is very friendly to people with lactose intolerance. At present, the processing technology of rice milk is divided into non-enzymatic method, enzymatic method and fermentation method. The non-enzymatic method has a complex process, the starch is easy to age, and the product is unstable, and it is easy to stratify. The enzymatic method is a process in which amylase, saccharifying enzyme or protease is used to enzymatically hydrolyze macromolecules such as starch and protein in cereal raw materials under certain conditions, which can increase the content of small molecular nutrients such as soluble sugar and amino acids, and the nutrition is easier to absorb and the stability is enhanced. However, pure enzyme preparations often lead to a single flavor of beverages and the taste is not easily accepted by the public. Adding ingredients such as white sugar, rice oil, sodium carboxymethyl cellulose, etc., although it can improve the taste of the product and further increase the stability, it loses its natural characteristics. Fermented rice milk is a beverage prepared by inoculating microorganisms in the raw materials and fermenting them. It is rich in nutrition and has a unique flavor. However, the strains currently used are mostly limited to lactic acid bacteria or yeast, the types are relatively single, and the early process still requires amylase and saccharifying enzyme for gelatinization; in addition, most of the composite strains need to be activated separately before being added, the process is complicated and the processing cycle is long. Summary of the invention

[0003] The invention aims to provide a flower-scented direct-throw rice milk fermented rice koji and a preparation method thereof.

[0004] A floral-scented direct-throw rice milk fermented rice koji is prepared by fermentation using glutinous rice flour, bran and bean skin as culture medium, inoculating with a composite strain consisting of Rhizopus arrhizus CMR21, Aspergillus oryzae CICC 2022, Fabianseberlindner yeast JGM9-1 and Lactobacillus plantarum RPC22; the Rhizopus arrhizus CMR21, whose scientific name is Rhizopus arrhizus, has been preserved in the China Center for Type Culture Collection in Wuhan, with a preservation number of CCTCC NO: M 20242064 and a preservation date of September 25, 2024.

[0005] The preparation method of the flower-scented direct-throw rice milk fermented rice koji is as follows:

[0006] 1) Base material preparation

[0007] a. Weigh the components according to the following mass ratio: glutinous rice: bran: bean skin: CaO: water = 6.0:1.0:1.0:0.025:4.8;

[0008] b. Take 90% of the total amount of water to fully dissolve CaO to prepare a CaO solution; sterilize the remaining 10% of the water and subsequently use it to dissolve the composite bacteria;

[0009] c. Slowly add the CaO solution to the solid mixture of bran, rice flour and bean skin, stir evenly while adding, let stand for 0.5-1.0h to allow the solid material to be fully soaked, and then sterilize to obtain a culture medium;

[0010] 2) Preparation of bacterial strains

[0011] Rhizopus arrhizus CMR21, Aspergillus oryzae CICC 2022, Fabianseberlindnerella JGM9-1 and Lactobacillus plantarum RPC22 were activated and expanded to obtain solid bacterial agents;

[0012] 3) Vaccination

[0013] Inoculum amount of bacterial species: Rhizopus arrhizus CMR21 0.55%, Aspergillus oryzae CICC 2022 0.30%, Fabianseber Lindnerella JGM9-1 0.12%, Lactobacillus plantarum RPC22 0.16%;

[0014] Weigh each bacterial agent according to the inoculation amount, dissolve it in the above-mentioned sterilized 10% water, mix it evenly, and then spray it evenly into the culture medium, stirring while spraying to make it fully mixed, to obtain the inoculation material;

[0015] 4) Fermentation

[0016] The inoculated materials are evenly piled on the fermentation rack (thickness 4-5cm), and the temperature is adjusted to 33-35℃ and the humidity is 90±2%; after 12-15 hours, the materials are evenly spread (thickness 2.0-2.5cm), the temperature is adjusted to 30±1℃, and the culture is continued. After that, the materials are turned over every 24 hours until the fermentation is completed. The total fermentation time is 72±2h;

[0017] 5) Drying and storage

[0018] The materials are collected, moved into an oven, and dried at 35-37° C. until the water content is below 10%, thereby obtaining a flower-scented direct-throw rice milk fermented rice koji; the materials are vacuum-packed and stored at 4-6° C. in the dark.

[0019] The invention discloses a kind of flower-scented direct-throw type rice milk fermentation rice koji, which contains Rhizopus arrhizus, Aspergillus oryzae, Fabian Seberlindner's yeast and Lactobacillus plantarum, is a multi-enzyme and multi-bacteria composite microorganism cooperative fermentation system, has strong saccharification and liquefaction ability, and the components such as starch and protein in the material are decomposed under the action of complex enzyme system, so that soluble sugar, amino acid, aromatic compound and the like are effectively released, thereby increasing the nutritional content and aroma of the rice milk prepared by the rice koji. The rice koji is direct-throw type, easy to use and simple to operate. In addition, the rice koji is based on sticky rice flour, bran and bean skin, etc., and realizes the high-value utilization of by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The colony morphology of CMR21 (left) and its microscopic morphology under light microscopy (middle) and electron microscopy (right).

[0021] Figure 2 This is the phylogenetic tree of strain CMR21 based on ITS rDNA gene sequence analysis.

[0022] Figure 3 It is a schematic diagram of the process flow of rice koji of the present invention. DETAILED DESCRIPTION

[0023] Example 1 Isolation, screening, identification and preservation of strains

[0024] 1 Materials and methods

[0025] 1.1 Materials

[0026] 1.1.1 Isolation samples and sources

[0027] (1) Wine mash: collected by laboratory staff from a winery in Fujian.

[0028] 1.1.2 Culture medium

[0029] (1) Rose Bengal culture medium: finished product, produced by Qingdao Haibo Biotechnology Co., Ltd.

[0030] (2) Malt extract medium: finished product, produced by Qingdao Haibo Biotechnology Co., Ltd.

[0031] (3) Fermentation medium: bran and water were mixed in a mass ratio of 1:0.6 and sterilized at 105°C for 30 min.

[0032] 1.2 Methods

[0033] 1.2.1 Strain isolation and purification

[0034] (1) Sample processing

[0035] Homogenize the mash sample by aseptic operation, take 5 g into 45 mL of sterile water, shake on a shaker at 150 r / min for 1-2 h, and set aside.

[0036] (2) Isolation and purification of bacterial strains

[0037] Using the 10-fold gradient dilution spreading method, spread the appropriate dilution gradient (so that the number of colonies on each plate is between 10 and 50) samples on the Bengal red culture medium plate, culture at 28°C for 2-3 days, observe the morphological characteristics of the colonies on the plate (such as color, size, integrity of the colony edge, hyphae length and spore production, etc.), pick out the strains that resemble Rhizopus, and inoculate them into another blank Bengal red culture medium plate for continued culture, repeat several times until the strain is completely purified, and transfer to the malt juice slant, culture at 28°C, and store in a 4°C refrigerator after the hyphae are fully grown and spores are produced for later use.

[0038] 1.2.2 Screening of strains with high saccharification and liquefaction capacity

[0039] (1) Preparation of bran koji

[0040] Scrape the spores of the isolated strain onto sterile water and disperse them with glass beads to make the spore concentration reach 1.0×10 6 The spore liquid was inoculated into the fermentation medium at a volume ratio of 5%, and cultured at 30°C for 3-4 days to obtain the bran koji sample, which was then dried at 35°C for later use.

[0041] (2) Determination of saccharification and liquefaction capacity

[0042] According to QB / T 4257-2011 "General Analysis Method for Brewing Daqu", the saccharifying power and liquefaction power of bran koji were determined.

[0043] (3) Flavor evaluation

[0044] The bran koji made from each isolated strain was inoculated into brown rice at a mass ratio of 1%, fermented at 55-60°C for 12h, filtered with a 60-80 mesh silk cloth to remove the brown rice residue, and the filtrate, i.e., brown rice milk, was obtained. The fermentation flavor was used as an indicator to evaluate the sensory properties of the rice milk, and strains with higher aroma metabolism capacity were selected. Among them, the sensory evaluation classified the samples according to the flavor characteristics, with the first level being a strong fermentation fragrance and floral fragrance, marked as "++++", the second level being a relatively strong fermentation fragrance and floral fragrance, marked as "+++", the third level being a general fermentation fragrance and floral fragrance, marked as "++", and the fourth level being a relatively light fermentation fragrance and floral fragrance, marked as "+". 12 trained professionals were invited to conduct independent evaluations, and the average values ​​were taken for statistical analysis.

[0045] 1.2.3 Strain identification

[0046] (1) Observation of colonies and bacterial characteristics

[0047] The target strain was inoculated on a malt juice plate and cultured at 28°C for 3 days, and the colony morphology was observed every day. At the same time, a small amount of culture was picked and placed under a microscope to observe the mycelium or spore morphology; the strain was preliminarily classified.

[0048] (2) Gene sequence analysis

[0049] Use an inoculation spatula to scrape the isolated strain colonies from the plate into a 1.5 mL centrifuge tube, freeze with liquid nitrogen and grind until the cell wall breaks, extract DNA according to the fungal extraction kit steps and test its purity, and perform PCR amplification and sequencing on qualified samples. Compare the obtained sequences online in databases such as NCBI and EZBIO, download strain-related gene sequences with different similarities, perform cluster analysis using MEGA software, and identify the strain species.

[0050] 2 Results and analysis

[0051] 2.1 Isolation and screening of strains with high saccharification and liquefaction capacity

[0052] Eleven pure strains of Rhizopus were isolated from the collected samples, numbered CMR11-16, CMR21, CMR22, CMR23, CMR31, and CMR32, and the saccharification and liquefaction abilities of the strains were significantly different (p < 0.05) (Table 1). CMR12, CMR13, CMR14, CMR15, CMR16, and CMR21 had strong saccharification and liquefaction abilities, especially CMR12, CMR13, and CMR21, whose saccharification and liquefaction abilities were significantly higher than those of other strains.

[0053] Further evaluation of the fermentation flavor of the three strains CMR12, CMR13 and CMR21 showed that the evaluation grades of each strain were "+++", "++", and "++++", respectively, that is, CMR21 had a strong fermentation aroma and floral aroma, followed by CMR12, and CMR13 had a general fermentation aroma and floral aroma. This shows that CMR21 is expected to improve the utilization rate of raw materials in the preparation of rice milk and enrich the aroma substances of rice milk.

[0054] Table 1 Saccharification and liquefaction ability of isolated strains

[0055]

[0056] 2.2 Analysis of morphology and genetic characteristics of excellent target strains

[0057] (1) Morphological characteristics of CMR21

[0058] like Figure 1As shown in the figure, on the wort plate, the colony of CMR21 is loose and flocculent, the hyphae grow rapidly, gradually spread from the center to the surroundings, and finally cover the entire surface of the plate; the hyphae are white at first, then turn gray-brown, and gray-black spores are produced at the top of the hyphae. The sporangium stalks of CMR21 are solitary or branched, the sporangium is spherical, and the color is yellow to dark brown; the spores are round to oval; the rhizoids are root-like and underdeveloped.

[0059] (2) Gene sequence analysis of CMR21

[0060] The ITS rDNA sequence of CMR21 is 655 bp in length, which was compared and a phylogenetic tree was constructed on NCBI, such as Figure 2 . CMR21 is clustered on the same branch as Rhizopus oryzae (scientific name Rhizopus arrhizus) and Rhizopus arrhizus, and the homology is as high as 100%; therefore, combined with the morphological characteristics, the fungus was identified as Rhizopus arrhizus. Rhizopus arrhizus CMR21 has been deposited in the China Center for Type Culture Collection in Wuhan, with the deposit number CCTCC NO: M20242064 and the deposit date of September 25, 2024.

[0061] Example 2 Study on the preparation process of rice koji

[0062] The measurement or evaluation method involved in this embodiment is as follows:

[0063] (1) Evaluation method of liquefaction and saccharification capacity

[0064] The rice koji is inoculated into brown rice for fermentation, and the saccharification and liquefaction capacity of the rice koji is indicated by the soluble solids (hereinafter referred to as solids) content of the brown rice milk. Specific operation: 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 after appropriate cooling, and water bath at 55-60°C for 10-12 hours; after squeezing and filtering, the brown rice milk is obtained, which is used to determine the soluble solids (hereinafter referred to as solids) content and other indicators.

[0065] (2) Determination of moisture content

[0066] The direct drying method in GB 5009.3-2016 “Determination of water content in food” was adopted.

[0067] (3) pH determination

[0068] The first method in GB 12456-2021 "Determination of total acid in food", i.e., the acid-base indicator titration method, is adopted.

[0069] (4) Bacterial count determination

[0070] The plate count method was used.

[0071] (5) Sensory evaluation method

[0072] 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 2, 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 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.

[0073] Table 2 Sensory evaluation standards for brown rice milk

[0074]

[0075] 1 Test materials

[0076] 1.1 Bacteria strains and sources

[0077] (1) Rhizopus oryzae CMR21, a self-isolated strain, was deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20242064.

[0078] (2) Lactobacillus plantarum RPC22, a self-isolated strain, with a deposit number of CCTCC NO: M 2019297, which has been disclosed in Chinese patent CN112553124B.

[0079] (3) Cyberlindnera fabianii JGM9-1, a self-isolated strain, with a deposit number of CCTCC NO: M 20221401, which has been disclosed in Chinese patent application CN117004499A.

[0080] (4) Aspergillus oryzae CICC 2022, purchased from China Industrial Microbiological Culture Collection Center.

[0081] 1.2 Culture medium

[0082] (1) Medium M1: 1 L 10° malt extract, 18-20 g agar, pH 5.4, sterilized at 121°C for 15 min.

[0083] (2) Medium M2: 2% glucose, 2% tryptone, 1% yeast extract, sterilized at 115°C for 15 min.

[0084] (3) Culture medium M3: 0.25% citric acid, 5% trypsin, 0.1% K2HPO4, 10% molasses, pH 6.5, sterilized at 121°C for 15 min.

[0085] (4) Medium M4: bran, water content 37.4%, sterilized at 105°C for 30 min.

[0086] 1.3 Raw materials

[0087] Bran, glutinous rice flour, and bean curd skin: commercially available, the moisture content of each raw material is controlled below 10%, and they are sealed and stored.

[0088] 2 Test methods

[0089] 2.1 Preparation of bacterial strains

[0090] (1) Activation and expansion of Rhizopus arrhizus: Inoculate Rhizopus arrhizus CMR21 on the slant of the culture medium M1 test tube and activate and culture at 30±2℃ for 4-5 days. Wash the slant spores with an appropriate amount of sterile water to obtain 1.0×10 6 The spore suspension is prepared by mixing spores of each mL and inoculating the spore suspension into a culture medium M4 at 10% (v / m) at 30±2° C. and 95±2% humidity for 70-72 hours. The culture material is collected and dried at 40° C. to obtain a solid bacterial agent of Rhizopus arrhizus CMR21, which is then stored in a refrigerator at -20° C. for later use.

[0091] (2) Activation and expansion of Aspergillus oryzae: The activation and expansion methods of Aspergillus oryzae CICC 2022 are the same as those of Rhizopus arrhizus CMR21.

[0092] (3) Activation and expansion of Fabianseberlindner yeast: Fabianseberlindner yeast JGM9-1 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) inoculum amount, 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 Fabianseberlindner yeast JGM9-1 inoculum granules, which were stored in a refrigerator at -20°C for future use.

[0093] (4) Activation and expansion of Lactobacillus plantarum: Lactobacillus plantarum RPC22 was inoculated into medium M3, activated and cultured at 30±2°C for 18-20 h, and then transferred to fresh medium M3 at a 10% (v / v) inoculum amount, expanded and cultured at 30±2°C for 18-20 h, and the cells were centrifuged, mixed with a protective agent, granulated, and vacuum-freeze-dried to prepare Lactobacillus plantarum RPC22 inoculum granules, which were stored in a refrigerator at -20°C for future use.

[0094] 2.2 Effects of different bacterial strain combinations on rice koji fermentation performance

[0095] (1) Bacterial strain combination test

[0096] Based on the previous experiments, Aspergillus oryzae CICC 2022, Rhizopus arrhizus CMR21, Fabianseberlindnerella JGM9-1 and Lactobacillus plantarum RPC22 were selected as the strains to be screened. They were inoculated into glutinous rice flour with a moisture content of 37.5% according to the following 6 different combinations. The inoculation amount of each strain in each combination is as follows:

[0097] ZH1: Aspergillus oryzae CICC2022 0.90% (referring to 0.90g CICC2022 inoculated into 100g culture material, the same below);

[0098] ZH2: Rhizopus arrhizus CMR21 0.80%;

[0099] ZH3: Aspergillus oryzae CICC 2022 0.90% + Fabianseber Lindnerella JGM9-1 0.12%;

[0100] ZH4: Rhizopus arrhizus CMR21 0.80% + Lindnerella fabianseber JGM9-1 0.12%;

[0101] ZH5: Aspergillus oryzae CICC 2022 0.45% + Rhizopus arrhizus CMR21 0.40% + Lindnerella fabianseber JGM9-1 0.12%;

[0102] ZH6: Aspergillus oryzae CICC 2022 0.45% + Rhizopus arrhizus CMR21 0.40% + Lindnerella fabianseber JGM9-1 0.12% + Lactobacillus plantarum RPC22 0.16%;

[0103] The inoculated material was cultured at 30°C and 95% humidity for 4 days to obtain rice koji. The saccharification and liquefaction capabilities of each treated rice koji and its contribution to flavor were measured to determine the appropriate bacterial strain combination.

[0104] The effects of different bacterial combinations on the fermentation performance of rice koji are shown in Table 3. The solidification of treatment ZH2 was slightly higher than that of treatment ZH1 (p>0.05), while the sensory evaluation score was just the opposite; this indicates that under the experimental conditions, CMR21 has a higher ability to produce amylase and saccharification enzymes than CICC 2022, and CICC 2022 produces better aroma. After adding Fabianseber Lindner yeast JGM9-1 to the two treatments, the solidification was significantly improved, especially in the ZH4 treatment (p<0.05); this indicates that yeast JGM9-1 can synergistically co-ferment with the two molds, especially CMR21, and has a positive effect on enzyme activity. Compared with treatment ZH5, the solidification of ZH6 treatment is not much different from that of ZH5, but the sensory score is higher than that of ZH5; this indicates that Lactobacillus plantarum RPC22 can also synergistically co-ferment with the other three strains, and its contribution is mainly in flavor formation.

[0105] Table 3 Fermentation performance of rice koji under different bacterial combinations

[0106]

[0107] (2) Test on the proportion of bacterial strains added

[0108] According to the above experiments, it can be found that Rhizopus arrhizus CMR21 and Aspergillus oryzae CICC 2022 are the main sources of amylase and saccharifying enzyme. Therefore, in order to further improve the saccharification and liquefaction ability of rice koji, the experiment further changed the inoculation ratio of the two and conducted experiments with different ratios of strains, namely:

[0109] CK: Rhizopus arrhizus CMR21 0.40%, Aspergillus oryzae CICC 2022 0.45%, Lindnerella fabianseber JGM9-1 0.12%, Lactobacillus plantarum RPC22 0.16%;

[0110] PB1: Rhizopus arrhizus CMR21 0.425%, Aspergillus oryzae CICC 2022 0.425%, Lindnerella fabianseber JGM9-1 0.12%, Lactobacillus plantarum RPC22 0.16%;

[0111] PB2: Rhizopus arrhizus CMR21 0.55%, Aspergillus oryzae CICC 2022 0.30%, Lindnerella fabianseber JGM9-1 0.12%, Lactobacillus plantarum RPC22 0.16%;

[0112] PB3: Rhizopus arrhizus CMR21 0.61%, Aspergillus oryzae CICC 2022 0.24%, Lindnerella fabianseber JGM9-1 0.12%, Lactobacillus plantarum RPC22 0.16%;

[0113] The inoculated material was cultured at 30°C and 95% humidity for 4 days to obtain rice koji. The saccharification and liquefaction capabilities of the rice koji in each treatment were measured to determine the appropriate addition ratio of each strain.

[0114] The results are shown in Table 4. As the inoculation ratio of CMR21 increased, the saccharification and liquefaction capacity of rice koji gradually increased and stabilized. Therefore, the PB2 treatment was an appropriate strain ratio, that is, the inoculation amount of CMR21 was 1.83 times that of CICC 2022. Under this condition, the solid content of rice milk was increased by 9.3% compared with the control.

[0115] Table 4 Effect of inoculation ratio on rice koji fermentation performance

[0116]

[0117]

[0118] 2.3 Effects of different materials and proportions on rice koji fermentation performance

[0119] (1) Nutritional screening

[0120] Sticky rice flour was used as the basic culture medium, and 5% bran, 5% husk, and 5% bean skin (based on the total weight of the culture medium) were added thereto, and the appropriate bacterial species combination and ratio obtained in 2.2 were inoculated (i.e., Rhizopus arrhizus CMR21 0.55%, Aspergillus oryzae CICC 2022 0.30%, Fabianseberlindner yeast JGM9-1 0.12%, and Lactobacillus plantarum RPC22 0.16%), and cultured at 30°C and 95% humidity for 4 days to obtain rice koji. The bacterial amount and saccharification and liquefaction capacity of different treatments were determined, and sensory evaluation was performed to clarify the nutrients suitable for matching with bran.

[0121] As shown in Table 5, compared with the control CK (basal culture medium), the bacterial amount of each treatment increased, among which the bacterial amount of the treatment with bran and bean skin was higher, about 2 times that of CK, and there was no significant difference between the two (p>0.05); from the perspective of saccharification and liquefaction capacity, the treatment effects of adding bran or bean skin were equivalent (p>0.05); in comparison, the effect of rice husk was smaller, and the bacterial agent containing rice husk would affect the taste of rice milk. It is speculated that bran and bean skin can provide more nutrients for the growth of strains, and also have the effect of loosening the culture matrix and thus improving the growth conditions of strains. Therefore, bran and bean skin can be selected as auxiliary materials for sticky rice flour in the future.

[0122] Table 5 Fermentation performance of rice koji treated with different materials

[0123]

[0124] (2) Material ratio test

[0125] Based on the above experiments, the proportion of rice flour, bran and bean skin was further optimized. The proportion of each component of the culture medium was changed as follows:

[0126] PL1: rice flour: bran: bean skin: water = 6.0: 0.6: 0.6: 4.4;

[0127] PL2: rice flour: bran: bean skin: water = 6.0:1.0:1.0:4.8;

[0128] PL3: glutinous rice flour: bran: bean skin: water = 6.0: 1.5: 1.5: 5.4;

[0129] The appropriate bacterial strain combination and ratio obtained in 2.2 were inoculated (i.e., Rhizopus arrhizus CMR21 0.55%, Aspergillus oryzae CICC 2022 0.30%, Fabianseberlindner yeast JGM9-1 0.12%, Lactobacillus plantarum RPC22 0.16%), and cultured at 30°C and 95% humidity for 4 days to obtain rice koji. The bacterial amount, saccharification and liquefaction capacity of different treatments were determined to clarify the appropriate addition amount of each material component.

[0130] The results are shown in Table 6. The bacterial count in PL2 treatment was the highest, close to 1.0×10 10 cfu / g, and its saccharification and liquefaction abilities are also the highest; therefore, the appropriate ratio of glutinous rice flour: bran: bean skin is 6.0:1.0:1.0.

[0131] Table 6 Effect of material ratio on rice koji fermentation performance

[0132]

[0133] 2.4 Culture condition test

[0134] (1) Effect of culture medium pH on rice koji performance

[0135] Based on the above experiments, the culture medium ratio was selected as follows: glutinous rice flour: bran: bean skin: water = 6.0:1.0:1.0:4.8, and CaO was added in different proportions to adjust the pH value of the material. After inoculation, the culture was carried out at a temperature of 30°C and a humidity of 95% for 4 days to obtain rice koji. The saccharification and liquefaction capacity of rice koji was measured to determine the appropriate pH value or CaO addition amount. The details are as follows:

[0136] PL2-1: glutinous rice flour: bran: bean skin: CaO: water = 6.0: 1.0: 1.0: 0: 4.8

[0137] PL2-2: glutinous rice flour: bran: bean skin: CaO: water = 6.0: 1.0: 1.0: 0.014: 4.8

[0138] PL2-3: glutinous rice flour: bran: bean skin: CaO: water = 6.0: 1.0: 1.0: 0.025: 4.8

[0139] PL2-4: glutinous rice flour: bran: bean skin: CaO: water = 6.0: 1.0: 1.0: 0.039: 4.8

[0140] As shown in Table 7, with the increase of CaO addition, the pH of the culture medium gradually increased, while the bacterial count and solid content increased first and then decreased. When the CaO addition ratio was the combination PL2-3, the bacterial count and solid content were the highest, and the pH of the material was close to neutral. The role of CaO in this experiment may be that CaO is converted into Ca(OH)2 after dissolving in water, thereby adjusting the pH value of the culture medium.

[0141] Table 7 Effect of CaO addition on rice koji fermentation performance

[0142]

[0143] (2) Effects of temperature, humidity and culture time on rice koji performance

[0144] Selecting culture medium formula is: sticky rice flour: bran: bean skin: CaO: water=6.0:1.0:1.0:0.025:4.8, after inoculation, carry out different culture condition processing, i.e. different temperatures (28 ℃, 30 ℃, 32 ℃), or different humidity (85%, 90%, 95%), or different incubation time (2d, 3d, 4d), after incubation ends, record rice koji saccharification, liquefaction ability, to determine suitable culture parameter. Wherein, during temperature gradient test, humidity is 95%, and time is 4d; during humidity gradient test, temperature is 30 ℃, and time is 3d; during incubation time test, temperature is 30 ℃, and humidity is 90%.

[0145] As shown in Tables 8-10, different temperatures, humidity or time have significant effects on strain growth and enzyme production. Among them, the strain grows best at 30°C and has the highest saccharification and liquefaction capabilities. 95% humidity conditions are more conducive to strain growth, but the difference in bacterial count and solidification under 90% conditions is not significant; therefore, from the perspective of economic cost, 90% humidity conditions can be selected. After 3 days of cultivation, the bacterial count basically reached its peak. Although the bacterial count remained basically unchanged on the 4th day, the solidification decreased by 0.5%, indicating that the mold began to produce spores in large quantities in the later stage and the enzyme activity decreased, so 3 days should be selected as the appropriate cultivation time.

[0146] Table 8 Effect of culture temperature on rice koji fermentation performance

[0147]

[0148] Table 9 Effect of environmental humidity on rice koji fermentation performance

[0149]

[0150] Table 10 Effect of culture time on rice koji fermentation performance

[0151]

[0152] Embodiment 3 A method for preparing a flower-scented direct-throw rice milk fermented rice koji

[0153] According to the results of Example 2, the best rice koji preparation process is as follows: 1. Preparation of base material

[0154] (1) Culture medium formula: Weigh the components according to the following mass ratio: rice flour: bran: bean skin: CaO: water = 6.0:1.0:1.0:0.025:4.8;

[0155] (2) 90% of the water is used to fully dissolve CaO to prepare a CaO solution; the remaining 10% of the water is sterilized at 121° C. for 30 min, cooled, and subsequently used to dissolve the mixed bacteria.

[0156] (3) Slowly add the CaO solution to the solid mixture of bran, glutinous rice flour and bean skin, stir evenly while adding, let it stand for 0.5-1.0 h to allow the solid material to be fully soaked, then sterilize at 105° C. for 30 min, cool, and obtain the culture medium.

[0157] 2. Preparation of bacterial strains

[0158] 2.1 Medium formulation

[0159] (1) Medium M1: 1 L 10° malt extract, 18-20 g agar, pH 5.4, sterilized at 121°C for 15 min.

[0160] (2) Medium M2: 2% glucose, 2% tryptone, 1% yeast extract, sterilized at 115°C for 15 min.

[0161] (3) Culture medium M3: 0.25% citric acid, 5% trypsin, 0.1% K2HPO4, 10% molasses, pH 6.5, sterilized at 121°C for 15 min.

[0162] (4) Medium M4: bran, water content 37.4%, sterilized at 105°C for 30 min.

[0163] 2.2 Activation and expansion of bacterial strains

[0164] (1) Activation and expansion of Rhizopus arrhizus: Inoculate Rhizopus arrhizus CMR21 on the slant of the culture medium M1 test tube and activate and culture at 30±2℃ for 4-5 days. Wash the slant spores with an appropriate amount of sterile water to obtain 1.0×10 6The spore suspension is prepared by mixing spores of each mL and inoculating the spore suspension into a culture medium M4 at 10% (v / m) at 30±2° C. and 95±2% humidity for 70-72 hours. The culture material is collected and dried at 40° C. to obtain a solid bacterial agent of Rhizopus arrhizus CMR21, which is then stored in a refrigerator at -20° C. for later use.

[0165] (2) Activation and expansion of Aspergillus oryzae: The activation and expansion methods of Aspergillus oryzae CICC 2022 are the same as those of Rhizopus arrhizus CMR21.

[0166] (3) Activation and expansion of Fabianseberlindner yeast: Fabianseberlindner yeast JGM9-1 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) inoculum amount, 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 Fabianseberlindner yeast JGM9-1 inoculum granules, which were stored in a refrigerator at -20°C for future use.

[0167] (4) Activation and expansion of Lactobacillus plantarum: Lactobacillus plantarum RPC22 was inoculated into medium M3, activated and cultured at 30±2°C for 18-20 h, and then transferred to fresh medium M3 at a 10% (v / v) inoculum amount, expanded and cultured at 30±2°C for 18-20 h, and the cells were centrifuged, mixed with a protective agent, granulated, and vacuum-freeze-dried to prepare Lactobacillus plantarum RPC22 inoculum granules, which were stored in a refrigerator at -20°C for future use.

[0168] 3. Vaccination

[0169] (1) Inoculum amount: Rhizopus arrhizus CMR21 0.55%, Aspergillus oryzae CICC 2022 0.30%, Fabianseber Lindnerella JGM9-1 0.12%, Lactobacillus plantarum RPC22 0.16%.

[0170] (2) Operation steps: Weigh each bacterial agent according to the inoculum amount, dissolve it in the above-mentioned sterilized 10% water, mix it evenly, and then spray it evenly into the sterilized culture medium, stirring while spraying to ensure that it is fully mixed to obtain the inoculum material.

[0171] 4. Fermentation

[0172] The inoculated material is evenly piled on the fermentation rack (thickness 4-5cm), the temperature is adjusted to 33-35℃, and the humidity is 90±2%; after 12-15h, the material is evenly spread (thickness 2.0-2.5cm), the temperature is adjusted to 30±1℃, and the culture is continued; thereafter, it is turned over every 24h until the fermentation is completed, and the total fermentation time is 72±2h.

[0173] 5. Drying and storage

[0174] The materials are collected, moved into an oven, and dried at 35-37° C. until the water content is below 10%, thereby obtaining a flower-scented direct-throw rice milk fermented rice koji; the materials are vacuum-packed and stored at 4-6° C. in the dark.

[0175] Example 4: Pilot test of rice koji and study on its application effect

[0176] The rice koji prepared in Example 3 was used for the rice koji pilot test; the commercial fermentation agent purchased in the market was used as a control, and the two fermentation agents (rice koji of the present invention and commercial fermentation agent) were respectively applied to the preparation of brown rice and black rice milk, and the utilization rate, juice yield and saccharification capacity of the two fermentation agents for different raw materials were detected, and sensory evaluation was performed to analyze the utilization effect of the fermentation agents on different raw materials. Among them, the raw material utilization rate (%) = (raw material dry weight - fermentation residue dry weight) / raw material dry weight × 100%, the juice yield (%) = filtrate weight / fermentation material total weight × 100%.

[0177] The method for preparing rice milk is as follows: soak the raw rice for 3-4 hours, drain the water, add water (twice the weight of the raw rice), sterilize at 105° C. for 30 minutes, add 1.0-1.5% of the weight of the raw rice prepared in Example 3 or commercial fermentation bacteria after appropriate cooling, and water bath at 55-60° C. for 10-12 hours; obtain rice milk by squeezing and filtering.

[0178] The application effects of the homemade rice koji of the present invention on different raw rice materials are shown in Tables 11 and 12. The utilization rate and sensory score of the two leavening agents for brown rice are the highest, followed by black rice; the two prepared rice milks both have a pleasant floral fragrance, as well as the typical aroma and color of the raw materials. Using the commercial leavening agent as a control, it can be seen that the raw material utilization rate, juice yield and solidification rate of the homemade rice koji are significantly higher than those of the control group (p < 0.05). It can be seen that the rice koji produced by this process has high saccharification and liquefaction capabilities, which can significantly improve the raw material utilization rate and juice yield, and save costs.

[0179] Table 11 Application effect of fermentation agent in brown rice

[0180]

[0181] Table 12 Application effect of fermentation agent in coix seed

[0182]

Claims

1. A flower-scented direct-injection rice milk fermented rice koji, characterized in that: The rice koji is prepared by inoculating a composite strain containing Rhizopus arrhizus CMR21 into a culture medium and fermenting it; the Rhizopus arrhizus CMR21, whose scientific name is Rhizopus arrhizus, has been preserved in the China Center for Type Culture Collection in Wuhan, with a preservation number of CCTCC NO: M20242064 and a preservation date of September 25, 2024.

2. A flower-scented direct-injection rice milk fermented rice koji, characterized in that: The culture medium comprises glutinous rice flour, bran and bean husk.

3. A flower-scented direct-injection rice milk fermented rice koji, characterized in that: The composite bacterial strain consists of Rhizopus arrhizus CMR21, Aspergillus oryzae CICC 2022, Fabianseberlindner yeast JGM9-1 and Lactobacillus plantarum RPC22.

4. The method for preparing a flower-scented direct-throw rice milk fermented rice koji as claimed in claim 1, characterized in that: The following steps are involved: 1) Base material preparation Weigh glutinous rice flour, bran, bean curd, CaO and water according to mass ratio, take a part of the water to fully dissolve CaO to prepare CaO solution, and sterilize the remaining water for standby use; add the CaO solution to the mixture of bran, glutinous rice flour and bean curd, stir evenly, let stand to allow the solid materials to be fully soaked, and then sterilize to obtain a culture medium; 2) Preparation of bacterial strains Rhizopus arrhizus CMR21, Aspergillus oryzae CICC 2022, Fabianseberlindnerella JGM9-1 and Lactobacillus plantarum RPC22 were activated and expanded to prepare solid bacterial agents; 3) Vaccination Solid inoculants of Rhizopus arrhizus CMR21, Aspergillus oryzae CICC 2022, Fabianseberlindnerella JGM9-1 and Lactobacillus plantarum RPC22 are taken according to the inoculum amount, dissolved in the sterilized water of step 1), then sprayed into the culture medium, mixed evenly, and obtained an inoculum material; 4) Fermentation The inoculated materials are evenly piled on the fermentation rack, and the temperature is adjusted to 33-35°C and the humidity is 90±2%. After 12-15 hours, the materials are evenly spread out, the temperature is adjusted to 30±1°C, and the culture is continued. After that, the materials are turned over every 24 hours until the fermentation is completed. The total fermentation time is 72±2 hours. 5) Drying and storage The materials are collected, moved into an oven, and dried until the water content is below 10%, thereby obtaining a flower-scented direct-throw rice milk fermented rice koji.

5. The method for preparing a flower-scented direct-throw rice milk fermented rice koji according to claim 4, characterized in that: In step 1), the water used to dissolve CaO accounts for 90% of the total water volume, and the remaining 10% of the water is sterilized and used to dissolve the bacterial agent in step 3).

6. The method for preparing a flower-scented direct-throw rice milk fermented rice koji according to claim 4, characterized in that: The sterilization in step 1) is performed at 105° C. for 30 min.

7. The method for preparing a flower-scented direct-throw rice milk fermented rice koji according to claim 4, characterized in that: The mass ratio of the base material components in step 1) is as follows: glutinous rice flour: bran: bean skin: CaO: water = 6.0: 1.0: 1.0: 0.025: 4.

8.

8. The method for preparing a flower-scented direct-throw rice milk fermented rice koji according to claim 4, characterized in that: Step 3) The inoculation amount of each bacterial agent is as follows: Rhizopus arrhizus CMR21 0.55%, Aspergillus oryzae CICC 2022 0.30%, Fabianseber Lindnerella JGM9-1 0.12%, and Lactobacillus plantarum RPC22 0.16%.

9. The method for preparing a flower-scented direct-throw rice milk fermented rice koji according to claim 4, characterized in that: In step 4), the material is evenly piled on the fermentation shelf 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.

10. The method for preparing red rice milk fermented rice koji based on biological fermentation according to claim 4, characterized in that: In step 5), the temperature of the drying process is 35-37° C., and the drying is performed until the moisture content is below 10%.

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