Aspergillus oryzae and its applications

Abstract

This invention relates to a strain of *Aspergillus oryzae* and its applications, belonging to the technical field of microbiology. The strain is named *Aspergillus oryzae* M6, which was deposited on April 12, 2023, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2023533, located at Wuhan University, Wuhan, China. *Aspergillus oryzae* M6 can be applied to the solid-state fermentation process of colored rice. During solid-state fermentation, it can simultaneously metabolize α-amylase, protease, cellulase, carboxymethyl cellulase, and pectinase, significantly shortening the ripening time of colored rice and delaying the increase in fatty acid value and peroxide value during storage.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to Aspergillus oryzae and its applications. Background Technology

[0002] Colored rice was widely found in its ancestral wild rice, but due to the linkage between pigment genes and genes related to grain shattering and seed germination, colored rice was simultaneously expressed along with easy grain shattering and low seed germination rates. Therefore, during seed domestication, colored rice was gradually eliminated by selection. However, colored rice has rich nutritional value, such as being rich in vitamins, trace elements, and flavonoids, and is therefore increasingly favored by people.

[0003] However, hulled colored rice is difficult to cook thoroughly, mainly because the gaps between starch granules are small, allowing water to easily penetrate the rice grains and resulting in incomplete starch gelatinization, thus prolonging the cooking time. Furthermore, the outer layer of colored rice is hard and contains more hemicellulose, cellulose, and lignin, making it difficult to soften during cooking and resulting in a hard texture that affects the taste. Therefore, it is necessary to provide a solution to improve these problems. Summary of the Invention

[0004] The purpose of this invention is to provide Aspergillus oryzae and its application. The Aspergillus oryzae can be applied to the solid-state fermentation process of colored rice processing. It can simultaneously metabolize α-amylase, protease, cellulase, carboxymethyl cellulase and pectinase, which can significantly shorten the cooking time of colored rice and delay the increase of fatty acid value and peroxide value of colored rice during storage.

[0005] Firstly, this invention provides a strain of Aspergillus oryzae, named Aspergillus oryzae M6, which was deposited on April 12, 2023, at the China Center for Type Culture Collection (CCTCC), accession number M2023533, located at Wuhan University, Wuhan, China. When Aspergillus oryzae M6 is applied to the solid-state fermentation process of black rice, it can simultaneously metabolize α-amylase, protease, cellulase, carboxymethyl cellulase, and pectinase, with the activities of these enzymes reaching 189.16 U / g, 212.86 U / g, 5.94 U / g, 15.55 U / g, and 17.85 U / g, respectively. This facilitates the decomposition of the surface layer of colored rice samples, allowing water to quickly penetrate the sample during cooking, thereby reducing the cooking time. Furthermore, the organic acids produced by Aspergillus oryzae M6 during fermentation can inhibit lipase activity, thereby reducing the increase in fatty acid value and improving storage time.

[0006] Secondly, the present invention also provides an application of any of the above-mentioned Aspergillus oryzae, wherein Aspergillus oryzae M6 can be used for solid-state fermentation in the processing of colored rice.

[0007] Optionally, the Aspergillus oryzae M6 is applied to the solid-state fermentation process in the processing of colored rice, and the solid-state fermentation environment is controlled at 36-38℃, the humidity is controlled at 88%-92%, and the solid-state fermentation time is controlled at 70-74h. Attached Figure Description

[0008] Figure 1 A is the X-ray diffraction pattern of the black rice sample in Comparative Example 1 and Example 1 of this invention;

[0009] Figure 1 B is the infrared spectrum of the black rice sample in Comparative Example 1 and Example 1 of this invention;

[0010] Figure 2 This is a graph showing the changes in fatty acid value and peroxide value of black rice samples during accelerated storage in Comparative Example 1 and Example 1 of this invention. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art. The terms "comprising" and similar expressions used herein mean that the element or object preceding the word covers the element or object listed after the word and its equivalents, but does not exclude other elements or objects.

[0012] This invention provides a *Aspergillus oryzae* strain, named *Aspergillus oryzae* M6, which was deposited on April 12, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: 2023533.

[0013] This invention also provides the application of Aspergillus oryzae M6 from the foregoing embodiments in the solid-state fermentation process of colored rice, which can simultaneously metabolize α-amylase, protease, cellulase, carboxymethyl cellulase and pectinase.

[0014] In some embodiments, the colored rice can be at least one of black rice, purple rice, and red rice.

[0015] In some embodiments, when Aspergillus oryzae M6 is applied to the solid-state fermentation of black rice, it can simultaneously metabolize α-amylase, protease, cellulase, carboxymethyl cellulase and pectinase, and the activities of the aforementioned enzymes reach 189.16 U / g, 212.86 U / g, 5.94 U / g, 15.55 U / g and 17.85 U / g, respectively.

[0016] Isolation of Aspergillus oryzae M6 (CCTCC NO: 2023533): Using a sterile spatula, 1g of naturally fermented soybean paste was mixed with 9mL of 0.9% physiological saline solution and stirred to dilute. After shaking, 1mL of the diluted soybean paste was mixed with 9mL of physiological saline solution and diluted to a dilution of 10. -2 The soybean paste dilution was prepared simultaneously with a dilution of 10. -3 10 -4 10 -5 10 -6 Diluent, and take 200 μL of 10 -3 10 -4 10 -5 10 -6 The diluted solution was evenly spread on Bengal Red agar medium containing chloramphenicol using a sterile spreader. After incubation at 28°C for 96 hours, typical mold single colonies were selected and streaked onto potato dextrose agar solid medium. After purification 2-3 times until single colonies were obtained, the purified mold strains were streaked onto potato dextrose agar solid slant medium to obtain Aspergillus oryzae strains for low-temperature preservation.

[0017] Mutagenesis: After the isolated Aspergillus oryzae strain was cultured to maturity, the Aspergillus oryzae plates were washed with sterile water to prepare a homogeneous spore suspension, and the concentration of the Aspergillus oryzae spore suspension was adjusted to 10. 8 CFU / mL, then 10 mL of spore suspension was evenly spread on the slide. The mutagenesis conditions were set as follows: aeration rate of 10 SLM, power of 100 W, and time of 0 s, 15 s, 30 s, 45 s, 60 s, 75 s, 90 s, and 120 s, respectively. After completing the atmospheric pressure and room temperature plasma mutagenesis treatment, the slide was washed in PBS and then resuspended in sterile plasma water for later use.

[0018] Screening: The mutagenized Aspergillus oryzae suspension was spread on casein agar and incubated at 30°C for 72 hours in the dark. Single colonies exhibiting vigorous mycelial growth and large clear zones compared to the unmutated Aspergillus oryzae strain were streaked onto soybean protein agar and incubated at 30°C for 48 hours. Single colonies with large diameters and vigorous growth were then selected and transferred to soybean protein agar for maturation and preservation. The strain was then activated and cultured to produce 1×10⁻⁶ cells / years. 6A spore suspension of CFU / mL was prepared and placed on a solid-state fermentation medium. Solid-state fermentation was carried out at 30℃ for 72 hours, and the activities of amylase, protease, cellulase, carboxymethyl cellulase, and pectinase were measured. Strains with normal growth and high enzyme activity were selected for preservation. The casein medium consisted of 10 g / L casein, 10 g / L beef extract, 2 g / L disodium hydrogen phosphate, 5 g / L sodium chloride, 15 g / L agar powder, and 1 L deionized water. The raw materials were autoclaved at 121℃ for 20 min. The soybean protein medium consisted of 10 g soybean protein isolate, 0.5 g MgSO4·7H2O, 30 g xylose, 1 g KH2PO4, and 15 g agar powder, dissolved in 1000 mL distilled water. After adjusting the pH to 6.4, the solution was sterilized at 121℃ for 15 min. The solid-state fermentation medium consisted of 97% cooked black rice and (NH4)2SO4. 3%, adjust the solid-to-water ratio to 1:0.8, and sterilize at 121 degrees Celsius for 15 minutes;

[0019] Identification: The bacterial strain was sent to Beijing Qingke Biotechnology Co., Ltd. for gene identification, and the gene sequence of Aspergillus oryzae M6 was obtained as follows:

[0020] .

[0021] Example 1

[0022] This embodiment 1 provides a method for preparing nutritious rice through solid-state fermentation, including the following steps:

[0023] S1. Inoculate Aspergillus oryzae M6 (CCTCC NO: 2023533) into potato dextrose agar medium and incubate at 37°C for 4-5 days. Adjust the Aspergillus oryzae M6 spore concentration to obtain an Aspergillus oryzae M6 content of 2-3 × 10⁻⁶. 6 CFU / mL Aspergillus oryzae M6 spore suspension;

[0024] S2. Take 10 kg of black rice and mix it with 2.5 L of potato glucose broth culture medium and balance the nutrients for 18 h. Then add 1 L of Aspergillus oryzae M6 spore suspension and stir evenly. Place it in a fermentation environment with a temperature of 37℃ and a humidity of 90% for solid fermentation for 72 h. During the fermentation process, turn the solid fermentation material once every 24 h. After the fermentation is completed, dry it at low temperature to obtain quick-cooking black rice.

[0025] Compare with Example 1

[0026] Comparative Example 1 is untreated black rice, and the type and batch of black rice are the same as those in Example 1.

[0027] Performance testing and analysis

[0028] (1) Determination of hydrolytic enzyme activity, soluble protein and polysaccharide

[0029] Following the methods of Chen G, Liu Y, Zeng J, et al. Enhancing three phenolic fractions of oats (Avena sativa L.) and their antioxidant activities by solid-state fermentation with Monascus anka and Bacillus subtilis[J]. Journal of Cereal Science, 2020, 93: 102-940. and Li WY, Zhao LL, He XL. Degradation potential of different lignocellulosic residues by Trichoderma longibrachiatum and Trichoderma afroharzianum under solid state fermentation[J]. Process Biochemistry, 2022, 112: 6-17., α-amylase, protease, cellulase, and pectinase in the samples were extracted and determined.

[0030] The sample was crushed and passed through a 60-mesh sieve. 1.0 g of black rice flour was weighed and added to 20 mL of sodium citrate buffer (50 mM, pH 5.5). The mixture was extracted at 30 °C for 30 min and then centrifuged at 4 °C for 10 min. The supernatant was collected as the crude enzyme solution of the fermentation substrate, and its enzyme activity was immediately measured.

[0031] α-Amylase activity: Take 250 μL of diluted crude enzyme solution, add 1.75 mL of 1% soluble starch solution (w / v), react in a 50℃ water bath for 10 min, then immediately add 4 mL of DNS reagent and boil in a water bath for 5 min, and finally bring the volume to 10 mL. After the sample cools to room temperature, measure the absorbance at 540 nm, and simultaneously plot a standard curve using glucose as a standard via the DNS method. Enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze starch to produce 1 μmol of glucose per minute under experimental conditions, and is expressed as U / g.

[0032] Protease activity: Mix 2 mL of crude enzyme solution with 2 mL of 1% casein solution, react in a 40℃ water bath for 15 min, stop the reaction by adding 4 mL of 0.4 mol / L trichloroacetic acid solution, let stand for 30 min, and then centrifuge at 12000 r / min for 5 min. Collect the supernatant and measure the absorbance at 280 nm, and plot a standard curve using tyrosine standards. Enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute under experimental conditions, and is expressed as U / g.

[0033] Cellulase activity: Take 1 mL of diluted crude enzyme solution, add 2 mL of sodium citrate buffer (0.1 mol / L, pH 4.8), and add 0.1 g of filter paper strip (1.0 × 6.0 cm) and 1 mL of 0.5% sodium carboxymethyl cellulose solution as reaction substrates. Measure the activities of filter paper cellulase (FPase) and carboxymethyl cellulase (CMCase). After reacting in a 50℃ water bath for 1 h, immediately add 4 mL of DNS reagent and react in a boiling water bath for 10 min, then bring the volume to 10 mL. After the sample cools to room temperature, measure the absorbance at 540 nm. Simultaneously, use glucose as a standard to plot a standard curve using the DNS method. Enzyme activity unit (U) is defined as the amount of enzyme required per minute to hydrolyze filter paper to produce 1 μmol of glucose under experimental conditions, expressed as U / g.

[0034] Pectinase activity: Take 1 mL of diluted crude enzyme solution, add 2 mL of sodium acetate buffer (0.04 mol / L, pH 4.6) and 1 mL of 1% pectin solution, react in a 50℃ water bath for 30 min, then immediately add 4 mL of DNS reagent and react in a boiling water bath for 10 min, and then bring the volume to 10 mL. After the sample cools to room temperature, measure the absorbance at 540 nm, and simultaneously plot a standard curve using D-galacturonic acid as a standard using the DNS method. Enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze pectin to produce 1 μmol of galacturonic acid per minute under experimental conditions, expressed as U / g.

[0035] The soluble proteins and polysaccharides in the samples were determined according to the method described in Shen D, Labreche F, Wu C, et al. Preparation and aroma analysis of flavonoid-rich ginkgo seeds fermented using rice wine starter[J]. FoodBioscience, 2021, 44: 101-459.

[0036] Soluble protein content: 1.0 g of rice flour was mixed with 0.05 mol / L NaOH solution (1:30 w / v), stirred in a water bath at 35 ℃ for 1.5 h, centrifuged, and the supernatant was brought to a final volume of 50 mL. Bovine serum albumin solutions of different concentrations were prepared, and the soluble protein content was determined using the Coomassie brilliant blue method.

[0037] Soluble polysaccharide content: 1.0g of rice flour was mixed with water (1:30w / v), stirred in a water bath at 35℃ for 1.5h, centrifuged and the supernatant was collected. Different concentrations of glucose solutions were prepared and the soluble polysaccharide content was determined by the phenol-sulfuric acid method.

[0038] All the above measurements were repeated three times. The data were analyzed by one-way ANOVA (Duncan's test) using SPSS 25.0. The significance level was p<0.05. The results are expressed as mean ± standard deviation.

[0039] The enzyme activity and soluble components of the black rice samples in Example 1 and Control Example 1 are shown in Table 1.

[0040] Table 1 Enzyme activity and soluble components

[0041]

[0042] Note: Data are expressed as mean ± standard deviation. Different letters in the same row indicate significant differences (p < 0.05).

[0043] As shown in Table 1, compared with the black rice sample in Control Example 1, the black rice sample fermented with Aspergillus oryzae M6 in Example 1 showed a significant increase in the activities of α-amylase, protease, cellulase, carboxymethyl cellulase, and pectinase, which increased by 3.54 times, 5.12 times, 2.86 times, 15.55 times, and 0.70 times, respectively.

[0044] (2) Gelatinization and cooking properties determination

[0045] The gelatinization characteristics of the samples were determined using a rapid viscosity analyzer (RVA) in accordance with the method described in Zhong Y, Xiang X, Zhao J, et al. Microwave pretreatment promotes the annealing modification of rice starch[J]. Food Chemistry, 2020, 304: 125432.

[0046] Weigh 3g of sample powder and add it to an aluminum cylinder containing 25mL of deionized water. Place the cylinder into the RVA groove, then select the standard program to stir, heat (50~95℃) and cool. The single run time is 12.5min. Measure the gelatinization characteristics of the sample.

[0047] The cooking quality and textural properties of the samples were determined according to the method described in Wu J, Chen J, Liu W, et al. Effects of aleurone layer on rice cooking: ahistorical investigation[J]. Food Chemistry, 2016, 191: 28-35.

[0048] Take 5g of sample rice grains and place them in 50mL of boiling water (98±1℃) to steam and start timing. After 15 minutes, randomly take 10 grains of rice, press them between two clean glass plates and squeeze to observe whether there is any ungelatinized starch core inside the rice grains. When 90% of the rice grains do not have a white core (ungelatinized starch), record the steaming time. At the same time, the water absorption rate should also be measured after the rice grains have reached the cooking time.

[0049]

[0050] Where: W0 represents the weight of the sample before cooking, g; W1 represents the weight of the sample after cooking, g; W i The weight is on a dry basis, in grams.

[0051] Take cooked rice (not exceeding the probe area) that has reached the required cooking time and place it on the texture analyzer testing platform while still hot. The measurement is performed using a two-cycle compression (TPA) mode. The probe speeds before, during, and after measurement are 1.0, 0.5, and 1.0 mm / s, respectively. The rice is compressed to 75% deformation using a 26 mm diameter cylindrical probe (P / 36). Each sample is measured 10 times. The instrument's built-in Texture Expert Exceed software automatically analyzes the texture curves to obtain the sample's hardness, adhesiveness, elasticity, cohesiveness, chewiness, and resilience.

[0052] All the above measurements were repeated three times. The data were analyzed by one-way ANOVA (Duncan's test) using SPSS 25.0. The significance level was p<0.05. The results are expressed as mean ± standard deviation.

[0053] The cooking quality, textural properties, and gelatinization properties of the black rice samples in Example 1 and Control Example 1 are shown in Table 2.

[0054] Table 2 Cooking quality, textural properties and gelatinization properties

[0055]

[0056]

[0057] Note: Data are expressed as mean ± standard deviation. Different letters in the same row indicate significant differences (p < 0.05).

[0058] As shown in Table 2, the cooking time of the black rice sample in Control Example 1 was 31.58 min, while the cooking time of the black rice sample fermented with Aspergillus oryzae M6 in Example 1 was reduced to 20.68 min. This indicates that solid-state fermentation with Aspergillus oryzae M6 can shorten the cooking time of black rice samples. The water absorption rate of the black rice sample in Control Example 1 was 125.21%, while the water absorption rate of the black rice sample in Example 1 increased to 160.90%. This indicates that solid-state fermentation with Aspergillus oryzae can improve the water absorption rate during cooking. This may be because water can quickly migrate into the interior of the cooked sample during cooking, enhancing the hydrogen bond interaction between starch and water. Furthermore, as shown in Table 1, after solid-state fermentation with Aspergillus oryzae M6 in Example 1, the activities of various proteases were significantly increased, which is beneficial for decomposing the outer layer of the cooked sample, thereby increasing the starch water absorption rate and reducing the cooking time.

[0059] Meanwhile, compared with the black rice sample in Control Example 1, the hardness, adhesiveness, elasticity, cohesiveness, chewiness and resilience of the black rice sample in Example 1 were significantly reduced (p<0.05). This may be because the cellulase and pectinase produced during the solid-state fermentation of Aspergillus oryzae M6 destroyed the fiber cortex of the sample, and the protease hydrolyzed the protein in the black rice sample, reducing the protein network structure and thus affecting the textural properties of the black rice sample.

[0060] Furthermore, compared with the black rice sample in Control Example 1, the peak viscosity, valley viscosity, disintegration value, final viscosity and recovery value of the black rice sample in Example 1 were significantly reduced (p<0.05). This may be because the amylase produced during the solid-state fermentation of Aspergillus oryzae M6 causes the starch granules to hydrolyze into short starch chains and small molecule sugars. At the same time, the reduction in protein content also reduces the gelatinization viscosity.

[0061] (3) Crystal structure determination

[0062] The long-range crystal structure was determined using a wide-angle X-ray diffractometer (XRD). The diffraction intensity scan range was 5°–35° (2θ), and the scan rate was 0.02° / sec. Diffraction patterns were acquired using Jade 6.0 software, and the relative crystallinity (%) was calculated based on the ratio of the crystalline region area to the total area of ​​the diffraction pattern.

[0063] The short-range ordered structure of the sample was analyzed using Fourier transform infrared spectroscopy (FTIR). The infrared spectral scanning range was 4000–400 cm⁻¹. -1 The resolution is 4cm. -1 After 64 scans, a 1200cm diameter was observed. -1 up to 800cm -1 The spectrum within the range is subjected to baseline correction, smoothing, and deconvolution processing.

[0064] Calculate 1047 / 1022cm -1 With 1020cm -1 / 995cm -1 The absorbance ratio (Method: Berti S, Jagus RJ, Flores S K. Effect of rice bran addition on physical properties of antimicrobial biocomposite films based on starch[J]. Food and Bioprocess Technology, 2021, 14(9): 1700-1711.).

[0065] The crystal structures of the black rice samples in Example 1 and Control Example 1 are as follows: Figure 1 As shown in Table 3, the crystallization characteristics of the black rice samples in Example 1 and Control Example 1 are shown in Table 3.

[0066] Table 3 Crystallization characteristics

[0067]

[0068] Note: Data are expressed as mean ± standard deviation. Different letters in the same row indicate significant differences (p < 0.05).

[0069] See Figure 1 A. The black rice sample (BR) in Comparative Example 1 has a typical starch type A crystallographic pattern with strong diffraction peaks near 15° and 23°, a continuous unseparated double peak near 17° and 18°, and a weak diffraction peak near 20°. The black rice sample (Fermented black rice, FBR) in Example 1 shows similar crystal characteristics, indicating that solid-state fermentation has no effect on the crystal form of black rice starch.

[0070] See Figure 1 As shown in B, in the infrared spectrum, 1047 cm⁻¹ -1 1020cm -1 and 995cm -1The nearby absorption peaks correspond to the hydrogen bond structures formed between starch crystalline regions, starch amorphous regions, and hydroxyl groups, respectively, at (1047 / 1020) cm⁻¹. -1 and (1020 / 995)cm -1 The peak intensity ratio can reflect the degree of order on the surface of starch granules, i.e., (1047 / 1020) cm. -1 The larger the peak intensity ratio, the higher the (1020 / 995)cm -1 The smaller the peak intensity ratio, the more ordered the short-range structure of the starch. Compared with the black rice sample (BR) in Control Example 1, the black rice sample (Fermented black rice, FBR) in Example 1 has a peak intensity ratio of (1047 / 1020) cm⁻¹. -1 The ratio gradually decreases, reaching (1020 / 995) cm. -1 The ratio gradually increased, indicating that the short-range order of the black rice sample in Example 1 was less than that in Control Example 1, suggesting that solid-state fermentation can reduce the short-range structural order of black rice.

[0071] (4) Determination of storage properties

[0072] The rancidity of oils in colored rice mainly occurs in two forms: hydrolysis and oxidation, which produce fatty acids and peroxides, respectively. Therefore, the degree of rancidity of oils is reflected by measuring the content of fatty acid value and peroxide value. Generally speaking, the higher the content of both, the worse the edible quality.

[0073] Accelerated storage experiment: The black rice sample (BR) from Control Example 1 and the black rice sample (FBR) from Example 1 were packaged in resealable bags and placed in a constant temperature and humidity chamber at 37°C and 75% relative humidity. Samples were taken at 0, 10, 20, 30, 40, and 50 days to determine their fatty acid value and peroxide value. The results are as follows: Figure 2 As shown.

[0074] Fatty acid value determination: Refer to the national standard GB / T 5510-2011 with slight modifications. The method is as follows: Take 1g of the crushed sample into a centrifuge tube, add 30mL of petroleum ether, and seal the centrifuge tube. Shake at 20℃ and 100r / min for 10min on a shaker, then centrifuge. Take 20mL of the supernatant into an Erlenmeyer flask, add 75mL of 50% ethanol and 5 drops of phenolphthalein solution. Using a microburette, titrate the lower ethanol solution with KOH standard titration solution (0.01mol / L) until a light pink color appears, continuing for about 3s. Replace the sample supernatant with 20mL of ethanol to perform a blank titration test. The result is expressed as mg / 100g (calculated as KOH), and the calculation formula is as follows:

[0075]

[0076] In the formula: c is the concentration of KOH standard titration solution (mol / L); m is the dry basis mass of the sample (g DW); V1 is the volume of KOH standard titration solution consumed by the sample (mL); V0 is the volume of KOH standard titration solution consumed by the blank (mL); 8415 is a constant represented by KOH, i.e. 56.1×1.5×100.

[0077] Peroxide value determination: The method was slightly modified from that of Wu J, McClements DJ, Chen J, et al. Improvement of storage stability of lightly milled rice using superheated steam processing[J]. Journal of Cereal Science, 2016, 71:130-137. 0.1 g of the pulverized sample was added to 9.8 mL of chloroform-methanol (7:3, v / v) solution, stirred in a 45℃ water bath for 10 min, cooled to room temperature, and centrifuged. All supernatant was diluted to 10 mL, 50 μL of ferric chloride solution was added and shaken well, followed by 50 μL of 30% potassium thiocyanate solution. After standing for 5 min, the absorbance was immediately measured at 500 nm. Using Fe... 3+ A standard curve is plotted using the solution as a standard, and the calculation formula is as follows:

[0078]

[0079] In the formula: A s A represents the absorbance of the sample. b λ is the blank absorbance; m is the reciprocal of the slope of the standard curve; m0 is the dry basis mass of the sample (g DW); 55.84 is the relative atomic mass of iron (g / mol).

[0080] See Figure 2 As shown, during storage, the fatty acid value and peroxide value of both samples gradually increased. Among them, the fatty acid value and peroxide value of black rice (BR) in control example 1 increased the fastest, followed by the black rice sample (FBR) in example 1. This may be because the organic acids produced by Aspergillus oryzae M6 during fermentation inhibited lipase activity, thereby reducing the increase in fatty acid value.

[0081] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A type of Aspergillus oryzae ( Aspergillus oryzae The application of M6 in colored rice processing is characterized by, The Aspergillus oryzae M6 was deposited at the China Center for Type Culture Collection on April 12, 2023, with accession number CCTCC NO: M2023533, and the deposit address is Wuhan University, Wuhan, China; the Aspergillus oryzae M6 is used for solid-state fermentation in the processing of colored rice.

2. The application according to claim 1, characterized in that, The Aspergillus oryzae M6 is applied to solid-state fermentation in the processing of colored rice, and can simultaneously metabolize α-amylase, protease, cellulase, carboxymethyl cellulase and pectinase, with the activities of the aforementioned enzymes reaching 189.16 U / g, 212.86 U / g, 5.94 U / g, 15.55 U / g and 17.85 U / g, respectively; the colored rice is black rice.

3. The application according to claim 1, characterized in that, The Aspergillus oryzae M6 is applied to the solid-state fermentation process in colored rice processing, controlling the ambient temperature of the solid-state fermentation environment at 36-38℃, the ambient humidity at 88%-92%, and the solid-state fermentation time at 70-74h.

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