Fermented roxburgh rose and oat tablet and preparation method thereof

Abstract

The invention discloses a fermented roxburgh rose and oat tablet and a preparation method thereof, and belongs to the technical field of fermented food. According to the invention, bacillus natto, bifidobacterium and aspergillus oryzae are adopted to ferment oat and are mixed with roxburgh rose juice for conversion, so that the sour and astringent taste of the roxburgh rose juice can be improved, the taste of a bacillus natto fermented product which is not popular with the public can be effectively improved, a high-value active ingredient nattokinase beneficial to cardiovascular health is generated, and the product quality is improved. The requirements of people for high-quality and large-health food can be met, high-valued application of the characteristic resource roxburgh rose in Guizhou can be achieved, and the high-quality roxburgh rose and oat tablet which is beneficial to human body health and rich in active ingredients such as nattokinase and the like is prepared.

Description

Technical Field

[0001] The present invention relates to the technical field of fermented foods, and particularly to a fermented Rosa roxburghii Tratt and oat tablet and a preparation method thereof. Background Art

[0002] Rosa roxburghii Tratt is rich in vitamins but lacks protein, while oats contain high levels of protein and lipids. The two are significantly complementary in nutrition, making the nutritional value of the final product more comprehensive and rich. However, the sour and astringent taste of Rosa roxburghii Tratt makes it unacceptable to most people, which to a certain extent limits its application. Therefore, it is of great practical significance and application prospects to provide a high-quality Rosa roxburghii Tratt and oat food that is rich in nutrition and popular among the public. Summary of the Invention

[0003] The object of the present invention is to provide a fermented Rosa roxburghii Tratt and oat tablet and a preparation method thereof to solve the problems existing in the above-mentioned prior art.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is a preparation method of a fermented Rosa roxburghii Tratt and oat tablet, comprising the following steps:

[0006] (1) Mix oats and soybean milk, inoculate with Aspergillus oryzae seed liquid for fermentation to obtain fermentation liquid I; add Rosa roxburghii Tratt juice to the fermentation liquid I for crushing treatment, then carry out closed conversion, freeze-dry, and grind into powder to obtain freeze-dried powder I;

[0007] (2) Mix oats and soybean milk, inoculate with BLH1 and GUTU09 seed liquids for fermentation to obtain fermentation liquid II; add Rosa roxburghii Tratt juice to the fermentation liquid II for crushing treatment, then carry out closed conversion, freeze-dry, and grind into powder to obtain freeze-dried powder II;

[0008] (3) Mix oats and soybean milk, inoculate with BLH1 and GUTU09 seed liquids for fermentation, freeze-dry, and grind into powder to obtain freeze-dried powder III;

[0009] (4) Combine the freeze-dried powders I, II, and III, mix and press into tablets to obtain the fermented Rosa roxburghii Tratt and oat tablet.

[0010] Another technical solution of the present invention is the fermented Rosa roxburghii Tratt and oat tablet prepared by the above preparation method.

[0011] Based on the above technical solutions, the present invention has the following technical effects:

[0012] The present invention ferments oats with Bacillus natto, Bifidobacterium, and Aspergillus oryzae and mixes them with the original juice of Rosa roxburghii Tratt, which can improve the sour and astringent taste of the Rosa roxburghii Tratt juice, effectively improve the unpopular taste of the products fermented by Bacillus natto, and generate nattokinase, a highly valuable active ingredient beneficial to cardiovascular health, thereby enhancing the product quality. This can not only meet people's needs for high-quality and great health foods, but also achieve the high-value application of the characteristic resource of Rosa roxburghii Tratt in Guizhou, and prepare a high-quality Rosa roxburghii Tratt oat tablet rich in active ingredients such as nattokinase and beneficial to human health. Description of the Drawings

[0013] Figure 1 is the total acid content.

[0014] Figure 2 is the change in nattokinase activity.

[0015] Figure 3 is the amino acid nitrogen content.

[0016] Figure 4 is the reducing sugar content.

[0017] Figure 5 is the polysaccharide content.

[0018] Figure 6 is the effect of fermentation and transformation on dietary fiber.

[0019] Figure 7 is the change in the viable count of Aspergillus oryzae.

[0020] Figure 8 is the change in the viable count of Bifidobacterium BLH1.

[0021] Figure 9 is the change in the viable count of Bacillus natto GUTU09.

[0022] Figure 10 is the effect of fermentation and transformation on infrared spectroscopy.

[0023] Figure 11 is the soluble protein content.

[0024] Figure 12 is the GABA content.

[0025] Figure 13 is the total phenol content.

[0026] Figure 14 is the total flavonoid content.

[0027] Figure 15 is the vitamin C content.

[0028] Figure 16It is the change in antioxidant capacity. Among them, the DPPH free radical scavenging rate (A); the ABTS free radical scavenging rate (B); the ferric ion reducing power (C).

[0029] Figure 17 It is the change in blood glucose lowering ability. Among them, the α-amylase activity inhibition rate (A); the α-glucosidase inhibition rate (B).

[0030] Figure 18 It is the change in blood lipid lowering ability. Among them, the cholesterol binding rate (A); the bile salt adsorption ability (B).

[0031] Figure 19 It is the angiotensin converting enzyme inhibition rate. Specific implementation manners

[0032] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0033] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or are already public.

[0034] The embodiments of the present invention provide a preparation method of a fermented Rosa roxburghii Tratt oat tablet, comprising the following steps:

[0035] (1) Mix oats and soybean milk, inoculate with Aspergillus oryzae seed liquid for fermentation to obtain fermentation broth I; add Rosa roxburghii Tratt juice to fermentation broth I for crushing treatment, then perform closed conversion, freeze-dry, and grind to obtain freeze-dried powder I;

[0036] (2) Mix oats and soybean milk, inoculate with BLH1 and GUTU09 seed liquids for fermentation to obtain fermentation broth II; add Rosa roxburghii Tratt juice to fermentation broth II for crushing treatment, then perform closed conversion, freeze-dry, and grind to obtain freeze-dried powder II;

[0037] (3) Mix oats and soybean milk, inoculate with BLH1 and GUTU09 seed liquids for fermentation, freeze-dry, and grind to obtain freeze-dried powder III;

[0038] (4) Combine freeze-dried powders I, II, and III, mix and press tablets to obtain the fermented Rosa roxburghii Tratt oat tablet.

[0039] In some specific implementation manners, in step (1), the mass ratio of the oats to the soybean milk is 1:1.5; the inoculation amount of the Aspergillus oryzae seed liquid is 4% (v:v), and the fermentation time is 24 h; the volume ratio of the fermentation broth I to the Rosa roxburghii Tratt juice is 2.5:1.5; the time for the closed conversion is 6 h, and the conditions are 27 °C and 170 rpm.

[0040] In some specific embodiments, in step (2), the mass ratio of oats to soy milk is 1:1.5; the inoculation amounts of the BLH1 and GUTU09 seed solutions are 4% (v:v), the fermentation time is 24 h; the ratio of the viable cell numbers of the BLH1 and GUTU09 seed solutions is 2.5:1; the volume ratio of the fermentation broth II to the roselle juice is 2.5:1.5; the time for the closed conversion is 6 h, and the conditions are 37 °C and 170 rpm.

[0041] In some specific embodiments, in step (3), the mass ratio of oats to soy milk is 1:1.5; the inoculation amounts of the BLH1 and GUTU09 seed solutions are 4% (v:v), the fermentation time is 24 h; the ratio of the viable cell numbers of the BLH1 and GUTU09 seed solutions is 2.5:1.

[0042] In some specific embodiments, in step (3), the mass ratio of the lyophilized powders I, II, and III is 1:1:1.

[0043] The embodiments of the present invention also provide fermented roselle oat tablets prepared by the preparation method.

[0044] Bacillus subtilis natto GUTU09 was isolated from Guizhou fermented soybean and preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M2021641, which has been disclosed in patent CN118743447A.

[0045] Bifidobacterium animalis subsp. lactis BLH1 was isolated from Guizhou red sour soup and preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M20221979, which has been disclosed in patent CN117305150A.

[0046] Aspergillus oryzae 3.042 was purchased from the Institute of Microbiology, Guangdong Academy of Sciences (preservation number: GDMCC 3.31).

[0047] Example 1

[0048] 1 Strain preparation

[0049] The activated Bacillus natto GUTU09 was inoculated into the corresponding seed solution. The culture conditions for GUTU09 were aerobic, 14 - 18 h, 37 °C, and 180 r / min. Bifidobacterium BLH1 was inoculated into the PTYG liquid culture medium. The culture conditions for BLH1 were anaerobic, static, 37 °C, and 48 h.

[0050] After culturing, the viable count of Bacillus natto was determined by casein plate counting, and that of Bifidobacterium was determined by improved MRS plate counting. Adjust the viable count of Bifidobacterium to approximately 6.25×10 8 CFU / mL, and adjust the viable count of Bacillus natto to approximately 2.5×10 8 CFU / mL for standby. Inoculate the Aspergillus oryzae activated on the PDB slant into the PDB seed liquid and culture for 24 h. After homogenizing the culture solution, it is ready for use.

[0051] 2 Sample preparation

[0052] Soybean milk: Wash the soybeans and soak them for 12 h. Add water to the soaked soybeans (the mass ratio of soybeans to water is 1:8) and make soy milk for standby.

[0053] Aspergillus oryzae-fermented and transformed sample: Add oats to soybean milk (the mass ratio of oats to soybean milk is 1:1.5), shake well, and sterilize (121 °C, 20 min). After sterilization, inoculate 4% (v:v) of the prepared Aspergillus oryzae seed liquid. Ferment for 24 h, take out and add rosa roxburghii juice (the volume ratio of the fermented sample to rosa roxburghii juice is 2.5:1.5) for crushing treatment, and then seal for transformation for 6 h (27 °C, 170 rpm). After the transformation is completed, freeze-dry, grind into powder, and press into tablets.

[0054] BLH1 and GUTU09 mixed-fermented and transformed sample: Add oats to soybean milk (the mass ratio of oats to soybean milk is 1:1.5), shake well, and sterilize (121 °C, 20 min). After sterilization, inoculate 4% (v:v) of the BLH1 and GUTU09 seed liquid (the viable count of the seed liquid is BLH1:GUTU09 = 2.5:1). Ferment for 24 h, take out and add rosa roxburghii juice (the volume ratio of the fermented sample to rosa roxburghii juice is 2.5:1.5) for crushing treatment, and then seal for transformation for 6 h (37 °C, 170 rpm). After the transformation, freeze-dry, grind into powder, and press into tablets. The sampling instructions are shown in Table 1.

[0055] Table 1 Sampling instructions

[0056]

[0057]

[0058] Note: The ratios in the table are mass ratios.

[0059] 3 Index determination

[0060] (1) Dilute the sample 10 times with deionized water and make a slurry. Measure it using a pH meter, and calibrate it before use. Place the sample on white paper and measure the color difference using a calibrated color difference meter: L* (brightness), a* (red - green intensity), and b* (yellow - blue intensity). Refer to the potentiometric titration method of the pH meter in the national standard of the People's Republic of China GB 12456 - 2021 "National Food Safety Standard - Determination of Total Acid in Foods", and express the total acid content in terms of acetic acid.

[0061] (2) Sensory evaluation

[0062] In a dedicated sensory food laboratory, randomly label 9 kinds of Rosa roxburghii oat tablets. A panel of 15 postgraduate students with rich sensory evaluation experience will conduct sensory evaluation from five aspects: odor, taste, color, appearance, and chewiness. The sensory scoring criteria are shown in Table 2, and the total sensory score is converted to a percentage system.

[0063] Table 2 Sensory scoring criteria

[0064]

[0065]

[0066] (3) The determination of nattokinase (NK) activity adopts the agarose fibrin plate method in the standard "DBS 44 / 013 - 2019". The standard curve equation is: y = -1.094 + 1.308x (R 2 = 0.9927). The determination of amino acid nitrogen refers to the national standard of the People's Republic of China GB5009.235 - 2016. The determination of reducing sugar and polysaccharide refers to the reference literature (Tian Xueyi, 2022). The determination of soluble and insoluble dietary fiber refers to the national standard of the People's Republic of China GB 5009.88 - 2023. Refer to the national standard of the People's Republic of China "GB 4789.2 - 2022" to determine the viable microbial count in the sample. Fourier transform infrared spectroscopy analysis refers to the research method in the reference literature (Guo Xilei et al., 2024). Use PeakFit v4 software for protein secondary structure simulation. The texture analysis refers to the research method in the reference literature with slight modification (Li Yao et al., 2018).

[0067] (4) Each sample is measured in parallel 3 times. The experimental results are expressed as mean ± standard deviation. The data is analyzed using Excel and SPSS26.0 software, and the charts are drawn using Origin 2022.

[0068] 4 Experimental results

[0069] 4.1 pH and color

[0070] After fermenting oats with GUTU09 and BLH1, L* and b* decreased significantly, while a* increased significantly. After adding and converting with prickly pear juice, L* decreased significantly, a* and b* increased. After freeze-drying and tabletting, L* increased significantly, while a* and b* showed no significant change. After fermenting oats with GUTU09 and BLH1, the pH value increased significantly. After adding prickly pear juice, the pH value decreased significantly, but after conversion, the pH value increased slightly.

[0071] After fermenting oats with Aspergillus oryzae, the L* and b* of the sample increased, while a* decreased significantly. After adding and converting with prickly pear juice, L* decreased significantly and a* increased significantly. b* decreased significantly after adding prickly pear juice, but after 6 hours of conversion, the b* value increased significantly. Then the sample was freeze-dried and tabletted, significantly increasing L* and b*, but significantly decreasing a*. After fermenting oats with Aspergillus oryzae, the pH decreased significantly. After adding prickly pear juice, the pH value decreased significantly, but after conversion, the pH value increased slightly, which is consistent with the conversion results of fermenting oats with GUTU09 and BLH1. This shows that the probiotic-fermented samples degraded the acidity of the prickly pear juice.

[0072] Fermentation with the three strains of bacteria and adding and converting with prickly pear juice would all decrease the L* value of the sample, increase the a* value of the sample, and the change in b* was relatively small. The effect of adding and converting prickly pear juice to the pH value of the sample fermented with GUTU09 and BLH1 was greater than that of adding and converting prickly pear juice to the pH value of the sample fermented with Aspergillus oryzae. Fermenting oats with GUTU09 and BLH1 could better convert the acidic substances in the prickly pear juice.

[0073] 4.2 Total acid

[0074] As Figure 1 shown, the change trend of the total acid content was consistent with that of the pH. After fermenting oats with Aspergillus oryzae, the total acid content of the sample increased significantly, which was due to the acid accumulation caused by the decomposition of the early raw materials, including organic acids, free fatty acids, and amino acids. After fermenting oats with GUTU09 and BLH1, the total acid content decreased slightly. Whether it was fermented with Aspergillus oryzae or converted with GUTU09 and BLH1, the total acid content decreased slightly. In the initial stage of fermentation, microorganisms might rapidly consume the available organic acids, resulting in a decrease in the total acid content. As fermentation progressed, the number of microorganisms increased, and some acids might be utilized by the microorganisms or converted into other metabolites. Comparing samples RB5, M5, and H, it was found that fermenting and converting with GUTU09 and BLH1 reduced the total acid content in the prickly pear oat tablets by approximately 49.2%, and fermenting and converting with Aspergillus oryzae could reduce the total acid content in the prickly pear oat tablets by approximately 10.5%. Fermentation and conversion with beneficial bacteria could reduce the total acid content, improve the sour taste of the prickly pear juice, and thus enhance the sensory characteristics of the prickly pear oat tablets.

[0075] 4.3 Sensory

[0076] The mixed fermentation and transformation of Aspergillus oryzae, Bacillus natto, and Bifidobacterium can improve the odor, color, appearance, and chewiness of the Rosa roxburghii Tratt-oat tablets. During the fermentation of oats by Aspergillus oryzae, various enzymes are produced to decompose the components in oats, which may produce odors such as fruity, fermented, and sweet scents. Aspergillus oryzae may produce pigments during its growth and metabolism, or the enzymes it secretes may cause reactions in the components of oats, leading to color changes. In addition, the fermentation of Aspergillus oryzae decomposes some components in oats, making its texture softer and the taste may be more delicate. The fermentation of Bacillus natto produces a special odor similar to natto, and Bifidobacterium produces some fermentation aromas, possibly accompanied by a faint sweet and sour taste. The Maillard reaction may be triggered during the fermentation of Bacillus natto, resulting in color changes. The fermentation of Bacillus natto causes the rupture of the oat cell wall, and components such as starch are decomposed, making the oats easier to chew. The fermentation of Bifidobacterium decomposes some dietary fiber and starch, etc., making the tablets softer and the taste more delicate. Bacillus natto and Bifidobacterium make the sample texture more delicate and the structure more compact, which may make the tablet surface smoother. Comparing B and RB5, it can be seen that the addition of Rosa roxburghii Tratt juice during transformation can significantly improve the overall sensory characteristics of the tablets, indicating that the transformation process can improve the unlikable taste in the products fermented by Bacillus natto (p<0.05). The mixed fermentation and transformation of Bacillus natto and Bifidobacterium can improve the sensory properties, but the taste of the tablets fermented by Aspergillus oryzae decreases. The main reason is that the oats tablets fermented by Aspergillus oryzae produce bitterness. The reason may be that the protease secreted by Aspergillus oryzae decomposes the proteins in oats, and some bitter short-chain peptides may be produced during this process. The mixed tablet MRBB not only has the highest sensory score but also neutralizes the benefits of the three beneficial bacteria in terms of nutrition and function, which reflects the synergistic effect of the three bacteria.

[0077] 4.4 Nattokinase activity

[0078] As Figure 2 shown, Bacillus natto GUTU09 ferments to produce nattokinase. The nattokinase activity of RB2 is 637.54 IU / g. The addition of Rosa roxburghii Tratt juice dilutes the sample, so the enzyme activity of RB3 decreases. After transformation, the nattokinase activity decreases to 316.48 IU / g. The pH values of RB2, RB3, and RB4 are 7.33, 5.15, and 5.31 respectively. The addition of Rosa roxburghii Tratt juice makes the pH of the sample deviate from the optimal pH of nattokinase, resulting in a decrease in enzyme activity. The enzyme activity of tablet B without the addition of Rosa roxburghii Tratt juice is 1861.73 IU / g, while the nattokinase activity of tablet RB5, which is greatly affected by Rosa roxburghii Tratt juice, is 1040.28 IU / g. The enzyme activity of the mixed tablet RBB is the highest, at 1578.56 IU / g.

[0079] 4.5 Amino acid nitrogen

[0080] As Figure 3, The amino acid nitrogen content of oats can be significantly increased by Aspergillus oryzae fermentation or the co-fermentation of GUTU09 and BLH1. After adding the juice of Rosa roxburghii Tratt, the amino acid nitrogen content slightly increases, and after conversion, it slightly decreases. The amino acid nitrogen contents of tablets M5, RB5, and H are 0.42, 0.53, and 0.27 g / 100 g respectively. The co-fermentation and conversion of GUTU09 and BLH1 increase the amino acid nitrogen content in the Rosa roxburghii Tratt-oat tablets by approximately 96.3%, and the fermentation and conversion of Aspergillus oryzae increase it by approximately 55.5%.

[0081] 4.6 Reducing sugars and polysaccharides

[0082] As Figure 4 , Comparing M5 and H, the fermentation and conversion of Aspergillus oryzae can increase the reducing sugar content in the Rosa roxburghii Tratt-oat tablets by approximately 5.2 times. Among them, the fermentation increases from 1.2 mg / g to 20.6 mg / g, the addition of the juice of Rosa roxburghii Tratt increases by 3.8 mg / g, and the conversion increases from 24.4 mg / g to 36.4 mg / g. The oats fermented by Aspergillus oryzae significantly increase the reducing sugars produced by the conversion of the juice of Rosa roxburghii Tratt, while the oats co-fermented by GUTU09 and BLH1 show no significant change in the reducing sugar content during the conversion of the juice of Rosa roxburghii Tratt. Generally speaking, microbial fermentation and conversion both increase the content of reducing sugars in the samples. The increase in reducing sugars can effectively improve the sour taste of the juice of Rosa roxburghii Tratt in the tablets, thereby enhancing the quality and quality of the Rosa roxburghii Tratt-oat tablets.

[0083] As Figure 5 shown, both Aspergillus oryzae fermentation or the co-fermentation of GUTU09 and BLH1 can increase the polysaccharide content of oats. In particular, the co-fermentation of GUTU09 and BLH1 increases the polysaccharide content in the tablets by approximately 1 time, from 10.0 mg / g to 20.4 mg / g. Rosa roxburghii Tratt contains a high content of polysaccharides, and the addition of the juice of Rosa roxburghii Tratt can increase the polysaccharides in the tablets. Microbial fermentation, the addition of the juice of Rosa roxburghii Tratt, and the conversion process can all increase the polysaccharide content in the tablets, improving the prebiotic functions such as weight maintenance and anti-aging of the tablets, thereby enhancing the quality of the tablets.

[0084] 4.7 Soluble / insoluble dietary fiber

[0085] As Figure 6 shown, after Aspergillus oryzae fermentation, the content of soluble cellulose increases, increasing by 1.37 times, and there is no significant change after conversion. After the co-fermentation of GUTU09 and BLH1, the content of soluble dietary fiber increases, but there is no significant change, and it decreases after conversion, but there is no significant change. After Aspergillus oryzae fermentation and conversion, the content of insoluble cellulose decreases, but there is no significant change. After the co-fermentation of GUTU09 and BLH1, the content of insoluble dietary fiber decreases, but there is no significant change, and it increases after conversion, but there is no significant change. After adding the juice of Rosa roxburghii Tratt for sealed conversion, the content of soluble dietary fiber decreases, which may be related to the change in pH in the samples after the addition of the juice of Rosa roxburghii Tratt.

[0086] 4.8 Viable Bacteria Count Changes

[0087] 4.8.1 Aspergillus oryzae

[0088] As Figure 7 shown, after fermentation by Aspergillus oryzae, the viable bacteria count in the sample increased significantly from 3.6 log CFU / g to 8.7 log CFU / g. After adding the prickly pear juice, the viable bacteria count decreased slightly because the sample was diluted. During the transformation process, the viable bacteria count of Aspergillus oryzae decreased from 8.3 log CFU / g to 8.0 log CFU / g. The viable bacteria count of Aspergillus oryzae in the final tablet M5 was 6.6 log CFU / g. The viable bacteria counts of Aspergillus oryzae in the other tablets MB, MRB, and MRBB were 5.6, 5.3, and 5.6 log CFU / g respectively, with no significant difference.

[0089] 4.8.2 Bifidobacterium BLH1

[0090] As Figure 8 shown, after fermentation by Bifidobacterium and Bacillus natto, the viable bacteria count of Bifidobacterium increased from 6.7 log CFU / g to 8.6 log CFU / g. After adding the prickly pear juice and during the transformation process, the viable bacteria count decreased slightly with no significant change. After freezing, drying, grinding, and tableting, the viable bacteria count of RB5 decreased significantly to 6.3 log CFU / g. The viable bacteria counts of the other tablets B, MB, MRB, RBB, and MRBB were 6.5, 5.4, 5.5, 6.3, and 5.2 log CFU / g respectively.

[0091] 4.8.3 Bacillus natto GUTU09

[0092] As Figure 9 shown, after co-fermentation by Bifidobacterium and Bacillus natto spores, the viable bacteria count of Bacillus natto increased significantly from 6.4 log CFU / g to 11.7 log CFU / g. After adding the prickly pear juice and during the transformation process, the viable bacteria count of Bacillus natto decreased slightly with no significant change. After making tablet RB5, the viable bacteria count was 11.0 log CFU / g. The viable bacteria counts of the other tablets B, MB, MRB, RBB, and MRBB were 11.3, 10.5, 11.0, and 10.5 log CFU / g respectively.

[0093] 4.9 Infrared

[0094] As Figure 10 , single bonds were detected in the high wavenumber region of 4000 - 2500 cm -1 , including the stretching vibrations of C-H (aliphatic, aromatic, and olefinic), O-H (alcohol or phenol), and N-H (amine). In the range of 2000 - 1500 cm -1Double bonds are detected in the region including the stretching vibrations of C=O (acids, esters, non-conjugated ketones, acid anhydrides, aldehydes), C=C (aromatic, alkenes). Triple bonds or cumulated double bonds are detected in the intermediate region of 2500 - 2000 cm-1, including the stretching vibrations of C≡N or N=C=O. A strong and broad absorption peak centered at 3380 cm -1 is for the stretching vibration of the functional group O-H. The peaks at 2924.6 cm -1 and 2852.8 cm -1 are for the symmetric and asymmetric stretching vibrations of the aliphatic functional group –CH2. The peak at 1743.6 cm -1 is the absorption peak of the carbonyl group (C=O) of triglycerides. The absorption peaks of other samples at 1743.6 cm -1 are weaker than that of sample K, indicating that fermentation and the addition of Rosa roxburghii juice have changed the fat structure of the samples. The absorption peaks between 1700 - 1600 cm -1 reflect the changes related to the secondary structure of proteins. The peak shape of sample K at 1640.1 cm -1 is stronger than that of other samples, indicating that fermentation and the addition of Rosa roxburghii juice have changed the secondary structure of proteins in the samples. The peak values from 1630 - 1100 cm -1 correspond to the stretching vibrations related to C-N and C=N in the aromatic ring. 950 - 750 cm -1 is the most commonly used analysis region for carbohydrates, providing important information about the α and β isomers of polysaccharides. In this example, the absorption peaks appearing near 854.2 cm -1 and 761.2 cm -1 are related to this. And for other samples except K, the intensities of these two absorption peaks have changed. Especially, the absorption peak intensity at 854.2 cm -1 has significantly weakened, indicating that fermentation and the addition of Rosa roxburghii juice may have changed the α and β isomers of polysaccharides in the samples.

[0095] 4.10 Secondary structure of proteins

[0096] The results show that Aspergillus oryzae fermentation and co-fermentation of GUTU09 and BLH1 do not significantly change the conformation of β-sheets in the samples (p > 0.05), but the conversion after co-fermentation of GUTU09 and BLH1 followed by the addition of Rosa roxburghii juice will reduce the proportion of this conformation (p < 0.05).

[0097] In addition, Aspergillus oryzae fermentation, co-fermentation with GUTU09 and BLH1, and transformation all increased the proportion of random coil conformation in the samples (p < 0.05). The random coil conformation is an irregular loose peptide chain structure, which can endow the protein molecule with certain flexibility and variability. This indicates that Aspergillus oryzae fermentation, co-fermentation with GUTU09 and BLH1, and transformation can increase the variability of the samples. The intuitive manifestation of the samples is that they become more delicate. The random coil conformation in Aspergillus oryzae fermentation may be the transformation from α-helix to random coil.

[0098] The addition of Rosa roxburghii juice also increased the random coil conformation in the samples (p < 0.05), which may be caused by the significant change in pH. Aspergillus oryzae fermentation decreased the proportion of α-helix conformation in the samples (p < 0.05), while co-fermentation with GUTU09 and BLH1 and the addition of Rosa roxburghii juice did not significantly change this conformation proportion (p > 0.05). The α-helix conformation endows the protein molecule with high stability, which is attributed to the generation of more non-covalent intramolecular interactions.

[0099] Finally, Aspergillus oryzae fermentation, co-fermentation with GUTU09 and BLH1, and the addition of Rosa roxburghii juice all decreased the proportion of β-turn conformation in the samples (p < 0.05). β-turn plays a key role in the conformational changes and molecular flexibility of proteins and often serves as an important structural unit connecting different secondary structures.

[0100] Generally speaking, both fermentation and transformation changed the β-turn and α-helix structures in the samples into β-sheet and random coil structures, making the secondary structure of the proteins in the samples change from ordered to disordered. The change in the secondary structure of proteins may be another main reason for the change in the content of soluble proteins in the samples.

[0101] 4.11 Texture properties

[0102] After the tablets were fermented and transformed, there were no significant changes in hardness, adhesiveness, cohesiveness, and gumminess (p > 0.05), while elasticity and chewiness changed significantly (p < 0.05). After Aspergillus oryzae fermentation, co-fermentation with GUTU09 and BLH1, and then transformation with the addition of Rosa roxburghii juice, the elasticity and chewiness of the samples decreased significantly (p < 0.05). In this example, both fermentation and transformation changed the β-turn and α-helix structures in the samples into β-sheet and random coil structures, making the secondary structure of the proteins change from ordered to disordered. And β-turn plays a key role in the conformational changes and molecular flexibility of proteins, which may be an important reason for the decrease in the elasticity of the tablets. For children, the elderly, or people with swallowing dysfunction, the decrease in the chewiness of the tablets is more conducive to swallowing. Lower chewiness means that the tablets are not easily broken or damaged during transportation and storage, improving the physical stability.

[0103] The results of this example show that the fermentation and transformation of beneficial bacteria effectively improved the sour taste of the Rosa roxburghii juice and the unpopular flavor of the natto bacteria fermentation product, and obtained a mixed tablet RBB with high nattokinase activity and a tablet MRBB with the best neutralized nutrition, function, and flavor.

[0104] Example 2

[0105] 1. The sample preparation and sampling were the same as in Example 1.

[0106] 2. Index determination

[0107] The DPPH free radical scavenging rate, ABTS free radical scavenging rate, ferric reducing ability (FRAP), inhibitory activity against α - amylase, inhibitory activity against α - glucosidase, and in vitro lipid - lowering ability of each sample were determined. The determination of soluble protein was carried out using a BCA protein concentration assay kit. Pre - column derivatization high - performance liquid chromatography with 2,4 - dinitrofluorobenzene (FDNB) was used for the determination of GABA content. The determination of total phenol content was carried out using the Folin - Ciocalteu reagent method (Tian Xueyi, 2022). The determination of total flavonoids was carried out using the method of the People's Republic of China l(NO3)3 method (Xu Changli, 2024). The determination of vitamin C was carried out using high - performance liquid chromatography, referring to the national standard of the People's Republic of China GB 5009.86—2016 "National Food Safety Standard - Determination of Ascorbic Acid in Foods". The determination of in vitro antioxidant activity referred to the master's thesis (Tian Xueyi, 2022). The determination of in vitro hypoglycemic activity referred to the master's thesis (Tian Xueyi, 2022). The cholesterol binding rate was based on the literature method (Fan Shengyu et al., 2024). The bile salt adsorption capacity was slightly modified according to the literature method (Cen Qin, 2024). The determination of the inhibitory rate of angiotensin - converting enzyme activity was carried out using high - performance liquid chromatography, referring to the literature method (Li Feifei, 2017). The determination of volatile flavor compounds referred to the national food safety standard of the People's Republic of China GB23200.8 - 2016. The determination of organic acids referred to the master's thesis (Wu Jialin, 2024). The determination method of free amino acids referred to the master's thesis (Xu Changli, 2024).

[0108] 3. Experimental results

[0109] 3.1 Soluble protein

[0110] As Figure 11 shown, after the addition of Rosa roxburghii juice, the soluble protein content in the sample increased significantly, which may be due to the significant change in the pH of the sample. Comparing sample K and sample H, it was found that the soluble protein content in the Rosa roxburghii oatmeal increased from 24.3 to 417.1 mg / g due to the addition of Rosa roxburghii juice. During the conversion of Rosa roxburghii juice by oats fermented with Aspergillus oryzae, the soluble protein content did not change significantly. During the conversion of Rosa roxburghii juice by oats fermented with the double bacteria GUTU09 and BLH1, the soluble protein content decreased significantly.

[0111] 3.2 GABA

[0112] As Figure 12 shown, after adding Rosa roxburghii juice, the content of GABA in the samples increased because Rosa roxburghii is rich in GABA. The GABA converted from Rosa roxburghii juice by Aspergillus oryzae-fermented oats increased, by 0.95 times. Comparing samples M5, RB5 and H, Aspergillus oryzae fermentation and conversion can increase by about 33.8%, and the double-fermentation and conversion of GUTU09 and BLH1 can increase by about 23.4%. The double-fermentation of GUTU09 and BLH1 will significantly reduce the content of GABA. Natto bacteria can produce unique viscous substances mainly composed of γ-polyglutamic acid (γ-PGA), and the reduction of GABA content after fermentation may be due to the production of these substances.

[0113] 3.3 Total phenols

[0114] As Figure 13 shown, the double-fermentation of GUTU09 and BLH1 significantly increased the total phenol content, by 1.78 times. Aspergillus oryzae fermentation and conversion can increase by about 29.6%. After adding Rosa roxburghii juice, the total phenol content increased significantly because Rosa roxburghii contains rich total phenols. The total phenol content in the tablets fermented by GUTU09 and BLH1 increased significantly, but the biggest factor affecting the total phenol content in the tablets is the addition of Rosa roxburghii juice.

[0115] 3.4 Total flavonoids

[0116] As Figure 14 shown, by comparing M5, RB5 and H, it was found that Aspergillus oryzae fermentation and conversion can increase by about 0.9 times, and the double-fermentation and conversion of GUTU09 and BLH1 have no significant effect on the total flavonoid content of Rosa roxburghii tablets. By comparing K and H, it can be seen that the addition of Rosa roxburghii juice increased the total flavonoid content in the oat tablets from 61.9 mg / 100 g to 385.1 mg / 100 g.

[0117] 3.5 Vitamin C

[0118] As Figure 15 , the source of vitamin C in the Rosa roxburghii oat tablets is mainly Rosa roxburghii juice. Finally, the vitamin C contents in the Rosa roxburghii oat tablets obtained by Aspergillus oryzae fermentation and the double-fermentation of GUTU09 and BLH1 are 14.3 and 22.5 mg / g respectively. Although the vitamin C in Rosa roxburghii juice was lost during the closed conversion process, the vitamin content in the tablets with Rosa roxburghii juice added is still much higher than that in the tablets without Rosa roxburghii juice added. Therefore, the addition of Rosa roxburghii juice in the present invention significantly improves the quality of the tablets.

[0119] 3.6 Antioxidation

[0120] The present invention determined the DPPH free radical scavenging rate (Figure 16 in A), the ABTS radical scavenging rate ( Figure 16 in B), and the ferric reducing antioxidant power ( Figure 16 in C) were used to study the changes in the antioxidant capacity of Rosa roxburghii Tratt oat tablets during fermentation and transformation. The antioxidant activity of the samples was enhanced by Aspergillus oryzae fermentation or co-fermentation with GUTU09 and BLH1. The total phenols of M1 and M2 were 0.383 and 0.481 mg / g, respectively, and the total phenol contents of RB1 and RB2 were 0.356 and 1.002 mg / g, respectively. After fermentation, the total phenol content increased, which was one of the reasons for the increase in antioxidant activity in the samples. The antioxidant capacity increased significantly after adding Rosa roxburghii Tratt juice because Rosa roxburghii Tratt juice contains abundant antioxidant substances, such as phenolic compounds, vitamins, superoxide dismutase (SOD), polysaccharides, and triterpenoids. [[ID=⑥]] [[ID=⑦]]

[0121] [[ID=⑧]]3.7 Hypoglycemic effect [[ID=⑨]] [[ID=⑩]]

[0122] [[ID=⑪]]The inhibition rate of α-amylase activity was as [[ID=⑫]] Figure 17 [[ID=⑬]]shown in A. Comparing samples M5, RB5, and H, it was found that Aspergillus oryzae fermentation and transformation could increase the inhibition rate of α-amylase activity by about 70.0%, and co-fermentation and transformation with GUTU09 and BLH1 could increase it by about 57.0%. As [[ID=⑭]] Figure 17 [[ID=⑮]]shown in B, the inhibition rate of α-glucosidase increased significantly from 8.6% to 73.6% after Aspergillus oryzae fermentation. Comparing M5 and H, it was found that Aspergillus oryzae fermentation and transformation could increase the inhibition rate of α-glucosidase by about 51.5%. The inhibition rate of α-glucosidase did not change significantly after co-fermentation with Bacillus natto and Bifidobacterium. After adding Rosa roxburghii Tratt juice, the inhibition rate of α-glucosidase increased significantly from 13.6% to 64.3%. The addition of Rosa roxburghii Tratt juice decreased the pH, and the pH of the samples changed during fermentation and transformation, deviating from its optimal pH. Therefore, the significant increase in the inhibition rate of α-glucosidase may be affected by the pH. In addition, flavonoids and phenolic substances can bind to the active centers of α-amylase and α-glucosidase or change their spatial conformations, thereby inhibiting the enzyme activity. The addition of Rosa roxburghii Tratt juice significantly increased the total phenol and total flavonoid contents, which may be another reason for the increase in the inhibition rate of α-glucosidase. [[ID=⑯]] [[ID=⑰]]

[0123] [[ID=⑱]]3.8 Hypolipidemic effect [[ID=⑲]] [[ID=⑳]]

[0124] [[ID=㉑]]As [[ID=㉒]] Figure 18 [[ID=㉓]]shown in A, the cholesterol binding rate did not change significantly after Aspergillus oryzae fermentation and after adding Rosa roxburghii Tratt juice. During the transformation process, the cholesterol binding rate decreased significantly from 62.4% to 40.7%. This may be because during the transformation process, some chemical changes occurred between Rosa roxburghii Tratt juice and Aspergillus oryzae-fermented oats, reducing the adsorption sites or weakening the adsorption ability for cholesterol. The cholesterol binding rate of the sample co-fermented with Bacillus natto and Bifidobacterium increased, but not significantly. As [[ID=㉔]] Figure 18In B, the bile salt adsorption capacity in the sample showed an upward trend after Aspergillus oryzae fermentation, addition of Rosa roxburghii juice, and the transformation process, but there was no significant change. The bile salt adsorption capacity of tablet M5 was 1.09 mg / g. The bile salt adsorption capacity in the sample increased significantly from 0.43 mg / g to 0.67 mg / g after fermentation with a mixed culture of Bacillus natto and Bifidobacterium, an increase of 55.8%. The bile salt adsorption capacities of the mixed tablets MB, MRB, RBB, and MRBB were between 0.89 and 1.01 mg / g.

[0125] 3.9 Blood pressure lowering

[0126] Such as Figure 19 , the angiotensin-converting enzyme inhibition rate did not change significantly after Aspergillus oryzae fermentation, and the inhibition rate increased by 21.2% after addition of Rosa roxburghii juice. The angiotensin-converting enzyme inhibition rate increased significantly from 43.6% to 54.4% after fermentation with a mixed culture of Bacillus natto and Bifidobacterium, an increase of 24.8%. The nattokinase activity of the tablets of the present invention was 637.5 IU / g after fermentation with Bacillus subtilis natto, and the nattokinase activity of tablet RB5 was 1040.3 IU / g. The angiotensin-converting enzyme inhibition rates of the mixed tablets MB, MRB, RBB, and MRBB were between 51.6% and 54.8%, showing a good blood pressure lowering effect.

[0127] 3.10 Changes in organic acids

[0128] After Aspergillus oryzae fermentation, the contents of various organic acids increased significantly (p < 0.05). For example, oxalic acid increased from 0.79 mg / g to 2.26 mg / g, and tartaric acid increased from 1.47 mg / g to 12.61 mg / g. During the fermentation process of Aspergillus oryzae, a large amount of organic acids were produced by its metabolic activities. It may be that Aspergillus oryzae contains relevant enzyme systems that can decompose substances such as polysaccharides and proteins in oats to generate organic acids. After addition of Rosa roxburghii juice, the contents of most organic acids decreased because the sample was diluted. However, during the transformation process, some organic acids increased significantly (p < 0.05). For example, formic acid increased from 0.11 mg / g to 7.63 mg / g, and lactic acid increased from 7.75 mg / g to 16.22 mg / g. After addition of Rosa roxburghii juice, more nutrients were provided for Aspergillus oryzae or some metabolic pathways were promoted, which may be the reason for the further increase in organic acids.

[0129] After fermentation by the mixed bacteria of Bacillus natto and Bifidobacterium, the content of organic acids also increased significantly. For example, the formic acid content increased from 1.61 mg / g to 8.64 mg / g, and the malic acid content increased from 0.70 mg / g to 10.20 mg / g, etc. When fermented by the mixed bacteria of Bacillus natto and Bifidobacterium, the two bacteria act synergistically. Bacillus natto can produce some enzymes to decompose the substrate, and Bifidobacterium uses the substrate for metabolism to produce organic acids. After adding Rosa roxburghii juice for conversion, the content of organic acids increased significantly (p<0.05), and the formic acid increased the most, from 3.08 mg / g to 25.65 mg / g. After adding Rosa roxburghii juice, it may have improved the growth environment of the mixed bacteria, promoted their metabolic activities, and increased the production of organic acids. The freeze-drying and tableting process may have little effect on the stability of organic acids and did not cause significant losses.

[0130] Compared with other tablets without Aspergillus oryzae fermentation components, the content of tartaric acid is relatively high in the tablets containing Aspergillus oryzae fermentation components. In addition, in the tablets containing the mixed fermentation components (RB5 and B) of Bacillus natto and Bifidobacterium, the contents of acetic acid and citric acid have certain advantages. The formic acid content in B is 28.02 mg / g, and the formic acid content in RB5 is 68.14 mg / g. RB5 is significantly higher than B (p<0.05). The reason may be that RB5 was converted after adding Rosa roxburghii juice. Rosa roxburghii juice is rich in sugars such as glucose and fructose, providing a rich carbon source for Bacillus natto and Bifidobacterium, and a large amount of pyruvate can be produced through glycolysis and other pathways, and then more formic acid is generated. Generally speaking, the mixed fermentation of Aspergillus oryzae, Bacillus natto and Bifidobacterium can all increase the content of organic acids in the Rosa roxburghii oat tablets, enrich the flavor, adjust the pH value to inhibit bacteria, improve the stability, promote nutrient conversion, and improve the quality of the tablets.

[0131] 3.11 Free Amino Acids

[0132] After fermentation and conversion by the mixed bacteria of Aspergillus oryzae, Bacillus natto and Bifidobacterium, the total amount of amino acids and the total amount of essential amino acids in the tablets increased significantly (p<0.05). Compared with tablet K, the total amount of amino acids in tablet M5 fermented and converted by Aspergillus oryzae increased by 3.69 times, and the total amount of essential amino acids increased by 5.96 times. The essential amino acids that increased more were valine, lysine and phenylalanine. The total amount of amino acids in tablet RB5 fermented and converted by the mixed bacteria of Bacillus natto and Bifidobacterium increased by 2.52 times, and the total amount of essential amino acids increased by 6.65 times. The essential amino acids that increased more were lysine and phenylalanine. It shows that the mixed fermentation and conversion of Aspergillus oryzae, Bacillus natto and Bifidobacterium have improved the quality of the Rosa roxburghii oat tablets.

[0133] Regarding umami, the addition of prickly pear juice significantly reduces the content of aspartic acid and significantly increases the content of glutamic acid (p < 0.05). Tablet M5 fermented and transformed by Aspergillus oryzae can significantly enhance the umami in the tablet, and both aspartic acid and glutamic acid are significantly increased (p < 0.05). Compared with tablet K, the content of aspartic acid has increased by 1.25 times, and the content of glutamic acid has increased by 2.71 times. For tablet RB5 fermented and transformed by the mixed bacteria of Bacillus natto and Bifidobacterium, the content of aspartic acid in umami decreases, while the content of glutamic acid increases by 1.17 times. The taste activity threshold of glutamic acid in the tablet is significantly increased after fermentation and transformation by Aspergillus oryzae, Bacillus natto and Bifidobacterium, indicating that the umami of the tablet has been greatly enhanced.

[0134] Regarding sweetness, fermentation and transformation by Aspergillus oryzae can increase sweetness, and all four sweet amino acids are significantly increased (p < 0.05). Among them, the most abundant is alanine, and the total amount of sweet amino acids has increased by 2.32 times compared with K. For tablet RB5 fermented and transformed by the mixed bacteria of Bacillus natto and Bifidobacterium, two sweet amino acids increase and two sweet amino acids decrease, and the total amount of sweet amino acids has no obvious change compared with K.

[0135] Regarding bitterness, the total amount of bitter amino acids in the oat tablets fermented and transformed by the mixed bacteria of Aspergillus oryzae, Bacillus natto and Bifidobacterium has increased significantly. For tablet M5 of oat tablets fermented and transformed by Aspergillus oryzae, the total amount of bitter amino acids has increased by 6.95 times compared with tablet K, and the most increased is arginine. For tablet RB5 of oat tablets fermented by the mixed bacteria of Bacillus natto and Bifidobacterium, the total amount of bitter amino acids has increased by 7.03 times, and the most increased is phenylalanine. From the content of phenylalanine in tablet B, it can be seen that the source of phenylalanine in tablet RB5 is mainly from the fermentation process of the mixed bacteria of Bacillus natto and Bifidobacterium. Comparing tablets K and H, the addition of prickly pear juice has increased the bitter amino acids in the prickly pear oat tablets by 2.73 times, and the most increased is arginine. Although phenylalanine and arginine are bitter, they can both improve the overall nutritional quality of the tablet.

[0136] Fermentation and transformation by the mixed bacteria of Aspergillus oryzae, Bacillus natto and Bifidobacterium can significantly improve the quality of prickly pear oat tablets. In terms of nutrition, it significantly increases the total amount of amino acids and essential amino acids, supplements key nutrients, and helps with growth and development and immune enhancement. In terms of flavor, it enhances umami and sweetness, enriches the taste. Although the bitter amino acids produced by fermentation increase the bitterness, they also improve the nutritional quality and participate in important physiological processes.

[0137] 3.12 Volatile flavor compounds

[0138] The results showed that there were 1,216 flavor substances in the Rosa roxburghii Tratt oatmeal tablets. The total contents of the volatile flavor substances in the 9 tablets, namely M5, RB5, B, K, H, MB, MRB, RBB, and MRBB, were 134.022, 510.501, 167.089, 127.023, 130.999, 172.581, 292.447, 225.030, and 209.248 μg / g, respectively. It can be seen from this that the co-fermentation of GUTU09 and BLH1 can significantly increase the content of volatile flavor compounds in the Rosa roxburghii Tratt oatmeal tablets, and the total amount increases by 2.9 times. The increase in the flavor substance content of the tablets fermented by Aspergillus oryzae is relatively small. The research results show that by comparing K and M5, it can be seen that the fermentation by Aspergillus oryzae increases the terpene volatile flavor substances; by comparing K and B, it can be seen that the co-fermentation of GUTU09 and BLH1 produces the most flavor compounds, which are heterocyclic compounds; by comparing B and RB5, it can be seen that the addition of Rosa roxburghii Tratt juice promotes the increase of ester compounds in the Rosa roxburghii Tratt oatmeal tablets.

[0139] The content of β-elemene in tablet M5 was the highest among the 9 tablets, which was 0.304 μg / g, indicating that this was one of the important characteristic flavor compounds produced by the fermentation of Aspergillus oryzae. The characteristic terpene compounds produced by the fermentation of Aspergillus oryzae also included β-bisabolene, aromadendrene, patchoulene, etc. Among them, there were 6 kinds of naphthalenes. Therefore, the fermentation of Aspergillus oryzae not only provided the Rosa roxburghii Tratt oatmeal tablets with a special fresh and chestnut flavor, but also improved the functional properties of the tablets. The co-fermentation of GUTU09 and BLH1 did not produce terpene characteristic compounds, but after its transformation with Rosa roxburghii Tratt juice, 4 characteristic terpene compounds, namely (S)-(+)-α-phellandrene, carene, (+)-4-pinene, and 3-carene, were produced, and the contents of these three substances were very high, all greater than 2 μg / g.

[0140] The heterocyclic compounds are the flavor compounds produced in the largest amounts by the mixed fermentation and conversion of GUTU09 and BLH1. There are 12 increased ones as follows, including 2-acetyl-3-methylpyrazine, methoxypyrazine, 2,5-dimethylpyrazine, 2-ethylpyridine, 2-methyl-1,3-dithiolane, 2,3,5-trimethylpyrazine, 2-ethyl-3-methylpyrazine, 2-(n-propyl)pyrazine, 4,6-dimethylpyrimidine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, and 2-ethyl-5-methylpyrazine. Among them, there are 9 kinds of pyrazines. These are the differential flavor substances between tablet RB5 and B, and they may be the main flavor substances that improve the less acceptable taste of natto bacteria fermentation products. The pyrazine compounds increased in this study all have alkyl groups. Research shows that the alkyl groups in pyrazines can improve the adsorption of proteins to pyrazines, which helps the stability and release of pyrazine flavor substances in tablets. Among them, 2,5-dimethylpyrazine is a typical pyrazine flavor compound, which can prevent fruit diseases and improve the quality of food in terms of enhancing its biological activity, color, flavor, etc. The substances increased by Aspergillus oryzae fermentation are coumarin and 2-(2-methylpropyl)pyridine.

[0141] The contents of coumarin in tablets M5, MB, MRB, and MRBB in this example are 0.121, 0.056, 0.058, and 0.033 μg / g respectively. When applied to daily diet, it will far not exceed the TDI standard. The contents of 3-propylpyridine and 2-aminopyridine increased after the mixed fermentation of GUTU09 and BLH1 and the addition of Rosa roxburghii juice for conversion. Whether it is the sample after Aspergillus oryzae fermentation or the double-bacteria fermentation of GUTU09 and BLH1, the contents of 2-acetyl-5-methylfuran and 1,2,3,6-tetrahydropyridine in the sample decreased after the addition of Rosa roxburghii juice for conversion.

[0142] The substances significantly reduced by Aspergillus oryzae fermentation include isophorone, 6-bromo-2-hexanone, and 1-methyl-2-pyrrolidone, etc. The ketone flavor compounds significantly increased after the mixed fermentation of GUTU09 and BLH1 are 1,3-dihydroxyacetone, and the significantly reduced one is 2,3-heptanedione. The ketone flavor compounds significantly increased after the mixed fermentation and conversion of GUTU09 and BLH1 are 1,3-dihydroxyacetone and 1-methyl-2-pyrrolidone, etc. 1,3-dihydroxyacetone is beneficial to human health.

[0143] The ester flavor compounds significantly increased after the mixed fermentation of GUTU09 and BLH1 and the addition of Rosa roxburghii juice for conversion include ethyl benzoate, etc. There are 16 ester flavor compounds with significantly increased contents after the mixed fermentation of GUTU09 and BLH1 and the addition of Rosa roxburghii juice for conversion. The total content of ester flavors increased from 20.64 μg / g to 34.56 μg / g. This may be because the addition of a large amount of acidic substances in Rosa roxburghii juice led to the increase of ester flavor compounds.

[0144] The obvious aldehyde flavor compounds increased by Aspergillus oryzae fermentation include phenylacetaldehyde, (E)-2-octenal, 2-octenal, etc. The obvious aldehyde flavor compounds increased after the mixed fermentation of GUTU09 and BLH1 include 4-oxodecanal, (E)-2-hexenal, etc. The obvious aldehyde flavor compounds significantly increased after the mixed fermentation of GUTU09 and BLH1 with the addition of Rosa roxbunghii juice conversion include benzaldehyde, phenylacetaldehyde, etc. Benzaldehyde is an aromatic aldehyde with caramel and roasted meat flavors, but some studies have shown that benzaldehyde is closely related to the formation of carcinogenic benzene. The daily intake of benzaldehyde set by the Food and Agriculture Organization of the United Nations (FAO / WHO) (1996) is 5 mg / kg·bw·d. The content range of benzaldehyde in the tablets of this study is 0.042 - 0.278 μg / g, which is far lower than this standard during consumption.

[0145] 127 volatile flavor compounds with OAV>1 were detected in nine kinds of tablets. The numbers of volatile flavor compounds with OAV>1 in M5, RB5, B, K, H, MB, MRB, RBB, and MRBB are 124, 110, 104, 103, 101, 118, 121, 109, and 120 respectively.

[0146] The results of principal component analysis show that the contribution rate of PC1 is 37.6%, the contribution rate of PC2 is 25.1%, and the total contribution rate is 62.7%. The flavors of RBB and RB5 are mainly driven by principal component 1, M5 and MB are mainly affected by principal component 2, and MRB and MRBB show the synergistic effect of the two principal components. The positions of samples B, K, and H show that they are greatly affected by the negative characteristic substances of principal component 1 and principal component 2, and the flavors are relatively much less, so the odors of tablets B, K, and H are light. The samples are aggregated near the center, indicating that these samples are relatively close to the overall characteristics of the sample group in flavor characteristics. Consistent with the experimental results, MRBB is composed of samples B, M5, RB5, etc. in equal proportions, echoing the result that the flavor characteristics of MRBB are relatively close to all the overall characteristics obtained from principal component analysis.

[0147] In the principal component analysis graph, the characteristic flavor compounds in the sample can be analyzed by the proximity of the flavor compound points and the sample points. The characteristic flavor compounds of M5 are D16 (2(5H)-furanone), B28 ((2E,4Z)-2,4-decadienal), B29 ((E,E)-2,4-decadienal); the characteristic flavor compound of MB is A7 (2-thienylmethyl mercaptan); the characteristic flavor compounds of B and K are F11 (p-cresol); the characteristic flavor compounds of H are D6 (5-ethyl-2(5H)-furanone), F11 (p-cresol); the characteristic flavor compound of RBB is E17 (δ-dodecalactone); the characteristic flavor compounds of RB5 are B16 (3-methylthio butyraldehyde), E22 (2-methylpropyl butyrate), F10 (guaiacol); the characteristic flavor compounds of MRB are B18 ((Z)-6-nonenal), D11 (tetrahydro-6-methyl-2H-pyran-2-one); the characteristic flavor compounds of MRBB are D4 (1-(2-thienyl)ethanone), C10 ((-)-carvyl acetate).

[0148] Example 3

[0149] 1. The sample preparation and sampling are the same as in Example 1.

[0150] 2. Index determination

[0151] The determination of cellulase activity refers to the agricultural industry standard of the People's Republic of China "NY / T 912-2004". The determination of superoxide dismutase (SOD) activity uses a detection kit (WST-1 method) (Solarbio Science & Technology Co., Ltd., Beijing, BC5165). The determination of β-glucosidase activity uses the p-nitrophenyl glucoside hydrolysis method (Wu Jialin, 2024). The determination of protease activity refers to the industry standard of the People's Republic of China "SB / T 10317-1999". The determination of esterase uses the p-nitrophenol colorimetric method (Xie Wei, 2023).

[0152] 3. Experimental results

[0153] 3.1 Cellulase activity

[0154] Aspergillus oryzae fermentation increased the cellulase activity in the sample to 0.25 U / g. The addition of Rosa roxburghii juice led to a decrease in enzyme activity to 0.02 U / g, and there was no significant change in cellulase activity during the conversion. The co-fermentation of GUTU09 and BLH1 increased the cellulase activity to 0.35 U / g, and the cellulase activity increased to 0.68 U / g during the conversion. Both Aspergillus oryzae fermentation and the dual fermentation of GUTU09 and BLH1 could produce cellulase. After the addition of Rosa roxburghii juice, the cellulase activity decreased. One reason was that the addition of Rosa roxburghii juice diluted the sample, and another reason might be the significant change in pH. The pH values of M2 and RB2 were 5.93 and 7.33 respectively, and the pH values of M3 and RB3 were 4.34 and 5.15 respectively. The pH in the sample was far from the optimal pH, which might be the main reason for the decrease in cellulase activity. After the conversion, the pH values of M4 and RB4 were 4.50 and 5.31 respectively, and the pH was closer to the optimal pH of cellulase, so the cellulase activity increased in M4 and RB4.

[0155] 3.2 β-Glucosidase activity

[0156] The results showed that both Aspergillus oryzae fermentation and the dual fermentation of GUTU09 and BLH1 could produce β-glucosidase. The pH values of M2 and RB2 were 5.93 and 7.33 respectively. The pH of RB2 deviated far from the optimal pH of β-glucosidase, which might lead to a decrease or even inactivation of enzyme activity, resulting in no detected enzyme activity, while the pH of M2 was closer to the optimal pH. However, after the addition of Rosa roxburghii juice, the pH values of M3 and RB3 were 4.34 and 5.15 respectively, which led to a decrease in the enzyme activity of M3 compared to M2, and the appearance of enzyme activity in RB3 compared to RB2. After the conversion, the pH values of M4 and RB4 were 4.50 and 5.31 respectively. The enzyme activity of M4 had no significant change compared to M3, while the enzyme activity of RB4 increased significantly by about 1 fold, which might be the result of the continuous production of enzymes by microorganisms. The influence of pH on β-glucosidase activity was significant. The addition of Rosa roxburghii juice had a positive effect on the β-glucosidase activity in RB5. Therefore, the addition of Rosa roxburghii juice could not only bring the fragrance of Rosa roxburghii to the product but also promote β-glucosidase to improve the flavor, aroma, and taste of the product.

[0157] 3.3 Protease activity

[0158] The results showed that both Aspergillus oryzae fermentation and the dual fermentation of GUTU09 and BLH1 could produce protease. After Aspergillus oryzae fermentation, the enzyme activity increased significantly from 3.8 U / g to 160.7 U / g, and after the dual fermentation of GUTU09 and BLH1, the protease enzyme activity increased significantly from 4.3 U / g to 35.8 U / g. Comparing M5, RB5, and H, it was found that Aspergillus oryzae fermentation and conversion could increase by about 66.5 U / g, and the dual fermentation and conversion of GUTU09 and BLH1 could increase by about 74.1 U / g.

[0159] 3.4 Esterase activity

[0160] After Aspergillus oryzae fermentation, the esterase activity increased significantly, from 0.15 U / g to 0.92 U / g, a 5.13-fold increase (p < 0.05). During the conversion process, the enzyme activity decreased significantly by 58%. The decrease in esterase activity during the conversion may be due to the decrease in pH from 5.9 to 4.3 after the addition of Rosa roxburghii juice. Although the pH increased to 4.5 during the conversion process, it was still far from the optimal pH of esterase. After co-fermentation with GUTU09 and BLH1, the enzyme activity increased significantly, from 0.18 U / g to 1.95 U / g. During the conversion, the enzyme activity increased significantly, from 1.07 U / g to 2.24 U / g, an increase of about 1-fold. This may be because the pH increased from 5.1 to 5.3, making it closer to the optimal pH of esterase. Generally speaking, the co-fermentation and conversion of GUTU09 and BLH1 can increase the esterase activity by about 1.4 times.

[0161] 3.5 SOD activity

[0162] The results showed that oats itself had SOD activity. After Aspergillus oryzae fermentation or co-fermentation with GUTU09 and BLH1, the SOD activity increased. After Aspergillus oryzae fermentation, the SOD enzyme activity increased by about 1.2 times. After co-fermentation with GUTU09 and Bifidobacterium, the SOD enzyme activity increased by about 3.2 times. The SOD activity of H was much higher than that of K. The decrease in SOD activity of RB3 relative to RB2 may be due to some chemical changes between a certain substance produced by the co-fermentation of GUTU09 and BLH1 and a certain substance in Rosa roxburghii juice, resulting in a decrease in SOD enzyme activity. The decrease in SOD activity during the conversion may be due to some inevitable exposure to light and oxygen during the experiment.

[0163] 4 Storage stability and shelf-life prediction

[0164] The sensory prediction results showed that the predicted storage times of Rosa roxburghii oat tablets MRBB at 4 °C, 25 °C and 37 °C were 84, 72 and 66 days respectively, and the predicted storage times of tablets RBB at 4 °C, 25 °C and 37 °C were 81, 73 and 69 days respectively.

[0165] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A preparation method of fermented Rosa roxburghii Tratt and oat tablets, characterized in that, It includes the following steps: (1) Mix oats and soy milk, inoculate with Aspergillus oryzae seed liquid for fermentation to obtain fermentation broth I; add Rosa roxburghii juice to fermentation broth I for crushing treatment, then carry out sealed conversion, freeze-dry, and grind to obtain freeze-dried powder I; (2) Mix oats and soy milk, inoculate with BLH1 and GUTU09 seed liquid for fermentation to obtain fermentation broth II; add Rosa roxburghii juice to fermentation broth II for crushing treatment, then carry out sealed conversion, freeze-dry, and grind to obtain freeze-dried powder II; (3) Mix oats and soy milk, inoculate with BLH1 and GUTU09 seed liquid for fermentation, freeze-dry, and grind to obtain freeze-dried powder III; (4) Combine freeze-dried powders I, II, and III, mix and press into tablets, which are the fermented Rosa roxburghii oat tablets.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of oats to soy milk is 1:1.5; the inoculation amount of the Aspergillus oryzae seed liquid is 4% (v:v), and the fermentation time is 24 h; the volume ratio of fermentation broth I to Rosa roxburghii juice is 2.5:1.5; the time for sealed conversion is 6 h, and the conditions are 27 °C and 170 rpm.

3. The preparation method according to claim 1, characterized in that In step (2), the mass ratio of oats to soy milk is 1:1.5; the inoculation amounts of BLH1 and GUTU09 seed liquids are 4% (v:v), and the fermentation time is 24 h; the viable bacteria number ratio of BLH1 and GUTU09 seed liquids is 2.5:1; the volume ratio of fermentation broth II to Rosa roxburghii juice is 2.5:1.5; the time for sealed conversion is 6 h, and the conditions are 37 °C and 170 rpm.

4. The preparation method according to claim 1, characterized in that In step (3), the mass ratio of oats to soy milk is 1:1.5; the inoculation amounts of BLH1 and GUTU09 seed liquids are 4% (v:v), and the fermentation time is 24 h; the viable bacteria number ratio of BLH1 and GUTU09 seed liquids is 2.5:

1.

5. The preparation method according to claim 1, wherein, In step (3), the mass ratio of freeze-dried powders I, II, and III is 1:1:

1.

6. The fermented Rosa roxburghii oat tablets prepared by the preparation method according to any one of claims 1-5.

Images