A method for converting sinigrin to p-hydroxyphenylacetonitrile in fried mustard seed

Abstract

The application belongs to the technical field of traditional Chinese medicine, and discloses a method for converting sinigrin in fried white mustard seed into p-hydroxyphenylacetonitrile. The method is realized through solid fermentation of probiotic bacteria. Through HPLC content determination, it is obtained that the degradation rates of sinigrin in fried white mustard seed after fermentation of lactobacillus plantarum, bacillus subtilis and saccharomyces cerevisiae are 73.11%, 19.35% and 14.21% respectively, and the conversion rates of p-hydroxyphenylacetonitrile are 69.29%, 6.75% and 14.37% respectively. Moreover, the precision, stability, repeatability and recovery test prove that the method has good precision, stability, repeatability and accuracy. The method will greatly promote the research on asthma disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a method for converting myosin into p-hydroxyphenylacetonitrile in fried myosin. BACKGROUND

[0002] Asthma is the most important chronic lung disease threatening public health worldwide. In recent years, the morbidity and mortality of asthma are on the rise. At present, the global incidence of asthma is 5% to 6%, and the total prevalence of asthma in developed countries is 7% to 8%, which is on the rise year by year. The incidence of asthma in China is 1% to 4%, and according to conservative estimates, there are more than 10 million asthma patients in China. The death caused by asthma accounts for 1 / 250 of the total global mortality. It is estimated that the global asthma patients will increase from 300 million to 400 million by 2025. With the acceleration of modern life pace, the complexity of social roles and the aggravation of environmental pollution, the incidence and prevalence of bronchial asthma in China are increasing year by year, which has brought a serious economic burden to the society. Therefore, seeking an efficient, inexpensive and low-toxicity treatment method to prevent or reduce acute attacks and improve the quality of life of patients has become a research hotspot in the medical field.

[0003] The clinical effect of "winter disease treated in summer" three-summer acupoint sticking is excellent in the treatment of chronic bronchial asthma, and the active ingredients of the main drug myosin include myosin, myosin thiocyanate, mustard acid and the like. However, the myosin in the main drug myosin is hydrolyzed into mustard oil by coexisting myosin enzyme, which has strong irritation to the skin mucosa and easily causes skin edema and blistering, resulting in poor safety of three-summer sticking. Modern research shows that the activity of myosin enzyme can be destroyed after the myosin is fried, the hydrolysis of myosin into mustard oil is prevented, the pungent nature of myosin is alleviated, and the content of p-hydroxyphenylacetonitrile, a cough-relieving component of myosin, is increased, so that the efficacy of myosin is further enhanced. If the myosin can be further converted into p-hydroxyphenylacetonitrile on the basis of fried myosin, the research on asthma disease will be greatly promoted. In view of this, the application provides a method for converting myosin into p-hydroxyphenylacetonitrile in fried myosin. SUMMARY

[0004] The application provides a method for converting myosin into p-hydroxyphenylacetonitrile in fried myosin.

[0005] In order to achieve the above purpose, the application adopts the following technical scheme:

[0006] The application provides a method for converting myosin into p-hydroxyphenylacetonitrile in fried myosin, which converts the myosin in fried myosin into p-hydroxyphenylacetonitrile through solid fermentation of probiotics.

[0007] Further, the probiotics include lactobacillus plantarum, bacillus subtilis or saccharomyces cerevisiae.

[0008] Further, the method specifically comprises the following steps:

[0009] Step 1, preparation of fried white rapeseed: take raw white rapeseed and heat to fry, until there is a popping sound and spicy gas is emitted, then take out and cool down, crush the fried white rapeseed, sieve, and obtain the fried white rapeseed; this step can destroy the myrosinase contained in the white rapeseed, preventing the hydrolysis of sinigrin by myrosinase during fermentation.

[0010] Step 2, solid-state fermentation of fried white rapeseed: take the fried white rapeseed above and add distilled water, mix uniformly, sterilize, cool down, inoculate probiotic bacteria liquid, mix uniformly with a sterile glass rod, constant temperature culture, take out and freeze-dry, and obtain the white rapeseed solid-state fermentation sample;

[0011] Step 3, preparation of test sample solution, and determination of the content of active ingredients of sinigrin and sinapine thiocyanate in white rapeseed.

[0012] Further, the frying temperature of the raw white rapeseed in step 1 is 180℃, and the sieve size is 40 mesh.

[0013] Further, in step 2, the solid-liquid ratio of fried white rapeseed to distilled water is 1:1.5; the sterilization temperature is 121℃, and the time is 15 min; the inoculation amount of probiotic bacteria liquid is 10%; the constant temperature culture temperature is 30℃, and the time is 168 h.

[0014] Further, the preparation process of the probiotic bacteria liquid in step 2 is as follows:

[0015] Dissolve the probiotic bacteria freeze-dried powder in the corresponding liquid medium, inoculate in the corresponding solid medium, activate and culture for 2 days; from the activated probiotic bacteria solid culture medium, inoculate single colonies in the corresponding liquid medium with an inoculation loop, and incubate at 30℃ for 3 days to obtain the probiotic bacteria liquid.

[0016] Further, the preparation method of the solid medium is as follows:

[0017] ① Bacillus subtilis activation culture medium: proteose peptone 5.0 g, beef extract 3.0 g, NaCl 5.0 g, agar 15.0 g, distilled water 1.0 L, pH value 7.0. Sterilize at 121℃ for 15 min, store in a 4℃ refrigerator, and use as needed;

[0018] ② Lactobacillus plantarum activation medium: tryptone 10.0g, sodium acetate 5.0g, diammonium citrate 2.0g, yeast powder 5.0g, Tween 80 1.0g, K2HPO4 (dipotassium hydrogen phosphate) 2.0g, MgSO4·7H2O 0.2g, glucose 5.0g, MnSO4·H2O 0.05g, beef extract powder 10.0g, CaCO3 20.0g, agar 15.0g, distilled water 1.0L, pH 6.8, sterilized by moist heat at 121℃ for 15min, stored at 4℃ for later use;

[0019] ③ Saccharomyces cerevisiae activation medium: Prepared with 1.0L of 5°Bé malt extract and 15.0g of agar, at natural pH, sterilized by moist heat at 121℃ for 15min, and stored in a refrigerator at 4℃ for later use;

[0020] The liquid culture medium is prepared as follows: the solid culture medium is sterilized by removing agar powder, while keeping other components unchanged. It is then sterilized by moist heat at 121°C for 15 minutes and stored in a refrigerator at 4°C for later use.

[0021] Furthermore, the preparation process of the test sample solution in step 3 is as follows: accurately weigh 0.2g of the white mustard seed solid fermentation sample obtained in step 2, accurately add 25mL of 70% methanol, weigh the sample, sonicate at 250W power and 40kHz frequency for 30 minutes, remove the sample, cool it, weigh it again, replenish the lost weight with 70% methanol, shake well, filter the sample, and collect the filtrate to obtain the final product.

[0022] Furthermore, the content determination in step 3 is performed by HPLC, with the specific chromatographic conditions as follows:

[0023] Column: Diamonsil(2)C 18 (4.6 mm × 250 mm, 5 μm); Mobile phase: acetonitrile (A) - 0.1% phosphoric acid solution (B), gradient elution (0–10 min, 4% A; 10–25 min, 4%–18% A; 25–40 min, 18–21% A; 40–50 min, 21–60% A; 50–60 min, 60–70% A); Column temperature: 30 °C, flow rate: 1.0 mL·min -1 Detection wavelength: 225nm; injection volume: 10μL;

[0024] The regression equation for glucosinolate was Y = 18569X + 242731, r = 0.9995, with a linear range of 91–910 μg / mL; the regression equation for sinigrin thiocyanate was Y = 15064X + 34543, r = 0.9997, with a linear range of 32.8–328 μg / mL; and the regression equation for p-hydroxyphenylacetonitrile was Y = 36023X + 107350, r = 0.9997, with a linear range of 18.8–188 μg / mL.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] This invention provides a method for converting glucosinolates in roasted white mustard seeds into p-hydroxyphenylacetonitrile through probiotic solid-state fermentation. Using this method, after fermentation with *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae*, the degradation rates of glucosinolates in roasted white mustard seeds are 73.11%, 19.35%, and 14.21%, respectively, while the conversion rates of p-hydroxyphenylacetonitrile are 69.29%, 6.75%, and 14.37%, respectively. Furthermore, precision, stability, repeatability, and recovery tests demonstrated that the method of this invention possesses good precision (RSDs for myrosinone, sinigrin thiocyanate, and p-hydroxyphenylacetonitrile were 1.12%, 2.17%, and 1.90%, respectively), stability (RSDs for myrosinone, sinigrin thiocyanate, and p-hydroxyphenylacetonitrile were 0.78%, 1.95%, and 1.60%, respectively), repeatability (RSDs for myrosinone, sinigrin thiocyanate, and p-hydroxyphenylacetonitrile were 0.82%, 1.81%, and 1.15%, respectively), and accuracy (average recoveries for myrosinone, sinigrin thiocyanate, and p-hydroxyphenylacetonitrile were 98.94%, 100.31%, and 100.02%, respectively; RSDs were 1.28%, 2.11%, and 2.10% (n=9)). Attached Figure Description

[0027] Figure 1 The figure shows the HPLC chromatogram of the mixed reference standards, containing 1-sinogenin, 2-sinogenin thiocyanate, and 3-p-hydroxyphenylacetonitrile.

[0028] Figure 2 This is the HPLC chromatogram of a white mustard seed sample solution.

[0029] Figure 3 This is the HPLC chromatogram of a solid-state fermentation sample solution of white mustard seeds.

[0030] Figure 4 This is the HPLC chromatogram of the blank solution.

[0031] Figure 5 The effect of different probiotic fermentation on the content of glucosinolates.

[0032] Figure 6 The effect of different probiotic fermentation on the content of sinigrin thiocyanate.

[0033] Figure 7 The effect of different probiotic fermentation on the content of p-hydroxyphenylacetonitrile. Detailed Implementation

[0034] The technical solutions of the present application will be described in detail below in combination with the embodiments of the present application and the drawings. It should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of variations and improvements can be made, which should also be considered as falling within the scope of protection of the present application.

[0035] 1. Instruments and reagents

[0036] Instruments: PH-070A biochemical incubator (Shanghai Yiheng Scientific Instruments Co., Ltd.); FW-100 ultramicro traditional Chinese medicine grinder (Beijing Yongguangming Medical Instrument Co., Ltd.); 14-1892 ultrasonic cleaner (Ningbo Xinzhi Biological Technology Co., Ltd.); 15-0259 freeze dryer (Ningbo Xinzhi Biological Technology Co., Ltd.); FA2104 electronic analytical balance (Shanghai Precision Instruments and Meter Co., Ltd.); Waters 2695 / 2998 high performance liquid chromatograph (Waters Corporation, USA); YXQ.SG4b·280 high pressure steam sterilization pot (Shanghai Medical Nuclear Instrument Factory); D500-1 medicine roaster (Henan Jetwin Machinery Equipment Co., Ltd.).

[0037] Reagents: white mustard seed (batch number: 210701, Anguo Anxing Chinese Herbal Pieces Co., Ltd.); sinigrin (batch number: wkq22030702, Sichuan Weikeqi Biological Technology Co., Ltd.); myrosine thiocyanate (batch number: 5868, Shanghai Shidand Standard Technology Service Co., Ltd.); p-hydroxyphenylacetonitrile (batch number: 111-RC-0010), allyl isothiocyanate (batch number: 1211-RB-0027), CATO Guangzhou Jiutu Technology Co., Ltd.; phosphoric acid (AR, batch number: 20210807, 500 mL / bottle), dipotassium hydrogen phosphate (AR, batch number: 20200902), glucose (AR, batch number: 20210517), manganese sulfate monohydrate (GR, batch number: 20210520), anhydrous sodium acetate (AR, batch number: 20210923), calcium carbonate (AR, batch number: 20210802), Tianjin Kemeluo Chemical Reagent Co., Ltd.; sodium chloride (AR, batch number: 20200802, Tianjin Beichen Fangzheng Reagent Co., Ltd.); beef extract powder (batch number: 20211021), agar powder (batch number: 20210903), Tween 80 (batch number: 202106), malt extract infusion (batch number: 20211021), Shanghai Ruichu Biological Technology Co., Ltd.; yeast extract (batch number: 4304391-02), proteose peptone (batch number: 3130821), OXOID, UK; diammonium hydrogen citrate (AR, batch number: C12326372), magnesium sulfate heptahydrate (AR, batch number: C12288818), Shanghai Maikelin Biological Technology Co., Ltd.; methanol (AR, batch number: 20220101), Tianjin Zhiyuan Chemical Reagent Co., Ltd.; acetonitrile (chromatographically pure, OCEANPAK, USA).

[0038] Fermentation strains: Lactobacillus plantarum (batch number: 21794); Saccharomyces cerevisiae (batch number: 1421); Bacillus subtilis (batch number: 20445), purchased from China Industrial Microbial Strain Preservation and Management Center (CICC).

[0039] 2. Experimental methods

[0040] 2.1 Preparation of strain activation medium

[0041] (1) Solid culture medium:

[0042] ① Bacillus subtilis activation medium: proteose peptone 5.0 g, beef extract 3.0 g, NaCl 5.0 g, agar 15.0 g, distilled water 1.0 L, pH value adjusted to 7.0. 121℃ wet heat sterilization for 15 min, stored in a 4℃ refrigerator, ready for use.

[0043] Lactobacillus plantarum activation medium: 10.0 g of tryptone, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 5.0 g of yeast powder, 1.0 g of Tween 80, 2.0 g of K2HPO4 (dipotassium hydrogen phosphate), 0.2 g of MgSO4.7H2O, 5.0 g of glucose, 0.05 g of MnSO4.H2O, 10.0 g of beef extract powder, 20.0 g of CaCO3, 15.0 g of agar, 1.0 L of distilled water, pH 6.8 (adjusted with 1 mol / L NaOH), 121°C moist heat sterilization for 15 min, stored in a 4°C refrigerator, and ready for use.

[0044] Saccharomyces cerevisiae activation medium: 1.0 L of 5°Bé wort, 15.0 g of agar, natural pH value, 121°C moist heat sterilization for 15 min, stored in a 4°C refrigerator, and ready for use.

[0045] (2) Liquid medium: The liquid medium was prepared by removing the agar powder from the above-mentioned solid medium, and the other components remained unchanged, 121°C moist heat sterilization for 15 min, stored in a 4°C refrigerator, and ready for use.

[0046] 2.2, Activation of strains: Bacillus subtilis freeze-dried powder, Lactobacillus freeze-dried powder, and Saccharomyces cerevisiae freeze-dried powder were respectively added to 0.5 mL of the above-mentioned liquid medium and completely dissolved, inoculated into the above-mentioned solid medium, and activated for 2 d.

[0047] 2.3, Preparation of liquid seed solution: Single colonies were picked from the activated solid medium of each strain with a inoculation loop and inoculated into the liquid medium of step 2.1. Incubate at 30°C for 3 d to obtain the fermentation liquid seed solution, which is the probiotic bacteria solution required for solid-state fermentation.

[0048] 2.4, Solid-state fermentation of fried white mustard seed:

[0049] Take 3000 g of raw white mustard seed and place it in a frying machine. Heat it at 180°C (or any temperature between 180-200°C) to destroy the myrosinase enzyme in the raw white mustard seed. When the sound of explosion and the spicy aroma are emitted, take it out and cool it down. Grind the fried white mustard seed and pass it through a 40-mesh sieve (or any mesh between 40-50 mesh) to obtain the fried white mustard seed sample, which is ready for use.

[0050] Take 50 g of the above white mustard seed stir-fried sample, place it in a 500 mL triangular flask, add distilled water at a ratio of 1:1.5, mix well, tightly plug the cotton plug, seal with cow leather paper, sterilize at 121℃ for 15 min, cool; inoculate the three probiotic bacteria Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus subtilis under item 2.3 with a bacteria inoculation amount of 10%, then mix well with a sterile glass rod, seal with 8 layers of gauze, and place in a biochemical incubator at a fermentation temperature of 30℃ for continuous constant temperature culture for 168 h, take out, freeze-dry, and obtain the white mustard seed solid-state fermentation sample.

[0051] 2.5, Establishment of content determination method

[0052] (1) Preparation of mixed reference solution: accurately weigh 9.10 mg of sinigrin reference substance, 3.28 mg of myrosinase thiocyanate reference substance, and 1.88 mg of p-hydroxyphenylacetonitrile reference substance into a 10 mL volumetric flask, dissolve and dilute to the mark with 70% methanol, shake well, and obtain; the concentrations of sinigrin, myrosinase thiocyanate, and p-hydroxyphenylacetonitrile reference substances are 0.910 mg·mL -1 , 0.328 mg·mL -1 , and 0.188 mg·mL -1 , respectively.

[0053] (2) Preparation of test solution: take about 0.2 g of the white mustard seed stir-fried sample and white mustard seed solid-state fermentation sample under item 2.4, accurately weigh, place in a conical flask with a plug, accurately add 25 mL of 70% methanol, weigh, ultrasonic treat (power 250 W, frequency 40 kHz) for 30 min, take out, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, and obtain.

[0054] (3) Chromatographic conditions: chromatographic column: Diamonsil (2) C18 (4.6 mm x 250 mm, 5 μm); mobile phase: acetonitrile (A)-0.1% phosphoric acid solution (B), gradient elution (0-10 min, 4% A; 10-25 min, 4%-18% A; 25-40 min, 18-21% A; 40-50 min, 21-60% A; 50-60 min, 60-70% A); column temperature: 30℃, flow rate: 1.0 mL·min-1, detection wavelength: 225 nm; injection volume: 10 μL;

[0055] Figures 1-4 The HPLC chromatograms of the mixed reference solution, white mustard seed stir-fried sample solution, white mustard seed solid-state fermentation sample solution, and blank solvent are shown in the above chromatographic conditions, and the separation degrees of sinigrin, myrosinase thiocyanate, and p-hydroxyphenylacetonitrile are good.

[0056] (4) Linearity test: 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, 1.00 mL, 1.20 mL, 1.60 mL, 2.00 mL of the mixed reference solution under item (1) were precisely pipetted into 2 mL volumetric flasks, diluted with methanol to the mark, and mixed to obtain reference solutions with different concentrations. 10 μL of each solution was injected into the liquid chromatograph for determination of the peak area integral value. The reference solution concentration (μg / mL) was used as the abscissa (X), and the peak area integral value was used as the ordinate (Y) for linear regression. The regression equation for sinigrin was Y = 18569X + 242731, r = 0.9995, and the linear range was 91-910 μg / mL; the regression equation for myronate thiocyanate was Y = 15064X + 34543, r = 0.9997, and the linear range was 32.8-328 μg / mL; and the regression equation for p-hydroxyphenylacetonitrile was Y = 36023X + 107350, r = 0.9997, and the linear range was 18.8-188 μg / mL.

[0057] (5) Precision test: 10 μL of the white mustard seed fermentation sample test solution under item (2) was precisely pipetted, and determination was performed according to the method under item (3). The peak area integral value (A) was determined by continuously repeating the injection 6 times. The RSD of sinigrin, myronate thiocyanate, and p-hydroxyphenylacetonitrile was 1.12%, 2.17%, and 1.90%, respectively, indicating that the precision of the instrument was good.

[0058] (6) Stability test: 10 μL of the white mustard seed fermentation sample test solution under item (2) was precisely pipetted, and determination was performed according to the method under item (3) by injecting at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h. The peak area integral value (A) was determined. The RSD of sinigrin, myronate thiocyanate, and p-hydroxyphenylacetonitrile was 0.78%, 1.95%, and 1.60%, respectively, indicating that the stability of the method was good.

[0059] (7) Reproducibility test: 6 portions of the same batch of white mustard seed fermentation sample were precisely weighed, and the test solution was prepared according to the method under item (2). Determination was performed according to the method under item (3) by injecting 10 μL of each portion. The peak area integral value was determined, and the content was calculated. The average content of sinigrin was 100.10 mg·g -1 , and the RSD was 0.82%. The average content of myronate thiocyanate was 15.73 mg·g -1 , and the RSD was 1.81%. The average content of p-hydroxyphenylacetonitrile was 3.73 mg·g -1 , and the RSD was 1.15%, indicating that the reproducibility of the method was good.

[0060] (8) Recovery Test: Nine samples of fermented white mustard used in the above repeatability test were weighed accurately, each approximately 0.1 g. Reference standards were added at proportions of 70%, 50%, and 30% of the known content, with three samples for each proportion. A certain amount of mixed reference standard solution of known concentration was accurately added to each sample. The test solution was prepared according to the method in section “(2)”, and then determined according to the method in section “(3)”. The peak area was recorded to calculate the recovery rate. The average recoveries of myrosin, sinigrin thiocyanate, and p-hydroxyphenylacetonitrile were 98.94%, 100.31%, and 100.02%, respectively; the RSDs were 1.28%, 2.11%, and 2.10% (n=9), respectively, indicating good method accuracy.

[0061] 6. Experiment on fermentation of roasted white mustard seeds with different probiotics

[0062] Nine samples of stir-fried white mustard seeds (section “2.4”) were taken, each weighing 50g. They were divided into three parallel groups. Each group was inoculated with three bacterial cultures (Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis) for solid-state fermentation according to the fermentation method for white mustard seeds in section “2.4”. Fermentation samples were collected at 0h, 6h, 12h, 24h, 36h, 48h, 72h, 96h, 120h, 144h, and 168h, respectively. The samples were freeze-dried, pulverized, and passed through a 40-mesh sieve. The test solution was prepared according to the method in section “2.5(2)” and determined under the chromatographic conditions in section “2.5(3)”.

[0063] (1) Effect of single-strain solid-state fermentation on glucosinolates

[0064] Depend on Figure 5 It was found that the glucosinolate content was 100.10 mg / g at the beginning of fermentation (0 h). With prolonged fermentation, all three probiotics—Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis—degraded glucosinolates, resulting in a significant decrease in glucosinolate content after fermentation (P<0.05). Lactobacillus plantarum had the greatest impact on the degradation of glucosinolates in roasted white mustard seeds, with a content of 26.91 mg / g and a degradation rate of 73.11% after 168 h of fermentation. Saccharomyces cerevisiae and Bacillus subtilis had relatively small effects on glucosinolate degradation, with degradation rates of 14.21% and 19.35%, respectively.

[0065] (2) Effect of single-strain solid-state fermentation on sinigrin thiocyanate content

[0066] Depend on Figure 6It was found that the sine thiocyanate content was 15.73 mg / g at the start of fermentation (0 h). The sine thiocyanate content of *Lactobacillus plantarum* gradually increased with fermentation time, reaching a maximum of 18.73 mg / g at 12 h, and then slowly decreased after 12 h. *Bacillus subtilis* fermentation from 0 to 168 h had no significant effect on the sine thiocyanate content. The effect of *Saccharomyces cerevisiae* on the sine thiocyanate content gradually increased with fermentation time, reaching 16.97 mg / g after 168 h, an increase of 17.17%.

[0067] (3) Effect of single-strain solid-state fermentation on the content of p-hydroxyphenylacetonitrile

[0068] Depend on Figure 7 It was found that the content of p-hydroxyphenylacetonitrile in all three strains increased significantly after 168 hours of fermentation with prolonged fermentation time. Compared with the other two strains, *Lactobacillus plantarum* fermentation had the most significant effect on the p-hydroxyphenylacetonitrile content, increasing from the initial 3.73 mg / g to 12.15 mg / g after 168 hours of fermentation, an increase of 69.29%. The p-hydroxyphenylacetonitrile content increased slowly with fermentations by *Saccharomyces cerevisiae* and *Bacillus subtilis*, with increase rates of 14.37% and 6.75%, respectively.

[0069] Analysis of the results of this experiment shows that the degradation rates of glucosinolates in roasted white mustard seeds after fermentation by *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* were 73.11%, 14.21%, and 19.35%, respectively, while the conversion rates of p-hydroxyphenylacetonitrile were 69.29%, 14.37%, and 6.75%, respectively. *Saccharomyces cerevisiae* fermentation increased the content of sinigrin thiocyanate in white mustard seeds by 17.17%. This experiment discovered that probiotic fermentation can degrade glucosinolates and convert them into the active ingredient p-hydroxyphenylacetonitrile, with minimal impact on the effective component sinigrin thiocyanate in white mustard seeds. Therefore, this study provides new evidence for the transformation of active ingredients through microbial fermentation.

Claims

1. A process for the conversion of sinigrin to p-hydroxyphenylacetonitrile in fried mustard seeds, characterized in that: The method is to convert sinigrin in fried white mustard seed into p-hydroxyphenylacetonitrile by solid fermentation of probiotic bacteria; The probiotic bacteria are Lactobacillus plantarum; The method specifically comprises the following steps: Step 1, preparation of fried white mustard seed: raw white mustard seed is heated for frying, and when there is a cracking sound and spicy gas is emitted, the fried white mustard seed is taken out, cooled, crushed, and sieved to obtain the fried white mustard seed; Step 2, solid fermentation of fried white mustard seed: the fried white mustard seed is weighed, mixed with distilled water, sterilized, cooled, inoculated with probiotic bacteria liquid, stirred with a sterile glass rod, and then incubated at a constant temperature, and the obtained sample is freeze-dried to obtain a white mustard seed solid fermentation sample; Step 3, preparation of a test sample solution from the white mustard seed solid fermentation sample of step 2, and then determination of the contents of sinigrin active ingredient p-hydroxyphenylacetonitrile and sinapine thiocyanate in white mustard seed; The preparation process of the probiotic bacteria liquid in step 2 is as follows: The probiotic bacteria freeze-dried powder is completely dissolved in a corresponding liquid medium, inoculated into a corresponding solid culture medium, and activated for 2 days; a single colony is picked from the activated probiotic bacteria solid culture medium with an inoculation loop, inoculated into a corresponding liquid medium, and incubated at 30°C for 3 days to obtain the probiotic bacteria liquid; The fermentation strain is Lactobacillus plantarum with a batch number of 21794, which is purchased from the China Industrial Microbial Culture Collection Center (CICC).

2. A process for the conversion of sinigrin to p-hydroxyacetonitrile in fried mustard seeds according to claim 1, characterized in that, The frying temperature of the raw white mustard seed in step 1 is 180-200°C, and the sieving mesh size is 40-50 mesh.

3. A process for the conversion of sinigrin to p-hydroxyacetonitrile in fried mustard seeds as claimed in claim 1, wherein, In step 2, the solid-liquid ratio of the fried white mustard seed to distilled water is 1:1.5; the sterilization temperature is 121°C, and the sterilization time is 15 min; the inoculation amount of the probiotic bacteria liquid is 10%; and the constant temperature incubation temperature is 30°C, and the incubation time is 168 h.

4. A process for the conversion of sinigrin to p-hydroxyacetonitrile in fried mustard seeds according to claim 2, characterized in that, The preparation method of the solid culture medium is as follows: Lactobacillus plantarum activation culture medium: 10.0 g of tryptone, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 5.0 g of yeast powder, 1.0 g of Tween 80, 2.0 g of potassium phosphate, 0.2 g of MgSO4.7H2O, 5.0 g of glucose, 0.05 g of MnSO4.H2O, 10.0 g of beef extract powder, 20.0 g of CaCO3, 15.0 g of agar, and 1.0 L of distilled water are used, and the pH value is 6.8; the mixture is sterilized at 121°C for 15 min, and then stored in a 4°C refrigerator for standby; The preparation method of the liquid culture medium is as follows: the agar powder is removed from the solid culture medium, and the other components remain unchanged; the mixture is sterilized at 121°C for 15 min, and then stored in a 4°C refrigerator for standby.

5. A process for the conversion of sinigrin to p-hydroxyacetonitrile in fried mustard seeds as claimed in claim 1, wherein, The preparation process of the test sample solution in step 3 is as follows: 0.2 g of the white mustard seed solid fermentation sample obtained in step 2 is precisely weighed, 25 mL of 70% methanol is precisely added, the weight is determined, the power is 250 W, the frequency is 40 kHz, and the mixture is ultrasonically treated for 30 min; then the mixture is taken out, cooled, weighed again, supplemented with 70% methanol to make up for the weight loss, shaken, filtered, and the filtrate is obtained.

6. A process for the conversion of sinigrin to p-hydroxyacetonitrile in fried mustard seeds as claimed in claim 1, wherein, The determination of the contents in step 3 is performed by HPLC, and the specific chromatographic conditions are as follows: Column: Diamonsil (2) C 18 Column: 4.6 mm x 250 mm, 5 μm; mobile phase: acetonitrile (A) - 0.1% phosphoric acid solution (B), gradient elution 0~10 min, 4% A; 10~25min, 4%~18%A; 25~40min, 18~21%A; 40~50min, 21~60%A; 50~60min, 60~70%A; Column temperature: 30 °C, flow rate: 1.0 mL min -1 detection wavelength: 225 nm; injection volume 10 μL; The regression equation of sinigrin was Y=18569X+242731, r=0.9995, the linear range was 91~910 μg / mL; the regression equation of myrosine thiocyanate was Y=15064X+34543, r=0.9997, the linear range was 32.8~328 μg / mL; the regression equation of p-hydroxyphenylacetonitrile was Y=36023X+107350, r=0.9997, the linear range was 18.8~188 μg / mL. The regression equation of sinigrin was Y=18569X+242731, r=0.9995, the linear range was 91~910 μg / mL; the regression equation of myrosine thiocyanate was Y=15064X+34543, r=0.9997, the linear range was 32.8~328 μg / mL; the regression equation of p-hydroxyphenylacetonitrile was Y=36023X+107350, r=0.9997, the linear range was 18.8~188 μg / mL.

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