Starter agent for mustard fermentation and preparation method and application thereof

Abstract

The present invention relates to the field of food technology, and in particular to a starter for mustard fermentation, and a preparation method and application thereof. The starter is prepared by fermenting Weissella greecea, Lactobacillus plantarum and Lactobacillus nenjiangensis. Experiments show that by fermenting mustard with the starter, the contents of 24 free amino acids and amino acid nitrogen and the types and contents of isothiocyanate compounds in the obtained fermented mustard are significantly improved, which effectively improves the flavor and quality of the product. At the same time, it has good anti-inflammatory and antioxidant effects, and has obvious advantages over traditional starters. The starter of the present invention is a direct-throw starter for mustard, which provides technical support for realizing the standardized and high-value production of fermented mustard and improving product quality and safety.

Description

Technical Field

[0001] The invention relates to the technical field of food, and in particular to a starter for mustard fermentation and a preparation method and application thereof. Background Art

[0002] Mustard (Brassicajuncea(L.)Czern.etCoss.) is an annual herbaceous plant of the genus Brassica. It is a famous specialty vegetable in my country. The main varieties include Chinese mustard, mustard, kohlrabi, oil mustard, large-leaf mustard, wrinkled mustard, and Shuidong mustard. It is one of the main raw materials for making kimchi. The main product types include instant noodle sauerkraut bags, fish sauerkraut, and Fuling mustard. The "earth pit sauerkraut" incident was exposed on March 15, 2022, indicating that the production technology and quality safety of traditional fermented mustard need to be improved. The industry mainly has the following drawbacks: there is a lack of special fermentation agents for mustard, and natural fermentation is basically adopted. The fermentation process control technology is backward, resulting in poor quality and unstable quality of fermented mustard, and lack of function, which seriously restricts the high-quality development of the mustard industry. Therefore, it is of great practical significance to provide a fermentation agent for fermenting and preparing mustard with enhanced flavor, flavor, and enhanced function, to achieve standardized and high-value production of fermented mustard, and to improve product quality and safety. Summary of the invention

[0003] In view of this, the present invention provides a fermentation agent for mustard fermentation and a preparation method and application thereof. The contents of 24 free amino acids and amino acid nitrogen and the types and contents of isothiocyanate compounds in the fermented mustard prepared by using the fermentation agent are significantly improved, and the fermented mustard has significant anti-inflammatory and antioxidant effects, which has obvious advantages over traditional fermentation agents.

[0004] The starter culture is made by fermenting Weissella greecea, Lactobacillus plantarum and Lactobacillus nenjiangensis.

[0005] Furthermore, the Weissella greecea is Weissella greecea 10d-17 with a preservation number of CGMCC No. 23497, the Lactobacillus plantarum is Lactobacillus plantarum W3-2 with a preservation number of CGMCC No. 25611, and the Lactobacillus Nenjiang is Lactobacillus Nenjiang 577 with a preservation number of CGMCC No. 25612.

[0006] Studies have shown that the contents of 24 free amino acids and amino acid nitrogen, as well as the types and contents of isothiocyanate compounds in the products obtained by fermenting mustard with starter cultures made from Weissella greece 10d-17 with a preservation number of CGMCCNo.23497, Lactobacillus plantarum W3-2 with a preservation number of CGMCCNo.25611, and Lactobacillus nenjiang 577 with a preservation number of CGMCCNo.25612 were significantly increased, and the products had good flavor, good quality, and good anti-inflammatory and antioxidant effects.

[0007] Further, in some embodiments, the fermentation agent is freeze-dried bacterial powder, and the total viable count is 2.13×10 11 ~1.76×10 12 CFU / g.

[0008] The invention also provides application of the leavening agent in mustard fermentation.

[0009] The present invention also provides a method for preparing the leavening agent, comprising:

[0010] Activating Weissella greecea, Lactobacillus plantarum and Lactobacillus nenjiangensis and inoculating them into a high-density culture medium for fermentation; centrifuging the fermentation liquid, collecting and washing the precipitate to obtain bacterial sludge;

[0011] The bacterial mud is mixed with a freeze-drying protective agent, and freeze-dried to obtain freeze-dried bacterial powder, which is the fermentation agent.

[0012] In some embodiments, the ratio of the effective bacterial counts of Weissella greecea, Lactobacillus plantarum and Lactobacillus nenjiangensis after activation is (0.5-2):(0.5-2):(0.5-2).

[0013] In some embodiments, the high-density liquid culture medium includes water and the following components in concentration: trehalose 10-20 g / L, black tiger freeze-dried powder 4-6 g / L, yeast extract 15-30 g / L, tryptone 10-20 g / L, manganese sulfate 0.50 g / L, magnesium sulfate 0.5 g / L, sodium acetate 5 g / L, diammonium hydrogen phosphate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, Tween 80 1 g / L, pH 6.0-6.5. In some specific embodiments, the concentration of each component is:

[0014] Trehalose 15g / L, Black Tiger freeze-dried powder 5g / L, yeast extract 15g / L, tryptone 15g / L, manganese sulfate 0.50g / L, magnesium sulfate 0.5g / L, sodium acetate 5g / L, diammonium hydrogen phosphate 2g / L, dipotassium hydrogen phosphate 2g / L, Tween 80 1g / L. Adjust pH to 6.0-6.5, sterilize at 121℃ for 15min.

[0015] Specifically, the preparation method of the black tiger freeze-dried powder includes:

[0016] The black tiger fruit particles are mixed with water to extract juice, and the juice is vacuum freeze-dried to obtain black tiger freeze-dried powder; the mass ratio of the black tiger fruit particles to water is 1:1;

[0017] The vacuum freeze drying specifically includes: pre-freezing at -70°C to -80°C for 2 to 6 hours; freeze drying at a vacuum degree of 0.01 to 0.2 Pa and a temperature of -100°C to -105°C for 20 to 50 hours, and controlling the moisture content of the freeze-dried powder to be 3 to 8%.

[0018] In some embodiments, the lyoprotectant comprises water and the following components in mass fractions: 6-10% skim milk powder, 6-12% xylo-oligosaccharide and 1-2% sodium L-glutamate;

[0019] The freeze drying specifically includes: pre-freezing at -70°C to -80°C for 2 to 6 hours; freeze drying at a vacuum degree of 0.01 to 0.2 Pa and a temperature of -100°C to -105°C for 20 to 50 hours, and controlling the moisture content of the freeze-dried powder to be 3 to 8%.

[0020] The present invention also provides a method for preparing fermented mustard, and the mustard is fermented using the starter agent of the present invention or the starter agent prepared by the preparation method of the present invention.

[0021] In some embodiments, the method for preparing fermented mustard specifically comprises:

[0022] The mustard is dried in the sun, salt is added to the mixture, the fermentation agent of the invention is inoculated, the mixture is sealed in a jar, and fermented to obtain fermented mustard.

[0023] In some embodiments, the drying is performed until the mustard greens lose 10% to 20% in weight.

[0024] In some embodiments, the mixing material is: based on the percentage of salt to the weight of the mustard greens after drying, 5%-8% of salt is added and mixed well.

[0025] In some embodiments, the fermentation temperature is 30-37° C. and the fermentation time is 6 to 12 days.

[0026] In some embodiments, the fermentation agent accounts for 0.1%-1% of the mass of the mustard.

[0027] The invention also provides fermented mustard prepared by the preparation method.

[0028] The starter is made by fermenting Weissella greecea, Lactobacillus plantarum and Lactobacillus nenjiangensis. Experiments show that by fermenting mustard with the starter, the contents of 24 free amino acids and amino acid nitrogen and the types and contents of isothiocyanate compounds in the fermented mustard are significantly improved, which effectively improves the flavor and quality of the product. At the same time, it has good anti-inflammatory and antioxidant effects, and has obvious advantages over traditional starters. The starter of the present invention is a direct-throw starter specifically for mustard, which provides technical support for realizing the standardized and high-value production of fermented mustard and improving product quality and safety.

[0029] Biological Deposit Description

[0030] Weissella 10d-17 (Weissellahellenica) of Greece was deposited on September 28, 2021 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 23497.

[0031] 577 (Lactobacillus nenjiangensis), deposited on August 29, 2022 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 25612.

[0032] W3-2 (Lactobacillus plantarum) was deposited on August 29, 2022 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 25611. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shows the content of interleukin IL-6 in each treatment group;

[0034] Figure 2 The contents of interleukin IL-1β in each treatment group are shown;

[0035] Figure 3 The contents of tumor necrosis factor TNF-α in each treatment group are shown;

[0036] Figure 4 Shows the relative ROS levels in different treatment groups;

[0037] Figure 5 Shows the effect of each treatment group on the survival rate of Drosophila in the Drosophila inflammation model;

[0038] Figure 6 Shown are the effects of various treatment groups on the survival rate of Drosophila in the Drosophila oxidative stress model. DETAILED DESCRIPTION

[0039] The present invention discloses a starter for mustard fermentation and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0040] In the present invention, the survival rate calculation method is:

[0041] After counting the live bacteria of the freeze-dried powder, the survival rate of lactic acid bacteria was calculated using the following formula:

[0042] Lactic acid bacteria survival rate = N1*W1 / N0*W0×100%

[0043] Where: N1 represents the number of viable bacteria per 1g of bacterial powder after freeze-drying (cfu / g);

[0044] N0 represents the number of viable bacteria per 1g of liquid sample before freeze-drying (cfu / g);

[0045] W1 represents the weight of the bacterial powder obtained after freeze-drying (g);

[0046] W0 represents the total weight of the bacterial solution before lyophilization (g).

[0047] The present invention will be further described below in conjunction with embodiments:

[0048] Example 1 Preparation of the starter of the present invention

[0049] (1) Activation of 10d-17, W3-2 and 577 strains:

[0050] The glycerol storage tubes of 10d-17 (Weissella greecea), W3-2 (Lactobacillus plantarum) and 577 (Lactobacillus nenjiangensis) stored at -80°C were taken out and thawed in a 4°C refrigerator. 1 mL of the preserved bacterial solution was respectively added to 100 mL of high-density liquid culture medium and 100 mL of MRS liquid culture medium, and cultured at 37°C for 12 h to 24 h. The concentration of the bacterial suspension was adjusted to 0.5 McFarland turbidimeter tube with sterile physiological saline. The bacterial suspensions of 10d-17, W3-2 and 577 were mixed in a volume ratio of 1:1:1 to prepare a composite bacterial strain activation solution.

[0051] (2) High-density culture medium:

[0052] Based on the weight of the high-density liquid culture medium, the composite bacterial activation liquid is inoculated in a fermentation tank containing a high-density liquid culture medium at an inoculation amount of 1% (m / m), stirred at 200r / min, and 15-20% (m / m) ammonia water is used as a neutralizer to control the pH of the fermentation liquid to 6.0-6.5, and the fermentation enrichment culture is carried out at 37°C for 24 hours. Take out 1mL, dilute it with 0.9% (m / m) physiological saline in a gradient manner, spread it on a plate, and count the viable bacteria in the fermentation liquid. The fermentation liquid is subjected to high-speed refrigerated centrifugation at 5000-8000r / min and 4°C for 6-10min to remove the supernatant, wash the precipitate with sterile physiological saline, centrifuge, remove the supernatant, and repeat the washing operation twice to obtain bacterial mud.

[0053] (3) Vacuum freeze drying

[0054] Add the same volume of freeze-drying protective agent as that of high-density liquid culture medium or ordinary MRS liquid culture medium to the bacterial mud and mix well, place it at -70℃--80℃ for pre-freezing for 2-6 hours, and finally put the frozen material into the freeze dryer for freeze drying. The freeze dryer parameters are set as follows: vacuum degree 0.01-0.2Pa, temperature -100℃--105℃, and drying time 20-50h. After obtaining the freeze-dried bacterial powder, seal it and store it in a 4℃ refrigerator. Take 1g of bacterial powder and dilute it with 0.9% saline for gradient dilution, and spread it on the plate for live bacteria count before freeze drying.

[0055] The formula of the high-density liquid culture medium (with water as solvent) is: trehalose 10g / L, black tiger freeze-dried powder 6g / L, yeast extract 30g / L, tryptone 12g / L, manganese sulfate 0.50g / L, magnesium sulfate 0.5g / L, sodium acetate 5g / L, diammonium hydrogen phosphate 1g / L, dipotassium hydrogen phosphate 1g / L, Tween 80 1g / L. Adjust the pH value to 6.0-6.5, and sterilize at 121°C for 15min.

[0056] The formula of the common MRS liquid culture medium (with water as solvent) is: peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, dipotassium hydrogen phosphate 2g / L, diammonium citrate 2g / L, sodium acetate 5g / L, glucose 20g / L, Tween 80 1g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.25g / L. The pH value is adjusted to 6.0-6.5, and sterilized at 121°C for 15min.

[0057] The black tiger is an evergreen woody vine wild plant of the genus Schisandrae Chinensis of the family Schisandraceae, and is also known as cold rice balls, vines of the mountain dragon, blood vine bubbles, Bufuna, etc. It is a folk medicine with a long history, contains a variety of nutrients, such as protein, amino acids, minerals, etc., and has anti-inflammatory, anti-oxidation, enhances the body's immunity, and promotes the growth of microorganisms. The black tiger freeze-dried powder is made by taking the surface fruit particles of the black tiger fruit and putting them into a juicer, according to water: fruit particles = 1:1, adding water to squeeze the juice, and after squeezing the juice, vacuum freeze drying is formed. The conditions for vacuum freeze drying are: the black tiger juice is pre-frozen at -70°C--80°C for 2-6h, and finally the frozen material is put into a freeze dryer for freeze drying. The freeze dryer parameters are set to: vacuum degree 0.01-0.2Pa, freeze drying time 20-50h, temperature -100°C--105°C, and the water content of the black tiger freeze-dried powder is controlled to be 3-8%.

[0058] The formula of the freeze-drying protective agent is: based on the weight of water (m / m), 6-10% skim milk powder, 6-12% xylo-oligosaccharide, and 1-2% sodium L-glutamate.

[0059] After being cultured in high-density liquid medium, the concentration of live bacteria in the fermentation broth reached 2.12-2.53×10 10 cfu·mL -1 After vacuum freeze drying, the bacterial survival rate was 94.29-95.63%, and the number of live bacteria in the powder was 1.44-1.76×10 12 CFU / g.

[0060] Comparative Example 2 Preparation of Starter

[0061] The difference from Example 1 is that step (2) is culture in ordinary culture medium, and other conditions are the same.

[0062] Based on the weight of the MRS liquid culture medium, the composite bacterial activation liquid is inoculated in a fermentation tank filled with the MRS liquid culture medium at an inoculation amount of 1% (m / m), stirred at 200r / min, and 15-20% (m / m) ammonia water is used as a neutralizer to control the pH of the fermentation liquid to 6.0-6.5, and the fermentation enrichment culture is carried out at 37°C for 24h. 1mL is taken out, gradiently diluted with 0.9% (m / m) physiological saline, coated on a plate, and the live bacteria count of the fermentation liquid is performed. The fermentation liquid is subjected to high-speed refrigerated centrifugation at 5000-8000r / min and 4°C for 6-10min to remove the supernatant, the precipitate is washed with sterile physiological saline, centrifuged, the supernatant is removed, and the washing operation is repeated twice to obtain bacterial mud.

[0063] After being cultured in ordinary culture medium, the concentration of live bacteria in the fermentation broth was 1.11×10 9 cfu·mL -1 After vacuum freeze drying, the bacterial survival rate was 85.18% and the number of viable bacteria in the powder was 2.13×1011 CFU / g.

[0064] Example 2 Preparation of fermented mustard

[0065] Fresh mustard greens → washing → drying → adding salt to mix → inoculating the direct-injection fermentation agent of Example 1 → sealing in a jar → fermentation → testing.

[0066] Among them, the drying, mixing, inoculation and fermentation processes are specifically as follows:

[0067] Drying: Drying the mustard until its weight is reduced by 10%-20%;

[0068] Mixing: Add 5%-8% salt based on the weight of the mustard after drying, and mix well;

[0069] Inoculation: Inoculate 0.1-1% fermentation agent based on the weight of the mustard after drying;

[0070] Fermentation: 30-37℃, fermentation for 10 days.

[0071] Test Example 1

[0072] (I) Experimental groups:

[0073] Test group: inoculated with 0.2% of the starter of Example 1 (a direct-injection starter prepared by culturing the strain combination of the present invention in a high-density liquid medium);

[0074] Control group 1: no inoculation, natural fermentation;

[0075] Control group 2: inoculated with 0.2% starter (a direct-injection starter prepared by culturing the strain combination of the present invention in a common MRS medium);

[0076] Control group 3: inoculated with 0.2% of the fermentation agent described in CN113337446A, in which the compound ratio of Lactobacillus plantarum: Lactobacillus brevis: Weissella mesenteroides was 2:1:1;

[0077] Control group 4: inoculated with 0.2% of the starter described in Example 1 of CN114634893A.

[0078] Preparation process of mustard greens:

[0079] Experimental group: Mustard inoculated with the starter culture of Example 1 of the present invention and fermented

[0080] Fresh mustard greens→wash→air-dry→add salt to mix→inoculate the direct-injection fermentation agent of Example 1 of the present invention→put into a jar and seal→ferment→test.

[0081] Control group 1: Mustard: Natural fermentation of mustard

[0082] Fresh mustard greens → wash → dry in the sun → add salt and mix → seal in a jar → ferment → test.

[0083] Control group 2: Mustard: Mustard was inoculated with a combination of 10d-17, W3-2 and 577 strains and fermented with a starter culture cultured in ordinary MRS medium

[0084] Fresh mustard greens → wash → dry in the sun → add salt and mix → inoculate with fermentation agent → seal in jar → ferment → test.

[0085] Control group 3 mustard: mustard was inoculated with the fermentation agent described in CN113337446A and fermented

[0086] Fresh mustard greens → washing → drying → adding salt to mix → inoculating the fermentation agent described in CN113337446A → sealing in a jar → fermentation → testing.

[0087] Control group 4: Mustard: Mustard was inoculated with CN114634893A fermentation agent and fermented

[0088] Fresh mustard greens → washing → drying → adding salt to mix → inoculating the starter of Example 1 of CN114634893A for fermentation → sealing in a jar → fermentation → testing.

[0089] Among them, in the experimental groups and control groups 1 to 4, in each step, drying, mixing, inoculation and fermentation are specifically as follows:

[0090] Drying: Dry the mustard until its weight decreases by 10%.

[0091] Mixing: Add 5% salt based on the weight of the mustard after drying, and mix well;

[0092] Inoculation: Based on the weight of the mustard after drying, inoculate 0.2wt% of the starter;

[0093] Fermentation: 37℃, fermentation 10d.

[0094] (II) Study on the content of amino acid nitrogen in fermented mustard

[0095] The amino acid nitrogen content of the inoculated fermented mustard (experimental group 1, control groups 1 to 4) was determined with reference to GB5009.235-2016. The results are shown in Table 1.

[0096] Table 1 Comparison table of amino acid nitrogen content

[0097]

[0098] The results show that after 10 days of fermentation, the amino acid nitrogen content of the direct-throwing fermentation agent (test group) of Example 1 prepared by high-density liquid culture medium inoculated with the present invention reaches 0.31g / 100g, followed by the direct-throwing fermentation agent (control group 2) of Example 2 prepared by ordinary MRS culture medium inoculated with the amino acid nitrogen content of 0.28g / 100g; while the amino acid nitrogen content of naturally fermented mustard (control group 1) is only 0.18g / 100g, and the amino acid nitrogen content of the fermentation agent described in CN113337446A (control group 3) and the fermentation agent described in CN114634893A (control group 4) is only 0.22g / 100g and 0.23g / 100g. It shows that fermenting mustard with the direct-throwing fermentation agent of the present invention can significantly increase the amino acid nitrogen content of the product.

[0099] (III) Determination of free amino acid content in fermented mustard

[0100] Sample pretreatment: Weigh 1.000g of the inoculated fermented mustard (experimental group), control group 1, control group 2, control group 3, and control group 4 samples in 10mL centrifuge tubes, add 5mL of 0.01mol / L hydrochloric acid, mix well, boil in water bath for 15min, ultrasonically extract for 10min, centrifuge at 4000rpm for 5min, and take the supernatant; add 4mL of 0.01mol / L hydrochloric acid to the precipitate, suspend it with ultrasonic for 10min, centrifuge, combine the supernatant, and make up to 10mL. Determination by membrane.

[0101] Online pre-column derivatization: Agilent's automatic online derivatization method was used to derivatize the primary amino acids with o-phthalaldehyde (OPA) and the secondary amino acids with fluorenylmethoxycarbonyl chloride (FMOC) before column detection. Chromatographic conditions: ZORBAX Eclipse AAA (4.6x75mm, 3.5μm); detection signal: UV 338nm (0-19min), 266nm (19.01-25min); mobile phase A: 40mmol / L sodium dihydrogen phosphate (pH7.8); mobile phase B: methanol / acetonitrile / water = 45 / 45 / 10; flow rate: 1.0mL / min;

[0102] Gradient elution process: 100% A: 0% B (0-1 min); 46% A; 57% B (23 min); 0% A: 100% B (27-34 min); 100% A: 0% B (40-41 min).

[0103] The contents of free amino acids in the inoculated fermented mustard (experimental group) and the control group are shown in Table 2.

[0104] Table 2 Free amino acid content (mg / g)

[0105]

[0106] As can be seen from Table 2, the fermented mustard (experimental group) prepared by inoculating the direct-throw starter of Example 1 of the present invention has the highest content of 24 free amino acids, reaching 10.3048 mg / g, and the fermented shepherd's purse (control group 2) prepared by inoculating the direct-throw starter of Example 2 prepared by culturing with the microbial combination of the present invention through ordinary MRS medium has the second highest content of 24 free amino acids, which is 7.8705 mg / g; the total content of 24 free amino acids in the naturally fermented mustard (control group 1) is only 5.7343 mg / g, and the contents of 24 free amino acids in the products of the starter described in CN113337446A (control group 3) and the starter described in CN113337446A (control group 4) are only 7.3286 mg / g and 6.3765 mg / g, respectively. Therefore, it is shown that the direct-injection fermentation agent (i.e., the fermentation agent of Example 1) prepared by culturing the microbial combination of the present invention in a high-density liquid medium has a significant amino acid production capacity (test group), and can be used to ferment mustard to enhance the freshness and flavor.

[0107] (IV) Determination of aroma substances in fermented mustard

[0108] The extraction head was aged at 270°C for 0.5 h.

[0109] Solid phase microextraction: take 2g of test group, control group 1, control group 2, control group 3, and control group 4 respectively into a 20mL headspace bottle, add 3mL of water, put in a small magnet, quickly tighten the bottle cap with a silicone rubber septum, preheat at 70℃ on a constant temperature magnetic heating stirrer for 15min, insert the aged extraction head into the headspace bottle and push the extraction head into the headspace about 0.7cm from the liquid surface, adsorb for 40min; inject and analyze for 2min.

[0110] Chromatographic conditions: Chromatographic column: RTX-5ms flexible quartz capillary column (30m×0.25mm×0.25μm); Carrier gas: high-purity He (99.999%), flow rate: 1.0mL / min; Injection port temperature: 250°C; Split injection. Heating program: Column temperature 50°C, increase to 180°C at 4°C / min, hold for 4min, increase to 250°C at 6°C, hold for 1min.

[0111] Mass spectrometry conditions: ion source: EI source; GC / MS interface temperature: 220°; ion source temperature: 200°C; electron energy: 70 eV, mass scanning range: 35-500 amu.

[0112] Retention index (RI): According to the above method, 40 The normal alkane mixed reference substance was subjected to GC-MS analysis, the retention time of each normal alkane was recorded, and the RI of each component was calculated according to the program. Calculation formula: t x is the retention time of the elution peak of the analyzed group; t n and t n+1 It is the retention time (min) of the elution peak of normal alkanes with carbon atoms n and n+1, and tn<tx<tn+1.

[0113] Qualitative analysis: Qualitative analysis was performed by similarity search between the pure mass spectra of each component and the NIST2017 standard library, and qualitative analysis was performed by comparison of the retention index RI values. The internal standard method was used to determine the percentage content of each component based on the compound with a similarity greater than 80% and the highest retention index RI value proximity.

[0114] Quantitative analysis: The internal standard method was used to quantitatively analyze the volatile substances in fermented mustard, with o-dichlorobenzene as the internal standard. The content of volatile substances was calculated as follows.

[0115] Calculation formula:

[0116] Where: C j ——Content of flavor substances to be tested, μg / kg;

[0117] A j ——The chromatographic peak area corresponding to the analyte in the sample;

[0118] A i ——Determination of chromatographic peak area by internal standard;

[0119] V——volume of internal standard, μL;

[0120] C i ——internal standard concentration, μg / mL;

[0121] M j ——Sample mass, g.

[0122] The contents of isothiocyanate compounds and other volatile aroma substances in the fermented mustard in the test group and each control group are shown in Table 3 and Table 4, respectively.

[0123] Table 3 Isothiocyanate compound content (μg / g)

[0124]

[0125] Isothiocyanate compounds are a class of compounds with unique aroma and rich biological activity in antibacterial, anti-cancer, anti-oxidation, etc. As can be seen from Table 3, 11 isothiocyanate compounds were detected in mustard after fermentation with the direct-throw starter of Example 1 of the present invention, which are allyl isothiocyanate, 3-butenyl isothiocyanate, methyl butyl isothiocyanate, sec-butyl isothiocyanate, cyclopropyl isothiocyanate, butyl isothiocyanate, isobutyl isothiocyanate, 3-methylthiopropyl isothiocyanate, isobutyl isothiocyanate, phenethyl isothiocyanate and sulforaphane, and the contents are 39.43, 50.11, 63.99, 94.30, 43.59, 64.62, 43.36, 32.59, 23.58, 53.17 and 31.89 μg / g, respectively. Although 11 isothiocyanate compounds were also detected in the control group 2 inoculated with the fermentation agent of Example 2, the contents were all lower than those in the test group (inoculated with the fermentation agent of Example 1). Only 5, 6, and 6 isothiocyanate compounds were detected in the control group 1, the control group 3, and the control group 4, respectively. The results show that the fermentation agents of Examples 1 and 2 of the present invention have a high ability to degrade glucosinolates into isothiocyanate compounds, and the mustard can significantly increase the types and contents of isothiocyanate compounds after fermentation with the direct-injection fermentation agent of the present invention, thereby enriching the anti-inflammatory and antioxidant functions of the fermented mustard, among which the fermentation agent of Example 1 has the best effect.

[0126] Test Example 2 Anti-inflammatory and Antioxidant Effect Test

[0127] (I) Anti-inflammatory cell experiment of fermented mustard:

[0128] Detection of anti-inflammatory ability of fermented mustard based on RAW264.7 cell inflammation model

[0129] Cell culture: RAW264.7 cells were cultured in 10% fetal bovine serum (FBS) + RPMI1640 medium at 37°C and 5% CO2. During digestion, the medium was removed, 2 mL of PBS was added for rinsing and then discarded, and then 1 mL of trypsin was added. After digestion for 60 seconds, the appropriate amount of medium was added, and the cells were blown off the wall to make a suspension, and the suspension was transferred to a new container.

[0130] Grouping: a blank group (only cell), a model group (cell+LPS), a test group (cell+LPS+mustard fermented with the starter of the present invention), a control group 1 (cell+LPS+mustard of control group 1), a control group 2 (cell+LPS+mustard of control group 2), a control group 3 (cell+LPS+mustard of control group 3), and a control group 4 (cell+LPS+mustard of control group 4). The preparation method of mustard of control groups 1 to 4 is the same as that of test example 1.

[0131] Experimental process: The fermented mustard is crushed, sieved, and filtered through a 0.22-micron filter membrane for later use. After the cell culture is stable, it is inoculated into a 48-well plate at a density of 80% to 90% per well. The plate is washed 3 times after adherence to the wall. According to the experimental grouping, fermented mustard is added or not for pretreatment for 4 hours. Subsequently, a lipopolysaccharide (LPS) solution with a concentration of 2.5 ng / ml prepared with 1640 culture medium is added according to the grouping. After stimulation for 12 hours, the supernatant is collected by centrifugation. The ELISA kit method is used to measure the content of interleukins (IL-1β and IL-6) and tumor necrosis factor (TNF-α) in the supernatant. The experimental results are as follows Figure 1-3 shown.

[0132] Depend on Figure 1-3 It can be seen that, based on the three inflammatory factor indicators, in the inflammatory model of lipopolysaccharide-stimulated mouse macrophages (RAW 264.7), mustard fermented with the starter of Example 1 of the present invention can effectively reduce the release of pro-inflammatory factors (IL-6, IL-1β, TNF-α), and the product has a good anti-inflammatory effect.

[0133] (II) Fermented mustard antioxidant cell test:

[0134] Experimental grouping: set up a blank group (only cell), a model group (cell + LPS), an experimental group (cell + LPS + mustard fermented with the starter of the present invention), a control group 1 (cell + LPS + mustard in control group 1), a control group 2 (cell + LPS + mustard in control group 2), a control group 3 (cell + LPS + mustard in control group 3), and a control group 4 (cell + LPS + mustard in control group 4).

[0135] Cell culture: Caco-2 was cultured in 10% fetal bovine serum (FBS) + MEM-ALPHA medium at 37°C and 5% CO2. During digestion, remove the medium, rinse with 2 mL PBS and discard, then add 1 mL pancreatin, digest for 30 seconds, aspirate and add an appropriate amount of medium, blow the cells off the wall to make a suspension, and transfer the suspension to a new container. After the cells have stably grown all over the cell bottle, inoculate a 96-well plate and grow for 24 hours. Be gentle when washing the plate, and blow off the cell clusters to break them into single cells.

[0136] After the cells are cultured to stability, they are digested with trypsin and inoculated into 96-well plates. After 48 hours, the experiment begins when each well is 80% to 90% full and in a polarized state. Add the prepared fermented mustard for pretreatment for 4 hours, then add H2O2 solution diluted with MEM-ALPHA medium to a final concentration of 500umol / l. After 2 hours of injury, use the DCFH-DA probe to detect the fluorescence level of reactive oxygen species (ROS). Add 60ul of DCFH-DA probe to each well, react in the dark for 20 minutes, and measure the wavelength under a fluorescence microplate reader with an excitation wavelength of 485nm and an emission wavelength of 530nm. Calculate according to the formula:

[0137]

[0138] The relative ROS levels in different groups are as follows Figure 4 shown.

[0139] Depend on Figure 4 It can be seen that in the oxidation model of human intestinal epithelial cells (Caco-2) stimulated by hydrogen peroxide, the mustard fermented by the starter culture of the present invention can effectively reduce the ROS level in damaged Caco-2 cells, and the mustard fermented by the starter culture of Example 1 of the present invention can significantly inhibit the oxidative stress damage of Caco-2 cells induced by H2O2, among which the mustard fermented by the starter culture of Example 1 (experimental group) has the best inhibitory effect.

[0140] (III) Anti-inflammatory / antioxidant effects of fermented mustard fruit fly model

[0141] (1) Establishment of anti-inflammatory / antioxidant Drosophila model

[0142] Adding dextran sulfate sodium DSS to the sucrose solution consumed by fruit flies causes damage to the intestines of fruit flies, directly damaging colon epithelial cells and inducing inflammatory reactions. This is used to establish an inflammatory model for anti-inflammatory effect testing. Adding paraquat (PQ) to the sucrose solution consumed by fruit flies induces the production of free radicals, inducing lipid peroxidation and causing oxidative damage. This is used to establish an oxidative stress model for antioxidant effect testing.

[0143] (2) Test reagents:

[0144] 5% sucrose solution: Dissolve 50 g glucose in 950 g sterile water.

[0145] Inoculation of fermented mustard sucrose solution: Dissolve 50 g of inoculated fermented mustard in 950 mL of 5% sucrose solution.

[0146] Naturally fermented mustard sucrose solution: 50 g of naturally fermented mustard was dissolved in 950 mL of 5% sucrose solution.

[0147] DSS sucrose solution: Dissolve 10 g DSS in 950 mL 5% sucrose solution.

[0148] PQ sucrose solution: 10 g PQ was dissolved in 950 mL 5% sucrose solution.

[0149] DSS+inoculated fermented mustard sucrose solution: 10 g DSS and 50 g inoculated fermented mustard according to Example 2 of the present invention were dissolved in 950 mL of 5% sucrose solution.

[0150] DSS + naturally fermented mustard sucrose solution: 10 g DSS and 50 g naturally fermented mustard were dissolved in 950 mL 5% sucrose solution.

[0151] PQ+inoculated fermented mustard sucrose solution: 10 g PQ and 50 g inoculated fermented mustard according to Example 2 of the present invention were dissolved in 950 mL of 5% sucrose solution.

[0152] PQ + naturally fermented mustard sucrose solution: 10 g PQ and 50 g naturally fermented mustard were dissolved in 1000 mL 5% sucrose solution.

[0153] The fermented mustard of the present invention is the fermented mustard obtained by inoculating the starter of Example 1 of the present invention.

[0154] (3) Experimental groups:

[0155] Model group: The fruit flies in the model group were fed with 5% sucrose solution for the first three days, and then with DSS sucrose solution or PQ sucrose solution from the fourth day. The feeding lasted for 7 days, and the survival status of the fruit flies was counted every day.

[0156] Experimental group: The fruit flies in the experimental group were fed with the fermented mustard sucrose solution of inoculation example 2 for the first three days, and were fed with PQ + fermented mustard sucrose solution of inoculation example 2 or DSS + fermented mustard sucrose solution of inoculation example 2 from the fourth day. The feeding lasted for 7 days, and the survival status of the fruit flies was counted every day.

[0157] Control group 1: The fruit flies in the experimental group were fed with mustard sucrose solution of control group 1 for the first three days, and then fed with PQ + mustard sucrose solution of control group 1 or DSS + mustard sucrose solution of control group 1 from the fourth day. The feeding lasted for 7 days, and the survival status of the fruit flies was counted every day.

[0158] Control group 2: The fruit flies in the experimental group were fed with mustard sucrose solution of control group 2 for the first three days, and then fed with PQ + mustard sucrose solution of control group 2 or DSS + mustard sucrose solution of control group 2 from the fourth day. The feeding lasted for 7 days, and the survival status of the fruit flies was counted every day.

[0159] Control group 3: The fruit flies in the experimental group were fed with mustard sucrose solution of control group 3 for the first three days, and then fed with PQ + mustard sucrose solution of control group 3 or DSS + mustard sucrose solution of control group 3 from the fourth day. The feeding lasted for 7 days, and the survival status of the fruit flies was counted every day.

[0160] Control group 4: The fruit flies in the experimental group were fed with the mustard sucrose solution of control group 4 for the first three days. From the fourth day, they were fed with PQ + mustard sucrose solution of control group 4 or DSS + mustard sucrose solution of control group 4. They were fed for 7 days, and the survival status of the fruit flies was counted every day.

[0161] (4) Anti-inflammatory effect of fermented mustard on DSS inflammation model of Drosophila

[0162] Anti-inflammatory effect of fermented mustard on Drosophila inflammation model Figure 5 shown.

[0163] from Figure 5 It can be seen that, compared with the model group and control groups 1 to 4, the fermented mustard prepared by the starter culture of the present invention can significantly improve the survival rate of DSS model fruit flies, indicating that the mustard has a more excellent anti-inflammatory effect after being fermented by the starter culture of the present invention.

[0164] (5) Antioxidant effect of fermented mustard on Drosophila oxidative stress model

[0165] Antioxidant effect of fermented mustard on Drosophila oxidative stress model Figure 6 shown.

[0166] from Figure 6 It can be seen that compared with the model group and the control group, the fermented mustard prepared by the starter of the present invention can significantly improve the survival rate of the Drosophila oxidative stress model, indicating that the mustard has a significant antioxidant effect after being fermented by the starter of the present invention.

[0167] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A leavening agent, characterized in that The Greek Weissella ( Weissella hellenica ) 10d-17, Lactobacillus plantarum with a deposit number of CGMCC No.25611 ( Lactobacillus plantarum ) W3-2 and Lactobacillus Nenjiangensis with a deposit number of CGMCC No.25612 ( Lactobacillus nenjiangensis )577 is obtained by fermentation.

2. The leavening agent according to claim 1, characterized in that The fermentation agent is freeze-dried bacterial powder, and the total number of viable bacteria is 2.13×10 11 ~1.76×10 12 CFU / g.

3. Use of the leavening agent according to any one of claims 1 to 2 in mustard fermentation.

4. The method for preparing the leavening agent according to any one of claims 1 to 2, characterized in that: include: Weissella greecea 10d-17, Lactobacillus plantarum W3-2 and Lactobacillus nenjiang 577 were activated and inoculated into high-density culture medium for fermentation; The fermentation liquid is centrifuged, and the precipitate is collected and washed to obtain bacterial sludge; The bacterial mud is mixed with a freeze-drying protective agent, and freeze-dried to obtain freeze-dried bacterial powder, which is the starter.

5. The preparation method according to claim 4, characterized in that: The ratio of the effective bacterial counts of the activated Weissella greecea 10d-17, Lactobacillus plantarum W3-2 and Lactobacillus nenjiang 577 is (0.5~2): (0.5~2): (0.5~2).

6. The preparation method according to claim 5, characterized in that: The high-density liquid culture medium includes water and components with the following concentrations: 10-20 g / L trehalose, 4-6 g / L black tiger freeze-dried powder, 15-30 g / L yeast extract, 10-20 g / L tryptone, 0.50 g / L manganese sulfate, 0.5 g / L magnesium sulfate, 5 g / L sodium acetate, 1-3 g / L diammonium hydrogen phosphate, 1-3 g / L dipotassium hydrogen phosphate, 1 g / L Tween 80, and a pH value of 6.0-6.

5.

7. The preparation method according to claim 6, characterized in that: The preparation method of the black tiger freeze-dried powder comprises: The black tiger fruit particles are mixed with water to extract juice, and the juice is vacuum freeze-dried to obtain black tiger freeze-dried powder; the mass ratio of the black tiger fruit particles to water is 1:1; The vacuum freeze drying specifically includes: pre-freezing at -70°C to -80°C for 2 to 6 hours; freeze drying at a vacuum degree of 0.01 to 0.2 Pa and a temperature of -100°C to -105°C for 20 to 50 hours, and controlling the moisture content of the freeze-dried powder to be 3 to 8%.

8. The preparation method according to claim 5, characterized in that: The freeze-drying protective agent comprises water and the following components in mass fractions: 6-10% skim milk powder, 6-12% xylo-oligosaccharide and 1-2% sodium L-glutamate; The freeze drying specifically includes: pre-freezing at -70°C to -80°C for 2 to 6 hours; freeze drying at a vacuum degree of 0.01 to 0.2 Pa and a temperature of -100°C to -105°C for 20 to 50 hours, and controlling the moisture content of the freeze-dried powder to be 3 to 8%.

9. A method for preparing fermented mustard, characterized in that: Fermenting mustard greens using the starter described in any one of claims 1 to 3 or the starter prepared by the preparation method described in any one of claims 5 to 8 comprises: drying the mustard greens, adding salt and mixing with materials, inoculating the starter, sealing the jar, and fermenting to obtain the fermented mustard greens.

10. The preparation method according to claim 9, characterized in that: The fermentation agent accounts for 0.1%-1% of the mass of the mustard.

11. The preparation method according to claim 9, characterized in that: The method comprises drying the mustard greens until the weight of the mustard greens is reduced by 10% to 20%; The salt mixing material is: to salt accounted for the percentage of weight of mustard after drying, add 5%-8% of salt, mix well; The fermentation temperature is 30-37°C and the fermentation time is 6-12 days.

12. Fermented mustard obtained by the preparation method according to any one of claims 9 to 11.

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