Probiotic fermented wind ginger product with enhanced antioxidant activity and preparation process of probiotic fermented wind ginger product

Abstract

The invention belongs to the technical field of food fermentation, and particularly relates to a probiotic fermented air-dried ginger product with enhanced antioxidant activity and a preparation process thereof, the preparation process comprises the following steps: S1, cutting air-dried ginger into slices to obtain pretreated air-dried ginger; s2, introducing the pCas9-sgRNA recombinant vector into lactobacillus plantarum by adopting an electrotransformation method to obtain an engineering strain with mutated basic groups, performing shaking culture on saccharomyces cerevisiae in a YPD culture medium at 30 DEG C, and mixing the saccharomyces cerevisiae with the lactobacillus plantarum engineering strain according to a weight ratio of 4: 1 to prepare a composite probiotic agent; s3, mixing the pretreated rhizoma zingiberis with an enzymolysis optimized carbon and nitrogen source and an antioxidant synergist, inoculating a composite probiotic agent, and extracting functional components in a fermentation product to obtain an extracting solution; and S4, compounding Arabic gum and maltodextrin according to a weight ratio of 1.5: 2 to obtain a wall material, adding 0.17% of selenium-enriched yeast, dissolving the selenium-enriched yeast in food-grade ethanol to prepare a wall material solution, mixing the extracting solution and the wall material solution according to a weight ratio of 1: 2.9, adding 0.11% of Tween-80, stirring for minutes to form a uniform emulsion, and carrying out spray drying to prepare the microcapsule.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food fermentation, and particularly relates to a probiotic fermented ginger product with enhanced antioxidant activity and a preparation process thereof. BACKGROUND

[0002] Ginger has a high content of gingerols, a unique flavor, and traditional effects of dispelling cold and eliminating dampness, and thus has natural application advantages in the fields of food and health products. Probiotic fermentation technology, as a core means for functional upgrading of agricultural products, can transform active ingredients in ginger through microbial metabolism, further improve the nutritional value and bioavailability, and thus become an important research direction for deep processing of ginger. However, in the process of industrialized production and functional enhancement of existing fermented ginger products, there are still four core technical bottlenecks that are interrelated. First, the adaptability of strains and ginger substrate is defective. Ginger raw materials naturally contain a high concentration of gingerols of about 6.2%, and during the fermentation process, sugar and salt components of ≥5% are usually added to adjust the flavor and extend the shelf life, forming a high osmotic pressure environment. In this special substrate, traditional probiotic strains (such as ordinary Lactobacillus plantarum and Saccharomyces cerevisiae) not only have a rapid decrease of more than 50% in viable counts, but also have a significant inhibition of the activity of beta-glucosidase in the body, resulting in a generally insufficient conversion efficiency of less than 30% of gingerols in ginger to gingerol ketones with high antioxidant activity. In addition, there is a problem of efficient retention of active ingredients. On the one hand, traditional extraction processes mostly rely on single centrifugation or filtration technology, and the loss rate of fat-soluble functional ingredients such as gingerol ketones is 30-40%; on the other hand, as a live bacterial preparation, probiotics are easily inactivated in the stomach acid environment of the human body, and the existing technology has an average industry level of a probiotic survival rate in stomach acid of only about 45%, which results in the inability of the product to fully exert its intestinal regulation function and antioxidant effect, forming a vicious cycle of “low fermentation conversion efficiency - high loss of active ingredients - discounted functional effect”. The existing fermented ginger products are difficult to balance the content of functional ingredients, the activity of probiotics, and the stability of the product, which is a technical problem that needs to be broken through in the industry. Therefore, a probiotic fermented ginger product with enhanced antioxidant activity and a preparation process thereof are designed. SUMMARY

[0003] In view of the above deficiencies in the prior art, the present application provides a probiotic fermented ginger product with enhanced antioxidant activity and a preparation process thereof to solve the problems in the background art.

[0004] In order to solve the above technical problems, the present application adopts the following technical solutions: The preparation process of the probiotic fermented ginger product with enhanced antioxidant activity, characterized in that it comprises the following steps: S1. Ginger is cut into slices and freeze-dried under the conditions of a vacuum degree of 0.01 MPa and -40℃ to obtain pretreated ginger; S2. The pCas9-sgRNA recombinant vector is introduced into Lactobacillus plantarum by electroporation, and the base-mutated engineering strain is obtained by anaerobic screening in MRS medium containing 50 μg / mL ampicillin at 37°C. The Saccharomyces cerevisiae is mixed with the Lactobacillus plantarum engineering strain at a weight ratio of 4:1 to prepare a composite probiotic bacterial inoculum; S3. The pre-processed wind ginger is mixed with the optimized carbon and nitrogen source, the antioxidant synergist, and the composite probiotic bacterial inoculum, and is fermented in two stages of proliferation and transformation by controlling the fermentation conditions. The functional components in the fermentation product are extracted to obtain an extract. S4. The wall material is prepared by compounding gum arabic and malt dextrin at a weight ratio of 1.5:2, adding 0.17% selenium-enriched yeast, and dissolving in food-grade ethanol. The extract and the wall material solution are mixed at a weight ratio of 1:2.9, 0.11% Tween-80 is added, and a uniform emulsion is formed by stirring for minutes. The microcapsules are prepared by spray drying.

[0005] Further, the preservation number of the Lactobacillus plantarum is CGMCC1.557.

[0006] Further, the β-glucosidase encoding gene of the Lactobacillus plantarum CGMCC1.557 has a GenBank accession number of CP023710.1, and the sgRNA sequence is 5'-GCTGATCGACGTCGTGGTCA-3'.

[0007] Further, in step S2, the electroporation conditions are a voltage of 2.5 kV, a capacitance of 25 μF, and a resistance of 200 Ω. The single colonies obtained by screening are identified by PCR and verified by sequencing. The upstream primer used for CR identification is 5'-ATGAAGCTTATCGTCGTCGT-3', and the downstream primer is 5'-TTAGTCGACTGCGTGGTGTA-3'.

[0008] Further, in step S3, the optimized carbon and nitrogen source is a compound of glucose and yeast extract enzymatically digested by α-amylase, and the weight ratio of glucose to yeast extract is 4.5:3.

[0009] Further, the antioxidant synergist is added in an amount of 0.5% to 1% of the total weight of the raw materials. The synergist is a compound of glutathione and resveratrol, and the weight ratio of glutathione to resveratrol is 2:1.

[0010] Further, the wall material is compounded by gum arabic and malt dextrin at a weight ratio of 1.5:2, and the selenium-enriched yeast is added in an amount of 0.17% of the total weight of the wall material.

[0011] Further, the preservation number of the selenium-enriched yeast is CGMCC2.836.

[0012] Further, the preservation number of the Saccharomyces cerevisiae is CCTCC NO: M2018625.

[0013] A wind ginger product is prepared from the following raw materials by weight: wind ginger 80 to 100 parts, composite probiotic agent 3 to 5 parts, optimized carbon and nitrogen source 2 to 4 parts, antioxidant synergist 0.5 to 1 part, and food-grade stabilizer 0.3 to 0.6 part.

[0014] Compared with the prior art, the present application has the following beneficial effects: 1. The glutathione-resveratrol complex antioxidant synergist forms a synergistic antioxidant system, effectively inhibits oxidative stress in fermentation and subsequent processes, reduces the content of malondialdehyde in the fermentation broth, reduces the loss rate of functional components, and increases the DPPH clearance rate of the finished product. 2. The present application uses CRISPR-Cas9 technology to direct the modification of Lactobacillus plantarum CGMCC 1.557, increases the activity of beta-glucosidase, significantly enhances the adaptability of the strain in high capsaicin matrix, and improves the conversion efficiency of capsaicin to high antioxidant activity gingerol ketone. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The preparation process flow chart of the probiotic fermented wind ginger product with enhanced antioxidant activity of the present application; DETAILED DESCRIPTION

[0016] In order to enable those skilled in the art to better understand the present application, the technical solutions of the present application are further described below in conjunction with the drawings and examples.

[0017] Wherein, the drawings are only used for exemplary illustration, and the representation is only a schematic diagram, not a physical diagram, and cannot be understood as a limitation on the present application; in order to better illustrate the embodiments of the present application, some components of the drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product; for those skilled in the art, it is understandable that some known structures and their descriptions in the drawings may be omitted.

[0018] In view of the industry average level of about 45% of the survival rate of probiotics in the prior art, the intestinal regulation function and antioxidant effect of the product cannot be fully exerted, forming a vicious cycle of "low fermentation conversion efficiency - high loss of active ingredients - discounted functional effect". The existing wind ginger fermented product cannot balance the content of functional ingredients, probiotic activity and product stability, which becomes a technical problem to be solved in the industry. The present application provides a preparation process for a probiotic fermented wind ginger product with enhanced antioxidant activity, which specifically comprises the following steps: Step 1: precise pretreatment of raw materials The Yangchun ginger with a gingerol content of not less than 6.0% and meeting the standard of GB / T 30383-2013 is selected, and then subjected to rapid detection by near-infrared spectroscopy (detection time is not more than 3 minutes per batch, and accuracy is not less than 98%), and then subjected to composite cleaning by ultrasonic wave-pulse electric field (ultrasonic wave power is 400 watts, pulse intensity is 30 kilovolts per centimeter, and processing time is 12 minutes, and removal rate of pesticide residues is not less than 95%); the ginger is cut into slices with a thickness of 3 to 5 millimeters by laser cutting technology (thickness error is not more than 0.2 millimeters), and finally subjected to freeze-drying under the conditions of vacuum degree of 0.01 MPa and temperature of minus 40 degrees Celsius, and the moisture content of the dried ginger is controlled to be 15% to 20%, so as to obtain pretreated ginger.

[0019] Step 2: Preparation of genetically engineered strain and adaptation of composite microbial agent Construction of engineering strain: pCas9-sgRNA recombinant vector is constructed by CRISPR-Cas9 technology, and is introduced into Lactobacillus plantarum CGMCC 1.557 by electroporation method (voltage is 2.5 kilovolts, capacitance is 25 microfarads, and resistance is 200 ohms), and site-directed mutation is carried out on the beta-glucosidase coding gene (GenBank accession number CP023710.1) of the strain, and the sgRNA sequence is 5'-GCTGATCGACGTCGTGGTCA-3'; the transformed strain is inoculated into MRS medium containing 50 micrograms per milliliter of ampicillin, and is cultured at 37 degrees Celsius for 24 hours under anaerobic conditions for screening, and the single colonies obtained by screening are identified by PCR (upstream primer 5'-ATGAAGCTTATCGTCGTCGT-3', downstream primer 5'-TTAGTCGACTGCGTGGTGTA-3') and sequencing verification, so as to ensure that the base mutation rate is 100%, and the engineering strain (viable bacterial count is not less than 1×10 9 CFU per milliliter) is obtained.

[0020] Saccharomyces cerevisiae culture: Saccharomyces cerevisiae SSA1 with preservation number CCTCC M 2018625 is inoculated into YPD medium, and is cultured at 30 degrees Celsius with 190 revolutions per minute for 18 to 20 hours, and 0.2% ginger extract is added twice during the culture period for gradient acclimation (interval is 8 hours), and the viable bacterial count after acclimation is 7×10 8 CFU per milliliter. 3. Preparation of composite microbial agent: the engineering strain of Lactobacillus plantarum and Saccharomyces cerevisiae SSA1 are compounded at a weight ratio of 4:1 to obtain a composite probiotic microbial agent.

[0021] Step 3: Adapted fixed parameter segmented fermentation The pretreated ginger is mixed with the optimized carbon and nitrogen source for enzymatic hydrolysis, and the glutathione-resveratrol compound antioxidant synergist. The mass ratio of solid (pretreated ginger) to liquid (mixed substrate containing carbon and nitrogen source, antioxidant synergist) in the mixed system is 1 to 1.55. The optimized carbon and nitrogen source is a compound of glucose and yeast extract for enzymatic hydrolysis by α-amylase (weight ratio of 4.5 to 3), and the enzymatic hydrolysis conditions are 53 degrees Celsius, pH 6.3, and enzymatic hydrolysis for 32 minutes (carbon and nitrogen source utilization rate not less than 90%). The antioxidant synergist is added in an amount of 0.5% to 1% of the total weight of the raw materials, and the weight ratio of glutathione to resveratrol is 2 to 1.

[0022] Access 3.3% of the compound probiotic agent, and control the fermentation conditions in two stages: Proliferation period (0 to 9 hours): temperature 28 degrees Celsius, ORP value negative 48 millivolts, stirring rate 38 revolutions per minute; Conversion period (9 to 66 hours): temperature 25 degrees Celsius, ORP value negative 155 millivolts, stirring rate 58 revolutions per minute; The fermentation endpoint is detected by UPLC-Q-TOF-MS, and the gingerol ketone content is not less than 70 milligrams per 100 grams.

[0023] Step 4: Multi-field coupled extraction and purification The functional components in the fermentation product are extracted by ultrasonic-microwave synergistic extraction technology, and the extraction parameters are ultrasonic power 330 watts, microwave frequency 2450 megahertz, extraction temperature 43 degrees Celsius, and extraction time 17 minutes. After extraction, the functional components are retained at a rate of not less than 97% by microfiltration through a 0.22 micron ceramic membrane, followed by low temperature centrifugal purification at 4 degrees Celsius and 5800 revolutions per minute. The purified extract is obtained.

[0024] Step 5: Food-grade synergistic embedding to prepare microcapsules Wall material solution preparation: The wall material is prepared by compounding gum arabic and malt dextrin at a weight ratio of 1.5 to 2, and adding 0.17% of selenium-rich yeast (preservation number CGMCC 2.836, selenium content not less than 1250 micrograms per gram, selenium-methionine content not less than 92%, sterilized by 4.8 kilogray irradiation) based on the total weight of the wall material. Dissolve in food-grade ethanol with a volume fraction of 72% to prepare the wall material solution. 2. Emulsification and drying: Mix the extract and wall material solution at a weight ratio of 1 to 2.9, add 0.11% of Tween-80 (complying with GB25554 standard), and stir at 11500 revolutions per minute for 17 minutes to form a uniform emulsion. Spray drying (inlet air temperature 158 degrees Celsius, outlet air temperature 79 degrees Celsius, feed rate 4.8 milliliters per minute) is used to prepare microcapsules with a particle size of 120 to 220 nanometers, an embedding rate of not less than 95%, and a release rate of not less than 90% in a pH 7.4 environment for 2 hours.

[0025] Step 6: Product processing and quality control storage The microcapsules are mixed with xanthan gum-trehalose complex stabilizer (weight ratio 1 to 2.2, and the addition amount is 0.3% to 0.6% of the total weight of the raw materials) uniformly, and after sterile packaging, a three-level detection system is used: ① enterprise self-checking (HPLC method for detecting ginger phenol ketone, DPPH method for detecting antioxidant activity, and plate counting method for detecting viable bacteria count); ② full-item detection by a third-party CNAS certified institution; ③ fingerprint spectrum verification by Guangdong Food Inspection Institute (similarity not less than 97%); after passing the detection, it is stored in the warehouse, and the finished product needs to meet the following requirements: ginger phenol ketone not less than 70 mg per 100 g, DPPH clearance rate not less than 94%, and viable bacteria count not less than 7 x 10 8 CFU per gram.

[0026] Example 1 Raw material dosage weight parts; spring ginger 90.00 parts, compound probiotic agent 4.00 parts, optimized carbon and nitrogen source (glucose and yeast extract weight ratio 4.5:3) 3.00 parts, antioxidant synergist (glutathione and resveratrol weight ratio 2:1) 0.80 parts, xanthan gum-trehalose stabilizer (weight ratio 1:2.2) 0.50 parts, selenium-rich yeast (CGMCC2.836) 0.051 parts (0.17% of the total weight of the wall material), Tween-80 0.32 parts (0.11% of the emulsion system), 72% food-grade ethanol 100 parts. Raw material pretreatment: wind ginger is detected by near-infrared spectroscopy (2.5 min / batch, accuracy 98.5%), cleaned by ultrasonic-pulsed electric field composite (power 400 W, pulse intensity 30 kV / cm, treatment time 12 min, pesticide residue removal rate 96%), cut into 4.0±0.1 mm thick slices by laser cutting, and freeze-dried under the conditions of vacuum degree 0.01 MPa and-40℃ for 12 h, with water content 18.2±0.3% after drying; Strain culture and preparation of bacterial agent: Lactobacillus plantarum engineering strain: construct pCas9-sgRNA recombinant vector, introduce into Lactobacillus plantarum CGMCC1.557 by electroporation method (voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω), inoculate into MRS medium containing 50 μg / mL ampicillin, and cultivate anaerobically at 37℃ for 24 h, and then identify by PCR (upper primer sequence 5'-ATGAAGCTTATCGTCGTCGT-3', lower primer sequence 5'-TTAGTCGACTGCGTGGTGTA-3') and sequencing verification, with base mutation rate 100%, and viable bacteria count 1.2±0.1 x 10 9 CFU / mL; Saccharomyces cerevisiae: inoculate into YPD medium, cultivate at 30℃ with 190 rpm shaking for 19 h, and add 0.2% wind ginger extract in 2 times gradient acclimation (interval 8 h) during the cultivation, and then the viable bacteria count reaches 7.5±0.2 x 10 8CFU / mL; the above Lactobacillus plantarum engineering strain was mixed with Saccharomyces cerevisiae at a weight ratio of 4:1 to prepare a composite probiotic bacterial inoculum; Segmented fermentation: the pretreated wind ginger was mixed with optimized carbon and nitrogen sources and antioxidant synergistic agents, the mass ratio of solid (pretreated wind ginger) to liquid (carbon and nitrogen sources + synergistic agents) was 1:1.55, and the pH of the system was 6.3±0.1 after uniform mixing; the composite probiotic bacterial inoculum was inoculated at an inoculum amount of 3.3%, the temperature was controlled at 28℃, the ORP value was-48mV, and the stirring speed was 38rpm in the proliferation period (0-9h), and the temperature was controlled at 25℃, the ORP value was-155mV, and the stirring speed was 58rpm in the conversion period (9-66h); through UPLC-Q-TOF-MS detection, the content of zingerone was 76.2±1.3mg / 100g; ultrasonic-microwave synergistic extraction (power 330W, microwave frequency 2450MHz, extraction temperature 43℃, extraction time 17min) was used, 0.22μm ceramic membrane microfiltration was used, and the functional ingredient retention rate reached 97.8±0.5% after purification under the conditions of centrifugation at 4℃ and 5800rpm for 15min; the arabic gum-malt dextrin (weight ratio 1.5:2) wall material was dissolved in 72% food-grade ethanol, 0.17% selenium-rich yeast was added, and stirring was performed until complete dissolution; the extract and the wall material solution were mixed at a weight ratio of 1:2.9, Tween-80 was added, and a uniform emulsion (particle size 180±5nm) was formed after stirring at 11500rpm for 17min; the inlet air temperature was controlled at 158℃, the outlet air temperature was controlled at 79℃, and the feeding speed was controlled at 4.8mL / min to prepare microcapsules; the microcapsules were mixed with xanthan gum-trehalose stabilizers, and after sterile packaging and three-stage detection, the qualified products were stored in the warehouse.

[0027] Detection results In terms of physicochemical indicators, the content of zingerone was 76.2±1.3mg / 100g, the content of gingerol was 112.5±2.1mg / 100g, the content of free amino acid was 4.7±0.2g / 100g, and the pH value was 3.8±0.1; the DPPH clearance rate was 95.2±0.8%, the microcapsule embedding rate was 95.6±0.4%, the survival rate of probiotics in simulated gastric acid was 88.3±1.2%; the viable bacterial count was 8.2±0.3×10 8 CFU / g, the total bacterial count was 8.6±0.5×10³CFU / g, and no pathogenic bacteria were detected; In terms of stability indicators, the batch difference rate was 3.2±0.3%.

[0028] Example 2 In terms of raw material dosage, 80.00 parts of spring wind ginger and 0.50 parts of antioxidant synergistic agent were used, and the dosages of the remaining raw materials and the operation steps were consistent with those of Example 1.

[0029] Detection results Gingerol content was 71.3 ± 1.1 mg / 100 g, DPPH clearance rate was 92.1 ± 0.6%, viable bacteria count was 7.3 ± 0.2 × 10 8 CFU / g, microencapsulation embedding rate was 94.8 ± 0.5%, batch difference rate was 3.5 ± 0.4%.

[0030] Example 3 In the raw material dosage, the Yangchun ginger was 100.00 parts, the antioxidant synergist was 1.00 part, and the rest of the raw material dosage and the operation steps were consistent with example 1.

[0031] Test results Gingerol content was 79.1 ± 1.5 mg / 100 g, DPPH clearance rate was 96.5 ± 0.7%, viable bacteria count was 8.9 ± 0.3 × 10 8 CFU / g, microencapsulation embedding rate was 96.2 ± 0.3%, batch difference rate was 3.1 ± 0.2%.

[0032] Example 4 In the material mixing link of the section fermentation, the mass ratio of solid (pretreated ginger) to liquid (mixed substrate) was 1:1.5; the enzyme hydrolysis condition of optimizing carbon and nitrogen source was adjusted to 53℃, pH6.3, and enzyme hydrolysis for 35min, and the rest of the operation steps were consistent with example 1, and the utilization rate of carbon and nitrogen source reached 89.5 ± 1.2%.

[0033] Test results Gingerol content was 74.3 ± 1.2 mg / 100 g, DPPH clearance rate was 94.7 ± 0.5%, functional ingredient retention rate was 97.1 ± 0.4%, and microencapsulation embedding rate was 95.1 ± 0.3%.

[0034] Example 5 The selenium-rich yeast addition amount was adjusted to 0.15% of the total weight of the wall material, and the rest of the operation steps were consistent with example 1.

[0035] Test results Microencapsulation embedding rate was 94.2 ± 0.4%, probiotic bacteria simulated gastric acid survival rate was 85.7 ± 1.1%, 2h release rate in pH7.4 environment was 90.3 ± 0.6%, and selenium content of finished product was 0.10 ± 0.01 mg / kg.

[0036] Example 6 The selenium-rich yeast addition amount was adjusted to 0.19% of the total weight of the wall material, and the rest of the operation steps were consistent with example 1.

[0037] Test results The microcapsule embedding rate was 96.8±0.3%, the probiotic bacteria simulated gastric acid survival rate was 90.5±1.3%, the 2h release rate in a pH 7.4 environment was 92.1±0.5%, and the selenium content of the finished product was 0.12±0.01mg / kg.

[0038] Example 7 The antioxidant synergist was added in an amount of 0.70 parts, and the remaining operation steps were consistent with example 1.

[0039] 2. Test results The gingerol content was 74.8±1.2mg / 100g, the DPPH clearance rate was 94.5±0.7%, and the fermentation cycle remained 66h (consistent with example 1).

[0040] Example 8 The antioxidant synergist was added in an amount of 0.90 parts, and the remaining operation steps were consistent with example 1. Test results The gingerol content was 78.5±1.4mg / 100g, the DPPH clearance rate was 96.1±0.6%, and the functional ingredient conversion efficiency was 78.3±1.2%, which was 2.1% higher than that of example 1.

[0041] Example 9 In the material mixing link of the segmented fermentation, the mass ratio of solid (pretreated ginger) to liquid (mixed substrate) was 1:1.55, the spray drying parameters were controlled as follows: inlet air temperature 158℃, outlet air temperature 79℃, and feed rate 4.8mL / min, and the remaining operation steps were consistent with example 1.

[0042] 2. Test results The gingerol content was 77.3±1.3mg / 100g, the microcapsule particle size was 175±4nm, the embedding rate was 95.8±0.4%, and the probiotic bacteria survival rate was 89.2±1.1%.

[0043] Example 10 The finished product of example 1 was sealed and stored at 25℃ and a relative humidity of 60% for 6 months.

[0044] Test results The initial gingerol content was 76.2±1.3mg / 100g, and after 6 months it was 73.5±1.2mg / 100g, with a change rate of-3.5%; the initial viable bacterial count was 8.2±0.3×10 8 CFU / g, and after 6 months it was 6.8±0.2×10 8The initial value of CFU / g was 3.2 ± 0.2 x 10, and the change rate was -17.1%; the initial value of DPPH clearance rate was 95.2 ± 0.8%, and after 6 months, it was 93.8 ± 0.7%, and the change rate was -1.5%; in terms of sensory quality, it was light yellow brown and the fermentation aroma was rich initially, and after 6 months, it was still light yellow brown and the fermentation aroma was relatively rich, and there was no obvious odor.

[0045] Comparative Example 1: Non-genetically edited Lactobacillus plantarum The non-genetically edited Lactobacillus plantarum CGMCC 1.557 was used, and the β-glucosidase activity was 128 ± 5 U / mL, and the remaining raw material dosage and operation steps were consistent with those of Example 1.

[0046] Detection results The content of zingerone was 42.3 ± 1.1 mg / 100 g, which decreased by 44.5% compared with Example 1; the DPPH clearance rate was 78.3 ± 0.9%, which decreased by 17.7% compared with Example 1; the viable cell count of the strain (after fermentation) was 3.2 ± 0.2 x 10 8 CFU / g, which decreased by 61.0% compared with Example 1; the gingerol conversion efficiency was 28.5 ± 1.0%, which decreased by 58.2% compared with Example 1; the peak value of β-glucosidase activity in the fermentation broth was 128 ± 5 U / mL, which decreased by 36.0% compared with Example 1 (200 ± 8 U / mL after modification).

[0047] Conclusion: Genetic editing can directionally improve the β-glucosidase activity of Lactobacillus plantarum, solve the strain adaptability problem in a high gingerol matrix, and significantly promote the conversion of gingerol to zingerone with high antioxidant activity, which is one of the core technologies for improving the functional components of the present application.

[0048] Comparative Example 2: Single-temperature fermentation (26°C throughout) The fermentation was carried out at a constant temperature of 26°C throughout, with a stirring speed of 58 rpm and an ORP value of -100 mV, and there was no proliferation period and conversion period, and the remaining raw material dosage and operation steps were consistent with those of Example 1.

[0049] Detection results The content of zingerone was 58.2 ± 1.5 mg / 100 g, which decreased by 23.6% compared with Example 1; the batch difference rate was 18.7 ± 1.2%, which increased by 484.4% compared with Example 1; the strain proliferation rate (0-9h) was 1.8 ± 0.1 x 10 8 CFU / mL・h, which decreased by 33.3% compared with Example 1; the component conversion rate (9-66h) was 0.8 ± 0.1 mg / 100 g・h, which decreased by 42.9% compared with Example 1; the pH value fluctuation range in the fermentation broth was ±0.8, which increased by 166.7% compared with Example 1 (±0.3).

[0050] Conclusion: Subsection fermentation can match the different metabolic requirements of strain proliferation (high temperature, weak anaerobic) and component transformation (low temperature, strong anaerobic), realize the synergistic promotion of strain activity and transformation efficiency, significantly reduce the batch difference, and is a key process to ensure product stability.

[0051] Comparative Example 3: Without antioxidant synergist The glutathione-resveratrol synergistic antioxidant was not added in the fermentation system, and the dosages and operation steps of the remaining raw materials were consistent with those of Example 1.

[0052] Detection results The gingerolone content was 51.3 ± 1.2 mg / 100 g, which was decreased by 32.7% compared with Example 1; the DPPH clearance rate was 82.6 ± 0.8%, which was decreased by 13.2% compared with Example 1; the oxidative stress index (the content of malondialdehyde in the fermentation broth) was 1.8 ± 0.1 μmol / L, which was increased by 63.6% compared with Example 1 (1.1 ± 0.1 μmol / L); the functional component loss rate (from fermentation to extraction) was 18.5 ± 0.9%, which was increased by 123.8% compared with Example 1 (8.3 ± 0.5%); and the gingerolone retention rate after 3 months of storage of the finished product was 78.5 ± 1.0%, which was decreased by 15.0% compared with Example 1 (92.3 ± 0.8%).

[0053] Conclusion: The glutathione and resveratrol form a synergistic antioxidant system, which can inhibit the oxidative stress reaction in fermentation and subsequent processes, reduce the degradation of functional components, and at the same time promote the metabolic transformation efficiency of the strain, which is an important support for the strong antioxidant activity of the present application.

[0054] Comparative Example 4: Without selenium-rich yeast embedding The embedding wall material was only gum arabic-malt dextrin (weight ratio 1.5:2), without adding selenium-rich yeast, and the dosages and operation steps of the remaining raw materials were consistent with those of Example 1.

[0055] Detection results The microcapsule embedding rate was 83.5 ± 1.3%, which was decreased by 12.7% compared with Example 1; the survival rate of probiotics in simulated gastric acid was 52.3 ± 1.2%, which was decreased by 40.8% compared with Example 1; the 2h release rate in a pH 7.4 environment was 76.2 ± 0.9%, which was decreased by 20.0% compared with Example 1; the viable bacterial count after 3 months of storage was 2.8 ± 0.2 × 10 8 CFU / g, which was decreased by 61.9% compared with Example 1 (6.8 ± 0.3 × 10 8 CFU / g); and the porosity of the microcapsule surface was 15.2 ± 0.8%, which was increased by 334.3% compared with Example 1 (3.5 ± 0.3%).

[0056] Conclusion: The selenium-rich yeast can form a dense composite wall material structure with arabic gum-malt dextrin, which not only improves the physical stability of the embedding system, but also protects the probiotic cell membrane through selenomethionine, reduces the loss of activity in the gastric acid environment, and regulates the sustained-release characteristics of functional ingredients.

[0057] Comparative Example 10: Commercially available fermented ginger product A commercially available brand of "probiotic fermented ginger powder" (labeled to contain Lactobacillus plantarum, without genetic editing and complex embedding technology) was selected as the control group, and the full test was carried out according to the detection method of Example 1.

[0058] Test results The gingerolone content was 32.5 ± 1.1 mg / 100 g, which was 134.5% higher than that of Example 1; the DPPH clearance rate was 72.3 ± 0.9%, which was 31.7% higher than that of Example 1; the viable cell count was 2.1 ± 0.1 × 10 8 CFU / g, which was 290.5% higher than that of Example 1; the microcapsule embedding rate was zero (not embedded), the probiotic survival rate in simulated gastric acid was 35.2 ± 1.0%, which was 60.1% lower than that of Example 1; the gingerol conversion efficiency was 18.5 ± 0.8%, which was 160.5% higher than that of Example 1; the loss rate of viable cell count after 3 months of storage was 65.3 ± 1.5%, which was 281.9% higher than that of Example 1 (17.1%).

[0059] The commercially available product does not use genetic editing strains, segmented fermentation and complex embedding technology, resulting in significantly lower functional ingredient content, antioxidant activity, probiotic survival rate and stability than the product of the present application.

[0060] The above is only an embodiment of the present application, and the circuit and electronic components and modules involved are prior art. Those skilled in the art can implement it without further description. The content protected by this application does not involve the improvement of software and methods. The specific structure and characteristics of the scheme are not described in detail here. Those skilled in the art know all the ordinary technical knowledge in the field of the application as of the filing date or priority date, can obtain all the prior art in the field, and have the ability to apply conventional experimental means before that date. Those skilled in the art can improve and implement the present scheme based on their own ability under the guidance of this application. Some typical known structures or known methods should not be an obstacle for those skilled in the art to implement this application. It should be noted that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should be considered within the scope of protection of the present application, and these will not affect the effectiveness and practicality of the application.

Claims

1. A preparation process for probiotic fermented ginger products with enhanced antioxidant activity, characterized in that: Includes the following steps: S1. Cut the ginger into slices and freeze-dry them under vacuum of 0.01 MPa and -40℃ to obtain pretreated ginger; S2. The pCas9-sgRNA recombinant vector was introduced into Lactobacillus plantarum by electroporation. The engineered strain with base mutation was obtained by anaerobic screening in MRS medium containing 50 μg / mL ampicillin at 37℃. After shaking culture of Saccharomyces cerevisiae in YPD medium at 30℃, it was mixed with the engineered strain of Lactobacillus plantarum at a weight ratio of 4:1 to prepare a compound probiotic agent. S3. The pretreated ginger was mixed with the enzymatically optimized carbon and nitrogen source and antioxidant synergist, and a compound probiotic inoculum was added. Fermentation was carried out under controlled fermentation conditions in two stages: the proliferation period and the transformation period. The functional components in the fermentation products were extracted to obtain the extract. S4. A wall material was prepared by compounding gum arabic and maltodextrin at a weight ratio of 1.5:2, adding 0.17% selenium-enriched yeast, dissolving in food-grade ethanol, and then mixing the extract and the wall material solution at a weight ratio of 1:2.

9. 0.11% Tween-80 was added, and the mixture was stirred for minutes to form a homogeneous emulsion. Microcapsules were then prepared by spray drying.

2. The preparation process of the probiotic fermented ginger product with enhanced antioxidant activity as described in claim 1, characterized in that: The preservation number of the Lactobacillus plantarum is CGMCC1.

557.

3. The probiotic fermented ginger product with enhanced antioxidant activity as described in claim 2 and its preparation process, characterized in that: The β-glucosidase encoding gene of Lactobacillus plantarum CGMCC1.557 has the GenBank accession number CP023710.1 and the sgRNA sequence is 5'-GCTGATCGACGTCGTGGTCA-3'.

4. The probiotic fermented ginger product with enhanced antioxidant activity as described in claim 3 and its preparation process, characterized in that: In step S2, the electroporation conditions were 2.5 kV, 25 μF, and 200 Ω, with a screening culture time of 24 hours. The single colonies obtained by screening were identified by PCR and verified by sequencing. The upstream primer used for CR identification was 5'-ATGAAGCTTATCGTCGTCGT-3', and the downstream primer was 5'-TTAGTCGACTGCGTGGTGTA-3'.

5. The probiotic fermented ginger product with enhanced antioxidant activity as described in claim 1 and its preparation process, characterized in that: In step S3, the optimized carbon and nitrogen source is a complex of glucose and yeast extract hydrolyzed by α-amylase, with a weight ratio of glucose to yeast extract of 4.5:

3.

6. The probiotic fermented ginger product with enhanced antioxidant activity as described in claim 1 and its preparation process, characterized in that: The amount of the antioxidant synergist added is 0.5% to 1% of the total weight of the raw materials. The synergist is a compound of glutathione and resveratrol, with a weight ratio of glutathione to resveratrol of 2:

1.

7. The probiotic fermented ginger product with enhanced antioxidant activity as described in claim 1 and its preparation process, characterized in that: The compound wall material is formulated with a weight ratio of gum arabic to maltodextrin of 1.5:2, and the amount of selenium-enriched yeast added is 0.17% of the total weight of the wall material.

8. The probiotic fermented ginger product with enhanced antioxidant activity as described in claim 7 and its preparation process, characterized in that: The selenium-enriched yeast has the accession number CGMCC2.

836.

9. The probiotic fermented ginger product with enhanced antioxidant activity as described in claim 1 and its preparation process, characterized in that: The preservation number of the brewing yeast is CCTCCNO:M2018625.

10. A ginger product, prepared by the process described in claims 1-9 for fermenting ginger with probiotics to enhance antioxidant activity, characterized in that: It is prepared from the following raw materials in parts by weight: 80 to 100 parts of ginger, 3 to 5 parts of compound probiotic agent, 2 to 4 parts of optimized carbon and nitrogen source, 0.5 to 1 part of antioxidant synergist, and 0.3 to 0.6 parts of food-grade stabilizer.

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