Method for obtaining garlic fermentation extract with high content of S-allyl-L-cysteine

Abstract

The invention provides a method for obtaining a garlic fermentation extract with high content of S-allyl-L-cysteine, and belongs to the technical field of biology. The bacillus subtilis capable of producing invertase is separated from a natto product, and the bacillus subtilis can be applied to food and is high in passage stability; the obtained invertase has transglutaminyl ability and thermal stability, the enzyme activity is not reduced due to the influence of conditions in the biosynthesis process, and gamma-glutamyl-S-allyl-L-cysteine (SAC) can be efficiently biotransformed to obtain S-allyl-L-cysteine (SAC) when the invertase is applied to preparation of the garlic extract. Compared with endogenous enzyme self-transformation, the method has the advantages that the time required for obtaining high-content SAC is remarkably shortened, and the SAC content in the garlic fermentation extract can reach 0.8%, is 200 times of the SAC content in fresh garlic, and is far higher than the market product level.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for obtaining a garlic fermentation extract with a high content of S-allyl-L-cysteine. Background Art

[0002] S-allyl-L-cysteine ​​(SAC) is a sulfur-containing amino acid found in aged garlic extracts. Studies have shown that SAC is an effective antioxidant, less harmful than other garlic components, and has multiple physiological effects such as liver protection, anti-tumor, anti-diabetes, and cholesterol-lowering. Glutamyl transpeptidase (GGT, EC 2.3.2.2) is an enzyme preparation ubiquitous in nature that catalyzes the following reactions: cleavage of the γ-glutamyl bond present in the donor substrate and then transfer of the γ-glutamyl moiety to another amino acid (transpeptidation) and / or a short peptide or water molecule (hydrolysis).

[0003] There are two main pathways for the synthesis of SAC in garlic. One pathway is the serine-thiol pathway, where serine and acetyl-CoA first synthesize O-acetylserine under the action of cysteine ​​synthetase (serine acetyltransferase SAT and O-acetylserine thiol lyase OASS), and then react with allyl thiol from unknown sources to generate S-allyl-cysteine; the second pathway is the glutathione / cysteine-allyl pathway, where γ-glutamyl-S-allyl-cysteine ​​(GSAC) is hydrolyzed to remove glutamyl under the action of glutamyl transpeptidase GGT to obtain S-allyl-cysteine. The content of SAC in natural garlic is only 0.003-0.04%, which is rare.

[0004] Under natural conditions, the harvested raw garlic cloves are washed and sliced, stored in a precisely controlled aqueous solution in a stainless steel tank, aged for 20 months without heating, and further concentrated to obtain aged garlic extract. Aged garlic extract has a high SAC content of 0.1% under the transformation of garlic's natural enzyme system, but the preparation cycle is long and the operation steps are complicated, which is not conducive to industrial intensive production.

[0005] In recent years, in order to fully increase the content of SAC in garlic extracts, researchers have conducted a lot of research on its enrichment methods. For example, in CN 102669668A, the activity of endogenous glutamyl transpeptidase in garlic is stimulated by immersion method, and the obtained SAC content is as high as 495μg / g DM; although this method has a simple process and a short cycle, the SAC content is low. CN201810765116.2 uses low-concentration alcohol ultrasonic extraction, and ultrafiltration and resin separation to obtain high-purity SAC, but the SAC yield is low and the organic solvent consumption is large. At present, the SAC content in garlic extracts prepared by extraction methods is often low and the yield is low. Therefore, it is extremely important to find a method for effectively extracting high-content SAC from garlic. Summary of the invention

[0006] The present invention provides a method for obtaining a garlic fermentation extract with a high content of S-allyl-L-cysteine. The method utilizes commercially available natto products to separate Bacillus subtilis that produces a thermostable invertase, enriches the invertase by fermentation, and utilizes a bioconversion method to obtain a garlic extract with a high SAC content. The method has innovative significance for key technologies for high-value processing and comprehensive utilization of garlic.

[0007] In order to achieve the above object, the present invention provides a method for obtaining a garlic fermented extract with a high content of S-allyl-L-cysteine, which comprises separating a fermentation strain capable of producing invertase from natto, fermenting and culturing the fermentation strain, separating and purifying the invertase, adding the obtained invertase to a garlic extract in proportion, drying after bioconversion, and obtaining a fermented garlic extract with a SAC content of up to 0.8%.

[0008] Preferably, the fermentation strain is Bacillus subtilis, and the converting enzyme is at least one of a protease and a glutamyl transpeptidase.

[0009] Preferably, the obtained fermentation bacteria are fermented and cultured by liquid submerged fermentation.

[0010] Preferably, the fermentation culture conditions are:

[0011] The culture temperature was 30-39°C, the pH was 6.5-7.5, the air flow rate was 2 vvm, the dissolved oxygen was controlled at 30-50% by dissolved oxygen association, and the feed rate was 0.15-0.3 g·L -1 ·h -1 , constant feeding rate.

[0012] As a preferred fermentation culture condition, the culture temperature is 37°C, the pH is controlled at about 7.0, the dissolved oxygen is controlled at 30%, and the feed rate is 0.2 g·L -1 ·h -1 , constant feeding rate.

[0013] Preferably, after the fermentation culture is completed, the method further includes removing the bacteria and insoluble impurities from the fermentation broth, and further purifying and concentrating the fermentation filtrate through ultrafiltration, so that the enzymatic activity of the obtained converting enzyme is higher than 100 U / g.

[0014] Preferably, the fermentation filtrate is purified by at least one or more of ammonium sulfate precipitation, two-phase aqueous extraction, and ultrafiltration.

[0015] Preferably, the ammonium sulfate precipitation is selected to have a saturation of 60%-80% to precipitate the target protein; the final concentrations of the two phases in the two-phase aqueous extraction are PEG1000 12%-18% and ammonium sulfate 12%-18% respectively; and the ultrafiltration membrane is selected to be 30000kDa for ultrafiltration concentration and desalting.

[0016] Preferably, the concentration of the garlic extract is 40-70° Bx, and the added ratio of the invertase is 0.05%-0.5% of the dry weight of the garlic extract.

[0017] Preferably, the source of the garlic extract is selected from at least one of fresh garlic, dehydrated garlic slices, dehydrated garlic powder, aged garlic slices, aged garlic powder and black garlic.

[0018] Preferably, the bioconversion conditions are pH 8.0-10.0, 45-65° C., and 150 rpm for 3-6 h.

[0019] The present invention also provides a garlic fermentation extract, which is prepared by the method described in any one of the above technical solutions.

[0020] Preferably, the moisture content is less than 5%, the particle size is 40-120 mesh, the SAC content is 0.76-0.82%, and the microbial indicators meet the internal requirements.

[0021] The present invention also provides an application of the Bacillus subtilis or the invertase according to the above technical solution in the bioconversion of garlic food processing to obtain a product with an S-allyl-L-cysteine ​​content of up to 0.8%.

[0022] Compared with the prior art, the advantages and positive effects of the present invention are:

[0023] The invention separates Bacillus subtilis capable of producing invertase from natto products, and the bacterium can be applied to food with high passage stability; the invertase obtained from the invertase has the ability to transglutamylate and thermal stability, and the enzyme activity will not be reduced due to the influence of conditions during the biosynthesis process. The invertase is applied to the preparation of garlic extract, and can efficiently bioconvert γ-glutamyl-S-allyl-L-cysteine ​​to obtain S-allyl-L-cysteine. Compared with endogenous enzyme self-conversion, the time required to obtain high-content SAC is significantly shortened, and the SAC content in the garlic fermentation extract can be as high as 0.8%, which is 200 times the SAC content in fresh garlic, and is much higher than the level of market products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A morphological diagram of the strain provided in the embodiment of the present invention;

[0025] Figure 2 An agarose gel electrophoresis diagram of the strain provided in the embodiment of the present invention;

[0026] Figure 3 The curve of the stability change of the strain provided in the embodiment of the present invention;

[0027] Figure 4 The enzyme activity assay standard curve provided in the embodiment of the present invention;

[0028] Figure 5 The curve of the change of invertase activity with fermentation time during the fermentation process provided by the embodiment of the present invention;

[0029] Figure 6 The optimum temperature diagram of the invertase provided in the embodiment of the present invention;

[0030] Figure 7 The optimal pH diagram of the invertase provided in the embodiment of the present invention;

[0031] Figure 8 The thermal stability change curve of the invertase provided in the embodiment of the present invention;

[0032] Fig. 9 Liquid chromatogram for detecting SAC and GSAC in garlic extract provided by the embodiment of the present invention, wherein A. SAC standard; B. GSAC standard; C. SAC and GSAC in garlic extract before biosynthesis; D. SAC in garlic extract after biosynthesis;

[0033] Fig.10 This is a curve of SAC content change in the biosynthesis process provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. In the following embodiments, the materials, reagents, strains, etc. used, unless otherwise specified, can be obtained through commercial channels.

[0035] Physiological and biochemical identification of isolated strains

[0036] The commercially available natto was dissolved and cultured on streaking plates to obtain a strain with the morphological characteristics of Bacillus colonies, which was Gram-positive, with buds that were approximately round and lateral. After a single colony was inoculated into LB medium and cultured at 37°C for 16 hours, the single colony was raised, with a rough, opaque surface and a dirty white color. The colony morphology is shown in the figure Figure 1 .

[0037] The strains were identified for physiological and biochemical characteristics (see Table 1, gel electrophoresis diagram see Figure 2 ), and PCR amplification of the strain was performed using 16S rRNA universal primers, 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT), and 16S rRNA sequencing was performed at Shanghai Sangon Biotechnology Co., Ltd. After sequencing and comparison, the strain was identified as Bacillus subtilis with GenBank accession number CP086007.1.

[0038] A single colony was inoculated into an LB shake flask and cultured at 37°C for 16 hours. Based on the absorbance at 410nm of the bacterial solution for the deglutamylation of γ-glutamyl-p-nitroaniline (γ-GPNA) to produce p-nitroaniline, a strain with high enzyme activity was obtained. After the strain was cultured, it was mixed with glycerol, and a strain with higher enzyme activity was selected and stored at -80°C. Then, its stability in passage was observed experimentally. The results are shown in Figure 3 .Depend on Figure 3 It can be seen that the enzyme production performance of this bacterium is stable. After 10 subcultures, the enzyme activity can still maintain 77% of the initial enzyme activity, and it can be used as a strain for subsequent fermentation tests.

[0039] Table 1 Identification results of physiological and biochemical characteristics of strains

[0040]

[0041] The culture medium, enzyme activity determination method, preparation and analysis method of garlic extract used in the following examples are described as follows.

[0042] Culture medium

[0043] Seed culture medium g / L: peptone 10, yeast extract powder 5, NaCl 10. If solid culture medium is prepared, add 2% agar.

[0044] Fermentation medium g / L: sucrose 10, yeast extract powder 15, peptone 15, potassium dihydrogen phosphate 1, magnesium sulfate heptahydrate 0.5, Tween-80 0.1.

[0045] Feed medium g / L: sucrose 5, yeast extract powder 10, peptone 10.

[0046] Invertase enzyme activity assay

[0047] γ-Glutamyl transpeptidase catalyzes the transfer of the γ-glutamyl group of the substrate γ-glutamyl-p-nitroaniline (γ-GPNA) to di-Glycine to generate p-nitroaniline. p-Nitroaniline has a special absorption at 410nm. Based on this characteristic, the concentration of p-nitroaniline generated by the reaction is measured to measure the activity of the enzyme.

[0048] The method for determining the transpeptidase activity is as follows: a system mixture containing 0.05 mol / L Tris-HCl (pH 9.0), 20 mmol / L di-Gly peptide, 2.5 mmol / L γ-glutamyl-p-nitroaniline (γ-GPNA) and 1 mL of enzyme solution diluted to an appropriate multiple is incubated at 60°C for 30 min (within the linear range), 1 mL of 1 mol / L HCl is added to terminate the reaction, and the absorbance of the mixture at 410 nm is measured. The presence of glycine can effectively inhibit the hydrolysis and autotranspeptidation reaction of γ-GPNA.

[0049] The preparation method of the p-nitroaniline standard curve is as follows: accurately weigh 4.3 mg of p-nitroaniline, place it in a 100 mL volumetric flask, add 50 mL of methanol, sonicate, let it stand and cool to room temperature, and then use 0.05 mol / L Tris-HCl solution with pH 9.0 to make up the volume, and gradually dilute it to concentrations of 0.311, 0.186, 0.125, 0.050, 0.020, 0.008, 0.003, and 0.001 mmol / L. As a standard solution, use an ultraviolet spectrophotometer to measure its absorbance at 410 nm, and use p-nitroaniline mmol / L as the horizontal axis and the absorbance at 410 nm as the vertical axis to make a standard curve. The mass concentration of p-nitroaniline is linearly related to the absorbance in the range of 0.0 to 0.1245 mmol / L, and the linear regression equation is y=8.5559x-0.001, and the correlation coefficient R 2 =0.9999. See the standard curve Figure 4 .

[0050] The amount of enzyme required to generate 1 μmol of p-nitroaniline per minute at 60°C, 30 min, and pH 9.0 was defined as one unit of enzyme activity.

[0051] Calculated from micromolar absorptivity

[0052]

[0053] Where:

[0054] △A / min: Absorbance change rate per minute

[0055] Vt: total reaction volume, ml

[0056] Vs: sample volume, ml

[0057] 1000: Change factor from U / ml to U / L

[0058] D: Sample dilution multiple

[0059] ε: micromolar absorption coefficient of p-nitroaniline at 410nm wavelength, 9870L / moL / cm

[0060] d: cuvette light path, 1cm

[0061] Preparation of garlic extract

[0062] For ease of explanation, the garlic products mentioned below are all prepared from dehydrated garlic slices. Dehydrated garlic slices are sterilized, dried, and crushed, 200 g of the material is weighed into a reaction container, 2000 mL of water is added, and the mixture is soaked and extracted at 60°C for 3 h, and the liquid is concentrated by rotary evaporation at 65°C to obtain a garlic extract with a concentration of 45°Bx.

[0063] HPLC analysis of SAC content

[0064] Chromatographic column: C18, 5μm, 4.6×250mm

[0065] Detector: UV

[0066] Mobile phase: A-methanol, B-1% V / V phosphoric acid aqueous solution

[0067] Flow rate: 1.0ml / min

[0068] Analysis wavelength: 201nm

[0069] Column temperature: 40°C

[0070] Sample volume: 20ul

[0071] Gradient analysis

[0072] Time / min 0 20 20.01 25 26 32 45 65 end A 15 15 25 25 50 50 15 15 B 85 85 75 75 50 50 85 85

[0073] Calculation formula:

[0074]

[0075] A S ——The peak area of ​​S-allyl-L-cysteine ​​in the sample solution;

[0076] A C ——Peak area of ​​S-allyl-L-cysteine ​​in standard solution;

[0077] C C ——Concentration of S-allyl-L-cysteine ​​in the standard solution, mg / ml;

[0078] V——volume of sample solution, ml;

[0079] W——The weight of the sample used to prepare the sample solution, mg.

[0080] Changes of enzyme activity with conditions

[0081] The activity changes of the invertase were observed under the gradient temperature (30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃), the pH of the buffer was controlled at 9.0, and the reaction time was 30min. It was found that the enzyme activity increased first and then decreased with the increase of temperature, and the highest point of enzyme activity appeared at 60℃. 60℃ was selected as the reaction temperature in the subsequent enzyme activity determination experiment. The change trend is shown in Figure 6 .

[0082] The activity changes of the invertase in a gradient pH environment (7, 7.5, 8, 8.5, 9, 9.5, 10) were observed. The reaction temperature was controlled at 60°C and the reaction time was 30 min. It was found that with the increase of pH, the enzyme activity first increased and then decreased. The highest enzyme activity appeared at pH 9.0. The pH value of the buffer solution for subsequent experiments was selected as 9.0. The trend of change is shown in Figure 7 .

[0083] The thermal stability of the invertase at different temperatures (45°C, 50°C, 55°C, 60°C, 65°C) for 5 hours was observed. As the reaction time increased, the activity of the invertase decreased to varying degrees at each temperature. The decrease range was 50°C < 55°C < 45°C < 60°C < 65°C from small to large. The enzyme activity at 50°C for 5 hours was still able to maintain 94% of the original activity. After 5 hours at 65°C, the invertase activity had lost 58%. Considering all factors, 50°C was selected as the subsequent enzyme conversion temperature. The trend of the thermal stability of enzyme activity is shown in Figure 8 .

[0084] Example 1

[0085] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.0, set the air flow rate to 2vvm, maintain the dissolved oxygen at 30% through dissolved oxygen association, and ferment at a constant temperature of 37℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. The enzyme activity change curve during the fermentation process is shown in Figure 5 It can be seen that during the fermentation period of 30h-50h, the enzyme activity of the fermentation broth was at a stable and high level. The enzyme activity of the fermentation broth was 202U / L at 50h. After 50h, the enzyme activity began to decay. Therefore, the fermentation end time under this condition was 50h.

[0086] After the fermentation, the bacteria and insoluble impurities are removed by plate and frame filtration and disc centrifuge, and the filtrate is further purified and concentrated. The target protein is precipitated by salting out, and the target protein is precipitated at 60%-80% ammonium sulfate saturation. After re-dissolution, 30000kDa is selected for ultrafiltration concentration and desalination, and spray drying is used to obtain a high-purity invertase, the enzyme activity of which can reach 145U / g.

[0087] Take 50g of garlic extract with a concentration of 45°Bx, add invertase at a ratio of 0.20% of dry matter, adjust pH to 9.0, react at 60°C and 150rpm, take samples regularly to detect SAC content, take them out after they remain unchanged, and boil them to inactivate the enzyme for 10 minutes. Vacuum drying to obtain fermented garlic extract, take samples and dissolve them, and then use HPLC to quantitatively biotransform SAC synthesis, where SAC and GSAC peak at about 12.6 and 27.2 minutes, respectively. Under this condition, 0.821% SAC was obtained within 3 hours, and GSAC was completely converted.

[0088] Among them, the HPLC chromatograms of SAC and GSAC contents in SAC standard, GSAC standard, garlic extract before and after biotransformation are shown in Fig. 9 It can be seen that after biotransformation, all GSAC in garlic extract is converted into SAC; the change of SAC content during biotransformation is shown in Fig.10 Under this condition, the SAC content reached 0.825% after 2h of biotransformation, and 0.821% after 3h of biotransformation. This method proves that this method can effectively obtain garlic fermentation extract with high content of S-allyl-L-cysteine.

[0089] Example 2

[0090] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.5, set the air flow rate to 2vvm, maintain the dissolved oxygen at 50% through dissolved oxygen association, and ferment at a constant temperature of 39℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 187U / L.

[0091] After the fermentation, the bacteria and insoluble impurities are removed by plate and frame filtration and disc centrifuge, and the filtrate is further purified and concentrated. The target protein is precipitated by salting out, and the target protein is precipitated at 60%-80% ammonium sulfate saturation. After re-dissolution, 30000kDa is selected for ultrafiltration concentration and desalination, and spray drying is used to obtain a high-purity invertase, the enzyme activity of which can reach 124U / g.

[0092] Take 50g of garlic extract with a concentration of 45°Bx, add invertase at a ratio of 0.5% of dry matter, adjust pH to 9.0, react at 60°C, 150rpm, take samples regularly to detect SAC content, take out when unchanged, boil and inactivate enzyme for 10min. Vacuum dry to obtain fermented garlic extract, take samples and dissolve them, and quantify SAC synthesis by biotransformation by HPLC. Under this condition, 0.799% SAC was obtained within 3 hours.

[0093] Example 3

[0094] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.0, set the air flow rate to 2vvm, maintain the dissolved oxygen at 30% through dissolved oxygen association, and ferment at a constant temperature of 37℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 202U / L.

[0095] After the fermentation, the bacteria and insoluble impurities are removed by plate and frame filtration and disc centrifuge, and the filtrate is further purified and concentrated. The target protein is precipitated by salting out, and the target protein is precipitated at 60%-80% ammonium sulfate saturation. After re-dissolution, 30000kDa is selected for ultrafiltration concentration and desalination, and spray drying is used to obtain a high-purity invertase, the enzyme activity of which can reach 145U / g.

[0096] Take 50g of garlic extract with a concentration of 45°Bx, add invertase at a ratio of 0.3% of dry matter, adjust pH to 9.0, react at 60°C, 150rpm, take samples regularly to detect SAC content, take out after it does not change, boil and kill enzyme for 10min. Vacuum dry to obtain fermented garlic extract, take samples and dissolve them, and then quantify SAC synthesis by biotransformation through HPLC. Under this condition, 0.819% SAC is obtained within hours, and GSAC is completely converted.

[0097] Example 4

[0098] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.0, set the air flow rate to 2vvm, maintain the dissolved oxygen at 30% through dissolved oxygen association, and ferment at a constant temperature of 37℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 202U / L.

[0099] After the fermentation, the bacteria and insoluble impurities were removed by plate and frame filtration and disc centrifuge, and the filtrate was further purified and concentrated. The GGT enzyme was crudely extracted by two-phase aqueous extraction. Ammonium sulfate with a final concentration of 15% and 15% PEG1000 were added to the fermentation supernatant. The mixture was allowed to stand at 4°C for 30 minutes to separate the lower phase. The lower phase extract was subjected to ultrafiltration, concentration and desalination using a 30000 ultrafiltration membrane at an operating pressure of 0.2MPa. After ultrafiltration, spray drying was performed to obtain a higher purity invertase, and the enzyme activity of the invertase could reach 156U / g.

[0100] Take 50g of garlic extract with a concentration of 45°Bx, add invertase at a ratio of 0.25% of dry matter, adjust pH to 9.0, react at 60°C, 150rpm, take samples regularly to detect SAC content, take out when unchanged, boil and inactivate enzyme for 10min. Vacuum dry to obtain fermented garlic extract, take samples and dissolve them, and quantify SAC synthesis by HPLC biotransformation. Under this condition, 0.820% SAC was obtained within 3 hours, and GSAC was completely converted.

[0101] Example 5

[0102] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.5, set the air flow rate to 2vvm, maintain the dissolved oxygen at 50% through dissolved oxygen association, and ferment at a constant temperature of 39℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 187U / L.

[0103] After the fermentation, the bacteria and insoluble impurities were removed by plate and frame filtration and disc centrifuge, and the filtrate was further purified and concentrated. The GGT enzyme was crudely extracted by two-phase aqueous extraction. Ammonium sulfate with a final concentration of 15% and 15% PEG1000 were added to the fermentation supernatant. The mixture was allowed to stand at 4°C for 30 minutes to separate the lower phase. The lower phase extract was subjected to ultrafiltration, concentration and desalination using a 30000 ultrafiltration membrane at an operating pressure of 0.2MPa. After ultrafiltration, spray drying was performed to obtain a higher purity invertase, and the invertase activity could reach 136U / g.

[0104] Take 50g of garlic extract with a concentration of 45°Bx, add invertase at a ratio of 0.5% of dry matter, adjust pH to 9.0, react at 60°C, 150rpm, take samples regularly to detect SAC content, take out when unchanged, boil and inactivate enzyme for 10min. Vacuum dry to obtain fermented garlic extract, take samples and dissolve them, and quantify SAC synthesis by biotransformation by HPLC. Under this condition, 0.795% SAC was obtained within 3 hours.

[0105] Example 6

[0106] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.0, set the air flow rate to 2vvm, maintain the dissolved oxygen at 30% through dissolved oxygen association, and ferment at a constant temperature of 37℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 202U / L.

[0107] After the fermentation, the bacteria and insoluble impurities were removed by plate and frame filtration and disc centrifuge, and the filtrate was further purified and concentrated. The GGT enzyme was crudely extracted by two-phase aqueous extraction. Ammonium sulfate with a final concentration of 12% and 12% PEG1000 were added to the fermentation supernatant. The mixture was allowed to stand at 4°C for 30 minutes to separate the lower phase. The lower phase extract was subjected to ultrafiltration, concentration and desalination using a 30000 ultrafiltration membrane at an operating pressure of 0.2MPa. After ultrafiltration, spray drying was performed to obtain a higher purity invertase, and the invertase activity could reach 126U / g.

[0108] Take 50g of garlic extract with a concentration of 45°Bx, add invertase at a ratio of 0.2% of dry matter, adjust pH to 9.0, react at 60°C, 150rpm, take samples regularly to detect SAC content, take out when unchanged, boil and inactivate enzyme for 10min. Vacuum dry to obtain fermented garlic extract, take samples and dissolve them, and quantify SAC synthesis by biotransformation by HPLC. Under this condition, 0.788% SAC was obtained within 3 hours.

[0109] Example 7

[0110] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.0, set the air flow rate to 2vvm, maintain the dissolved oxygen at 30% through dissolved oxygen association, and ferment at a constant temperature of 37℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 202U / L.

[0111] After the fermentation, the bacteria and insoluble impurities were removed by plate and frame filtration and disc centrifuge, and the filtrate was further purified and concentrated. The GGT enzyme was crudely extracted by two-phase aqueous extraction. Ammonium sulfate with a final concentration of 18% and 18% PEG1000 were added to the fermentation supernatant. The mixture was allowed to stand at 4°C for 30 minutes to separate the lower phase. The lower phase extract was subjected to ultrafiltration, concentration and desalination using a 30000 ultrafiltration membrane at an operating pressure of 0.2MPa. After ultrafiltration, spray drying was performed to obtain a higher purity invertase, and the invertase activity could reach 106U / g.

[0112] Take 50g of garlic extract with a concentration of 45°Bx, add invertase at a ratio of 0.2% of dry matter, adjust pH to 9.0, react at 60°C, 150rpm, take samples regularly to detect SAC content, take out when unchanged, boil and inactivate enzyme for 10min. Vacuum dry to obtain fermented garlic extract, take samples and dissolve them, and quantify SAC synthesis by biotransformation by HPLC. Under this condition, 0.762% SAC was obtained within 3 hours.

[0113] Comparative Example 1

[0114] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.0, set the air flow rate to 2vvm, maintain the dissolved oxygen at 30% through dissolved oxygen association, and ferment at a constant temperature of 37℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 202U / L.

[0115] After the fermentation, the bacteria and insoluble impurities are removed by plate and frame filtration and disc centrifuge, and the filtrate is further purified and concentrated. The target protein is precipitated by salting out, and the target protein is precipitated at 60%-80% ammonium sulfate saturation. After re-dissolution, 30000kDa is selected for ultrafiltration concentration and desalination, and spray drying is used to obtain a high-purity invertase, the enzyme activity of which can reach 145U / g.

[0116] Take 50g of garlic extract with a concentration of 45°Bx, add invertase at a ratio of 0.01% of dry matter, adjust pH to 9.0, react at 60°C, 150rpm, take samples regularly to detect SAC content, take out when unchanged, boil and inactivate enzyme for 10min. Vacuum dry to obtain fermented garlic extract, take samples and dissolve them, and quantify SAC synthesis by biotransformation by HPLC. Under this condition, 0.342% SAC was obtained within 3 hours.

[0117] Comparative Example 2

[0118] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.0, set the air flow rate to 2vvm, maintain the dissolved oxygen at 30% through dissolved oxygen association, and ferment at a constant temperature of 37℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 202U / L.

[0119] After the fermentation, the bacteria and insoluble impurities are removed by plate and frame filtration and disc centrifuge, and the filtrate is further purified and concentrated. The target protein is precipitated by salting out, and the target protein is precipitated at 60%-80% ammonium sulfate saturation. After re-dissolution, 30000kDa is selected for ultrafiltration concentration and desalination, and spray drying is used to obtain a high-purity invertase, the enzyme activity of which can reach 145U / g.

[0120] Take 50g of garlic extract with a concentration of 45°Bx, add invertase at a ratio of 0.75% of dry matter, adjust pH to 9.0, react at 60°C, 150rpm, take samples regularly to detect SAC content, take out when unchanged, boil and inactivate enzyme for 10min. Vacuum dry to obtain fermented garlic extract, take samples and dissolve them, and quantify SAC synthesis by biotransformation by HPLC. Under this condition, 0.744% SAC was obtained within 3 hours.

[0121] Comparative Example 3

[0122] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.0, set the air flow rate to 2vvm, maintain the dissolved oxygen at 30% through dissolved oxygen association, and ferment at a constant temperature of 37℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 202U / L.

[0123] After the fermentation, the bacteria and insoluble impurities are removed by plate and frame filtration and disc centrifuge, and the filtrate is further purified and concentrated. The target protein is precipitated by salting out, and the target protein is precipitated at 60%-80% ammonium sulfate saturation. After re-dissolution, 30000kDa is selected for ultrafiltration concentration and desalination, and spray drying is used to obtain a high-purity invertase, the enzyme activity of which can reach 145U / g.

[0124] Take 50g of garlic extract with a concentration of 20°Bx, add invertase at a ratio of 0.2% of dry matter, adjust pH to 9.0, react at 60°C, 150rpm, take samples regularly to detect SAC content, take out when unchanged, boil and inactivate enzyme for 10min. Vacuum dry to obtain fermented garlic extract, take samples and dissolve them, and quantify SAC synthesis by biotransformation by HPLC. Under this condition, 0.587% SAC was obtained within 3 hours.

[0125] Comparative Example 4

[0126] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.0, set the air flow rate to 2vvm, maintain the dissolved oxygen at 30% through dissolved oxygen association, and ferment at a constant temperature of 37℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 202U / L.

[0127] After the fermentation, the bacteria and insoluble impurities are removed by plate and frame filtration and disc centrifuge, and the filtrate is further purified and concentrated. The target protein is precipitated by salting out, and the target protein is precipitated at 60%-80% ammonium sulfate saturation. After re-dissolution, 30000kDa is selected for ultrafiltration concentration and desalination, and spray drying is used to obtain a high-purity invertase, the enzyme activity of which can reach 145U / g.

[0128] Take 50g of garlic extract with a concentration of 75°Bx, add invertase at a ratio of 0.05% of dry matter, adjust pH to 9.0, react at 60°C, 150rpm, take samples regularly to detect SAC content, take out when unchanged, boil and inactivate enzyme for 10min. Vacuum dry to obtain fermented garlic extract, take samples and dissolve them, and quantify SAC synthesis by biotransformation by HPLC. Under this condition, 0.692% SAC was obtained within 3 hours.

[0129] Comparative Example 5

[0130] Inoculate the glycerol tube stored at -80℃ into LB liquid medium at 1% inoculation amount, and culture at 37℃ for 12h to obtain the first-level seed liquid; inoculate the first-level seed liquid into the second-level seed medium at 5% inoculation amount, and culture at 37℃ for 16h to obtain the second-level seed liquid; inoculate the second-level seed liquid into the fermentation medium at a ratio of 5% inoculation amount, and set the fermentation conditions as follows: use ammonia water to automatically control the pH at around 7.0, set the air flow rate to 2vvm, maintain the dissolved oxygen at 30% through dissolved oxygen association, and ferment at a constant temperature of 37℃. After the dissolved oxygen rebounds, the growth rate is 0.2g·L -1 ·h -1 Feed addition was performed. During the fermentation process, the bacterial liquid was collected regularly, the supernatant was centrifuged, and the enzyme activity was tested. The fermentation was stopped before the enzyme activity decayed. Under this condition, the enzyme activity of the fermentation liquid was 202U / L.

[0131] After the fermentation, 50 g of garlic extract with a concentration of 45°Bx was taken, added to the fermentation liquid at a ratio of 1:1 by dry weight, adjusted to pH 9.0, reacted at 60°C and 150rpm, and sampled regularly to detect the SAC content. After it remained unchanged, it was taken out and boiled to inactivate the enzyme for 10 minutes. The fermented garlic extract was vacuum dried, sampled and dissolved, and then quantitatively biotransformed by HPLC for SAC synthesis. Under this condition, 0.194% SAC was obtained within 3 hours.

Claims

1. A method for obtaining a garlic fermentation extract having a high content of S-allyl-L-cysteine, characterized in that: A fermentation strain capable of producing invertase is separated from natto, the fermentation strain is fermented and cultured, and the invertase is separated and purified. The invertase is added to a garlic extract in proportion, and the extract is dried after bioconversion to obtain a fermented garlic extract with a SAC content of up to 0.8%.

2. The method according to claim 1, characterized in that The fermentation bacteria is Bacillus subtilis, and the converting enzyme is at least one of protease and glutamyl transpeptidase.

3. The method according to claim 1, characterized in that The obtained fermentation bacteria are fermented and cultured by using a liquid submerged fermentation method; The fermentation culture conditions are as follows: The culture temperature was 30-39°C, the pH was 6.5-7.5, the air flow rate was 2 vvm, the dissolved oxygen was controlled at 30-50% by dissolved oxygen association, and the feed rate was 0.15-0.3 g·L -1 ·h -1 , constant feeding rate.

4. The method according to claim 1, characterized in that: After the fermentation culture is completed, the method further includes removing the bacteria and insoluble impurities from the fermentation broth, and further purifying and concentrating the fermentation filtrate through an ultrafiltration step, so that the enzyme activity of the obtained converting enzyme is higher than 100 U / g.

5. The method according to claim 4, characterized in that The concentration of the garlic extract is 40-70° Bx, and the added ratio of the invertase is 0.05%-0.5% of the dry weight of the garlic extract.

6. The method according to claim 1, characterized in that The biotransformation conditions were pH React at 8.0-10.0, 45-65°C, 150 rpm for 3-6 hours.

7. The method according to any one of claims 1 to 6, characterized in that: The raw material source of the garlic extract is selected from at least one of fresh garlic, dehydrated garlic slices, dehydrated garlic powder, aged garlic slices, aged garlic powder and black garlic.

8. A garlic fermentation extract, characterized in that The method is prepared by any one of claims 1 to 7.

9. The garlic fermented extract according to claim 8, characterized in that Its moisture content is less than 5%, the particle size is 40-120 mesh, the SAC content is 0.76-0.82%, and the microbial indicators meet the internal requirements.

10. Use of the Bacillus subtilis or the invertase according to claim 2 in the bioconversion of garlic food processing to obtain a product with an S-allyl-L-cysteine ​​content of up to 0.8%.

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