Compound enzyme and preparation method thereof

Abstract

The invention relates to the technical field of food processing, in particular to a compound enzyme and a preparation method thereof. Schisandra chinensis and figs are used as raw materials, a compound enzyme preparation is prepared through solid-state fermentation of aspergillus oryzae and aspergillus niger, the compound enzyme preparation is used for enzymolysis, yeast and lactic acid bacteria are inoculated to enzymatic hydrolysate in a segmented mode for liquid-state fermentation, and the compound enzyme is prepared. The invention provides a closed-loop process of solid enzyme production, liquid enzymolysis and residue recycling, figs and schisandra chinensis are fermented synergistically, and aspergillus oryzae and aspergillus niger are used for solid fermentation to self-produce a compound enzyme, so that the yield of active ingredients such as lignan and GABA in the product is greatly improved, and the method has great significance in improving the efficacy of the enzyme; and residues are recycled, so that waste of raw materials is reduced, and green production and resource circulation are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, in particular to a composite enzyme and a preparation method thereof. Background Art

[0002] Enzymes, a new generation of health drinks, are gaining popularity among consumers. Enzymes are made from vegetables, fruits, and traditional Chinese medicines that are both edible and medicinal. After a series of processes involving juicing or extraction, enzymes are fermented with yeast, lactic acid bacteria, and other strains. The resulting fermentation liquid is rich in nutrients such as sugars, organic acids, minerals, vitamins, phenols, and terpenes, as well as important enzymes and other bioactive substances.

[0003] Schisandra chinensis, a traditional Chinese medicinal herb with both medicinal and edible properties, has been shown to calm the nerves, provide antioxidant benefits, and lower blood sugar and blood pressure. Its antioxidant properties can also slow aging and reduce the prevalence of diseases caused by cellular oxidation. Therefore, the integration of Schisandra chinensis as a raw material with modern enzyme production processes has become a key focus within the industry.

[0004] Existing technologies, such as Chinese patent CN118680245A, rely heavily on commercial enzymes (e.g., cellulase, pectinase, and amylase) to hydrolyze the raw materials. However, these exogenous enzymes are poorly targeted at the structural structure of Schisandra chinensis lignans (e.g., cross-linked phenylpropane units), resulting in insufficient lignan dissolution rates (typically <0.8 mg / mL). Furthermore, commercial enzyme preparations are expensive and unsuitable for large-scale production. Furthermore, Schisandra chinensis has a bitter taste, leading to poor user acceptance. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a composite enzyme to solve the problem in the prior art that the exogenous enzyme has poor compatibility with Schisandra chinensis, resulting in a low release rate of lignans and imbalanced taste of single fermentation. At the same time, the present invention will also provide a method for preparing a composite enzyme, using Schisandra chinensis and fig as raw materials, and making a composite enzyme preparation through solid-state fermentation of Aspergillus oryzae and Aspergillus niger, using the composite enzyme preparation for enzymatic hydrolysis, and then inoculating the enzymatic hydrolyzate in sections with yeast and lactic acid bacteria for liquid fermentation.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing a composite enzyme, comprising the following steps:

[0008] (1) grinding and mixing dried Schisandra chinensis and dried figs, or beating and mixing fresh Schisandra chinensis and fresh figs to prepare a mixed raw material;

[0009] (2) adding bran and a nitrogen source to the mixed raw materials, adjusting the water content to prepare a solid-state fermentation matrix, sterilizing and inoculating a spore suspension of Aspergillus oryzae and Aspergillus niger, and performing solid-state fermentation;

[0010] (3) adding the solid-state fermentation product to a buffer solution to extract the crude enzyme solution, and obtaining a composite enzyme preparation after centrifugal filtration;

[0011] (4) After sterilizing the new mixed raw material, the composite enzyme preparation of step (3) is used for enzymatic hydrolysis:

[0012] (5) inoculating the enzymatic hydrolysate obtained in step (4) with yeast and lactic acid bacteria in sections for liquid fermentation;

[0013] (6) Filtering and sterilizing the fermentation liquid of step (5) to obtain the final product, i.e., the composite enzyme.

[0014] The present invention adopts Aspergillus oryzae and Aspergillus niger solid-state fermentation to produce compound enzymes. The two enzyme spectra complement each other. The cellulase secreted by Aspergillus oryzae preferentially degrades the cellulose microfibrils in the cell wall of Schisandra chinensis; the β-glucosidase secreted by Aspergillus niger specifically cuts the lignan glycosidic bonds (such as C-7 glucoside), releasing the active ingredient in the form of aglycone, greatly improving the lignan free rate. In addition, the present invention uses cheap bran as a matrix for solid-state fermentation to produce enzymes, which can greatly reduce costs. In addition, through the synergistic fermentation of figs, figs are rich in natural sugars and can be used as a carbon source to accelerate yeast to start fermentation. In addition, natural fructose is converted into esters by yeast during the liquid fermentation stage, which can mask the bitter taste of Schisandra chinensis and make the taste better.

[0015] As a preferred technical solution of step (1), the mass ratio of dried schisandra chinensis to dried figs or fresh schisandra chinensis to fresh figs is 2:8 to 5:5, preferably 3:7. The schisandra chinensis is selected after the seeds are removed to prevent the oil in the seeds from interfering with the fermentation.

[0016] On the one hand, the lignans in Schisandra chinensis are mainly present in the lignified cell walls, which require sufficient cellulase / β-glucosidase to break down. The content of Schisandra chinensis will affect the lignan yield; on the other hand, the high viscosity of figs will reduce the porosity of the solid-state fermentation matrix. Schisandra chinensis contains wood fiber, which can maintain the porosity of the matrix, and the porosity will affect the fermentation efficiency; therefore, a reasonable ratio of figs and Schisandra chinensis can achieve the optimal balance between fermentation efficiency and lignan yield.

[0017] As a preferred technical solution for step (2), the bran is added in an amount of 15-25 wt %; the nitrogen source is selected from at least one of ammonium sulfate, soybean meal, and urea, and is added in an amount of 1-2 wt %. The addition of the bran provides porosity and trace elements to the solid-state fermentation matrix, and the nitrogen source promotes mycelial growth and the expression of genes related to extracellular enzyme synthesis.

[0018] As a preferred technical solution of step (2), the water content of the solid-state fermentation substrate is adjusted to 55-60% using 0.1 M pH 5.0 acetate buffer.

[0019] As a preferred technical solution of step (2), after adjusting the water content, the solid-state fermentation matrix is ​​stirred until there is no agglomeration, and then sterilized at 120-125° C. for 15-30 minutes and cooled to 30° C. for use.

[0020] As a preferred technical solution of step (2), the spore inoculation ratio of Aspergillus oryzae to Aspergillus niger is 1:1 to 3:1, preferably 2:1.

[0021] As a preferred technical solution of step (2), the concentration of the spore suspension is 1×10 7 spores / mL; the total inoculum amount of the spore suspension is 3-6 v / w%.

[0022] The spore suspension can be prepared by the following method: Aspergillus oryzae and Aspergillus niger are inoculated into a culture medium and cultured respectively, and after the spores mature, the spores are eluted with an eluent, and then diluted to 1×10 7 spores / mL, and spore suspension was obtained by mixing according to the proportion.

[0023] Specifically, the culture medium is selected from PDA slant culture medium, malt extract agar culture medium or potato dextrose agar; the eluent is selected from bactericidal physiological saline supplemented with 0.05% Tween 80 and 0.1% peptone.

[0024] As a preferred technical solution for step (2), the solid-state fermentation temperature is 30±2° C.; the fermentation time is 60 to 84 hours; the ventilation volume during the fermentation stage is maintained at about 0.3 VVM, and the relative humidity is controlled at 85-90%.

[0025] As a preferred technical solution for step (2), a termination operation is performed after the fermentation is completed: immediate hot air drying at 60° C. for 2 hours (to inactivate the bacteria and retain enzyme activity).

[0026] As a preferred technical solution of step (3), the buffer solution is 0.1M pH 5.0 acetate buffer solution, and the solid-state fermentation product to buffer solution ratio is 1:3 to 1:8, preferably 1:5; after adding the buffer solution, the mixture is shaken (200 rpm) at 40°C for 2 h.

[0027] As a preferred technical solution for step (3), the centrifugal speed is 8000-10000 rpm, and the centrifugation time is 15 min; the enzyme is then sterilized with a 0.45 μm microporous membrane to obtain a crude enzyme solution, which is stored at 4°C for 7 days, or stored at -20°C with 10% glycerol for a long term.

[0028] As a preferred technical solution of step (3), the cellulase activity of the complex enzyme preparation is ≥150 U / g and / or the β-glucosidase activity is ≥100 U / g.

[0029] As a preferred technical solution of step (4), the addition amount of the complex enzyme preparation is 20-30 v / w%; the enzymatic hydrolysis temperature is 50±2° C.; and the enzymatic hydrolysis time is 2-3 h.

[0030] As a preferred technical solution of step (4), the enzyme is inactivated after the enzymatic hydrolysis is completed, and the pH is adjusted to 5.5-6.5.

[0031] As a preferred technical solution of step (5), the yeast is selected from at least one of Saccharomyces cerevisiae, Saccharomyces bayanus, and Kluyveromyces marxianus; and the lactic acid bacteria is selected from at least one of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus paracasei.

[0032] As a preferred technical solution of step (5), the inoculation amount of the yeast or lactic acid bacteria is 3-8 v / v%, preferably 5 v / v%.

[0033] As a preferred technical solution of step (5), the fermentation time of the yeast is 12 to 36 hours; the fermentation time of the lactic acid bacteria is 48 to 60 hours.

[0034] As a preferred technical solution for step (6), the filtration process first uses diatomaceous earth for pre-filtration and then uses a 0.22 μm membrane for sterilization.

[0035] As a preferred technical solution for step (6), after the fermentation broth is filtered and dried, a portion of the residue is reused in step (2) to replace bran, and the rest can be processed into a dietary fiber additive. The amount of residue reused is preferably 20-40% of the weight of the bran. The reuse ratio of the residue should not be too high, otherwise the porosity of the solid-state fermentation matrix will drop sharply, and the enzyme activity yield will drop significantly. The optimal reuse ratio is 30%.

[0036] The second aspect of the present invention provides a composite enzyme obtained by the above preparation method.

[0037] As described above, the composite enzyme and preparation method of the present invention have the following beneficial effects:

[0038] 1. The present invention uses Aspergillus oryzae and Aspergillus niger to produce composite enzymes through solid-state fermentation. The two enzyme spectra are complementary. The cellulase secreted by Aspergillus oryzae preferentially degrades cellulose microfibrils in the cell wall of Schisandra chinensis; the β-glucosidase secreted by Aspergillus niger specifically cuts the lignan glycosidic bonds (such as C-7 glucoside), greatly improving the yield of lignans in the product.

[0039] 2. The present invention uses figs and schisandra chinensis to synergistically ferment. The natural sugars in the figs can serve as a carbon source to accelerate yeast fermentation, and the natural fructose is converted into esters by yeast during the liquid fermentation stage, which can mask the bitter taste of schisandra chinensis and make the taste better. The reasonable ratio of the two is controlled to achieve an optimal balance between fermentation efficiency and lignan yield.

[0040] 3. The present invention innovatively proposes a closed-loop process of "solid-state enzyme production-liquid enzymatic hydrolysis-residue recycling", which can increase the yield of active ingredients such as lignans, quercetin, γ-aminobutyric acid (GABA), polyphenols, etc. in the enzyme, which is of great significance to improving the efficacy of the enzyme; moreover, the process of the present invention uses cheap bran to make homemade enzyme preparations, which are more compatible with Schisandra chinensis and can also reduce production costs; in addition, the residue is recycled to reduce raw material waste, thereby realizing green production and resource circulation. DETAILED DESCRIPTION

[0041] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0042] Example 1

[0043] This embodiment provides a composite enzyme, the preparation method of which includes the following steps:

[0044] (1) 300 g of seeded dried Schisandra chinensis and 700 g of dried figs were ultrafinely ground, passed through a 300-mesh sieve, and mixed to prepare a mixed raw material;

[0045] (2) 500 g of the mixed raw material was added with 100 g of bran and 7.5 g of ammonium sulfate, and 0.1 M pH 5.0 acetate buffer was added to adjust the total water content to 55-60% to prepare a solid fermentation matrix, which was sterilized by high-pressure steam sterilization at 121°C for 20 minutes and then cooled to 30°C;

[0046] The spore suspensions of Aspergillus oryzae (CICC 2012) and Aspergillus niger (CGMCC 3.316) (concentration of 1×10 7 spores / mL) were mixed at a volume ratio of 2:1, and 50 mL was inoculated into the solid-state fermentation matrix for solid-state fermentation at a temperature of 30°C for 72 h. The mixture was stirred aseptically every 12 hours and the fermentation was terminated after completion.

[0047] (3) The solid-state fermentation product was added to 5 volumes of 0.1 M, pH 5.0 acetate buffer, extracted at 40°C with shaking (200 rpm) for 2 hours, centrifuged, the supernatant removed, and sterilized with a 0.45 μm microporous membrane to obtain a light yellow crude enzyme solution, i.e., the complex enzyme preparation;

[0048] (4) After sterilizing 500 g of the mixed raw material of step (1), 0.1 M pH 5.0 acetate buffer was added to make the total water content reach 50%, and 100 mL of the complex enzyme preparation was added for enzymatic hydrolysis. The enzymatic hydrolysis temperature was 50 ° C. and the temperature was kept constant at 100 rpm for 2 hours. After the enzymatic hydrolysis was completed, the enzyme was heated at 85 ° C for 15 minutes to inactivate the enzyme. After cooling, the pH was adjusted to 6 with 1 M NaOH;

[0049] (5) Take 100 mL of enzymatic hydrolysate and inoculate 5 mL of cerevisiae yeast (CICC 1001) and ferment at 28°C for 24 h. Then inoculate 3 mL of Lactobacillus plantarum (CICC 23121) and 2 mL of Lactobacillus acidophilus (CICC 6005) and ferment at 37°C for 48 h until the pH is ≤ 3.8.

[0050] (6) The fermentation broth of step (5) is pre-filtered with diatomaceous earth and then sterilized by passing through a 0.22 μm membrane. The filtrate is pasteurized to obtain the final product, namely the composite enzyme (the residue is recovered after drying).

[0051] Example 2

[0052] This embodiment provides a composite enzyme, which is different from that of Example 1 only in that: in step (2), the volume ratio of the spore suspension of Aspergillus oryzae (CICC 2012) and Aspergillus niger (CGMCC 3.316) is 1:1.

[0053] Example 3

[0054] This embodiment provides a composite enzyme, which is different from that of Example 1 only in that: in step (2), the volume ratio of the spore suspension of Aspergillus oryzae (CICC 2012) and Aspergillus niger (CGMCC 3.316) is 3:1.

[0055] Example 4

[0056] This embodiment provides a composite enzyme, which is different from that of Example 1 only in that: in step (4), 50 mL of composite enzyme preparation is added to 500 g of mixed raw materials for enzymatic hydrolysis.

[0057] Example 5

[0058] This embodiment provides a composite enzyme, which is different from that of Example 1 only in that: in step (4), 150 mL of composite enzyme preparation is added to 500 g of mixed raw materials for enzymatic hydrolysis.

[0059] Example 6

[0060] A composite enzyme, the preparation method of which comprises the following steps:

[0061] (1) 300 g of seeded dried Schisandra chinensis and 700 g of dried figs were ultrafinely ground, passed through a 300-mesh sieve, and mixed to prepare a mixed raw material;

[0062] (2) 500 g of the mixed raw material was added with 70 g of bran, 7.5 g of ammonium sulfate, and 30 g of the residue obtained after filtration in step (6) of Example 1, and 0.1 M acetate buffer (pH 5.0) was added to adjust the total water content to 55-60% to prepare a solid-state fermentation matrix. The mixture was sterilized by high-pressure steam at 121° C. for 20 minutes and then cooled to 30° C.

[0063] Spore suspensions of Aspergillus oryzae (CICC 2012) and Aspergillus niger (CGMCC 3.316) (concentration: 1 × 107 spores / mL) were mixed at a volume ratio of 2:1, and 50 mL was inoculated into a solid-state fermentation matrix for solid-state fermentation at 30°C for 72 h. The mixture was stirred aseptically every 12 h and the fermentation was terminated after completion.

[0064] (3) The solid-state fermentation product was added to 5 volumes of 0.1 M, pH 5.0 acetate buffer, extracted at 40°C with shaking (200 rpm) for 2 hours, centrifuged, the supernatant removed, and sterilized with a 0.45 μm microporous membrane to obtain a light yellow crude enzyme solution, i.e., the complex enzyme preparation;

[0065] (4) After sterilizing 500 g of the mixed raw material of step (1), 0.1 M pH 5.0 acetate buffer was added to make the total water content reach 50%, and 100 mL of the complex enzyme preparation was added for enzymatic hydrolysis. The enzymatic hydrolysis temperature was 50 ° C. and the temperature was kept constant at 100 rpm for 2 hours. After the enzymatic hydrolysis was completed, the enzyme was heated at 85 ° C for 15 minutes to inactivate the enzyme. After cooling, the pH was adjusted to 6 with 1 M NaOH;

[0066] (5) Take 100 mL of enzymatic hydrolysate and inoculate 5 mL of cerevisiae yeast (CICC 1001) and ferment at 28°C for 24 h. Then inoculate 3 mL of Lactobacillus plantarum (CICC 23121) and 2 mL of Lactobacillus acidophilus (CICC 6005) and ferment at 37°C for 48 h until the pH is ≤ 3.8.

[0067] (6) The fermentation broth from step (5) is pre-filtered with diatomaceous earth and then sterilized by passing through a 0.22 μm membrane. The filtrate is pasteurized to obtain the final product, namely the composite enzyme (the residue is recovered after drying).

[0068] Comparative Example 1

[0069] This comparative example provides a composite enzyme, the preparation method of which comprises the following steps:

[0070] (1) 150 g of seeded dried Schisandra chinensis and 350 g of dried figs were ultrafinely ground, passed through a 300-mesh sieve, and mixed to prepare a mixed raw material;

[0071] (4) Add 0.1 M pH 5.0 acetate buffer to 500 g of the sterilized mixed raw material to make the total water content reach 50%, add 10,000 U of cellulase (Sigma C2730), 7,500 U of pectinase (Sigma P2611) and 5,000 U of β-glucosidase (Sigma 49291) to 100 mL of 0.1 M pH 5.0 acetate buffer, and add the mixed solution to the mixed raw material for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 50°C and the temperature is constantly shaken (100 rpm) for 2 hours. After the enzymatic hydrolysis is completed, the enzyme is heated at 85°C for 15 minutes to inactivate the enzyme. After cooling, the pH is adjusted to 6 with 1 M NaOH;

[0072] (5) Take 100 mL of enzymatic hydrolysate and inoculate 5 mL of cerevisiae yeast (CICC 1001) and ferment at 28°C for 24 h. Then inoculate 3 mL of Lactobacillus plantarum (CICC 23121) and 2 mL of Lactobacillus acidophilus (CICC 6005) and ferment at 37°C for 48 h until the pH is ≤ 3.8.

[0073] (6) The fermentation broth of step (5) is pre-filtered with diatomaceous earth and then sterilized by passing through a 0.22 μm membrane. The filtrate is pasteurized to obtain the final product, namely the composite enzyme (the residue is recovered after drying).

[0074] Experimental testing

[0075] The enzyme activities of the composite enzyme preparations prepared in Examples 1 to 3 and 6 were detected, and the active ingredients of the composite enzymes prepared in Examples 1 to 6 and Comparative Example 1 were detected. The test results are shown in Tables 1 and 2, respectively.

[0076] Table 1. Enzyme activity of the composite enzyme preparations in Examples 1 to 3 and 6

[0077] Detection method Example 1 Example 2 Example 3 Example 6 Cellulase activity (U / g) DNS Method 162 150 155 160 β-glucosidase activity (U / g) PNPG method 112 92 89 110 Pectinase activity (U / g) Galacturonic acid colorimetric method 128 115 135 128

[0078] Table 2. Yield of active ingredients of composite enzymes in Examples 1 to 6 and Comparative Example 1

[0079]

[0080] In summary, the present invention uses Schisandra chinensis and Fig as raw materials, and makes a homemade composite enzyme preparation through solid-state fermentation of Aspergillus oryzae and Aspergillus niger. The composite enzyme preparation is used for enzymolysis, and then the enzymolysis liquid is segmented and inoculated with yeast and lactic acid bacteria for liquid fermentation. The present invention proposes a closed-loop process of "solid-state enzyme production-liquid enzymolysis-residue reuse", in which Fig and Schisandra chinensis are fermented in a coordinated manner, and Aspergillus oryzae and Aspergillus niger are used for self-production of composite enzymes through solid-state fermentation, which greatly improves the yield of active ingredients such as lignans and GABA in the product, which is of great significance for improving the efficacy of the enzyme; and the residue is reused to reduce the waste of raw materials and realize green production and resource recycling. Therefore, the present invention effectively overcomes the various shortcomings in the prior art and has a high industrial utilization value.

[0081] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for preparing a composite enzyme, characterized in that: The following steps are involved: (1) grinding and mixing dried Schisandra chinensis and dried figs, or beating and mixing fresh Schisandra chinensis and fresh figs to prepare a mixed raw material; (2) adding bran and a nitrogen source to the mixed raw materials, adjusting the water content to prepare a solid-state fermentation matrix, sterilizing and inoculating a spore suspension of Aspergillus oryzae and Aspergillus niger, and performing solid-state fermentation; (3) adding the solid-state fermentation product to a buffer solution to extract the crude enzyme solution, and obtaining a composite enzyme preparation after centrifugal filtration; (4) After sterilizing the new mixed raw material, the composite enzyme preparation of step (3) is used for enzymatic hydrolysis: (5) inoculating the enzymatic hydrolysate obtained in step (4) with yeast and lactic acid bacteria in sections for liquid fermentation; (6) Filtering and sterilizing the fermentation liquid of step (5) to obtain the final product, i.e., the composite enzyme.

2. The preparation method according to claim 1, characterized in that In step (1), the mass ratio of dried Schisandra chinensis and dried figs or fresh Schisandra chinensis and fresh figs is 2:8 to 5:

5.

3. The preparation method according to claim 1, characterized in that In step (2), the added amount of the bran is 15-25 wt%; the nitrogen source is selected from at least one of ammonium sulfate, soybean meal powder, and urea, and the added amount of the nitrogen source is 1-2 wt%.

4. The preparation method according to claim 1, characterized in that In step (2), the spore inoculation ratio of Aspergillus oryzae to Aspergillus niger is 1:1 to 3:

1.

5. The preparation method according to claim 4, characterized in that In step (2), the solid-state fermentation temperature is 30±2° C. and the fermentation time is 60 to 84 hours.

6. The preparation method according to claim 1, characterized in that In step (3), the buffer solution is 0.1M acetate buffer solution with a pH of 5.0, and the material-liquid ratio of the solid-state fermentation product to the buffer solution is 1:3 to 1:

8.

7. The preparation method according to claim 1, characterized in that In step (4), the addition amount of the complex enzyme preparation is 20-30 v / w%; the enzymatic hydrolysis temperature is 50±2° C.; and the enzymatic hydrolysis time is 2-3 h.

8. The preparation method according to claim 1, characterized in that In step (5), the yeast is selected from at least one of Saccharomyces cerevisiae, Saccharomyces bayanus, and Kluyveromyces marxianus; and the lactic acid bacteria is selected from at least one of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus paracasei.

9. The preparation method according to claim 1, characterized in that In step (6), after the fermentation liquid is filtered, the residue is dried and a portion thereof is recycled to step (2) to replace the bran. The recycled amount of the residue is 20-40% of the mass of the bran.

10. A composite enzyme, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 9.

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