Preparation method of viable bacterium type Chinese wolfberry enzyme

Abstract

The invention discloses a preparation method of a viable bacterium type Chinese wolfberry enzyme, and belongs to the field of enzyme preparation. The preparation method comprises the following steps: inoculating lactobacillus rhamnosus and lactobacillus paracasei into Chinese wolfberry juice, and fermenting to obtain viable bacterium type fermented Chinese wolfberry juice; and adding auxiliary materials into the viable bacterium type fermented Chinese wolfberry juice, and sterilizing in a water bath to obtain the viable bacterium type Chinese wolfberry enzyme. According to the method, by optimizing the strain compounding proportion, the inoculum size, the fermentation time, the sterilization temperature of the lycium barbarum enzyme and the like, a simple and efficient method for preparing the viable bacterium type lycium barbarum enzyme is provided, and a foundation is laid for improving the potential value of lycium barbarum. After being sterilized in a water bath at the temperature of 40 DEG C, the viable bacterium type Chinese wolfberry enzyme obtained by the method can be ensured to contain high active substances such as flavones and polyphenols and have good antioxidant activity.

Description

Technical Field

[0001] The invention relates to the field of enzyme preparation, and in particular to a method for preparing live bacteria type wolfberry enzyme. Background Art

[0002] Wolfberry is a food that can be used as both medicine and food, rich in bioactive substances such as phenols, flavonoids, and polysaccharides. However, wolfberry is not suitable for direct use in the production of juice due to its high sugar content, unbalanced sugar-acid ratio, and unique Chinese medicine flavor. Wolfberry enzyme refers to a product containing specific bioactive ingredients that is made from wolfberry as raw material and fermented by microorganisms. Therefore, the use of lactic acid bacteria to ferment wolfberry enzyme can not only improve the taste of wolfberry juice drinks and enrich the product flavor, but also significantly improve the nutritional value of the product.

[0003] At present, most studies use single-strain lactic acid bacteria to ferment wolfberry juice and pasteurize it. However, single-strain fermentation enzyme products have a single flavor and unstable quality, and sterilization often destroys its bioactive substances and the number of live bacteria, thereby weakening the nutritional value of the product. Therefore, it is urgent to screen compound lactic acid bacteria suitable for fermenting wolfberry enzymes, optimize the preparation process of wolfberry enzymes, and further explore the sterilization temperature of live bacteria type wolfberry enzymes, in order to provide a theoretical basis for the development of a new live bacteria type enzyme. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a live bacteria type wolfberry enzyme to solve the problems existing in the above-mentioned prior art. By optimizing the composite ratio of bacterial strains, inoculation amount and fermentation time, as well as the sterilization temperature of wolfberry enzyme, a simple and efficient preparation of live bacteria type wolfberry enzyme is provided, laying the foundation for improving the potential value of wolfberry.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a composite bacteria for preparing live bacteria type wolfberry enzyme, wherein the composite bacteria consists of Lactobacillus rhamnosus and Lactobacillus paracasei.

[0007] Preferably, the Lactobacillus rhamnosus is Lactobacillus rhamnosus LGG, and the Lactobacillus paracasei is Lactobacillus paracasei CICC-6244.

[0008] The present invention also provides a method for preparing live bacteria type wolfberry enzyme, comprising the steps of inoculating the composite bacteria into wolfberry juice for fermentation.

[0009] Preferably, the preparation method comprises the following steps:

[0010] Mixing wolfberry dried fruit with water to extract juice, adding sucrose, and sterilizing to obtain wolfberry juice;

[0011] Inoculating Lactobacillus rhamnosus and Lactobacillus paracasei into the wolfberry juice at 1%-5% of the total volume of the wolfberry juice, fermenting at 32-38° C. for 12-60 hours, and then performing solid-liquid separation to obtain the supernatant to obtain live bacteria fermented wolfberry juice;

[0012] Auxiliary materials are added to the live bacteria type fermented wolfberry juice, and the mixture is sterilized in a water bath to obtain the live bacteria type wolfberry enzyme.

[0013] Preferably, the mass ratio of the wolfberry dried fruit to water is 1:(2-5), the sucrose is added at 3%-5% of the total weight of the mixed juice, and the sterilization is performed at 90° C. for 15-20 min.

[0014] Preferably, the Lactobacillus rhamnosus and Lactobacillus paracasei are both inoculated into the wolfberry juice in the form of bacterial liquid, and the volume ratio of the bacterial liquid of Lactobacillus rhamnosus to the bacterial liquid of Lactobacillus paracasei is (1-3):(1-3).

[0015] Preferably, the volume ratio of the bacterial solution of Lactobacillus rhamnosus to that of Lactobacillus paracasei is 2:3; and / or the optimal inoculation amount of the bacterial solution of Lactobacillus rhamnosus and that of Lactobacillus paracasei is 3.6%; and / or the optimal fermentation time is 47.8h.

[0016] Preferably, the auxiliary materials include any one of rose powder and jasmine powder, and erythritol;

[0017] Every 100g of the live bacteria type fermented wolfberry juice contains 1g of rose powder and 6g of erythritol, or every 100g of the live bacteria type fermented wolfberry juice contains 2g of jasmine powder and 6g of erythritol.

[0018] Preferably, the water bath sterilization condition is 40° C. sterilization for 15 min.

[0019] The invention also provides a live bacteria type wolfberry enzyme, which is prepared by the preparation method.

[0020] The present invention discloses the following technical effects:

[0021] The present invention uses Lactobacillus rhamnosus and Lactobacillus paracasei in fermented wolfberry juice, optimizes the bacterial composition ratio, inoculation amount and fermentation time, and finally screens out the best fermentation conditions: inoculation amount 3.6%, composition ratio 2:3, fermentation time 47.8h. Under the best conditions, wolfberry juice is fermented to obtain live bacteria type wolfberry fermented juice, and then rose powder or jasmine powder and erythritol are added, and the addition amount is optimized to obtain the best live bacteria type wolfberry enzyme formula: (1) rose wolfberry enzyme: 1g rose powder and 6g erythritol are added to 100g fermented wolfberry juice; (2) jasmine wolfberry enzyme: 2g jasmine powder and 6g erythritol are added to 100g fermented wolfberry juice. The wolfberry enzyme sensory scores of these two formulas are 94 and 91 points respectively. It can be seen that the present invention provides a preparation method of live bacteria type wolfberry ferment, which is simple and efficient, and provides a high-quality wolfberry enzyme formula, and excavates and develops the potential value of wolfberry.

[0022] After being sterilized in a 40° C. water bath, the live bacteria type wolfberry enzyme prepared by the present invention can ensure that it contains relatively high levels of active substances such as flavonoids, polyphenols and good antioxidant activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Effects of strain inoculation ratio on the content of active substances and antioxidant capacity of fermented wolfberry juice; a: superoxide anion scavenging rate and DPPH free radical scavenging rate, b: total phenolic and total flavonoid contents;

[0025] Figure 2 Effects of strain inoculation amount on the content of active substances and antioxidant capacity of fermented wolfberry juice; a: superoxide anion scavenging rate and DPPH free radical scavenging rate, b: total phenolic and total flavonoid contents;

[0026] Figure 3 The effect of strain fermentation time on the content of active substances and antioxidant capacity of fermented wolfberry juice; a: superoxide anion scavenging rate and DPPH free radical scavenging rate, b: total phenolic and total flavonoid content;

[0027] Figure 4 The influence of strain inoculation ratio, inoculation amount and fermentation time on the fuzzy comprehensive evaluation value; a: strain inoculation ratio, b: inoculation amount, c: strain fermentation time;

[0028] Figure 5 The results of response surface experiment. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The strains involved in the following examples: Lactobacillus rhamnosus LGG was purchased from Beina Biotechnology; Lactobacillus casei CICC-6244 was purchased from China Industrial Microbiological Culture Collection Center.

[0035] Example 1

[0036] 1. Activation of lactic acid bacteria

[0037] Lactobacillus rhamnosus LGG and Lactobacillus paracasei CICC-6244 were inoculated into MRS broth at a 2% (v / v) inoculation rate, cultured at 37°C for 24 h, subcultured for three generations, centrifuged at 8000 rpm for 10 min at 4°C, resuspended in sterile saline, and the bacterial solution concentration was adjusted to 10 9CFU / mL.

[0038] 2. Preparation of wolfberry juice fermented by lactic acid bacteria

[0039] The wolfberry dried fruit was rehydrated with water in a mass ratio of 1:3 to extract juice, 5% sucrose was added, sterilized at 90°C for 15 min, and then cooled to room temperature. Lactobacillus rhamnosus and Lactobacillus paracasei were added at a volume of 3% (v / v) of the total volume of wolfberry juice. 鼠李糖乳杆菌 :v 类干酪乳杆菌 =1:2) was inoculated into wolfberry juice, and after fermentation at 37°C for 48 hours, the supernatant was centrifuged at 4000 r / min to obtain fermented wolfberry juice. The natural fermentation group (CK) without inoculation of lactic acid bacteria was used as the control.

[0040] 3. Single Factor Test

[0041] According to the preparation process of fermented wolfberry juice, single factor experiments were carried out on the compound ratio of lactic acid bacteria, inoculation size and fermentation time, with total phenols, total flavonoids, DPPH free radical scavenging rate and superoxide anion scavenging rate as evaluation indicators.

[0042] Strain compounding ratio: the fixed compound lactic acid bacteria inoculation amount is 3%, the fermentation time is 48h, and the volume ratio of Lactobacillus rhamnosus LGG to Lactobacillus paracasei CICC-6244 is 1:1, 1:2, 1:3, 2:1 and 3:1.

[0043] Inoculation amount: The fixed lactic acid bacteria compound ratio is 1:2, the fermentation time is 48h, and the inoculation amount is 1%, 2%, 3%, 4% and 5%.

[0044] Fermentation time: The compound ratio of lactic acid bacteria was fixed at 1:2, the inoculation amount was 3%, and the fermentation time was 12h, 24h, 36h, 48h and 60h.

[0045] 4. Establishment of fuzzy comprehensive evaluation model

[0046] Total phenol, total flavonoids, DPPH free radical scavenging rate and superoxide anion scavenging rate are used as sample evaluation factor sets, and their corresponding variance relative contribution rates are used as weights. The corresponding weights are linearly weighted, and their corresponding weights are 25%, 25%, 24% and 26%, respectively. The evaluation set data is dimensionlessly transformed by the extreme value method, and the dimensionlessly transformed evaluation factor set Xi and the corresponding factor weight set K are normalized by the score calculated by the multiplication operator to obtain the fuzzy comprehensive evaluation value (Z).

[0047] 5. Response surface experiment

[0048] According to the single factor test results of fermented wolfberry juice, based on Design-Expert 8.0.6 software and the Box-Behnken central combination design principle, three factors, namely fermentation time (A), strain inoculation amount (B) and compound ratio (C), were selected. The fuzzy evaluation value (Z) was used as the evaluation index to design experiments to further optimize the fermentation conditions.

[0049] 6. Preparation of different wolfberry enzyme products

[0050] Fermented wolfberry juice was prepared using the process optimized by response surface methodology.

[0051] Rose wolfberry enzyme: The fermented wolfberry juice was prepared by the optimized process, and different amounts of rose powder and erythritol were added to the fermented wolfberry juice to prepare wolfberry enzyme (see Table 2).

[0052] Jasmine wolfberry enzyme: Fermented wolfberry juice was prepared by optimized process, and jasmine wolfberry enzyme was prepared by adding different amounts of jasmine powder and erythritol to the fermented wolfberry juice (see Table 2).

[0053] And sensory evaluation was carried out on wolfberry enzymes with different formulas to screen out the best wolfberry enzyme formula.

[0054] 7. Determination of the sterilization temperature of wolfberry enzyme

[0055] The wolfberry enzyme was placed in a water bath at 40℃, 45℃, 50℃, and 55℃ for 15 minutes. After cooling, the total phenols, total flavonoids, DPPH free radical scavenging rate, and superoxide anion scavenging rate were measured.

[0056] 8. Determination of active substances and antioxidant capacity

[0057] 8.1 Determination of total phenols and total flavonoids

[0058] The total phenol content in the fermented wolfberry juice was determined by the Folin phenol method. The total phenol standard curve equation was y = 0.061x + 0.0511, R 2 =0.9992. The content of total flavonoids was determined by aluminum nitrate colorimetry. The standard curve equation of total flavonoids was y=0.0037x+0.0538, R 2 =0.9998.

[0059] 8.2 Determination of DPPH free radical scavenging activity

[0060] Take 1.0mL of the sample to be tested and add 1.0mL of 0.2mmol / L DPPH-anhydrous ethanol solution, react at room temperature in the dark for 30min, centrifuge at 3500r / min for 10min, and measure the absorbance of the supernatant at 517nm. The clearance rate is calculated according to the following formula:

[0061]

[0062] Wherein: Ai is 1.0 mL sample plus 1.0 mL DPPH-anhydrous ethanol, Aj is 1.0 mL sample plus 1 mL PBS, Ac is 1.0 mL PBS plus 1.0 mL DPPH-anhydrous ethanol.

[0063] 8.3 Determination of superoxide anion radical scavenging ability

[0064] Take 2.0mL Tris-HCl buffer (150mmol / L, pH=8.0), add 0.6mL of the sample to be tested, preheat in a water bath at 25℃, then add 0.4mL of pyrogallol preheated in a water bath at 25℃, and measure its absorbance at 325nm after 20min in a water bath at 25℃. The clearance rate is calculated according to the following formula:

[0065]

[0066] Wherein: A11 is sample and pyrogallol, A10 is sample and distilled water, A01 is pyrogallol, and A00 is water.

[0067] 9. Results and Analysis

[0068] 9.1 Single factor test results

[0069] like Figure 1-Figure 4 As shown in the figure, the optimal strain combination ratio was V through single factor test and fuzzy evaluation. 鼠李糖乳杆菌LGG∶ V 类干酪乳杆菌CICC-6244 =1:2, inoculation amount is 3%, and fermentation time is 48h.

[0070] 9.2 Response surface experiment results

[0071] like Figure 5 As shown, the response surface results showed that the optimal fermentation conditions were inoculation volume 3.6%, compound ratio 2:3, and fermentation time 47.8h.

[0072] 9.3 Recipe Optimization

[0073] Table 1 Sensory evaluation table

[0074]

[0075]

[0076] Table 2 Formulation optimization

[0077]

[0078] Rose wolfberry enzyme: 1g rose powder and 6g erythritol were added to 100g fermented wolfberry juice, and the sensory score was 94. Jasmine wolfberry enzyme: 2g jasmine powder and 6g erythritol were added to 100g fermented wolfberry juice, and the sensory score was 91.

[0079] 9.4 Sterilization temperature

[0080] Based on the content of active ingredients and antioxidant capacity of rose wolfberry enzyme, the sterilization temperature was finally determined to be 40°C.

[0081] Table 3 Contents of active ingredients and antioxidant capacity of wolfberry enzymes at different sterilization temperatures

[0082]

[0083] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composite bacteria for preparing live-bacteria wolfberry enzyme, characterized in that: The composite bacteria consists of Lactobacillus rhamnosus and Lactobacillus paracasei.

2. The composite bacteria according to claim 1, characterized in that The rhamnosus lactobacillus is rhamnosus lactobacillus LGG, and the paracasei lactobacillus is paracasei lactobacillus CICC-6244.

3. A method for preparing live bacteria type wolfberry enzyme, characterized in that: The method comprises the steps of inoculating the composite bacteria according to claim 1 or 2 into wolfberry juice for fermentation.

4. The preparation method according to claim 3, characterized in that: The preparation method comprises the following steps: Mixing wolfberry dried fruit with water to extract juice, adding sucrose, and sterilizing to obtain wolfberry juice; Inoculating Lactobacillus rhamnosus and Lactobacillus paracasei into the wolfberry juice at 1%-5% of the total volume of the wolfberry juice, fermenting at 32-38° C. for 12-60 hours, and then performing solid-liquid separation to obtain the supernatant to obtain live bacteria fermented wolfberry juice; Auxiliary materials are added to the live bacteria type fermented wolfberry juice, and the mixture is sterilized in a water bath to obtain the live bacteria type wolfberry enzyme.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the wolfberry dried fruit to water is 1:(2-5), the sucrose is added in an amount of 3%-5% according to the total weight of the mixed juice, and the sterilization is performed at 90° C. for 15-20 minutes.

6. The preparation method according to claim 4, characterized in that: The Lactobacillus rhamnosus and Lactobacillus paracasei are both inoculated into the wolfberry juice in the form of bacterial liquid, and the volume ratio of the bacterial liquid of the Lactobacillus rhamnosus to the bacterial liquid of the Lactobacillus paracasei is (1-3):(1-3).

7. The preparation method according to claim 6, characterized in that: The volume ratio of the bacterial solution of Lactobacillus rhamnosus to that of Lactobacillus paracasei is 2:3; and / or the optimal inoculation amount of the bacterial solution of Lactobacillus rhamnosus and that of Lactobacillus paracasei is 3.6%; and / or the optimal fermentation time is 47.8h.

8. The preparation method according to claim 4, characterized in that: The auxiliary materials include any one of rose powder and jasmine powder, and erythritol; Every 100g of the live bacteria type fermented wolfberry juice contains 1g of rose powder and 6g of erythritol, or every 100g of the live bacteria type fermented wolfberry juice contains 2g of jasmine powder and 6g of erythritol.

9. The preparation method according to claim 4, characterized in that: The water bath sterilization condition is 40° C. for 15 min.

10. A live bacteria type wolfberry enzyme, characterized in that: Prepared by the preparation method according to any one of claims 3 to 9.

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