LU102402B1

LU102402B1 - Preparation method and application for plant enzymes fermenting tibetan kefir

Info

Publication number: LU102402B1
Application number: LU102402A
Authority: LU
Inventors: Hongwei Wang; Huayi Suo; Yu Zhang
Original assignee: Univ Southwest
Priority date: 2020-11-24
Filing date: 2021-01-08
Publication date: 2021-07-09
Also published as: CN112401232A

Abstract

The present invention relates a preparation method and application for plant enzymes fermenting tibetan kefir that includes the specific steps of activating tibetan kefir, activating lactobacillus acidophilus and streptococcus thermophilus, and taking the fruit ready to be fermented with cutting into pieces, adding crystal sugar and white sugar to adjust sugar acid after enzymatic inactivation, and cooling to room temperature after sterilization, then adding the activated tibetan kefir, lactobacillus acidophilus and streptococcus thermophilus, which are fermented at 30-32°C for 48-96 hours, then transferred to 0-10°C and preserved for 12-24 hours to obtain the enzyme. Based on the background of the industrialization for agricultural product processing and the comprehensive health, the present invention simplifies the three-stage fermentation of traditional enzyme production and utilizes the tibetan kefir mainly containing yeast, lactobacillus, and lactococcus, to synergistically ferment fruit with the streptococcus thermophilus and the lactobacillus acidophilus to produce enzymes, which synergistically promote the fermentation process.

Description

BL-5189 LU102402

PREPARATION METHOD AND APPLICATION FOR PLANT ENZYMES FERMENTING TIBETAN KEFIR

TECHNICAL FIELD OF THE INVENTION The invention belongs to the technical field of microbial fermentation, and relates to preparation method and application for plant enzymes fermenting tibetan kefir.

DESCRIPTION OF THE PRIOR ART Enzyme is a special functional food fermented from various beneficial bacteria by using single or mixed fruit and vegetables as raw material. Enzymes not only preserve the nutrients contained in the raw materials, but also produce products with biologically active ingredients, such as antioxidant activity. Traditional enzymes are natural fermentation products, the fermentation of which takes a long time, and it is easy to grow miscellaneous bacteria, which affects the growth of beneficial bacteria, so that it is unfavourable to efficient production. While people gradually increase their health awareness, enzymes have become more popular drinks in recent years because of their multiple health functions. At present, the domestic enzyme market has just started, without industry standard, so manufacturers produce enzymes with instable processing level, also without the lack of irregular production. The existing industrial production for enzymes mostly adapts three-stage fermentation, namely alcohol fermentation, acetic fermentation, and lactic acid fermentation, the process of which takes a long time with high production cost, which also leads to high selling price for enzymes. In different fruit-planting bases, the added value is low only depending on the fresh materials, with serious waste. In addition, the price of fresh fruit is unstable in the market, and the fruit planting is greatly affected by uncertain factors, causing huge economic losses to fruit farmers. The fruit is deeply processed for enzymes to improve the industrialization for processing agricultural products. However, for the enzymes prepared with different raw materials or different methods, their nutritional ingredients have their own advantages, so a large number of researchers continue to optimize the fermentation process in order to obtain the best content of each functional ingredient, taste and active effect.

Tibetan kefir is a unique and rare species derived from Tibet's snowfields as a gelatinous 1

BL-5189 LU102402 lump in milky white, growing with irregular shapes, most of which are like spherical rice grains. Tibetan kefir has curled surface and grows quickly in the milk, forming cauliflower-like lumps, which are elastic and have a sticky feeling when pinched by hand. Tibetan kefir is similar to snow lotus in appearance, so it is also called "Tibetan Snow lotus". The yogurt made from tibetan kefir contains a large number of beneficial live bacteria, which can produce metabolites that are beneficial to the human body, and has various physiological health functions. All different sources and environmental difference will have a partial impact on the microbial composition and structure of the dominant flora of tibetan kefir. However, on the whole, the tibetan kefir is mainly composed of lactobacillus, lactococcus and yeast.

SUMMARY OF THE INVENTION In view of this, an object of the present invention is to provide a preparation method and application for plant enzymes fermenting tibetan kefir, and the application of the enzyme obtained by the preparation method and the health care products prepared by the enzyme.

To achieve the above object, the present invention provides the following technical solutions: I. A preparation method and application for plant enzymes fermenting tibetan kefir includes the following steps: a. activating tibetan kefir; b. activating lactobacillus acidophilus and streptococcus thermophilus; c. taking the fruit ready to be fermented with cutting into pieces, adding crystal sugar and white sugar to adjust sugar acid after enzymatic inactivation, and cooling to room temperature after sterilization, then adding the activated tibetan kefir, lactobacillus acidophilus and streptococcus thermophilus, which are fermented at 30-32°C for 48-96 hours, then transferred to 0-10°C and preserved for 12-24 hours to obtain the enzyme.

Further, activating tibetan kefir in step a is to inoculate the preserved tibetan kefir in sterilized milk at 30-34°C to be cultured for 24-48 hours, then take out the tibetan kefir and add new sterilized milk with repeating activating for 4-7 generations.

Further, the inoculation amount for said inoculating is 3-5% (w/w).

Further, activating lactobacillus acidophilus and streptococcus thermophilus in step b is to 2

BL-5189 | 102402 inoculate the bacterial strain on a solid culture medium to be activated and cultured at 40-43°C for 2-3 generations, and then inoculate the activated bacterial colony into MRS liquid culture medium to be cultured at 37°C for 24 hours.

MRS liquid culture medium: beef extract (10.0g), yeast extract (5.0g), peptone (10.0g), glucose (or other sugar) (20.0g), K:HPO, (2.0g), sodium acetate(5.0g), MgSO, 7H,0 (0.2g), MnSO, (0.2g), Tween-80 (1.0g), diammonium citrate(2.0g), agar(15.0g). Distilled water is added to 1000ml, and pH is 6.2-6.6 before sterilization.

Further, the solid culture medium includes 1% of yeast extract (w/v), 1.5% of glucose, 0.3% of beef extract, 0.5% of peptone, 1.0% of CaCOs, 1 ml of Tween 80, and 2% of agar with pH

6.5-7.0.

Further, the enzyme inactivation is performed with heating at 75°C for 5 minutes.

Further, in step c, the proportion of fruit, crystal sugar, and white sugar is 1:0.2~0.4:0.3~0.5 by mass fraction.

Further, in step c, the inoculation amount of tibetan kefir is 0.2-0.8%, the inoculation amount of lactobacillus acidophilus is 0.1-0.2%, and the inoculation amount of streptococcus thermophilus is 0.1-0.2%.

Further, the proportion of lactobacillus acidophilus and streptococcus thermophilus is 1:1-1.5.

Further, the proportion of the ratio of lactobacillus acidophilus and streptococcus thermophilus is 1:1.

Further, in step c, the inoculation amount of tibetan kefir is 0.4-0.6%.

Further, in step c, the inoculation amount of tibetan kefir is 0.4%.

Further, the fruit is citrus.

IL. The enzyme obtained by the above preparation method for plant enzymes fermenting tibetan kefir.

HI. The application of the enzyme obtained by the above preparation method for the health care products.

The beneficial effect of the present invention is that based on the background of the industrialization for agricultural product processing and the comprehensive health, the present invention simplifies the three-stage fermentation of traditional enzyme production and utilizes 3

BL-5189 LU102402 the tibetan kefir mainly containing yeast, lactobacillus, and lactococcus, to synergistically ferment fruit with the streptococcus thermophilus and the lactobacillus acidophilus to produce enzymes, which synergistically promote the fermentation process. A method has been obtained to produce better-quality plant enzymes through one-time fermentation of the tibetan kefir, streptococcus thermophilus and lactobacillus acidophilus, by a large number of experiments to verify and compare the type and compound proportion of the strains on the performance of the fruit after fermentation, which provides diversified product channels for fruit deep-processing and improves the industrialization of agricultural products processing.

BRIEF DESCRIPTION OF THE DRAWINGS In order to make the object, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for description: FIG.1 shows the effect of fermentation time on the enzyme acidity; FIG.2 shows the effect of fermentation time on the soluble solids of enzyme; FIG.3 shows the effect of fermentation time on enzyme activity; FIG.4 shows the effect of the tibetan kefir originated from different growing areas on amino acids of enzyme.

DETAILED DESCRIPTION OF THE INVENTION Hereinafter, the preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental methods without specific conditions in the examples usually follow the conventional conditions or the conditions recommended by the manufacturer.

The tibetan kefir used in the embodiments were all self-collected, from Mozhugongka, Dangxiong, and Yangbajing of Tibet, Hengshui of Hebei, Baotou of Nei Monggol, Zhengzhou of Henan, and Jiamusi of Heilongjiang, respectively. The commercial enzyme strains were purchased from Angel's fruit and vegetable enzymes.

A preparation method and application for plant enzymes fermenting tibetan kefir includes the following steps: a. activating tibetan kefir: the tibetan kefir preserved in sterilized milk at 4°C is washed 2-3 4 | |

BL-5189 LU102402 times with sterile water, and then inoculated into sterilized milk with an amount of 3-5%, and cultivated at 30-32°C for 20-28 hours, then the tibetan kefir is taken out and added new sterilized milk, after being repeated activation for 4-7 generations, the tibetan kefir is separated into solid and liquid in asepsis for later use.

b. activating lactobacillus acidophilus and streptococcus thermophilus: the preserved lactobacillus acidophilus and streptococcus thermophilus strains are respectively inoculated on the solid culture medium that includes 1% of yeast extract (w/v), 1.5% of glucose, 0.3% of beef extract, 0.5% of peptone, 1.0% of CaCO3, 1 ml of Tween 80, and 2% of agar with pH 6.5-7.0 and is autoclaved at 115°C for 25min, and activated and cultured at 40-43°C for 2-3 generations, then the activated colonies are respectively inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours, with centrifugation of 10000-16000r/min at 4°C for 5-10min to collect the supernatant, so the bacterial fluid of lactobacillus acidophilus and streptococcus thermophilus are obtained respectively. MRS liquid culture medium: beef extract (10.0g), yeast extract (5.0g), peptone (10.0g), glucose (or other sugar) (20.0g), K:HPO4 (2.0g), sodium acetate(5.0g), MgSO, 7H:0 (0.2g), MnSO, (0.2g), Tween-80(1.0g), diammonium citrate(2.0g), agar(15.0g). Distilled water is added to 1000ml, and pH is 6.2-6.6 before sterilization.

c. cutting 1 part of fruit (citrus, apple, pear, cantaloupe, etc.) into pieces by mass fraction, and heating it at 75°C for 5 minutes to inactivate enzyme, then adding 0.2-0.4 part of crystal sugar and 0.3-0.5 part of white sugar to be pasteurized at 60°C for 30min, then quickly cooling to room temperature, then adding the strains prepared for activation in the above steps respectively, according to different inoculation amounts (mass percentages) such as 0.2-0.8% of tibetan kefir,

0.1-0.2% of bacterial fluid of lactobacillus acidophilus, and 0.1-0.2% of bacterial fluid of streptococcus thermophilus, wherein in the bacterial fluid of lactobacillus acidophilus, the proportion of the lactobacillus acidophilus and the bacterial fluid is 1:1-1.5, preferably 1:1, finally, obtaining the enzyme by fermenting for 48-96 hours at 20-25°C and storing at 4°C for 12-24 hours, with preferable inoculation amount of tibetan kefir of 0.4-0.6%, more preferable

0.4%.

d. filtering the fermented enzymes, then putting them into aseptic packages and sealing them for storage.

BL-5189 LU102402 Embodiment 1 A preparation method and application for plant enzymes fermenting tibetan kefir includes the following steps: a. activating tibetan kefir originated from Dangxiong of Tibet: the tibetan kefir preserved in sterilized milk at 4°C is washed 2-3 times with sterile water, and then inoculated into sterilized milk with an amount of 3-5%, and cultivated at 32°C for 24 hours, then the tibetan kefir is taken out and added new sterilized milk, after being repeated activation for 4-7 generations, the tibetan kefir is separated into solid and liquid in asepsis for later use.

b. activating lactobacillus acidophilus and streptococcus thermophilus: the preserved lactobacillus acidophilus and streptococcus thermophilus strains are respectively inoculated on the solid culture medium that includes 1% of yeast extract (w/v), 1.5% of glucose, 0.3% of beef extract, 0.5% of peptone, 1.0% of CaCO4, 1 ml of Tween 80, and 2% of agar with pH 6.5-7.0 and is autoclaved at 115°C for 25min, and activated and cultured at 40-43°C for 2-3 generations, then the activated colonies are respectively inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours, with centrifugation of 10000-16000r/min at 4°C for 5-10min to collect the supernatant, so the bacterial fluid of lactobacillus acidophilus and streptococcus thermophilus are obtained respectively. MRS liquid culture medium: beef extract (10.0g), yeast extract (5.0g), peptone (10.0g), glucose (or other sugar) (20.0g), K,HPO4 (2.0g), sodium acetate(5.0g), MgSO, 7H,0 (0.2g), MnSO4 (0.2g), Tween-80(1.0g), diammonium citrate(2.0g), agar(15.0g). Distilled water is added to 1000ml, and pH is 6.2-6.6 before sterilization.

c. cutting 1 part of citrus into pieces by mass fraction, and heating it at 75°C for 5 minutes to inactivate enzyme, then adding 0.3 part of crystal sugar and 0.5 part of white sugar to be pasteurized at 60°C for 30min, then quickly cooling to room temperature, then adding the strains prepared for activation in the above steps respectively, according to different inoculation amounts (mass percentages) such as 0.4% of tibetan kefir, 0.1% of bacterial fluid of lactobacillus acidophilus, and 0.1% of bacterial fluid of streptococcus thermophilus, finally, obtaining the enzyme by fermenting for 96 hours at 32°C and storing at 4°C for 12 hours.

6

BL-5189 LU102402 Embodiment 2 A preparation method and application for plant enzymes fermenting tibetan kefir includes the following steps: a. activating tibetan kefir originated from Yangbajing of Tibet: the tibetan kefir preserved in sterilized milk at 4°C is washed 2-3 times with sterile water, and then inoculated into sterilized milk with an amount of 3-5%, and cultivated at 32°C for 24 hours, then the tibetan kefir is taken out and added new sterilized milk, after being repeated activation for 4-7 generations, the tibetan kefir is separated into solid and liquid in asepsis for later use.

b. activating lactobacillus acidophilus and streptococcus thermophilus: the preserved lactobacillus acidophilus and streptococcus thermophilus strains are respectively inoculated on the solid culture medium that includes 1% of yeast extract (w/v), 1.5% of glucose, 0.3% of beef extract, 0.5% of peptone, 1.0% of CaCO3, 1 ml of Tween 80, and 2% of agar with pH 6.5-7.0 and is autoclaved at 115°C for 25min, and activated and cultured at 40-43°C for 2-3 generations, then the activated colonies are respectively inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours, with centrifugation of 10000-16000r/min at 4°C for 5-10min to collect the supernatant, so the bacterial fluid of lactobacillus acidophilus and streptococcus thermophilus are obtained respectively. MRS liquid culture medium: beef extract (10.0g), yeast extract (5.0g), peptone (10.0g), glucose (or other sugar) (20.0g), K,HPO4 (2.0g), sodium acetate(5.0g), MgSO4 7H2,0 (0.2g), MnSO, (0.2g), Tween-80(1.0g), diammonium citrate(2.0g), agar(15.0g). Distilled water is added to 1000ml, and pH is 6.2-6.6 before sterilization.

c. cutting 1 part of citrus into pieces by mass fraction, and heating it at 75°C for 5 minutes to inactivate enzyme, then adding 0.2 part of crystal sugar and 0.3 part of white sugar to be pasteurized at 60°C for 30min, then quickly cooling to room temperature, then adding the strains prepared for activation in the above steps respectively, according to different inoculation amounts (mass percentages) such as 0.4% of tibetan kefir, 0.1% of bacterial fluid of lactobacillus acidophilus, and 0.15% of bacterial fluid of streptococcus thermophilus, finally, obtaining the enzyme by fermenting for 96 hours at 30-32°C and storing at 4°C for 12 hours.

7

BL-5189 LU102402 Embodiment 3 A preparation method and application for plant enzymes fermenting tibetan kefir includes the following steps: a. activating tibetan kefir originated from Maizhokunggar of Tibet: the tibetan kefir preserved in sterilized milk at 4°C is washed 2-3 times with sterile water, and then inoculated into sterilized milk with an amount of 3-5%, and cultivated at 32°C for 24 hours, then the tibetan kefir is taken out and added new sterilized milk, after being repeated activation for 4-7 generations, the tibetan kefir is separated into solid and liquid in asepsis for later use.

b. activating lactobacillus acidophilus and streptococcus thermophilus: the preserved lactobacillus acidophilus and streptococcus thermophilus strains are respectively inoculated on the solid culture medium that includes 1% of yeast extract (w/v), 1.5% of glucose, 0.3% of beef extract, 0.5% of peptone, 1.0% of CaCOs, 1 ml of Tween 80, and 2% of agar with pH 6.5-7.0 and is autoclaved at 115°C for 25min, and activated and cultured at 40-43°C for 2-3 generations, then the activated colonies are respectively inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours, with centrifugation of 10000-16000r/min at 4°C for 5-10min to collect the supernatant, so the bacterial fluid of lactobacillus acidophilus and streptococcus thermophilus are obtained respectively. MRS liquid culture medium: beef extract (10.0g), yeast extract (5.0g), peptone (10.0g), glucose (or other sugar) (20.0g), K,HPO4 (2.0g), sodium acetate(5.0g), MgSO, 7H,0 (0.2g), MnSO4 (0.2g), Tween-80(1.0g), diammonium citrate(2.0g), agar(15.0g). Distilled water is added to 1000ml, and pH is 6.2-6.6 before sterilization.

c. cutting 1 part of citrus into pieces by mass fraction, and heating it at 75°C for 5 minutes to inactivate enzyme, then adding 0.3 part of crystal sugar and 0.4 part of white sugar to be pasteurized at 60°C for 30min, then quickly cooling to room temperature, then adding the strains prepared for activation in the above steps respectively, according to different inoculation amounts (mass percentages) such as 0.5% of tibetan kefir, 0.15% of bacterial fluid of lactobacillus acidophilus, and 0.15% of bacterial fluid of streptococcus thermophilus, finally, obtaining the enzyme by fermenting for 96 hours at 20-25°C and storing at 4°C for 12 hours.

In the fermentation process of step c, sampling is done every 24 hours to measure the | |

BL-5189 LU102402 acidity, soluble solids and enzyme activity. The test results are shown in FIGS. 1-3, respectively. The acidity is determined by potentiometric titration and measured with tartaric acid. The determination of soluble solids is in accordance with GB 12295-1990 "Determination of Soluble Solids of Fruit and Vegetable Products”. The enzyme activity is determined according to the Marklund method of determining SOD enzyme activity by pyrogallol. It can be seen from the results that the fermentation time is preferably 3 days.

Embodiment 4 The citrus enzymes fermented from the tibetan kefir sampled from different regions is compared with the citrus enzymes fermented from the purchased commercial enzyme strains.

The specific preparation method of the enzyme is as follows: a. activating tibetan kefir originated from Dangxiong County, Tibet: the tibetan kefir preserved in sterilized milk at 4°C is washed 2-3 times with sterile water, and then inoculated into sterilized milk with an amount of 3-5%, and cultivated at 32°C for 24 hours, then the tibetan kefir is taken out and added new sterilized milk, after being repeated activation for 4-7 generations, the tibetan kefir is separated into solid and liquid in asepsis for later use.

b. activating lactobacillus acidophilus and streptococcus thermophilus: the preserved lactobacillus acidophilus and streptococcus thermophilus strains are respectively inoculated on the solid culture medium that includes 1% of yeast extract (w/v), 1.5% of glucose, 0.3% of beef extract, 0.5% of peptone, 1.0% of CaCOs, 1 ml of Tween 80, and 2% of agar with pH 6.5-7.0 and is autoclaved at 1150 for 25min, and activated and cultured at 40-43°C for 2-3 generations, then the activated colonies are respectively inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours, with centrifugation of 10000-16000r/min at 4°C for 5-10min to collect the supernatant, so the bacterial fluid of lactobacillus acidophilus and streptococcus thermophilus are obtained respectively. MRS liquid culture medium: beef extract (10.0g), yeast extract (5.0g), peptone (10.0g), glucose (or other sugar) (20.0g), K,HPO, (2.0g), sodium acetate(5.0g), MgSO, 7H,0 (0.2g), MnSO4 (0.2g), Tween-80(1.0g), diammonium citrate(2.0g), agar(15.0g). Distilled water is added to 1000ml, and pH is 6.2-6.6 before sterilization.

c. cutting 1 part of citrus into pieces by mass fraction, and heating it at 75°C for 5 minutes to inactivate enzyme, then adding 0.3 part of crystal sugar and 0.4 part of white sugar to be 9

BL5189 LU102402 pasteurized at 60°C for 30min, then quickly cooling to room temperature, then adding the strains prepared for activation in the above steps respectively, according to different inoculation amounts (mass percentages) such as 0.5% of tibetan kefir, 0.15% of bacterial fluid of lactobacillus acidophilus, and 0.15% of bacterial fluid of streptococcus thermophilus, finally, obtaining the enzyme by fermenting for 72 hours at 32°C and storing at 4°C for 12 hours.

In step a, the tibetan kefirs were originated from Yangbajing of Tibet, Heilongjiang, Nei Monggol, Hebei, and Henan for comparative tests, and the purchased commercial enzyme strains were used instead of the compound fermentation bacteria of tibetan kefir, lactobacillus acidophilus and streptococcus thermophilus as a comparison for fermentation.

Each sample is taken to test its acidity, soluble solids and enzyme activity, and the test results are shown in Table 1. Each enzyme sample is tested for the type and content of amino acids, and the test results are shown in Table 2 and FIG. 4, and the horizontal ordinate is indicated by the abbreviation of region’s name. It can be seen from FIG. 4 that although the types and content of amino acids of the enzymes prepared by the tibetan kefir strains from deferent region are slightly different, they are roughly the same. Among them, there are 13 kinds of amino acids in the enzymes prepared by tibetan kefir strains from Heilongjiang, which is the most, and there are 9 kinds of amino acids in the enzymes prepared by commercial bacteria, which is the least.

Table 1 Growing Area Acidity (g/L) Soluble Solids (%) Enzyme Activity (U/mL) Tibet 6.4+0.16 17.93+0.05 209.02+9.2 Heilongjiang 7.75+0.37 19.5+0.41 183.94-+34.83 Nei Monggol 8.72+0.21 16.5+0.41 197.72+20.07 Heibei 7.76+0.88 15.03+0.05 161.19+5.49 Heinan 7.96+0.11 14.93+0.05 159.13+16.75

BL-5189 LU102402 Commercial 5.89+0.2 12.1+0.08 128.26+8.14 Table 2 [ee a 8 ww | wn || oa pr | wel an ow am |e | ee |e ee |e] ee ew ||| re | ww] oe | mon |e “he eel we] | en | an |e In combination with significance and sensory analysis, it is found that the citrus enzymes fermented from the tibetan kefir samples collected in Tibet, Heilongjiang, and Nei Monggol are better than those fermented from the commercial enzymes.

Besides producing ethanol, yeast is also metabolized to produce aroma components such as alcohol, ester, and acid. Besides producing lactic acid and providing esterified substrates, lactobacillus acidophilus can also provide non-volatile lactic acid for acetic acid fermentation, the precursor of some flavour substances produced by its own metabolism play an important role in the flavour of final products. Therefore, the yeast, lactobacillus, and lactococcus contained in the tibetan kefir, lactobacillus acidophilus and streptococcus thermophilus are synergistically fermented to produce a higher proportion of flavour substances, such as ethyl acetate and 3-methyl-1-butanol. Alcohol, (R)-limonene, etc. The products have the special fragrance that the raw material should have. It is reported in the literature that co-fermenting foods with multi-bacteria has positive and negative effects on the final flavour of the food, and the type and 11

BL-SIE LU102402 compound proportion of strains determine the flavour of the final product together. Embodiment 5 As for the anti-oxidant activity of the enzyme prepared in the example, The effect of enzymes cleaning up DPPH and the effect of enzymes cleaning up O7 are examined.

1. The test method with the effect of cleaning up DPPH: 2.0mL of sampling solution is mixed with 2.0mL of DPPH solution (0.1mol/L in ethanol), and the mixed solution is left in a dark room to react for 30min, then the absorbance is measured at 517nm. DPPH: clearance (%)=[1-(A1-A0)/A2]x100, where A1 is the absorbance value of DPPH+sampling solution, AO is the absorbance value of sampling solutiontethanol, and A2 is the absorbance value of DPPH+ethanol.

2. The test method with the effect of cleaning up O, : the pyrogallol autooxidation is adopted for determination. 2.5mL of sampling solution is mixed with 4mL of Tris-HCI buffer (0.05mol/L, pH8.2), and the mixed solution reacts in a water bath at 250 for 20min, then 1mL of pyrogallol is added, and the solution is left at 250) for Smin, then the reaction is stop by

0.1mLHCI(10jmol/L) and the absorbance is measured at 320nm.

O1 Clearance rate (%)=[1-(A1-A2)/A3]x100, where Al is the absorbance value without sampling liquid; A2 is the absorbance value of replacing pyrogallol with ultrapure water; A3 is the absorbance value of adding the sampling solution.

Finally, the results of the clearance rates determined in each example are shown in Table 3.

Table 3 Sample Strain Source DPPH Clearance (%) O, ‘Clearance (%) “Embodimentl Tibet 948 9562 Embodiment 2 Tibet 94.56 94.11 Embodiment 3 Tibet 92.84 95.48 Embodiment 4 Tibet 97.00 96.58 Embodiment 4 Heilongjiang 94.24 94.18 Embodiment 4 Nei Monggol 92.06 93.27 Embodiment 4 Heibei 93.98 93.93 12

| BL-S189 | U102402 Embodiment 4 Heinan 91.86 89.51 Embodiment 4 Commercial 76.91 62.48 The anti-oxidant activity of the enzymes produced by synergistically fermenting the yeast, lactobacillus, and lactococcus contained in the tibetan kefir, lactobacillus acidophilus and streptococcus thermophilus is high.

Finally, it should be noted that the above preferred embodiments are only used to describe the technical solutions of the present invention, but not to limit it.

Although the present invention has been described in detail through the above preferred embodiments, the person skilled in the technical field of the invention should understand that various changes can be made to it in form and details without departing from the scope defined by the claims of the present invention. 13

Claims

BL-5189 LU102402 CLAIMS

1. A preparation method for plant enzymes fermenting tibetan kefir, comprising the following steps: a. activating tibetan kefir; b. activating lactobacillus acidophilus and streptococcus thermophilus; c. taking fruit to be fermented, cutting into pieces, adding crystal sugar and white sugar to adjust sugar acid after enzyme deactivation treatment, and cooling to room temperature after sterilization, then adding the activated tibetan kefir, lactobacillus acidophilus and streptococcus thermophilus strains, fermenting at 30-32°C for 48-96 hours, then storing at 0-10°C for 12-24 hours to obtain the enzymes.

2. The preparation method for plant enzymes fermenting tibetan kefir according to claim I, wherein the activating tibetan kefir in step a is performed by inoculating the preserved tibetan kefir in sterilized milk, and culturing at 30-34°C for 24-48 hours, then taking out the tibetan kefir and adding new sterilized milk, and repeatedly activating for 4-7 generations.

3. The preparation method for plant enzymes fermenting tibetan kefir according to claim 2, wherein the inoculation amount for said inoculating is 3-5% (w/w).

4. The preparation method for plant enzymes fermenting tibetan kefir according to claim 1, wherein the activating lactobacillus acidophilus and streptococcus thermophilus in step b is performed by inoculating the strains on a solid culture medium, performing activated culture at 40-43°C for 2-3 generations, and then inoculating the activated bacterial colonies into MRS liquid culture medium at 37°C for 24 hours.

5. The preparation method for plant enzymes fermenting tibetan kefir according to claim 4, wherein the solid culture medium comprises: 1% (w/v) of yeast extract, 1.5% of glucose, 0.3% of beef extract, 0.5% of peptone, 1.0% of CaCO;, 1 mL of Tween 80, 2% of agar, and has a pH value of 6.5-7.0.

14

BL-5189 LU102402

6. The preparation method for plant enzymes fermenting tibetan kefir according to claim 1, wherein the ratio in mass parts in the step c as follows: fruit: crystal sugar: white sugar = 1:

0.2-0.4: 0.3-0.5.

7. The preparation method for plant enzymes fermenting tibetan kefir according to claim 1, wherein in step c, the inoculation amount of tibetan kefir is 0.2-0.8%, the inoculation amount of lactobacillus acidophilus is 0.1-0.2%, and the inoculation amount of streptococcus thermophilus is 0.1-0.2%.

8. The preparation method for plant enzymes fermenting tibetan kefir according to claim 1, wherein the ratio of lactobacillus acidophilus and streptococcus thermophilus is 1:1-1.5.

9. The preparation method for plant enzymes fermenting tibetan kefir according to any one of claims 1-8, wherein the fruit is citrus.

10. The use of enzymes obtained by the preparation method for plant enzymes fermenting tibetan kefir according to any one of claims 1-9 in the preparation of health care products. |