Actinidia arguta enzyme as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: uniformly mixing dry actinidia arguta powder, white granulated sugar and sterile water according to a certain ratio to prepare a fermentation stock solution, then adding bifidobacterium, wine yeast and streptococcus thermophilus, and carrying out combined fermentation at the fermentation temperature of 30-35 DEG C, so as to obtain the actinidia arguta enzyme. The inoculum sizes of the bifidobacterium, the wine saccharomycetes and the streptococcus thermophilus are all 1%-2%, the inoculum sizes are all the volume ratio of a strain seed solution to a fermentation stock solution, and sterilization is performed after fermentation is finished to obtain the actinidia arguta enzyme. The raw materials for preparing the actinidia arguta enzyme are simple and safe, the fermentation process is controllable, the three specific strains including the bifidobacterium, the wine yeast and the streptococcus thermophilus are adopted for combined fermentation, the finished product is excellent in sense, high in antioxidant activity and high in total phenol content, and a novel method for preparing the actinidia arguta enzyme is provided.

Description

Technical Field

[0001] The invention relates to the technical field of food fermentation, in particular to an Actinidia arguta enzyme and a preparation method and application thereof. Background Art

[0002] Actinidia arguta is one of the most widely distributed wild fruit trees in China, distributed in Northeast China, North China, Shandong, Northwest China and the Yangtze River Basin, among which the resources are the most abundant in the three provinces of Northeast China. Actinidia arguta contains a variety of nutrients such as amino acids, sugars, pectin, proteins, fats, vitamins, niacin, mineral elements, unsaturated fatty acids, lipids, flavonoids, phenols, trace elements, etc. It has the functions of resisting bacterial infection immunity, inhibiting rotavirus, lowering cholesterol, preventing cardiovascular diseases, reducing lipid peroxidation, regulating blood lipids, helping to promote the expansion of small arteries, dilate cardiovascular, improve blood circulation, and prevent thrombosis.

[0003] Fresh and ripe kiwifruit has a short shelf life and is not suitable for storage and transportation. It is usually made into juice or preserved fruit. Its biologically active substances are greatly lost during the processing and storage process. Moreover, if it is not deeply processed, these products will retain the unique sour and astringent taste of kiwifruit, which cannot meet the nutritional and taste needs of modern people.

[0004] Fermented kiwifruit beverage is a functional food containing specific biologically active ingredients (including polysaccharides, oligosaccharides, proteins and peptides, amino acids, and vitamins) obtained by fermenting mature fruits with microbial strains. Chinese patent document CN201711296975.3 discloses a method for making kiwifruit enzyme, which uses gradient freezing to process fresh fruits, heats them several times when making juice, and filters out the pomace, destroying heat-sensitive vitamins, active enzymes, and other substances, and losing part of the pomace raw materials before fermentation. It is first fermented with brewer's yeast for 8-10 hours, then added with Lactobacillus acidophilus for fermentation for 24-30 hours, and then placed at 4°C for 10-12 hours for post-fermentation, and then directly sterilized after mixing. The fermentation time is short and it cannot produce high content of biologically active substances. Summary of the invention

[0005] The purpose of the present invention is to provide an Actinidia arguta enzyme with excellent sensory properties, strong antioxidant activity and high total phenol content, as well as a preparation method and application thereof, which is specifically achieved by the following technical scheme.

[0006] A method for preparing kiwifruit enzyme comprises the following steps: preparing kiwifruit dry powder into a fermentation stock solution, then adding bifidobacterium, wine yeast and thermophilic streptococcus for joint fermentation, wherein the fermentation temperature is 30-35°C, the inoculation amounts of the bifidobacterium, wine yeast and thermophilic streptococcus are all 1%-2%, and the inoculation amounts are all the volume ratio of the bacterial seed solution to the fermentation stock solution, and sterilizing after the fermentation to obtain the kiwifruit enzyme.

[0007] The method for preparing the kiwifruit dry powder comprises the following steps: selecting kiwifruits of uniform size and without external damage, vacuum freezing and freeze-drying the kiwifruits to make their water content ≤5%, and then crushing the kiwifruits to obtain dry powder.

[0008] The method of preparing the fermentation stock solution from the dried kiwifruit powder comprises the following steps: mixing the dried kiwifruit powder, white sugar and sterile water in a mass ratio of (0.7-1.3): (0.7-1.3): (7.7-8.3). Preferably, the mass ratio of the three is 1:1:8.

[0009] Furthermore, the volume of the fermentation liquid in the fermentation container is two-thirds of the container volume, and the fermentation time is 10 to 60 days. Preferably, the fermentation time is 20 to 50 days. Further, the fermentation time can be 30 days or more than 60 days to obtain the highest content of total phenols.

[0010] Preferably, the inoculation amount of bifidobacteria is 1.5%, the inoculation amount of wine yeast is 2%, and the inoculation amount of thermophilic streptococcus is 2%.

[0011] The invention also provides an Actinidia arguta enzyme, which is obtained by adopting the above-mentioned preparation method.

[0012] The above preparation method can also be used to increase the total phenol content of kiwifruit enzyme and improve the antioxidant activity of kiwifruit enzyme. The antioxidant activity includes reducing power, superoxide anion free radical scavenging ability, hydroxyl free radical scavenging ability, DPPH free radical scavenging ability and SOD enzyme activity.

[0013] The kiwifruit enzyme prepared by the present invention can be used in beverages, nutritional products, foods and other products, and is not limited to the dosage form of the final product.

[0014] The advantages of the present invention are:

[0015] The invention prepares the kiwifruit enzyme with simple raw materials and a controllable fermentation process. The invention adopts three specific bacterial species, namely bifidobacterium, wine yeast and thermophilic streptococcus, for joint fermentation. The finished product has high sensory evaluation and good taste. Not only the unique flavor substances and beneficial components of the kiwifruit are preserved, but the microorganisms can also metabolize and produce amino acids, polypeptides, phenols and other substances required by the human body during the fermentation process. The antioxidant activity of the enzyme, including reducing power, superoxide anion free radical scavenging ability, hydroxyl free radical scavenging ability, DPPH free radical scavenging ability and SOD enzyme activity, is improved. The invention provides a new method for preparing the kiwifruit enzyme and also provides a new idea for further deep processing of the kiwifruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Attached Figure 1 The effect of different bacterial inoculation amounts on total phenol content.

[0017] Attached Figure 2 The surface graphs of the interaction of various bacterial factors on the total phenol content. (a) is the surface graph of the interaction of wine yeast and bifidobacterium inoculation on the total phenol content, (b) is the surface graph of the interaction of thermophilic streptococcus and wine yeast inoculation on the total phenol content, and (c) is the surface graph of the interaction of thermophilic streptococcus and bifidobacterium inoculation on the total phenol content.

[0018] Attached Figure 3 It is a curve chart of SOD enzyme activity changes. DETAILED DESCRIPTION

[0019] The specific implementation modes provided by the present invention are described in detail below with reference to the accompanying drawings.

[0020] 1 Materials and methods

[0021] 1.1 Materials and reagents

[0022] Wild Actinidia arguta is picked at the experimental base of Shenyang Agricultural University.

[0023] Bifidobacterium (Chuanxiu Bifidobacterium 10 bacteria powder);

[0024] Wine yeast (Angel wine yeast rw);

[0025] Streptococcus thermophilus (50 billion cfu / g of Streptococcus thermophilus).

[0026] Tryptone, yeast extract, beef extract, diammonium hydrogen citrate, glucose, Tween-80, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, yeast extract, sodium carbonate, gallic acid, potassium hemoglobin, trichloroacetic acid, ferric chloride, sodium salicylate, anhydrous ethanol (all biochemical reagents or analytical grade, Sinopharm Chemical Reagent Co., Ltd.); white sugar (food grade, COFCO Tunhe Sugar Co., Ltd.); citric acid (food grade, Weifang Yingxuan Industrial Co., Ltd.); DPPH (analytical grade, Tianjin Zhonglian Chemical Reagent Co., Ltd.).

[0027] 1.2 Main instruments and equipment

[0028] Quintix3102 electronic balance (Sartorius Scientific Instruments Co., Ltd.); P9 double-beam UV-visible spectrophotometer (Shanghai Mepda Instrument Co., Ltd.); M1 glucose-lactic acid analyzer (Shenzhen Silman Technology Co., Ltd.); H1850 high-speed centrifuge (Hunan Xiangyi Centrifuge Instrument Co., Ltd.); HCB-1300V clean bench (Haier Biomedical Co., Ltd.); BXM-60VF vertical pressure steam sterilizer (Shanghai Boxun Medical Biological Instrument Co., Ltd.).

[0029] 1.3 Test methods

[0030] 1.3.1 Processing process and key points of kiwifruit enzyme processing

[0031] Raw material pretreatment (picking, cleaning, freeze-drying, crushing) → fermentation stock solution → adding microbial strains in proportion → mixing → fermentation → sterilization → filling → finished product.

[0032] Raw material pretreatment: select kiwifruit of uniform size and without external damage, vacuum freeze-dry the kiwifruit (water content ≤ 5%), grind the kiwifruit after freeze-drying, and divide the grinded kiwifruit dry powder into parts for use.

[0033] 1.3.1.1 Preparation of fermentation stock solution

[0034] Place the dried Actinidia arguta powder, white sugar and sterile water in a sterilized fermentation container in a certain proportion, with the height of the original liquid being at two-thirds of the height of the container.

[0035] 1.3.1.2 Inoculation and fermentation

[0036] The cultured bifidobacterium, wine yeast, and thermophilic streptococcus seed liquid are inoculated into the fermentation stock solution in a certain proportion and mixed after inoculation. The fermentation culture temperature is 30°C ~ 35°C. During the culture process, bubbles and bacterial contamination are observed. During the process, samples need to be taken and tested after centrifugation (double-beam UV-visible spectrophotometer is used to detect OD value and bacterial growth curve; M1 glucose-lactic acid analyzer is used to detect the value of residual sugar in the culture medium and the amount of lactic acid accumulated during the fermentation process). Observe the indicators. If there is no significant change in the indicators for 7 consecutive days, the fermentation is terminated.

[0037] Bifidobacterium seed liquid culture medium: 0.5% tryptone, 1% yeast extract, 0.5% beef extract, 1% glucose, 0.04% dipotassium hydrogen phosphate, 0.002% magnesium sulfate, 0.008% sodium chloride, 0.0080% calcium chloride, 0.04% sodium bicarbonate, 0.004% manganese sulfate, 1L sterile water, pH 7.0.

[0038] Wine yeast seed liquid culture medium: 2% glucose, 1% tryptone, 1% yeast extract, 1% beef extract, 1L sterile water, natural pH.

[0039] Streptococcus thermophilus seed liquid culture medium: yeast extract 1%, tryptone 1%, glucose 1%, dipotassium hydrogen phosphate 0.02%, Tween-80 0.05%, sodium acetate 1%, purified water 1L, natural pH.

[0040] Preparation of seed liquid culture medium: Add glucose, trypsin, beef extract, magnesium sulfate and other components in proportion according to the culture medium formula, and make up to the specified volume with sterile water.

[0041] Repackaging: Pour the prepared seed liquid culture medium into a conical flask with a filling volume of 100mL / (250mL). When repackaging, do not let the culture medium touch the bottle mouth to avoid contamination. Wrap it with high-pressure polypropylene transparent plastic film.

[0042] Sterilization: Place the packaged instruments and seed liquid culture medium in a pulse sterilizer for sterilization. After completion, place them in a clean bench or biological safety cabinet for use.

[0043] Inoculation: Add bacterial powder into liquid seed culture medium at a ratio of 1% (mass / volume).

[0044] Culture conditions: shake in a constant temperature shaker at 30℃~35℃, and obtain seed culture solution after 5~7 days of culture.

[0045] The inoculation amount of Bifidobacterium (0.5%, 1.0%, 1.5%, 2.0% and 2.5%), the inoculation amount of wine yeast (0.5%, 1.0%, 1.5%, 2.0% and 2.5%), and the inoculation amount of thermophilic Streptococcus (0.5%, 1.0%, 1.5%, 2.0% and 2.5%) were used as the main influencing factors. The changes in total phenols and sensory evaluation of the products were explored through single-factor experiments, and the three bacteria were used to ferment the stock solution. The antioxidant activity, total phenol content and sensory evaluation of the final prepared enzyme were measured to determine the appropriate inoculation amount of the strains. The inoculation amount of the strains is the volume ratio of the strain seed solution to the fermentation stock solution.

[0046] 1.3.1.3 Sensory evaluation

[0047] 30 students were selected to give sensory evaluation in the same environment. After trying each sample, they were required to rinse their mouths with water and rest for 1 minute before tasting the next sample to prevent the occurrence of taste crossover and taste dullness due to continuous tasting of similar drinks. The enzymes were scored sensoryly in terms of color, aroma and taste. The scores were divided into four levels from high to low, with a full score of 10 points, 8 to 10 points (including 8) for excellent, 6 to 8 points (including 6) for good, 4 to 6 points (including 4) for qualified, 2 to 4 points (including 2) for poor, and 0 to 2 points (including 0) for extremely poor. Finally, according to the proportion of color 30%, aroma 25%, and taste 45%, the sum of the three scores is the final score.

[0048] Odor evaluation

[0049] 8-10 (including 8)...Excellent (no change in aroma, very obvious fermentation aroma)

[0050] 6~8 (including 6)...Good (the aroma changes slightly, and the fermentation aroma is obvious)

[0051] 4-6 (including 4) ... qualified (the aroma has changed, but the fermentation aroma characteristics can still be identified)

[0052] 2~4 (including 2)…poor (the aroma changes and it is difficult to identify the characteristics of fermentation aroma)

[0053] 0~2 (including 0) ... Very bad (the aroma changes drastically and the fermentation aroma characteristics cannot be identified)

[0054] Color and state of the sample

[0055] 8 to 10 (including 8) ... Excellent (no change in color or condition)

[0056] 6~8 (including 6) ... Good (slight color difference)

[0057] 4 to 6 (including 4) ... qualified (color is different, but within the acceptable range; no change in state)

[0058] 2~4 (including 2) ... Poor (significant color difference, or obvious turbidity, precipitation, stratification, etc.)

[0059] 0~2 (including 0) ... Very bad (serious discoloration, or serious turbidity, precipitation, stratification, etc.)

[0060] Taste

[0061] 8-10 (including 8) ... excellent (excellent taste, sweet and sour, no astringency)

[0062] 6-8 (including 6) ... good (good taste, moderate sweetness and sourness)

[0063] 4-6 (including 4) ... Pass (average taste, moderate sweetness and sourness, slightly astringent)

[0064] 2~4 (including 2) ... Poor (poor taste, slightly sour, slightly astringent)

[0065] 0~2 (including 0)... Very bad (bad taste, sour, and astringent)

[0066] 1.4 Determination of total phenol content

[0067] The total phenol content was measured by the Folin phenol method.

[0068] 1.5 Antioxidant activity assay

[0069] The antioxidant activity mainly tests reducing power, superoxide anion free radical scavenging ability, hydroxyl free radical scavenging ability, DPPH free radical scavenging ability, and SOD enzyme activity. Determination method reference:

[0070] YILDIRIM A, MAVI A, KARA A A. Determination of antioxidant and antimicrobial activities of Rumes crispus L. Extracts[J]. Journal of Agricultural and Food Chemistry, 2001, 49(8): 4083-4089.

[0071] YEN GC, CHEN H Y. Antioxidant activity of various tea extracts inrelation to their antimutagenicity[J]. Journal of Agricultural and FoodChemistry, 1995, 43(1): 27-32.

[0072] LIU W, XU J, JING P, et al. Preparation of a hydroxypropyl Ganodermalucidum polysaccharide and its physicochemical properties[J]. Food Chemistry, 2010, 122(4): 965-971.

[0073] BLOIS M S. Antioxidant determination by the use of a stable freeradical[J]. Nature, 1958, 181: 1199-1200.

[0074] Xu Yajuan, Zhao Yanjing, Hu Hong. Study on the determination of superoxide dismutase activity by pyrogallol autooxidation method [J]. Journal of South-Central University for Nationalities. Natural Science Edition, 2006, 32(6): 1207-1209.

[0075] 1.6 Data processing and analysis

[0076] Origin 2021 software was used to complete the single-factor experimental drawing, and Design-Expert13 software was used to complete the orthogonal experimental design and data processing and analysis.

[0077] 2 Results and analysis

[0078] 2.1 Results of single factor test of bacterial species

[0079] The mass ratio of Actinidia arguta dry powder, white sugar and sterile water in the fermentation stock solution was 0.7∶1.3∶8.3. Only one strain was inoculated in the fermentation stock solution. Other conditions were the same and the fermentation was carried out at 30℃~35℃ for 30 days. The test results are shown in Table 1 and Figure 1 .

[0080] Table 1 Sensory evaluation after 30 days of fermentation with a single strain

[0081]

[0082] In Table 1, blank refers to no added bacteria. The blank group was fermented for 30 days, and the color was lighter, there were more sediments, the taste was sour, and the taste was astringent. The overall evaluation showed that the taste was not ideal. When fermented by wine yeast, the color was lighter, the sediment was less than that of the blank group, the astringency was weaker, there was no sourness, but the taste was mellower, giving people the feeling of fruit wine with lower alcohol content, and the direction of fermented wine. When fermented by Bifidobacterium, the color was lighter, the sediment was less than that of the blank group, the taste was slightly sour, slightly astringent, and the sweetness was heavier, which may be due to incomplete fermentation. When fermented by Streptococcus thermophilus, the color was lighter, the taste was sour and sweet, there was a sense of astringency, and the sediment was less than that of the blank group.

[0083] Depend on Figure 1 It can be seen that under the same fermentation conditions, the total phenol content produced by the metabolism of bifidobacteria showed a trend of first increasing and then decreasing with the increase of inoculation amount, and the total phenol content was the highest at 1.5%, which was 0.623 mg / mL. The total phenol content produced by the metabolism of wine yeast showed a trend of first increasing, then decreasing, and then increasing with the increase of inoculation amount, and the total phenol content was the highest at 1.0%, which was 0.513 mg / mL. The total phenol content produced by the metabolism of thermophilic streptococci showed a trend of first increasing and then decreasing with the increase of inoculation amount, and the total phenol content was the highest at 1.0%, which was 0.623 mg / mL.

[0084] 2.2 Results of response surface optimization experiment on inoculation amount of different bacterial species

[0085] According to the Box-Behnken central composite experimental design principle and the results of the single-factor experiment, a three-factor three-level response surface experiment was designed using Design-Expert 13 with the inoculum size of Bifidobacterium (A), wine yeast inoculum size (B) and thermophilic Streptococcus (C) as independent variables and the total phenol content (Y) as the response value. The inoculum size was selected as 1.0%, 1.5% and 2.0% to determine the optimal ratio of bacterial species. The response surface experiment design table is shown in Table 2.

[0086] Table 2 Experimental design table of different bacterial inoculation amounts

[0087]

[0088] The results of response surface test and regression analysis are shown in Tables 3 and 4.

[0089] Table 3 Response surface test results of different bacterial species proportions

[0090]

[0091]

[0092] As can be seen from Table 3, when the inoculation amount of the three bacteria is within the range of 1% to 2%, the sensory score of the enzyme after 30 days of fermentation is excellent, the taste of multi-bacteria fermentation is slightly sour and sweet, without astringency, the sediment is less than that of single-bacteria fermentation, the color is lighter, there is no mellow smell, and the unique smell of Actinidia arguta is retained. Among them, when the inoculation amount of Bifidobacterium is 1.5%, the inoculation amount of wine yeast is 2.0%, and the inoculation amount of thermophilic Streptococcus is 2.0%, the total phenol content is the highest, which is 0.936mg / mL, and the sensory score is also the highest.

[0093] Table 4 Regression analysis and results of different bacterial species inoculation amounts

[0094]

[0095]

[0096] The data were processed and analyzed by variance analysis in Table 4. A, B, C, AB, BC, A 2 , C 2 There were significant differences in the items (p<0.001); AC, B 2 The difference was not significant. 2 =0.9940, indicating that the model has good correlation, and the value of the lack-of-fit term is 0.1105>0.05, so the experimental data is credible.

[0097] The total phenol content model was analyzed using Design-Expert 13 software: Y1 = 7.32 + 0.7175A + 0.7925B + 0.665C + 0.6925AB - 0.1125AC - 0.4725BC - 0.3173A 2 -0.0373B 2 +1.1C 2 The model p value is less than 0.01, so the response surface model is meaningful and reaches a very significant level.

[0098] The three-dimensional response surface between the two bacterial strain factors was obtained from the Design-Expert 13 response surface analysis software. Figure 2 The surface plot can directly reflect the effect of the interaction of each factor on the total phenol content. The steeper the slope of the surface, the greater the effect of the factor on the total phenol content. The total phenol content first increased and then decreased with the increase of the three factors. Among them, the interaction between the inoculation amount of Bifidobacterium and the inoculation amount of wine yeast, and the inoculation amount of wine yeast and the inoculation amount of thermophilic Streptococcus was extremely significant, and the effect on the total phenol content was extremely significant.

[0099] 2.3 Antioxidant activity assay results

[0100] During the fermentation process, the antioxidant activity was tested and analyzed, and the results are shown in Table 5 (total phenol content test results comparison), Table 6 (reducing power test results comparison), Table 7 (superoxide anion free radical scavenging ability test results comparison), Table 8 (hydroxyl free radical scavenging ability test results comparison), Table 9 (DPPH·free radical scavenging ability test results comparison). In Tables 5-9, ① wine yeast group means that only wine yeast is inoculated, and the inoculation amount is 2.0%; ② Bifidobacterium group means that only Bifidobacterium is inoculated, and the inoculation amount is 1.5%; ③ Streptococcus thermophilus group means that only Streptococcus thermophilus is inoculated, and the inoculation amount is 2.0%; ④ Wine yeast + Bifidobacterium means that two bacteria are inoculated, and the wine yeast inoculation amount is 2.0% plus the bifidobacterium inoculation amount is 1.5%; ⑤ Bifidobacterium + Streptococcus thermophilus means that two bacteria are inoculated, and the bifidobacterium inoculation amount is 1.5% plus the thermophilic streptococcus inoculation amount is 2.0%; ⑥ Wine yeast + thermophilic streptococcus means inoculation of two bacteria, with a wine yeast inoculation amount of 2.0% plus a thermophilic streptococcus inoculation amount of 2.0%; ⑦ The control group means no bacteria are added; ⑧ The experimental group means the use of three bacteria for joint fermentation, with a bifidobacterium inoculation amount of 1.5%, a wine yeast inoculation amount of 2.0% and a thermophilic streptococcus inoculation amount of 2.0%. The other fermentation conditions of groups ①-⑧ were the same, wherein the fermentation temperature was 30°C to 35°C, and the mass ratio of arugula arguta dry powder, white sugar and sterile water in the fermentation stock solution was 1:1:8.

[0101] Table 5 Comparison of total phenol content test results

[0102]

[0103] Table 6 Comparison of reducing power test results

[0104]

[0105] Table 7 Comparison of superoxide anion free radical scavenging ability test results

[0106]

[0107] Table 8 Comparison of hydroxyl radical scavenging ability test results

[0108]

[0109] Table 9 Comparison of DPPH free radical scavenging ability test results

[0110]

[0111] As can be seen from the above table, under the same fermentation time, the total phenol content and antioxidant activity of the three bacteria combined fermentation products of Actinidia arguta were significantly higher than those of the products without adding bacteria, and the total phenol content and antioxidant activity were also significantly higher than those of the products using only one or two bacteria. The total phenol content of the experimental group products increased by 162% compared with the control group at 60 days of fermentation; the reducing power increased by 41.1% at 50 days of fermentation; at 50 days of fermentation, the superoxide anion free radical scavenging ability increased by 89.4%; at 50 days of fermentation, the hydroxyl free radical scavenging ability increased by 26.0%; at 40 days of fermentation, the DPPH free radical scavenging ability increased by 48.2%.

[0112] 2.4 Changes in SOD enzyme activity

[0113] The three bacteria were combined to produce kiwifruit enzyme. The mass ratio of kiwifruit powder, white sugar and sterile water in the fermentation stock solution was 1.3∶0.7∶7.3. The inoculation amount of Bifidobacterium was 1.5%, the inoculation amount of wine yeast was 2.0% and the inoculation amount of thermophilic Streptococcus was 2.0%. The fermentation was carried out at 30℃~35℃. The changes of SOD enzyme activity were shown in Table 2. Figure 3 The control is a product without added bacteria.

[0114] pass Figure 3 It can be seen that the SOD enzyme activity is basically the same at the beginning of fermentation. As the fermentation time increases, the SOD enzyme activity of the natural control product without adding bacteria gradually increases, and the SOD enzyme activity of the multi-bacteria fermentation enzyme first increases and then decreases. The use of three bacteria for combined fermentation can improve the SOD enzyme activity of the kiwifruit enzyme. At 30 days of fermentation, the SOD enzyme activity of the multi-bacteria fermentation enzyme was the highest, at 335U / mL, and the SOD enzyme activity increased by 32%.

[0115] 3 Conclusion

[0116] The experiment uses Actinidia arguta as the main raw material, and through single factor and response surface experiments, the multi-bacteria fermentation process of Actinidia arguta is determined. The combined fermentation of bifidobacteria, wine yeast, and thermophilic streptococci with an inoculation amount of 1% to 2% can significantly improve the total phenol content, antioxidant activity, and SOD enzyme activity of Actinidia arguta enzyme. When the inoculation amount of bifidobacteria is 1.5%, the inoculation amount of wine yeast is 2.0%, and the inoculation amount of thermophilic streptococci is 2.0%, under the condition of 30℃ to 35℃, compared with the product without adding bacteria, the enzyme fermented by Actinidia arguta multi-strains has significantly improved total phenol content, reducing power, superoxide anion free radical scavenging ability, DPPH free radical scavenging ability, and SOD enzyme activity. The product of the combined fermentation of the three bacteria has a better sensory evaluation than the product prepared by the bacteria alone. The present invention provides the market with an Actinidia arguta enzyme with excellent sensory, strong antioxidant activity, and high total phenol content, and the enzyme can be further used in the preparation of other products such as beverages, nutritional products, and foods.

[0117] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing Actinidia arguta enzyme, characterized in that: The kiwifruit dry powder is made into a fermentation stock solution, and then bifidobacterium, wine yeast and thermophilic streptococcus are added for joint fermentation at a fermentation temperature of 30°C to 35°C. The inoculation amounts of the bifidobacterium, wine yeast and thermophilic streptococcus are all 1% to 2%, and the inoculation amounts are all the volume ratio of the bacterial seed solution to the fermentation stock solution. After the fermentation is completed, sterilization is performed to obtain the kiwifruit enzyme.

2. The preparation method according to claim 1, characterized in that: The method for preparing the kiwifruit dry powder comprises the following steps: selecting kiwifruits of uniform size and without external damage, vacuum freezing and freeze-drying the kiwifruits to make their water content ≤5%, and then crushing the kiwifruits to obtain dry powder.

3. The preparation method according to claim 1, characterized in that: The method of preparing the fermentation stock solution from the kiwifruit dry powder comprises the following steps: mixing the kiwifruit dry powder, white sugar and sterile water in a mass ratio of (0.7-1.3): (0.7-1.3): (7.7-8.3) to obtain the fermentation stock solution.

4. The preparation method according to claim 1, characterized in that: The volume of the fermentation stock solution in the fermentation container is two-thirds of the container volume, and the fermentation time is 10 to 60 days.

5. The preparation method according to claim 1, characterized in that: The inoculation amount of the bifidobacterium is 1.5%, the inoculation amount of the wine yeast is 2%, and the inoculation amount of the thermophilic streptococcus is 2%.

6. An Actinidia arguta enzyme, characterized in that: The method is obtained by the preparation method according to any one of claims 1 to 5.

7. A product, characterized in that Contains the actinidia arguta enzyme as described in claim 6.

8. Application of the preparation method according to claim 1 in increasing the total phenol content of Actinidia arguta enzyme.

9. Application of the preparation method according to claim 1 in improving the antioxidant activity of Actinidia arguta enzyme.

10. The use according to claim 9, characterized in that: The antioxidant activity includes reducing power, superoxide anion free radical scavenging ability, hydroxyl free radical scavenging ability, DPPH·free radical scavenging ability and SOD enzyme activity.

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