Preparation method of pitaya enzyme

Abstract

The invention belongs to the technical field of food processing, and particularly relates to a preparation method of pitaya enzyme. Fresh red-pulp pitaya is used as a raw material and matched with a phellinus igniarius traditional Chinese medicinal material for compound fermentation to prepare the pitaya enzyme. The pitaya enzyme prepared by the preparation method of the pitaya enzyme has the advantages of high stability, high SOD (superoxide dismutase) enzyme activity and remarkable antibacterial effect, is an ideal production process of a composite pitaya enzyme product, not only can effectively improve the economic benefit of pitaya, but also can promote the development and application of medicinal and edible products, and has a wide application prospect. And the market value is high.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a method for preparing pitaya enzyme. Background Art

[0002] Pitaya (Hylocereus undatus) is a cultivar of Hylocereus undatus of the Cactaceae family and the genus Hylocereus undatus. It is a tropical and subtropical fruit. Pitaya is sweet and flat in nature. Its main nutrients include protein, dietary fiber, vitamin B2, vitamin B3, vitamin C, iron, phosphorus, calcium, magnesium, potassium, etc. Pitaya is a low-energy fruit rich in water-soluble dietary fiber. It has the effects of losing weight, lowering cholesterol, preventing constipation, and colorectal cancer. Pitaya also contains plant albumin, which is rare in general fruits and vegetables. This albumin will combine with heavy metal ions in the human body to play a detoxifying role. Fruit and vegetable enzymes are products made by fermenting fruits and vegetables. They can not only retain the nutrients of fruits and vegetables, but also produce more beneficial substances through the fermentation process, which is more conducive to improving the value of fruits and vegetables. In order to better improve the nutritional value of pitaya, more and more pitaya products have emerged.

[0003] Patent document CN115119933A discloses a pitaya enzyme and its fermentation preparation process. The preparation process specifically comprises the following steps: first peel the pitaya to obtain the pulp, crush it to obtain pulp, add the pulp to a fermentation device, add sugar to adjust the sugar content, add a protective agent and heat it to above 90°C, sterilize it after heat preservation to obtain a fermentation raw material, add the first strain of bacteria when it is cooled to 35°C or below, let it stand for fermentation, and obtain a primary fermentation product; add sugar to the primary fermentation product to adjust the sugar content, mix well, add the second strain of bacteria, and ferment with stirring at intervals to obtain the product. This method reduces the degradation of anthocyanins during the fermentation process and reduces the inhibitory effect of anthocyanins on the activity of fermented bacteria by adding a protective agent to combine with anthocyanins in pitaya juice, thereby improving the fermentation efficiency.

[0004] Patent document CN111657491A discloses a pitaya peel enzyme and a preparation method thereof. The method uses pitaya peel as raw material, adds highly active yeast, xylitol and calcium lactate, and ferments the pitaya peel enzyme. Compared with traditional enzymes, the sugar content is significantly reduced and the calcium content is increased, so that the pitaya peel enzyme can produce a strong rose aroma, meet people's demand for enzyme flavor and taste without adding artificial flavors, and realize the diversification of nutritional elements in enzyme products.

[0005] Patent document CN106071566A discloses a biological drink that uses pitaya juice as a carrier to cultivate Phellinus linteus. The method is to use ripe pitaya to squeeze juice and use it as a liquid culture medium, then inoculate Phellinus linteus strains and form another special combination through deep fermentation - pitaya Phellinus linteus liquid strain drink. The drink not only has the nutrients of pitaya, but also contains the nutrients of Phellinus linteus.

[0006] At present, the product development of pitaya is still imperfect, the production system of pitaya producing areas is also imperfect, and there is little research and development on pitaya compound functional products. Therefore, it is of great economic benefit to research and develop a pitaya functional food with high stability and significant health effects. Summary of the invention

[0007] In order to solve the defects of the prior art, the present invention provides a method for preparing pitaya enzyme. The present invention uses fresh red flesh pitaya as raw material and combines it with Chinese medicinal materials such as Phellinus igniarius to prepare pitaya enzyme. The pitaya enzyme prepared by the method for preparing pitaya enzyme provided by the present invention has the advantages of high stability, high SOD enzyme activity and good antibacterial effect.

[0008] The present invention provides a method for preparing pitaya enzyme, comprising the following steps:

[0009] Step S1, selecting ripe fresh red dragon fruit, peeling, cutting and beating to obtain dragon fruit pulp, adding pectinase and cellulase to the dragon fruit pulp, enzymolyzing at 45-55° C. for 2-3 h, filtering and removing seeds to obtain dragon fruit juice;

[0010] Step S2, adjusting the initial sugar content of the pitaya juice obtained in step S1 to 14-20% to obtain pitaya juice, adding the superfine powder of Phellinus linteus to the pitaya juice, stirring evenly, and sterilizing to obtain a mixed solution;

[0011] Step S3, inoculating the fermentation strain into a liquid culture medium and culturing for 24 to 48 hours, taking the culture solution and shaking it evenly, placing it in a sterile centrifuge tube, centrifuging it at a speed of 2000 to 4000 rpm for 8 to 15 minutes, removing the supernatant, adding an equal amount of sterile water, vortex mixing, adjusting the concentration of the bacterial solution, and obtaining a seed solution;

[0012] Step S4, inoculating the seed liquid obtained in step S3 into the mixed liquid obtained in step S2 for fermentation, centrifuging, taking the supernatant, filtering, and bottling to obtain the product.

[0013] Furthermore, in the step S1, the amount of pectinase added is 0.2% (w / v) of the pitaya pulp, and the amount of cellulase added is 0.1% (w / v) of the pitaya pulp.

[0014] Furthermore, the amount of Phellinus linteus superfine powder added in step S2 is 0.2-0.3% of the weight of pitaya juice.

[0015] Furthermore, the fermentation bacteria in step S3 include yeast, Bacillus subtilis and Lactobacillus plantarum.

[0016] The fermentation strains are all commercially available products, the yeast is the yeast numbered DBY4742, the Lactobacillus plantarum is the Lactobacillus plantarum numbered ATCC8014, and the Bacillus subtilis is purchased from Guangdong Provincial Microbiological Collection Center.

[0017] Furthermore, the preparation of the fermentation strains: 100 mL of liquid culture medium is prepared, the yeast is inoculated into a YPDA culture medium for culture, the Bacillus subtilis is inoculated into an LB culture medium for culture, and the Lactobacillus plantarum is inoculated into an MRS broth culture medium for culture.

[0018] Furthermore, the OD value of the bacterial liquid concentration in step S3 is equal to 1.

[0019] Furthermore, the inoculation order of the seed liquid in step S4 is: inoculating the yeast seed liquid into the mixed liquid obtained in step S2 for fermentation, sterilizing at 90-95° C. for 4-6 minutes after the fermentation is completed to obtain a first fermentation liquid; inoculating the Bacillus subtilis seed liquid into the first fermentation liquid for fermentation, and directly inoculating the Lactobacillus plantarum seed liquid for fermentation without sterilization after the fermentation is completed.

[0020] Furthermore, the inoculation order of the seed liquid in step S4 is specifically as follows: inoculating the yeast seed liquid into the mixed liquid obtained in step S2, fermenting it at a temperature of 27 to 29° C. for 14 to 18 hours, and sterilizing it at 90 to 95° C. for 4 to 6 minutes after the fermentation is completed to obtain the first fermentation liquid; inoculating the Bacillus subtilis seed liquid into the first fermentation liquid, fermenting it at a temperature of 33 to 35° C. for 14 to 18 hours, and directly inoculating the Lactobacillus plantarum seed liquid without sterilization after the fermentation is completed, and fermenting it at a temperature of 33 to 35° C. for 14 to 18 hours.

[0021] Furthermore, the inoculation amounts of the yeast seed solution, the Bacillus subtilis seed solution and the Lactobacillus plantarum seed solution are all 4-6% of the volume of the mixed solution.

[0022] Furthermore, the step S4 further comprises a step of adding modified trehalose after filtration, and the amount of modified trehalose added is 0.1-0.2% w / v of the filtrate after filtration.

[0023] Furthermore, the preparation method of the modified trehalose is:

[0024] Dissolve trehalose in water to prepare a trehalose solution with a mass concentration of 45-50%, add a pH regulator to adjust the pH value of the trehalose solution to 8-9, then add octenyl succinic anhydride, the amount of octenyl succinic anhydride added is 2-4% of the volume of the trehalose solution, stir and react for 2-3 hours at a temperature of 50-55°C and a pH of 8-9, add a pH regulator to adjust the pH value to 5-6 to terminate the reaction, concentrate until no water is evaporated, crystallize, and dry to obtain the product.

[0025] In addition, the present invention also provides the use of the pitaya enzyme prepared by the preparation method of the pitaya enzyme in the preparation of functional foods with antioxidant effects.

[0026] The inventor has been committed to the development and research of pitaya products in order to better develop products that combine pitaya with Chinese medicinal materials. The inventor first proposed the product idea of ​​fermenting pitaya with Chinese medicinal materials Phellinus igniarius, which is helpful for the development of medicine and food products. To this end, the inventor optimized the initial sugar content of pitaya juice, fermentation strains, strain inoculation amount, fermentation time, fermentation temperature and other key factors to seek a suitable process for producing pitaya mulberry igniarius enzyme. During the screening test, the inventor found that using yeast, Bacillus subtilis and Lactobacillus plantarum as fermentation bacteria can produce pitaya enzymes with high total acid, reducing sugar, total sugar and SOD enzyme activity.

[0027] Furthermore, the inventor unexpectedly discovered in in-depth research that the order of inoculation of yeast, Bacillus subtilis and Lactobacillus plantarum has an impact on the effective ingredients of the enzyme, especially the SOD enzyme activity of the enzyme. When yeast is first inoculated with the mixed solution for fermentation, sterilization is performed after the fermentation is completed, and then Bacillus subtilis is inoculated for fermentation, and Lactobacillus plantarum is directly inoculated for fermentation without sterilization after the fermentation is completed, the content of total acid, reducing sugar, total sugar and SOD enzyme activity of pitaya enzyme can be effectively increased.

[0028] Furthermore, the inventors also found that by adding a specific amount of mulberry linterus superfine powder to pitaya for composite fermentation, the mulberry linterus superfine powder can improve the ability of yeast, Bacillus subtilis and Lactobacillus plantarum to produce SOD enzyme by fermentation, especially can promote the ability of Bacillus subtilis to produce SOD enzyme by fermentation, thereby further increasing the SOD enzyme content of pitaya enzyme and enhancing the antioxidant effect of pitaya enzyme.

[0029] In addition, it has been found that the pH value, SSC (soluble solid content) and sugar-acid ratio, SOD enzyme activity and ascorbic acid and other active ingredients of pitaya mulberry linteus complex enzyme decreased significantly during storage. In order to better improve the stability of pitaya enzyme SOD enzyme activity. The inventor proposed to add trehalose as a stabilizer for SOD enzyme, but the stabilization effect did not achieve the expected effect. After in-depth research, the inventor found that adding trehalose to octenyl succinic anhydride for modification can not only improve the antioxidant properties of trehalose, but also improve the dispersibility of trehalose. Adding the modified trehalose to pitaya enzyme can quickly disperse into the enzyme system, which can not only promote the adhesion and binding of modified trehalose to SOD enzyme, but also prevent ascorbic acid from oxidative degradation, and effectively improve the stability of pitaya enzyme.

[0030] In summary, compared with the prior art, the pitaya enzyme prepared by the preparation method of the pitaya enzyme provided by the present invention has the advantages of high stability, high SOD enzyme activity and significant antibacterial effect. It is a more ideal production process of a composite pitaya enzyme product, which helps to improve the economic benefits of pitaya. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The chart shows the reducing sugar, total sugar, total acid and sensory evaluation results of pitaya enzymes produced at different fermentation times.

[0032] Figure 2 The chart shows the reducing sugar, total sugar, total acid and sensory evaluation results of pitaya enzymes produced at different fermentation temperatures.

[0033] Figure 3 The chart shows the reducing sugar, total sugar, total acid and sensory evaluation results of pitaya enzymes produced with different bacterial inoculation amounts.

[0034] Figure 4 The chart shows the reducing sugar, total sugar, total acid and sensory evaluation results of pitaya enzymes prepared with different initial sugar contents. DETAILED DESCRIPTION

[0035] The present invention is further described below by describing specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention. The materials involved in the present invention are all food grade and can be obtained by commercial or conventional technical means in the art.

[0036] Exploratory experiment 1: Exploring the fermentation process of pitaya enzyme

[0037] 1. Test steps:

[0038] 1. Raw material pretreatment:

[0039] Select fresh red-fleshed pitaya with high maturity, peel, cut and pulp to obtain pitaya pulp, add 0.2% (w / v) pectinase and 0.1% (w / v) cellulase of pitaya pulp, enzymolyze at 50°C for 2h, filter, remove seeds and obtain pitaya juice.

[0040] 2. Adjust the ingredients:

[0041] Adjust the sugar content of pitaya juice to the set value to obtain pitaya juice, weigh 100g of pitaya juice, add 0.3g of ultrafinely crushed mulberry linterus, mix well, and sterilize at 95℃ for 5min.

[0042] 3. Optimization of strain fermentation conditions:

[0043] 3.1. Preparation of strains:

[0044] Prepare 100 mL of liquid culture medium, inoculate yeast into YPDA medium for culture, inoculate Bacillus subtilis into LB medium for culture, inoculate Lactobacillus plantarum into MRS broth medium for culture, and culture at the strain culture temperature for 36 hours. Shake the culture solution before use, take the required liquid into a sterile centrifuge tube, centrifuge at 3000 rpm for 10 minutes, remove the supernatant, add an equal amount of sterile water, vortex mix, and adjust the OD value of the bacterial solution concentration to 1.

[0045] 3.2. Vaccination order:

[0046] The yeast was first inoculated for fermentation, and then sterilized at 90°C for 5 minutes; secondly, Bacillus subtilis was inoculated, and after fermentation, Lactobacillus plantarum was directly inoculated for fermentation without sterilization. The fermentation time of the fermentation strains (yeast, Bacillus subtilis and Lactobacillus plantarum) was the same as the inoculation amount.

[0047] 3.3. Control of fermentation conditions:

[0048] The fermentation conditions mainly include temperature, time, inoculation amount and initial sugar content. The most suitable fermentation conditions are obtained through single factor-orthogonal test.

[0049] (1) Fermentation time: under the same conditions, the fermentation temperature of yeast was 28°C, the fermentation temperature of Bacillus subtilis and Lactobacillus plantarum was 37°C, the inoculation amount was 3% (3 mL of bacterial solution was inoculated under the condition of OD value of 1), the initial sugar content was 16%, and the fermentation time of each bacteria was controlled to be 8 h, 12 h, 16 h, 20 h, and 24 h for fermentation.

[0050] (2) Fermentation temperature: under the same conditions, the fermentation time was 12 h, the inoculation amount was 3%, the initial sugar content was 16%, and the fermentation temperatures of yeast, Bacillus subtilis and Lactobacillus plantarum were controlled at 24°C, 30°C, 30°C; 26°C, 32°C, 32°C; 28°C, 34°C, 34°C; 30°C, 36°C, 36°C; and 32°C, 38°C, 38°C, respectively.

[0051] (3) Inoculation amount: Under the same conditions, the fermentation time was 12 h, the yeast fermentation temperature was 28°C, the Bacillus subtilis and Lactobacillus plantarum fermentation temperature was 37°C, the initial sugar content was 16%, and the inoculation amounts were controlled to be 1%, 3%, 5%, 7%, and 9% respectively for fermentation.

[0052] (4) Initial sugar content: Under the same conditions, the fermentation time was 12 h, the yeast fermentation temperature was 28° C., the fermentation temperature was 37° C. for Bacillus subtilis and Lactobacillus plantarum, the inoculation amount was 3%, and the initial sugar content was controlled to be 14%, 16%, 18%, 20%, and 22% for fermentation.

[0053] 4. Determine the reducing sugar, total sugar, and total acid of pitaya enzymes prepared with different parameters, and conduct a sensory evaluation of pitaya enzymes.

[0054] 4.1. Determination of reducing sugar and total sugar: Refer to the instructions of the kit for testing.

[0055] 4.2. Total acid determination method:

[0056] Refer to the pH potentiometric titration method in the national standard GB 12456-2021, calculated in lactic acid.

[0057] Analysis steps: Centrifuge the fermentation broth at 3500r / min for 10min, take 5mL of the supernatant and add 45mL of water into a 150mL beaker. After preheating the acidometer, calibrate the pH meter using pH=6.86 and pH=9.18 buffer solutions. Place the beaker containing the sample solution on a magnetic stirrer and immerse the acidometer electrode. Start the stirrer and quickly titrate with 0.01mol / L sodium hydroxide standard titration solution. Observe the pH change of the solution until the pH of the solution reaches 8.2. Record the volume of the sodium hydroxide standard titration solution consumed. Do three parallel tests and a blank experiment. The calculation formula is as follows.

[0058]

[0059] Where:

[0060] c--Concentration of sodium hydroxide standard titration solution, mol / L;

[0061] V1--The volume of sodium hydroxide standard titration solution consumed when titrating the test solution, mL;

[0062] V2--The volume of sodium hydroxide standard titration solution consumed during the blank test, mL;

[0063] k--conversion coefficient of acid: based on lactic acid, lactic acid is 0.090.

[0064] F--dilution multiple of the test solution;

[0065] V--Volume of the sample, mL.

[0066] 4.3. Sensory evaluation method:

[0067] Refer to the sensory scoring table in Table 1 for scoring and evaluation.

[0068] Table 1 Sensory score table of pitaya mulberry linterus enzyme

[0069]

[0070]

[0071] Note: Tasting order: first taste the smell of the Phellinus igniarius dragon fruit enzyme, then observe the color and texture, and finally taste it.

[0072] The specific method is: invite 11 volunteers (5 men and 6 women) to form an evaluation team after training, and use a comprehensive evaluation method to perform sensory evaluation on the color, texture, smell and taste of pitaya mulberry enzyme. The sensory evaluation standard of pitaya mulberry enzyme is shown in Table 1. The average value is calculated based on the tasting score results of each taster. The data with individual tasting errors exceeding the average value by more than 10 points should be discarded, and the average value should be recalculated after discarding. Finally, the average value of the tasting score is used as the pitaya enzyme tasting score, and the calculation result is rounded to an integer.

[0073] 2. Test results:

[0074] The test results are as follows Figure 1 to Figure 4 shown. Figure 1 The reducing sugar, total sugar, total acid and sensory evaluation results of pitaya enzymes produced at different fermentation times are shown in the figure. Figure 2 The reducing sugar, total sugar, total acid and sensory evaluation results of pitaya enzymes produced at different fermentation temperatures are shown in the figure. Figure 3 The chart shows the reducing sugar, total sugar, total acid and sensory evaluation results of pitaya enzymes produced with different inoculation amounts of bacteria. Figure 4 The chart shows the reducing sugar, total sugar, total acid and sensory evaluation results of pitaya enzymes prepared with different initial sugar contents.

[0075] from Figure 1 to Figure 4It can be seen that the preferred fermentation time in the pitaya enzyme preparation method provided by the present invention is: 14 to 18 hours; the fermentation temperature is: the fermentation temperature of yeast is 27 to 29°C, the fermentation temperature of Bacillus subtilis is 33 to 35°C, and the fermentation temperature of Lactobacillus plantarum is 33 to 35°C; the inoculation amount of the fermentation strains such as yeast, Bacillus subtilis and Lactobacillus plantarum is: 4 to 6%; the initial sugar content of pitaya juice is: 17 to 19%.

[0076] Exploratory test 2: Sensory evaluation test of pitaya enzyme

[0077] 1. Test method:

[0078] Refer to the preparation method of pitaya enzyme in exploratory experiment 1, the specific fermentation time, fermentation temperature, inoculation amount and initial sugar content are shown in Table 2. Sensory evaluation was performed on the pitaya enzyme prepared according to the parameters in Table 2 to screen the best preparation process. The sensory evaluation method refers to 4.3 in exploratory experiment 1.

[0079] Table 2

[0080]

[0081] 2. Test results:

[0082] The test results are shown in Tables 3 and 4.

[0083] Table 3

[0084]

[0085] Table 4

[0086]

[0087]

[0088] It can be seen from Tables 3 and 4 that the optimal parameters of the pitaya enzyme provided by the present invention are: fermentation time is 14 hours, fermentation temperature: the fermentation temperature of yeast is 27°C, the fermentation temperature of Bacillus subtilis is 33°C, the fermentation temperature of Lactobacillus plantarum is 33°C, the inoculation amount is 6%, and the initial sugar content is 18%.

[0089] Example 1, preparation of pitaya enzyme

[0090] Step S1, selecting ripe fresh red dragon fruit, peeling, cutting and beating to obtain dragon fruit pulp, adding pectinase and cellulase to the dragon fruit pulp, enzymolysis at 50° C. for 2 h, wherein the amount of the pectinase added is 0.2% w / v, and the amount of the cellulase added is 0.1% w / v, filtering, removing the seeds, and obtaining dragon fruit juice;

[0091] Step S2, adjusting the initial sugar content of the pitaya juice obtained in step S1 to 18% to obtain pitaya juice, adding 0.3 g of superfine phellinus linteus powder to 100 g of pitaya juice, stirring evenly, and sterilizing at 95° C. for 5 min to obtain a mixed solution;

[0092] Step S3, the fermentation strain is inoculated in a liquid culture medium and cultured, 100 mL of the liquid culture medium is configured, the yeast colony is inoculated into a YPDA culture medium for culture, Bacillus subtilis is inoculated into an LB culture medium for culture, and Lactobacillus plantarum is inoculated into an MRS broth culture medium for culture, and the culture temperature is cultivated for 36 h. Before use, the culture solution is shaken, the required liquid is taken into a sterile centrifuge tube, and the rotation speed is 3000 rpm. Centrifuge for 10 min, remove the supernatant, add an equal amount of sterile water, vortex mix, and adjust the OD value of the bacterial solution concentration to be equal to 1 to obtain a seed solution;

[0093] Step S4, inoculating the seed liquid obtained in step S3 into the mixed liquid obtained in step S2 for fermentation, inoculating 6% of the yeast seed liquid by volume of the mixed liquid into the mixed liquid obtained in step S2, fermenting at a temperature of 27° C. for 14 hours, sterilizing at 90° C. for 5 minutes after the fermentation is completed, and obtaining a first fermentation liquid; inoculating 6% of the Bacillus subtilis seed liquid by volume of the mixed liquid into the first fermentation liquid, fermenting at a temperature of 33° C. for 14 hours, and directly inoculating 6% of the plant lactobacillus seed liquid by volume of the mixed liquid without sterilization after the fermentation is completed, and fermenting at a temperature of 33° C. for 14 hours. Centrifuging, taking the supernatant, filtering, and filling to obtain.

[0094] Example 2: Preparation of modified trehalose

[0095] Dissolve trehalose in water to prepare a trehalose solution with a mass concentration of 45%, add a pH adjuster to adjust the pH value of the trehalose solution to 9, then add octenyl succinic anhydride, the amount of octenyl succinic anhydride added is 3% of the volume of the trehalose solution, stir and react for 2 hours at a temperature of 50°C and a pH of 9, add a pH adjuster to adjust the pH value to 5 to terminate the reaction, concentrate until no water is evaporated, crystallize, and dry to obtain.

[0096] Example 3, Preparation of pitaya enzyme

[0097] Step S1, selecting ripe fresh red dragon fruit, peeling, cutting and beating to obtain dragon fruit pulp, adding pectinase and cellulase to the dragon fruit pulp, enzymolysis at 50° C. for 2 h, wherein the amount of the pectinase added is 0.2% w / v, and the amount of the cellulase added is 0.1% w / v, filtering, removing the seeds, and obtaining dragon fruit juice;

[0098] Step S2, adjusting the initial sugar content of the pitaya juice obtained in step S1 to 18% to obtain pitaya juice, adding 0.3 g of superfine phellinus linteus powder to 100 g of pitaya juice, stirring evenly, and sterilizing at 95° C. for 5 min to obtain a mixed solution;

[0099] Step S3, the fermentation strain is inoculated in a liquid culture medium and cultured, 100 mL of the liquid culture medium is configured, the yeast colony is inoculated into a YPDA culture medium for culture, Bacillus subtilis is inoculated into an LB culture medium for culture, and Lactobacillus plantarum is inoculated into an MRS broth culture medium for culture, and the culture temperature is cultivated for 36 h. Before use, the culture solution is shaken, the required liquid is taken into a sterile centrifuge tube, and the rotation speed is 3000 rpm. Centrifuge for 10 min, remove the supernatant, add an equal amount of sterile water, vortex mix, and adjust the OD value of the bacterial solution concentration to be equal to 1 to obtain a seed solution;

[0100] Step S4, inoculating the seed liquid prepared in step S3 into the mixed liquid prepared in step S2 for fermentation, inoculating 6% of the volume of the yeast seed liquid into the mixed liquid prepared in step S2, fermenting at a temperature of 27° C. for 14 hours, sterilizing at 90° C. for 5 minutes after the fermentation is completed, to obtain a first fermentation liquid; inoculating 6% of the volume of the mixed liquid of Bacillus subtilis seed liquid into the first fermentation liquid, fermenting at a temperature of 33° C. for 14 hours, directly inoculating 6% of the volume of the mixed liquid of Lactobacillus plantarum seed liquid without sterilization after the fermentation is completed, fermenting at a temperature of 33° C. for 14 hours, centrifuging, taking the supernatant, filtering to obtain a filtrate, and then adding modified trehalose, the amount of the modified trehalose added is 0.1% w / v of the filtrate after filtration, and the modified trehalose is the modified trehalose prepared in Example 2, filling, and obtaining.

[0101] Comparative Example 1: Preparation of pitaya enzyme

[0102] The difference from Example 1 is that no superfine phellinus linteus powder is added, and the remaining steps are similar to those in Example 1.

[0103] Comparative Example 2: Preparation of pitaya enzyme

[0104] The difference from Example 1 is that the superfine phellinus powder is replaced by superfine tuckahoe powder, and the remaining steps are similar to those in Example 1.

[0105] Comparative Example 3: Preparation of pitaya enzyme

[0106] The difference from Example 1 is that in step S4, the inoculated yeast seed solution, Bacillus subtilis seed solution and Lactobacillus plantarum seed solution are added to the mixed solution at the same time and fermented at a temperature of 30° C. for 14 hours, the inoculation amount of the fermentation strain is 6%, and the remaining steps are similar to those in Example 1.

[0107] Comparative Example 4: Preparation of pitaya enzyme

[0108] The difference from Example 3 is that the stabilizer is trehalose, and the remaining steps are similar to those in Example 3.

[0109] Test Example 1: Efficacy test of pitaya enzyme

[0110] 1. Test method:

[0111] The ABTS free radical scavenging ability and antibacterial ability of the pitaya enzymes prepared in Example 1 and Comparative Example 1 were tested.

[0112] 1.1. Determination of ABTS free radical scavenging ability:

[0113] Use PBS buffer (5mmol / L sodium dihydrogen phosphate and disodium hydrogen phosphate) with pH 7.4 to prepare 7mmol / LABTS solution and 2.45mmol / L potassium persulfate solution respectively. Mix equal volumes of the two and keep them at room temperature in the dark for 12 hours. Then dilute them 25 times with buffer for use (prepare and use immediately). Take 3mL of the above dilution in a test tube, add 1mL of the sample diluted 50 times, react in a water bath at 30℃ for 20min, measure the absorbance value A1, and measure the absorbance value A0 at 734nm with distilled water as a blank tube.

[0114]

[0115] 1.2. Determination of antibacterial ability:

[0116] Foodborne pathogens including Salmonella typhimurium ATCC14028, Escherichia coli ATCC12229, Pseudomonas aeruginosa ATCC10415 and Staphylococcus aureus ATCC6538 were cultured in nutrient broth at 37°C for 24 hours. The antibacterial activity of the pitaya enzyme prepared in Example 1 and Comparative Example 1 was determined using a 96-well microtiter plate. 50 mg of the lyophilized pitaya enzyme prepared in Example 1 and Comparative Example 1 was dissolved in 1 mL of sterile distilled water to prepare the sample. 100 μL of the pitaya enzyme prepared in Example 1 and Comparative Example 1 and 100 μL of each containing 10 μL of the pitaya enzyme were added to the 96 wells. 6 CFU / mL of nutrient solution for foodborne pathogens. Incubate at 37℃ for 24h, and measure the absorbance at 600nm using an enzyme-linked immunosorbent assay to determine the antibacterial activity. The test was carried out three times, and the growth inhibition rate was calculated as follows:

[0117]

[0118] 2. Test results:

[0119] The test results are shown in Table 5.

[0120] Table 5 Efficacy test of pitaya enzyme

[0121]

[0122] It can be seen from Table 5 that the ABTS free radical scavenging ability of the pitaya enzyme prepared by the present invention is greater than 78, and it has a good antibacterial effect.

[0123] Test Example 2: SOD enzyme activity test of pitaya enzyme

[0124] 1. Test method:

[0125] The SOD enzyme activity of the pitaya enzymes prepared in Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 was detected. The SOD enzyme activity was detected according to the kit instructions.

[0126] 2. Test results:

[0127] The test results are shown in Table 6.

[0128] Table 6 SOD enzyme activity test data of pitaya enzyme

[0129]

[0130]

[0131] As can be seen from Table 6, the pitaya enzyme prepared by the present invention has a relatively high content of SOD enzyme, which can effectively neutralize free radicals in the human body, thereby exerting a good antioxidant effect.

[0132] Test Example 3: Stability Test of Pitaya Enzyme

[0133] 1. Test method:

[0134] The SOD enzyme activity of the pitaya enzymes prepared in Example 1, Example 3 and Comparative Example 4 was measured after being placed at 45° C. for 30 days and 60 days. The SOD enzyme activity was detected according to the kit instructions.

[0135] 2. Test results:

[0136] The test results are shown in Table 7.

[0137] Table 7 Stability test results of pitaya enzyme

[0138]

[0139] As shown in Table 7, the pitaya enzyme prepared in Example 3 of the present invention has high stability. After being placed at 45°C for 60 days, the SOD enzyme activity of the enzyme remains basically unchanged, which can effectively ensure the antioxidant effect of the pitaya enzyme. After Example 1 and Comparative Example 4 were placed at 45°C for 60 days, the SOD enzyme activity in the enzyme continued to decrease, indicating that the modified trehalose added in the present invention can protect the SOD enzyme activity in the pitaya enzyme for a long time.

[0140] 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 familiar with 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for preparing pitaya enzyme, characterized in that: The following steps are involved: Step S1, selecting ripe fresh red dragon fruit, peeling, cutting and beating to obtain dragon fruit pulp, adding pectinase and cellulase to the dragon fruit pulp, enzymolyzing at 45-55° C. for 2-3 h, filtering and removing seeds to obtain dragon fruit juice; Step S2, adjusting the initial sugar content of the pitaya juice obtained in step S1 to 14-20% to obtain pitaya juice, adding the superfine powder of Phellinus linteus to the pitaya juice, stirring evenly, and sterilizing to obtain a mixed solution; Step S3, inoculating the fermentation strain into a liquid culture medium and culturing for 24 to 48 hours, taking the culture solution and shaking it evenly, placing it in a sterile centrifuge tube, centrifuging it at a speed of 2000 to 4000 rpm for 8 to 15 minutes, removing the supernatant, adding an equal amount of sterile water, vortex mixing, adjusting the concentration of the bacterial solution, and obtaining a seed solution; Step S4, inoculating the seed liquid obtained in step S3 into the mixed liquid obtained in step S2 for fermentation, centrifuging, taking the supernatant, filtering, and bottling to obtain the product.

2. The method for preparing pitaya enzyme according to claim 1, characterized in that, In the step S1, the amount of pectinase added is 0.2% w / v of the pitaya pulp, and the amount of cellulase added is 0.1% w / v of the pitaya pulp.

3. The method for preparing pitaya enzyme according to claim 1, characterized in that, The amount of superfine phellinus linteus powder added in step S2 is 0.2-0.3% of the weight of pitaya juice.

4. The method for preparing pitaya enzyme according to claim 1, characterized in that, The fermentation bacteria in step S3 are yeast, Bacillus subtilis and Lactobacillus plantarum.

5. The method for preparing pitaya enzyme according to claim 1, characterized in that, The OD value of the bacterial liquid concentration in step S3 is equal to 1.

6. The method for preparing pitaya enzyme according to claim 1, characterized in that, The seed liquid inoculation sequence in step S4 is as follows: the yeast seed liquid is inoculated into the mixed liquid obtained in step S2 for fermentation, and after the fermentation is completed, it is sterilized at 90-95° C. for 4-6 minutes to obtain a first fermentation liquid; the Bacillus subtilis seed liquid is inoculated into the first fermentation liquid for fermentation, and after the fermentation is completed, the Lactobacillus plantarum seed liquid is directly inoculated without sterilization for fermentation.

7. The method for preparing pitaya enzyme according to claim 6, characterized in that: The inoculation order of the seed liquid in step S4 is specifically as follows: inoculating the yeast seed liquid into the mixed liquid obtained in step S2, fermenting at a temperature of 27-29° C. for 14-18 hours, and sterilizing at 90-95° C. for 4-6 minutes after the fermentation is completed to obtain the first fermentation liquid; inoculating the Bacillus subtilis seed liquid into the first fermentation liquid, fermenting at a temperature of 33-35° C. for 14-18 hours, and directly inoculating the Lactobacillus plantarum seed liquid without sterilization after the fermentation is completed, and fermenting at a temperature of 33-35° C. for 14-18 hours.

8. The method for preparing pitaya enzyme according to claim 6, characterized in that, The inoculation amounts of the yeast seed liquid, the bacillus subtilis seed liquid and the lactobacillus plantarum seed liquid are all 4-6% of the volume of the mixed liquid.

9. The method for preparing pitaya enzyme according to claim 1, characterized in that, The step S4 further comprises adding modified trehalose after filtration, wherein the amount of modified trehalose added is 0.1-0.2% w / v of the filtrate after filtration.

10. The method for preparing pitaya enzyme according to claim 9, characterized in that: The preparation method of the modified trehalose is: Dissolve trehalose in water to prepare a trehalose solution with a mass concentration of 45-50%, add a pH regulator to adjust the pH value of the trehalose solution to 8-9, then add octenyl succinic anhydride, the amount of octenyl succinic anhydride added is 2-4% of the volume of the trehalose solution, stir and react for 2-3 hours at a temperature of 50-55°C and a pH of 8-9, add a pH regulator to adjust the pH value to 5-6 to terminate the reaction, concentrate until no water is evaporated, crystallize, and dry to obtain the product.

Images