Preparation method of active lactobacillus beverage co-fermented by litchi juice and pericarp

Abstract

The invention discloses a preparation method of a litchi juice and peel co-fermented active lactobacillus beverage, which comprises the following steps: cleaning fresh litchis, peeling, denucleating, juicing, filtering and centrifuging to obtain clear litchi juice, and sterilizing; performing vacuum freeze drying on the separated litchi peel, crushing into powder, and sieving; inoculating activated lactobacillus plantarum into the clear litchi juice, adding litchi peel powder, carrying out ultrahigh pressure treatment, uniformly shaking, placing in an incubator, and carrying out static fermentation for 24 hours; and stopping the fermentation to obtain the litchi juice and pericarp co-fermented active lactobacillus beverage. The active lactic acid bacteria beverage co-fermented by the litchi juice and pericarp obtained by the preparation method disclosed by the invention can reduce the sugar content of the litchi, along with rapid proliferation of lactic acid bacteria, the sugar content of the litchi can be reduced, the flavor of the litchi is improved by increasing the sour taste, the antioxidant activity of the litchi juice is improved, the storage stability is improved, and the shelf life of the litchi juice is prolonged. Meanwhile, the nutritional value of the fermented fruit juice can be remarkably increased.

Description

Technical Field

[0001] The invention belongs to the field of food science and technology, and relates to a method for preparing an active lactic acid bacteria beverage, in particular to a method for preparing an active lactic acid bacteria beverage prepared by co-fermentation of lychee juice and peel. Background Art

[0002] Litchi is an important fruit cultivated in tropical and subtropical regions. It is rich in nutrients and delicious. Litchi pulp contains a variety of nutrients, such as carbohydrates, proteins, lipids, vitamins and minerals. However, the high water content and high sugar content of lychees make them difficult to store after picking, and they are prone to browning and rotting. Litchi is mainly eaten fresh, and deep-processed products account for only 13%, and post-harvest losses are as high as 20% (Lu Sushan, Tu Zhenkai, Zhao Xihe, et al. Research Status of Litchi Processing Technology in my country [J]. Modern Agricultural Equipment, 2022, 43(02):14-17+48.). Therefore, it is urgent to develop a deep processing solution that can enhance the commercial value of lychees to cope with the current storage and processing problems.

[0003] Jizui, Guiwei, Huaizhi and Nuomici are famous Chinese lychee varieties, each with its own characteristics and deeply loved by consumers. Jizui lychee is named because the tip of the fruit resembles a chicken's beak. The flesh is thick, juicy, sweet and fragrant. Guiwei lychee is characterized by crystal clear flesh, sweet taste, light osmanthus fragrance, small core and thick flesh. Huaizhi lychee is characterized by medium-sized fruit, crisp flesh, sweet and sour, and unique flavor. Nuomici lychee is characterized by soft and glutinous flesh, juicy and sweet taste, small core or no core, and excellent taste. These lychee varieties are not only suitable for fresh consumption, but also can be used for deep processing, such as juice, dried fruit, fermented beverages, etc., with high economic value and market potential.

[0004] In recent years, as consumers have recognized the nutritional benefits of lactic acid bacteria fermented products, lactic acid bacteria fermented fruit and vegetable juices have also been popular among consumers. Lychees are rich in sugar, which provides sufficient nutrients for the growth of lactic acid bacteria. However, not all lychee varieties are suitable for the development of functional fermented beverages. More importantly, there are significant variety differences in the content of phytochemical components and antioxidant activity among different lychee fruits. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing an active lactic acid bacteria beverage prepared by co-fermentation of lychee juice and lychee peel.

[0006] The method for preparing the active lactic acid bacteria beverage obtained by co-fermentation of lychee juice and peel of the present invention comprises the following steps: A. Wash fresh lychees, peel, remove the core, squeeze the juice, filter, centrifuge to obtain lychee juice, sterilize and set aside; B. vacuum freeze-drying the lychee peel separated in step A, crushing it into powder, sieving it, and setting it aside; C. Inoculate the activated Lactobacillus plantarum L100 at a rate of 1.5% (v / v) into the lychee juice obtained in step A, add the lychee peel powder obtained in step B at a mass volume ratio of 0.25%, treat with ultrahigh pressure, shake well, and place in a 34°C incubator for static fermentation for 24 hours; D. Take samples every 6 hours to measure physical and chemical indicators. When the total viable bacteria count reaches at least 10.0 lg CFU / ml, the fermentation is terminated to obtain the active lactic acid bacteria beverage co-fermented with lychee juice and peel.

[0007] According to a further feature of the preparation method of the present invention, in step A, the lychee is selected from one of the following lychee varieties: Jizui lychee, Guiwei lychee, Huaizhi lychee and Nuomici lychee.

[0008] Preferably, the lychee is a lychee.

[0009] According to a further feature of the preparation method of the present invention, in step A, the juice is filtered using a 100-mesh filter cloth, and the filtered juice is centrifuged at 4500 r / min for 5 minutes.

[0010] According to a further feature of the preparation method of the present invention, in step A, the sterilization treatment is performed at 85° C. for 10 minutes.

[0011] According to a further feature of the preparation method of the present invention, in the step B, the sieving is through a 60-mesh sieve.

[0012] According to a further feature of the preparation method of the present invention, in the step C, the viable count of the activated Lactobacillus plantarum L100 is at least 6.5 lg CFU / ml.

[0013] According to a further feature of the preparation method of the present invention, in step C, the ultrahigh pressure treatment is 400 MPa for 10 minutes.

[0014] The method for preparing an active lactic acid bacteria beverage by co-fermentation of lychee juice and peel of the present invention has the following beneficial effects: (1) lychees with high juice yield are selected from different varieties of lychee fruits for the preparation of lactic acid bacteria fermented beverages; (2) the sugar content of lychees can be reduced by fermentation with Lactobacillus plantarum, and the sour taste can be increased to enhance the flavor of lychees; the process of preparing lychee juice fermented with Lactobacillus plantarum is optimized to obtain the optimal fermentation process; (3) adding part of the peel to the lychee juice for co-fermentation in combination with ultra-high pressure food processing technology is conducive to the rapid proliferation of lactic acid bacteria, improves the antioxidant activity of the lychee juice, and increases the storage stability. (4) Lychee peel is rich in active substances such as polyphenols, and adding lychee peel can significantly increase the nutritional value of the fermented juice. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Showing the juice yield of different litchi juices.

[0016] Figure 2 Showing the total phenolic content of litchi peel.

[0017] Figure 3 Showing the total flavonoids content of litchi peel.

[0018] Figure 4 Showing the total anthocyanin content of litchi peel.

[0019] Figure 5 Displays the changes in the number of live bacteria during the fermentation process.

[0020] Figure 6 Shows the changes in total phenolic content during fermentation.

[0021] Figure 7 Showing the changes in total flavonoid content during fermentation.

[0022] Figure 8 Shows the change in total sugar content during fermentation.

[0023] Fig. 9 Shows the change in total acid content during fermentation.

[0024] Fig.10 Shows the change in pH during fermentation.

[0025] Fig.11 Shows the change in total soluble solids content during the fermentation process.

[0026] Fig.12 Showing the effect of fermentation temperature on total phenolics.

[0027] Fig.13 Showing the effect of fermentation temperature on total SOD activity.

[0028] Fig.14Shows the effect of fermentation temperature on DPPH radical scavenging rate.

[0029] Fig.15 Showing the effect of fermentation temperature on the scavenging rate of ABST free radicals.

[0030] Fig.16 Showing the effect of fermentation time on total phenolics.

[0031] Fig.17 Showing the effect of fermentation time on total SOD activity.

[0032] Fig.18 Shows the effect of fermentation time on DPPH radical scavenging rate.

[0033] Fig.19 Showing the effect of fermentation time on the scavenging rate of ABST free radicals.

[0034] Fig. 20 Shows the effect of inoculum dose on total phenols.

[0035] Fig.21 Shows the effect of inoculum dosage on total SOD activity.

[0036] Fig. 22 Shows the effect of inoculum dosage on DPPH radical scavenging rate.

[0037] Fig.23 The effect of inoculum dose on ABST free radical scavenging rate (b) is shown.

[0038] Fig.24 Shown is the effect of initial pH on total phenolics.

[0039] Fig.25 Showing the effect of initial pH on total SOD activity.

[0040] Fig.26 Showing the effect of initial pH on DPPH radical scavenging rate.

[0041] Fig. 27 The effect of initial pH on the scavenging rate of ABST free radicals is shown.

[0042] Fig.28 Shows the changes in the number of viable bacteria during the fermentation process in different treatment groups.

[0043] Fig.29 Showing the changes in total phenolic content in different treatment groups.

[0044] Fig.30 Shows the changes in total SOD activity in different treatment groups.

[0045] Fig.31 Shows the changes in total sugar and reducing sugar in different treatment groups.

[0046] Fig.32 Shows the changes in α-amylase inhibition rate in different treatment groups.

[0047] Fig.33 Shows the changes in α-glucosidase inhibition rate in different treatment groups.

[0048] Fig.34 Showing the changes in extracellular polysaccharide content in different treatment groups.

[0049] Fig.35 The effects of high pressure treatment and heat treatment on the browning degree of litchi fermented juice before and after fermentation and during storage are shown.

[0050] Fig.36 The effects of high pressure treatment and heat treatment on the browning degree of litchi fermented juice (with peel added) before, during and after fermentation and during storage are shown.

[0051] Fig.37 The effects of high pressure treatment and heat treatment on the browning degree of litchi fermented juice and litchi fermented juice (with added peel) samples before, during and after fermentation and during storage are shown. DETAILED DESCRIPTION

[0052] Embodiment 1: screening of litchi varieties Main Materials: (1) Four litchi varieties, namely, “Jizui”, “Guiwei”, “Huaizhi” and “Niumici”, were purchased from Jiangnan Fruit Wholesale Market in Guangzhou, Guangdong Province.

[0053] (2) Lychee peel: The water in the peel of the four types of lychee was freeze-dried in a vacuum freeze dryer, pulverized into powder using a grinder and passed through a 60-mesh sieve, and stored frozen at -20°C for further analysis.

[0054] (3) Bacterial strain: Lactobacillus plantarum LP-100 (LP-100) is a strain preserved in our laboratory. Commercial strains (such as those purchased from Guangdong Yikewei Biotechnology Co., Ltd.) can also be used.

[0055] Experimental methods: (1) Four kinds of fresh lychees purchased from the market were washed, peeled, and pitted. Lychee juice was then produced using a juicer. The juice was then filtered using a 100-mesh filter cloth and centrifuged (4500 r / min, 5 min) to obtain clear juice.

[0056] (2) Dissolve the four types of lychee juice obtained in step A and sterilize (85°C, 10 min). Cool to room temperature and place on a sterile operating table. Inoculate the activated Lactobacillus plantarum L100 into the lychee juice at a rate of 2.0% (v / v), shake well and place in a 34°C incubator for static fermentation for 0-48 h. Take samples every 6 h to measure physical and chemical indicators. The number of viable cells of the activated Lactobacillus plantarum L100 is at least 6.5 lg CFU / ml.

[0057] (3) Take 200 g of each of the four types of litchi fruits, peel and remove the core, and use a juicer to squeeze the juice. Use 100-mesh gauze to filter the filtered juice. Weigh the filtered juice and measure its juice yield. Repeat the measurement three times for each group of samples. The calculation formula is as follows:

[0058] (4) Determination of total phenol content in fermented samples: The litchi fermented juice sample was centrifuged at 5000 r / min for 5 min. 100 µL of the supernatant of the litchi fermented juice was taken into a 10 mL glass test tube. 500 µL of Folin phenol reagent was added and vortexed to mix. The mixture was reacted at room temperature for 5 min. 2.0 mL of 15% Na2CO3 solution was added and the volume was made up to 10 mL with pure water. The mixture was allowed to stand in the dark for 30 min. Finally, the absorbance was measured at 765 nm. The total phenol content was expressed as gallic acid equivalent.

[0059] (5) Determination of total flavonoids in samples: The fermented litchi juice samples were centrifuged at 5000 r / min for 5 min and diluted to an appropriate multiple. Accurately pipette 1 mL of each sample into a 10 mL glass test tube, and add each reagent in the order of adding reagents as described above. The sample was allowed to stand at room temperature for 20 min. After the treatment, the absorbance was measured at a wavelength of 510 nm, and the total flavonoid content in the sample was calculated according to the rutin standard curve. The method for extracting total flavonoids from litchi peel was the same as the method for extracting total phenols from litchi peel. The total flavonoid content of litchi peel was determined according to the above method, and the content was expressed as rutin equivalent.

[0060] (6) Determination of total anthocyanin content in fermentation samples: The method for extracting anthocyanins from litchi peel is the same as the method for extracting total phenols from litchi peel in step (4). The pH differential method was used. 1 mL of the extract sample was added to 9 mL of 0.025 mol / L pH 1.0 KCl buffer and 0.4 mol / L pH 4.5 CH3COONa buffer, respectively. The absorbance values ​​at 510 nm and 700 nm were measured, respectively, and the net absorbance A was calculated. The total anthocyanin content in litchi peel was calculated as follows:

[0061] Where: A = (A 510-A 700 ) pH1.0 -(A510-A700) pH4.5 ; M is the molecular mass (the molecular weight of cyanidin-3-glucoside is 449.2); FD is the dilution factor; V is the volume of the extract; ε is the molar extinction coefficient (26900 L mol-1 cm-1 for cyanidin-3-glucoside); m is the sample mass; 0.552 is the optical path length of the 96-well plate (in cm).

[0062] (7) The pH value of litchi juice before fermentation was measured using a pH meter. The pH value of litchi juice at different time points was measured, and each group of samples was measured three times.

[0063] (8) Determination of total sugar in fermentation samples: Dilute the sample to be tested to an appropriate multiple. Use a pipette to accurately draw 500 µL of the diluted supernatant into a 10 mL glass test tube, then add 500 µL of 6 mol / L HCl solution, seal the tube, heat and hydrolyze in a 100°C metal bath for 30-40 min, quickly cool to room temperature with running water, add 1 drop of phenolphthalein indicator, and use a concentration of 6 mol·L -1 Titrate the NaOH solution until the solution turns light red, and dilute to 10 mL with pure water as the total sugar determination sample solution. Take 200 µL of the total sugar determination sample solution, add 500 µL of the DNS reagent prepared in advance, mix well, and boil in a water bath for 5 min. Immediately remove the running water and cool to room temperature. After shaking well, measure the absorbance at 540 nm. The total sugar content is expressed as glucose equivalent.

[0064] (9) The soluble solids content (Brix) was directly measured using a handheld refractometer, and each group of samples was measured three times.

[0065] Experimental results: (1) Comparison of juice yield like Figure 1 As shown in the figure, the juice yield of different litchi varieties varies from 54.84 ± 1.01 to 61.22 ± 1.11%, among which the juice yield of litchi Jizui is the highest at 61.22 ± 1.11%. The other three varieties are Huaizhi, Guiwei and Nuomici from high to low.

[0066] (2) Processing characteristics of litchi peel Litchi peel also contains a large number of bioactive phenolic compounds in free and bound forms. This study investigated the contents of total phenols, total flavonoids and total anthocyanins in the peels of four types of litchi. Figure 2As shown in the figure, the total phenol content in litchi peel ranged from 8.22 ± 0.12 to 1.26 ± 0.15 mg / g, with the highest content in Huaizhi peel being 11.26 ± 0.15 mg / g, and the lowest content in Guiwei litchi peel. Figure 3 As shown in the figure, the total flavonoids content ranged from 2.81 ± 0.11 to 4.12 ± 0.13 mg / g, with the highest content in the lychee peel and the lowest content in the guiwei lychee peel. Figure 4 As shown, the content of total anthocyanins varied from 0.25 ± 0.05 to 1.03 ± 0.05 mg / g, with Huaizhi peel having the highest content and Guiwei litchi peel having the lowest content.

[0067] (3) Changes in the number of live bacteria of LP-100 during the fermentation of different litchi juices like Figure 5 As shown in the figure, LP-100 has good environmental adaptability in the four types of lychee juices, and the number of viable bacteria increases rapidly within 6-30 hours, and the number of viable bacteria can reach 10.0 lg CFU / ml at 30 hours. However, within 12-36 hours of fermentation, the growth rate and number of viable bacteria in Huaizhi lychee juice are significantly lower than those in the other three types of lychee juices.

[0068] (4) Changes in the content of total phenols and total flavonoids in LP-100 during different litchi juice fermentation processes like Figure 6 As shown in the figure, as the fermentation progressed, the overall change in the total phenol content in the four types of lychee juices showed a trend of first increasing and then decreasing. The total phenol content of the chicken mouth lychee juice was the highest compared to the other three, and it remained at the highest level during the 0-48 h fermentation process. Figure 7 As shown in the figure, the overall change of total flavonoids content in litchi juice during the fermentation process showed a trend of first decreasing and then maintaining stability, and the total flavonoids content in chicken-mouth litchi juice also remained at the highest level.

[0069] (5) Comparison of LP-100's ability to reduce blood sugar and produce acid in different litchi juices In order to explore the ability of Lactobacillus plantarum LP-100 to reduce sugar and produce acid in four different litchi juices, the residual sugar content and total acid content at different time points during the fermentation process were measured. Figure 8As shown in the figure, as the fermentation progressed from 0 to 48 h, the total sugar content of the four types of lychee juices continued to decrease. The three varieties of Guiwei, Nuomici, and Huaizhi were similar. The fermentation was relatively slow from 0 to 18 h, and the total sugar consumption was relatively small. The fermentation speed accelerated from 18 to 36 h, and the sugar consumption was relatively large. It gradually stabilized from 36 to 48 h. The fermentation of "Jizui" was relatively slow from 0 to 12 h, but the total sugar consumption rate was significantly accelerated after 12 h, and the total sugar decreased significantly faster after 18 h than the other three. The acid production of LP-100 during the fermentation process is shown in the figure. Fig. 9 As shown in the figure, as the fermentation progressed, the total acid content increased. The total acid content increased rapidly from 0 to 30 h, and the total acid content continued to increase after 30 h of fermentation, but the rate was relatively slow. The acid production rate of LP-100 in Chicken Mouth and Guiwei after 12 h was significantly faster than that of the other two. From a comprehensive analysis, the initial sugar content of "Chicken Mouth" was moderate, but the residual sugar content was the lowest, and the ability to reduce sugar and produce acid was strong, making it suitable for fermentation by Lactobacillus plantarum LP-100.

[0070] (6) Effect of LP-100 on pH and solid content of different litchi juices The changes of pH value during fermentation Fig.10 As shown in the figure, the initial pH values ​​of the four types of lychee juices were between 5.1 and 5.2. As the fermentation progressed, acid was continuously produced during the process, and the pH values ​​of the four types of juices showed a downward trend. After 30 hours, the pH value stabilized at about 3.6. Fig.11 As shown in the data, the Huaizhi lychee juice remained relatively stable during the fermentation process, while the soluble solids content of the Nuomici, Guiwei and Jizui lychee juices decreased to varying degrees, with a more obvious decrease from 0 to 12 h and remained relatively stable from 12 to 48 h.

[0071] Taking all these circumstances and economic factors into consideration, among the four types of lychees purchased, the chicken-bill lychee is the most suitable for processing into lychee juice with high nutritional added value and rich in active lactic acid bacteria, which is fermented together with the peel and juice.

[0072] Example 2: Study on Optimization of Litchi Juice Fermentation Process by Lactobacillus Plantarum Main Materials: (1) Chicken beak lychee juice (2) Bacterial strain: Lactobacillus plantarum LP-100 is a strain preserved in our laboratory Experimental methods: (1) Determination of total phenols: Refer to the method in Example 1 (2) Evaluation of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging ability: The fermentation broth was reacted with DPPH solution in dark conditions for 30 min, and the absorbance was measured at 517 nm to calculate the DPPH free radical scavenging rate. The calculation formula is as follows (1): Where A1 and A0 are the absorbance of the sample and the control, respectively. (1) (3) Evaluation of 2,2'-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) free radical scavenging ability: The fermentation broth was reacted with ABTS solution in dark conditions for 10 min, and the absorbance was measured at 734 nm to calculate the ABTS free radical scavenging rate. The calculation formula is as follows (2): Where A1 and A0 are the absorbance of the sample and the control, respectively. ABTS (2) (4) Determination of total superoxide dismutase (SOD) activity: Determination was performed according to the method provided in the SOD kit (Nanjing Jiancheng) manual, and the method was appropriately improved. The amount of SOD corresponding to the SOD inhibition rate of 50% per milliliter of sample was taken as a SOD activity unit (U). The main determination steps are as follows: (a) Sample pretreatment: Fermented litchi juice was treated with ultrasound at 300 W for 2 min with a time interval of 2 s. The sample was then centrifuged at 6000 r / min for 5 min and the supernatant was collected for later use.

[0073] (b) Preparation of relevant reagents: The color developing solution is prepared in a volume ratio of reagent 5: reagent 6: glacial acetic acid = 3:3:2. To reduce errors, reagent 2, reagent 3 and reagent 4 application solution are mixed in advance in a volume ratio of 1:1:1.

[0074] (c) The sample was kept in a 37°C water bath for 10 min and the operation was performed according to Table 1.

[0075] Table 1: Order of reagent addition

[0076] The color developer was added, mixed using a vortexer, and then allowed to stand at room temperature for 10 min. The absorbance was measured at 550 nm.

[0077] (d) Total SOD activity was calculated as follows: Total SOD activity (U / mL) ÷ 50% × reaction system dilution factor × sample dilution factor The following steps were used to optimize the fermentation process: (1) Effect of fermentation temperature on LP-100 fermented litchi juice Use pre-activated LP-100 for inoculation (the number of viable bacteria is at least 6.5 lg CFU / ml), ensure the same fermentation level conditions, inoculation amount 2.0%, fermentation time 24 h, initial pH value 5.5, and control the fermentation temperature at 28, 31, 34, 37, 40, 43, and 46 ℃ for fermentation. The total phenol content, total SOD activity, DPPH free radical scavenging rate, and ABST free radical scavenging rate of litchi fermented juice at different temperatures were determined, and each group of treatments was repeated three times to determine the optimal fermentation temperature of litchi juice.

[0078] Experimental results: Fig.12 As shown in the figure, when the fermentation temperature was 34 ℃, the total phenol content in the litchi fermented juice was the highest at 27.18±0.15 mg / 100mL. As the fermentation temperature increased, the total phenol content in the litchi fermented juice showed a trend of first increasing and then decreasing. Fig.13 As shown in the results, the total SOD activity of fermented litchi juice increased first and then decreased with the increase of fermentation temperature, ranging from 80.28 ± 0.27 to 110.71 ± 0.23 U / mL. The SOD activity in fermented litchi juice reached its maximum at 34°C, and then with the increase of fermentation temperature, when the fermentation temperature was greater than 34°C, the total SOD activity continued to decrease.

[0079] Effect of fermentation temperature on scavenging capacity of DPPH and ABST in fermented litchi juice Fig.14 ,15, with the increase of fermentation temperature, the DPPH scavenging ability and ABST scavenging ability of fermented litchi juice gradually increased; when the fermentation temperature was 34℃, the scavenging rates of the two reached the highest, 42.34 ± 0.03% and 85.62 ± 0.02%, respectively. When the temperature range was 34-40℃, the DPPH scavenging ability and ABST scavenging ability had a small change trend.

[0080] (2) Effect of fermentation time on LP-100 fermented litchi juice According to the fermentation conditions mentioned above, the same fermentation level was ensured, and the fermentation time was controlled to be 0, 12, 18, 24, 30, 36, and 42 h. The total phenol content, total SOD activity, DPPH free radical scavenging rate, and ABST free radical scavenging rate of litchi fermented juice at different times were determined. Each group of treatments was repeated three times to determine the optimal fermentation time of litchi juice.

[0081] Experimental results: The total phenol content in litchi fermented juice increased first and then decreased with the extension of fermentation time, and reached a maximum of 27.55 ± 0.13 mg / 100mL at 24 h. When the fermentation time was greater than 30 h, the total phenol content decreased faster. Fig.16As shown in Figure 2, compared with 0 h, as the fermentation time increased, the DPPH·scavenging rate of fermented litchi juice gradually increased, and the main changes occurred in 0-18 h, while 18-42 h was relatively stable. Fig.17 As shown in the figure, compared with 0 h, the total SOD activity in the fermented litchi juice gradually increased with the progress of fermentation. When the fermentation time was 24 h, the enzyme activity reached a maximum of 106.18 ± 0.12 U / mL.

[0082] like Fig.18 As shown in the figure, after 18 h of fermentation, the DPPH clearance rate increased by nearly 25%. Fig.19 The change of ABST· clearance rate shown in the figure shows that during 0-18 h, as the fermentation progresses, the ABST· clearance capacity decreases, and then there is an upward trend during 18-30 h, and it remains relatively stable during 30-42 h. Based on the above index results, 24 h was selected as the optimal fermentation time of Lactobacillus plantarum LP-100 in chicken mouth lychee juice, and 18-30 h was selected for response surface analysis. Based on the above index results, 24 h was selected as the optimal fermentation time of Lactobacillus plantarum LP-100 in chicken mouth lychee juice, and 18-30 h was selected for response surface analysis.

[0083] (3) Effect of inoculation amount on LP-100 fermentation of litchi juice According to the fermentation conditions mentioned above, the same fermentation level was ensured, and the inoculation amount was controlled to be 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% for fermentation. The total phenol content, total SOD activity, DPPH free radical scavenging rate, and ABST free radical scavenging rate of litchi fermented juice under different inoculation amounts were determined, and each group of treatments was repeated three times to determine the optimal inoculation amount of litchi juice.

[0084] Experimental results: Fig. 20 ,21, fermented at different inoculum concentrations for the same fermentation time, the total phenol content at the end of fermentation was different. When the inoculum concentration was 1.5%, the total phenol content of fermented litchi juice reached a maximum of 28.17 ± 0.27 mg / 100mL. When the inoculum concentration was further increased, the total phenol content gradually decreased. The total SOD activity also showed a trend of increasing first and then decreasing with the increase of the inoculum concentration. When the inoculum concentration was 1%, the total SOD activity reached a maximum of 97.99 ± 0.80 U / mL.

[0085] Effects of inoculum dose on DPPH·scavenging capacity and ABST·scavenging capacity Fig. 22, 23, when the inoculum dose was 1.5-2.5%, the DPPH clearance rate was relatively high, and the effect of the inoculum dose on the DPPH clearance ability was relatively small; the ABST clearance rate generally showed a downward trend with the increase of the inoculum dose, but remained relatively stable when the inoculum dose was in the range of 1.5-2.5%.

[0086] (4) Effect of initial pH value on LP-100 fermented litchi juice According to the fermentation conditions mentioned above, the same fermentation level was ensured, and the pH value of litchi juice was adjusted using 2 mol / L citric acid solution and 2 mol / L sodium carbonate solution, and the initial pH values ​​were controlled to be 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 for fermentation. The total phenol content, total SOD activity, DPPH free radical scavenging rate, and ABST free radical scavenging rate of litchi fermented juice at different initial pH values ​​were determined, and each treatment group was repeated three times to determine the optimal initial pH value of litchi juice.

[0087] Experimental results: pH is a significant factor affecting the growth of microorganisms. Generally, acidic conditions are more conducive to the growth of Lactobacillus plantarum. At the same time, pH also has a great influence on the accumulation of products. Fig.24 , 25 showed that when the initial pH value of litchi juice was 5-6, the total phenol content and total SOD activity were at a relatively high level. When the pH value was 4-4.5, Lactobacillus plantarum LP-100 may have poor survival ability and produce less SOD through metabolism.

[0088] Effects of initial pH on DPPH clearance and ABST clearance Fig.26 , 27, as shown in Figure 2, with the increase of pH, the DPPH· scavenging rate and ABST· scavenging rate generally showed a decreasing trend. The scavenging rate of the two free radicals by fermented lychee juice was better when the initial pH value was lower, which may be because the antioxidants in lychee juice were more stable at a lower pH. The DPPH· scavenging rate was relatively stable at a pH of 5.5-6.0, while the ABST· scavenging rate was relatively stable at a pH of 5-5.5.

[0089] (5) Response plane analysis experiment of litchi juice fermentation process Based on the single factor test as shown in Table 2, the Box-Behnken test design was performed using Design-Expert software.

[0090] Table 2: Factors and levels of response surface experiment for fermentation process optimization

[0091] Experimental results: Considering that there may be complex interactions between these parameters, the results of single-factor experiments were used as the basis, and total phenol content and total SOD activity were used as experimental response values ​​to explore the effects of the interaction between the fermentation time (A), fermentation temperature (B), inoculation amount (C), initial pH value (D) and the optimal fermentation combination conditions on the LP-100 fermentation process of lychee juice. Through Box~Behnken design experiments and response surface analysis, the experimental design and results are shown in Table 3.

[0092] Table 3: Box~Behnken test design and results

[0093] The obtained data were subjected to regression analysis using software, and the results are shown in Table 4.

[0094] Table 4: Box-Behnken experimental design results regression model variance analysis

[0095] Design-Expert 8.0 response surface software was used to perform regression analysis on the test results data in Table 4, that is, the measured experimental data were fitted using multivariate linear regression technology to obtain the second-order response surface regression equation.

[0096] The regression equation of total phenol content and total SOD activity in fermented litchi juice is as follows: (1) Total phenol content = +29.15 -0.37A +0.45B +0.52C -0.69D- 1.66AB +1.32AD +1.06BD -2.76 CD -2.51A 2 -1.98B 2 -3.15 C 2 -2.69D 2 (2) Total SOD activity = +103.96 +0.97A -0.62 B +0.64C +0.021D -2.05AC -1.97BC -5.31A 2 -7.92B 2 -3.74C 2 -3.59D 2 A, B, C, and D represent fermentation time, fermentation temperature, inoculation amount, and initial pH, respectively. The equation is used to indicate that there is a significant relationship between fermentation temperature, fermentation time, inoculation amount, pH value, and total phenol content and total SOD activity of fermented litchi juice ( P<0.0001). The F values ​​of the regression models for total phenol content and total SOD activity were 17.86 and 27.42, respectively, indicating that there was a significant relationship between the independent variable and the dependent variable. Therefore, these two models can be used to predict the total phenol content and total SOD activity in fermented litchi juice.

[0097] Table 5: Experimental data analysis of the prediction model

[0098] The model was analyzed by ANOVA. As shown in Table 5, the regression model equation of total phenol and total SOD activity is P The values ​​are all <0.01 and extremely significant, while the lack of fit terms of the two models P The values ​​are 0.2818 and 0.8651, both greater than 0.05, which are not significant, indicating that the coefficients of the established model are significantly fitted to the response data. As shown in Table 5, the determination coefficient R 2 All greater than 0.93, adjustment coefficient R 2 Adj was greater than 0.87, indicating that several experimental factors had significant effects on total phenolic content and total SOD activity in litchi fermented juice. CV is a measure of model reproducibility as the ratio of the residual variation of the data to the mean size.

[0099] The model was optimized and solved using Design-Expert statistical software. The optimal process conditions for litchi juice fermentation were recommended to achieve the highest total phenol content and total SOD activity at the end of fermentation. The fermentation time was 23.98 h, the fermentation temperature was 33.95 ℃, the inoculation amount was 1.55%, and the initial pH was 5.45. Under these conditions, the theoretical values ​​of total phenol content and total SOD activity after fermentation were 29.23 mg GAE / 100 mL and 103.95 U / mL, respectively. According to the actual operating conditions in the experiment, the parameters of the recommended fermentation process were adjusted to: fermentation time 24 h, fermentation temperature 34 ℃, inoculation amount 1.5%, and initial pH 5.5. The model was verified, and the actual total phenol content and total SOD activity were 28.89 ± 0.21 mg GRE / 100 mL and 105.35 ± 0.94 U / mL, respectively, which were very close to the theoretical values. The optimal fermentation process conditions for litchi juice fermentation obtained through the response surface method optimization analysis are feasible. Therefore, the optimal process route for fermenting litchi juice was finally selected as the fermentation time of 24 h, the fermentation temperature of 34 ℃, the inoculation amount of 1.5%, and the initial pH value of 5.5.

[0100] In summary, the optimal fermentation process of Lactobacillus plantarum LP-100 for lychee juice is: fermentation time 24 h, fermentation temperature 34 ℃, inoculation amount 1.5%, and initial pH value 5.5. The optimal fermentation process was verified, and it was found that the regression model was feasible and could be used to predict the total phenol content and total SOD activity in the fermented lychee juice at the end of fermentation. The research results can provide a scientific basis for the development of functional lychee fermented juice and the comprehensive utilization of lychee resources.

[0101] Example 3: Research process of ultra-high pressure pre-treatment and heat treatment on the quality of fermented lychee juice (including peel) Experimental groups: (1) Heat treatment and fermentation of litchi juice (2) Lychee juice ultra-high pressure fermentation group (3) Lychee juice (+0.25% lychee peel) heat treatment fermentation group (4) Lychee juice (+0.25% lychee peel) ultra-high pressure fermentation group The heat treatment conditions were 85 °C for 10 min and the ultra-high pressure treatment conditions were 400 MPa for 10 min.

[0102] Experimental methods: (1) Determination of total phenols: the same method as in Example 1.

[0103] (2) Determination of total SOD activity: the same as the determination method in Example 2.

[0104] (3) Total sugar determination: Same as the determination method in Example 1 (4) Determination of reducing sugar: 3,5-dinitrosalicylic acid colorimetric method (DNS) was used for determination, referring to the method of Liao Xiangbing et al. (Liao Xiangbing, Chen Xiaoming, Xiao Wei et al. Wavelength selection for quantitative determination of reducing sugar by DNS method [J]. Chinese Agricultural Science Bulletin, 2017, 33(15): 144-149.).

[0105] (5) Evaluation of hydroxyl radical scavenging ability: The test was carried out according to the instructions of the hydroxyl radical scavenging rate test kit (Source Leaf Biotechnology), and appropriate improvements were made. The main steps are as follows: (a) Fermented litchi juice was centrifuged at 5000 r / min for 5 min and the supernatant was collected for later use.

[0106] (b) Set up undamaged tubes, damaged tubes, and sample tubes according to the table below. Add reagents in sequence according to Table 6. Set up 3 parallel sample groups.

[0107] Table 6: Order of reagent addition

[0108] (c) After each tube is shaken and placed on a 37°C shaker for 1 hour, 200 µL of the solution in each tube is added to a 96-well plate and the absorbance at 536 nm is measured and recorded as A1, A2, and A3, respectively. The calculation is:

[0109] In the above formula, A1 is the absorbance value of the undamaged tube; A2 is the absorbance value of the damaged tube; A3 is the absorbance value of the sample tube.

[0110] (6) Determination of α-amylase and α-glucosidase inhibition rate 1) Sample processing: Litchi fermented juice was centrifuged at 6000 r / min for 5 min and the supernatant was taken for later use 2) Determination of α-amylase inhibition rate: 100 µL of litchi fermentation juice supernatant and 200 µL of α-amylase solution with an enzyme activity of 2 U / mL were mixed and reacted in a 37°C water bath for 15 min. α-amylase was dissolved in PBS. Then 100 µL of soluble starch solution with a mass concentration of 1% was added and mixed in a 37°C water bath for 15 min. Then 100 µL of DNS reagent was added and placed in boiling water to terminate the reaction. After 5 min, it was immediately cooled and the volume was adjusted to 10 mL. The absorbance value was measured at 504 nm and recorded as sample A. The control group sample was replaced with PBS, and the absorbance value was recorded as control A. Calculate according to the following formula:

[0111] 3) Determination of α-glucosidase inhibition rate: Take 100 µL of the sample to be tested and add it to 500 µL PBS, mix well, then add 200 µL of α-glucosidase solution and place in a water bath at 37 °C for 15 min. Then add 400 µL PNPG solution (20mmol / L), continue to place in a water bath for 10 min, and finally add 1 mL of 1 mol / L Na2CO3 solution to terminate the reaction. The absorbance value at 405nm is measured and recorded as A sample. The control group sample is replaced with PBS, and the absorbance value is recorded as A control. Calculate according to the following formula:

[0112] (7) Determination of extracellular polysaccharide content Extraction of exopolysaccharide (EPS) from fermented litchi juice: After the fermented juice was shaken, 500 µL of fermented litchi juice was mixed with 300 µL of 40% trichloroacetic acid, placed in a -20 °C refrigerator for 20 min, then placed at 4 °C for 1 h, centrifuged at 12,000 rpm for 10 min to remove bacteria and foreign proteins, and after centrifugation, 200 µL of supernatant was mixed with 1 mL of ice-cold anhydrous ethanol and placed at 4 °C for 2 h, and then centrifuged at 12,000 rpm / min for 10 min to obtain polysaccharide precipitate, and then washed with 1 mL of 75% ethanol. Finally, 1 mL of pure water was used to dissolve the extracted polysaccharide precipitate.

[0113] (8) Determination of browning degree The fermented litchi juice was centrifuged at 12000 rpm / min for 10 min and the supernatant was taken. The absorbance value was measured at 420 nm to characterize the browning degree. The reference sample was distilled water and the measurement was repeated three times.

[0114] Experimental results: (1) Changes in viable bacterial count The changes of viable bacteria count during fermentation in different treatment groups are shown in Figure 2. Fig.28 As shown in the figure, after different treatments, the change trend of the number of live bacteria in each group is consistent. From 0 to 24 h, the nutrients in litchi juice are rich, and Lactobacillus plantarum continues to grow. At the end of 24 h fermentation, the number of live bacteria in each group can reach 10.0 lg CFU / mL. However, from 0 to 12 h, the number of live bacteria and its growth rate in the ultra-high pressure treatment fermentation group were significantly faster than those in the heat treatment group, which shows that the growth rate of Lactobacillus plantarum LP-100 in litchi juice is accelerated after ultra-high pressure treatment.

[0115] (2) Changes in total phenol content and total SOD activity The changes of total phenol content after fermentation Fig.29 As shown in the figure, after fermentation, the total phenol content showed an increasing trend, and the total phenol content of the ultra-high pressure fermentation group increased by 15.12% compared with the non-fermentation group ( P <0.05). The total phenol content of the ultra-high pressure treated peel fermentation group increased by 9.76% compared with the heat treated peel fermentation group ( P <0.05). This indicates that compared with heat treatment before fermentation, ultra-high pressure treatment is more conducive to the release of phenolic substances during the fermentation process. In addition, the total phenol content of the ultra-high pressure treated peel fermentation group and the heat treated peel fermentation group increased by 19.83% and 19.30% respectively compared with the corresponding non-peel fermentation group. This is because the peel contains many bound phenolic substances, which will be released into the system under the action of LP-100. The changes in the total SOD activity of litchi fermented juice are shown in Figure 2. Fig.30 As shown in Figure 2, it can be found that compared with the ultrahigh pressure pre-treatment, the total SOD activity of litchi juice after heat treatment decreased by 14.4% ( P <0.05). This indicates that heat treatment will directly lead to a decrease in the total SOD activity of litchi juice. After fermentation, compared with the unfermented group, the total SOD activity of each fermentation group showed different degrees of improvement ( P <0.05), the total SOD activity of the ultra-high pressure fermentation group was 15.46% higher than that of the heat treatment fermentation group ( P <0.05), which indicates that after ultrahigh pressure treatment, LP-100 produced more SOD during the fermentation process.

[0116] (3) Changes in total sugar and reducing sugar The changes of total sugar and reducing sugar in litchi juice before and after fermentation Fig.31 As shown in the results, ultra-high pressure treatment did not significantly affect the total sugar content and reducing sugar content in litchi juice. Before fermentation, the total sugar content of litchi juice was 236.76 ± 1.52 g / L, and the reducing sugar content was 64.93 ± 2.39 g / L. After fermentation, the total sugar content decreased by about 55.7%, and the reducing sugar content increased by 24.33% ( P <0.05).

[0117] (4) Changes in α-amylase and α-glucosidase inhibition rates α-Amylase is an important enzyme in the digestion of starch and glycogen and is a useful strategy for treating abnormal carbohydrate intake. α-Glucosidase is present on the surface of the intestinal cell cavity and is secreted in the small intestine. It is a key enzyme that catalyzes the hydrolysis of disaccharides (maltose and sucrose) into monosaccharides (glucose and fructose). For the two blood sugar lowering indicators, the changes in α-amylase and α-glucosidase inhibition rates before and after fermentation are shown in Figure 2. Fig.32 , 33, after fermentation, compared with the non-fermented group, the α-amylase inhibition ability of each fermentation group was significantly enhanced (P<0.05), and the α-amylase inhibition rate of the ultra-high pressure fermentation group was 30.05% higher than that of the heat treatment fermentation group. The α-glucosidase inhibition rate also increased significantly after fermentation. Compared with the corresponding non-fermented group, the α-glucosidase inhibition rates of the ultra-high pressure fermentation group and the heat treatment fermentation group increased by 257.83% and 228.32%, respectively. In addition, compared with the corresponding non-fermented group, the α-glucosidase inhibition rates of the ultra-high pressure peel fermentation group and the heat treatment peel fermentation group increased by 89.04% and 51.50%, respectively.

[0118] (5) Changes in free radical scavenging rate As shown in Table 7, the DPPH radical scavenging rates of each group increased to varying degrees after fermentation. The DPPH· scavenging rate of the ultra-high pressure treatment group was higher than that of the heat treatment group but not significantly. After the fermentation, the DPPH· scavenging rate of the ultra-high pressure treatment fermentation group increased by 24.94% compared with the heat treatment group. The DPPH· scavenging rate of the ultra-high pressure treatment peel fermentation group was the highest, and the DPPH· scavenging rate increased by 202.46% compared with the heat treatment non-fermentation group. Although the ABST· free radical scavenging rate of fermented lychee juice decreased compared with that before fermentation, the scavenging rate was still above 80%. The hydroxyl radical scavenging rate increased after fermentation. The ultra-high pressure treatment fermentation group had a 17.27% higher OH· scavenging capacity than the heat treatment fermentation group. The hydroxyl radical scavenging rate of the ultra-high pressure treatment peel fermentation group was as high as 90.10%, which was more than 1 times that of the heat treatment non-fermentation group. At the same time, the free radical scavenging capacity of the fermentation group after adding peel was improved compared with the fermentation group without peel. Comprehensive analysis shows that ultra-high pressure pre-treatment and fermentation after adding peel are beneficial to improving the scavenging ability of fermented lychee juice on these three free radicals.

[0119] Table 7: Changes in free radical scavenging rate before and after fermentation

[0120] (6) Changes in extracellular polysaccharide content The changes of extracellular polysaccharide content after fermentation Fig.34 As shown, the EPS content of the ultra-high pressure fermentation group and the heat treatment fermentation group was 2.98 times and 2.38 times higher than that before fermentation, respectively. The extracellular polysaccharide content in the fermented juice of the ultra-high pressure pre-treatment fermentation group was significantly higher than that of the heat treatment group ( P <0.05).

[0121] (7) Effect of ultrahigh pressure on browning degree during storage like Figure 35-37 As shown in the figure, from the browning degree before fermentation, the browning degree of the heat treatment group is significantly greater than that of the ultra-high pressure treatment group, indicating that the heat treatment process directly causes the browning of litchi juice, while non-heat treatment processes such as ultra-high pressure will not cause browning. The browning degree after storage for 21 days after fermentation is significantly less than that of the unfermented group. From this point of view, the browning process can be slowed down after fermentation, and fermentation after ultra-high pressure treatment can also inhibit the progress of browning to a certain extent compared with fermentation after heat treatment. After adding the peel, the dissolution of the coloring substance in the peel causes the litchi juice to appear reddish brown, which is the reason why the browning degree is higher than that of the group without peel, but the browning degree of the litchi juice is also reduced after fermentation. Therefore, ultra-high pressure pre-treatment can achieve the purpose of maintaining the color of litchi juice by slowing down the browning process.

[0122] Example 4: Preparation of active lactic acid bacteria beverage co-fermented with lychee juice and peel Based on the above-mentioned embodiments 1 to 3, a method for preparing an active lactic acid bacteria beverage co-fermented with lychee juice and peel is determined, comprising the following steps: A. Wash fresh lychees, peel, remove the core, squeeze the juice, filter, centrifuge to obtain lychee juice, sterilize and set aside; B. vacuum freeze-drying the lychee peel separated in step A, crushing it into powder, sieving it, and setting it aside; C. Inoculate the activated Lactobacillus plantarum L100 at a rate of 1.5% (v / v) into the lychee juice obtained in step A, add the lychee peel powder obtained in step B at a mass volume ratio of 0.25%, treat with ultrahigh pressure, shake well, and place in a 34°C incubator for static fermentation for 24 hours; D. Take samples every 6 hours to measure physical and chemical indicators. When the total viable bacteria count reaches at least 10.0 lg CFU / ml, the fermentation is terminated to obtain the active lactic acid bacteria beverage co-fermented with lychee juice and peel.

[0123] Preferably, in step A, the following litchi varieties can be used: Jizui litchi, Guiwei litchi, Huaizhi litchi and Nuomici litchi. More preferably, Jizui litchi is used.

[0124] Preferably, in step A, the juice is filtered using a 100-mesh filter cloth, and the filtered juice is centrifuged at 4500 r / min for 5 minutes.

[0125] Preferably, in step A, the sterilization treatment is performed at 85° C. for 10 minutes.

[0126] Preferably, in step B, the sieving is through a 60-mesh sieve.

[0127] Preferably, in step C, the viable count of activated Lactobacillus plantarum L100 is at least 6.5 lg CFU / ml.

[0128] Preferably, in step C, the ultrahigh pressure treatment is 400 MPa for 10 minutes.

[0129] According to the experimental results of the above embodiments, the active lactic acid bacteria beverage obtained by co-fermenting lychee juice and peel by the preparation method of the present invention can reduce the sugar content of lychee with the rapid proliferation of lactic acid bacteria, increase the sour taste to enhance the flavor of lychee, improve the antioxidant activity of lychee juice, increase storage stability, and significantly increase the nutritional value of the fermented juice.

Claims

1. A method for preparing an active lactic acid bacteria beverage co-fermented with lychee juice and peel, characterized in that: The following steps are involved: A. Wash fresh lychees, peel, remove the core, squeeze the juice, filter, centrifuge to obtain lychee juice, sterilize and set aside; B. vacuum freeze-drying the lychee peel separated in step A, crushing it into powder, sieving it, and setting it aside; C. Inoculate the activated Lactobacillus plantarum L100 at a rate of 1.5% (v / v) into the lychee juice obtained in step A, add the lychee peel powder obtained in step B at a mass volume ratio of 0.25%, treat with ultrahigh pressure, shake well, and place in a 34°C incubator for static fermentation for 24 hours; D. Take samples every 6 hours to measure physical and chemical indicators. When the total viable bacteria count reaches at least 10.0 lg CFU / ml, the fermentation is terminated to obtain the active lactic acid bacteria beverage co-fermented with lychee juice and peel.

2. The preparation method according to claim 1, characterized in that: In step A, the lychee is selected from one of the following lychee varieties: Jizui lychee, Guiwei lychee, Huaizhi lychee and Nuomici lychee.

3. The preparation method according to claim 2, characterized in that: The lychee is the chicken-mouth lychee.

4. The preparation method according to claim 1, characterized in that: In the step A, the juice is filtered with a 100-mesh filter cloth, and the filtered juice is centrifuged at 4500 r / min for 5 minutes.

5. The preparation method according to claim 1, characterized in that In the step A, the sterilization treatment is performed at 85° C. for 10 minutes.

6. The preparation method according to claim 1, characterized in that: In the step B, the sieving is through a 60-mesh sieve.

7. The preparation method according to claim 1, characterized in that: In the step C, the viable count of the activated Lactobacillus plantarum L100 is at least 6.5 lg CFU / ml.

8. The preparation method according to claim 1, characterized in that: In the step C, the ultrahigh pressure treatment is performed at 400 MPa for 10 minutes.

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