Method for preparing sour cherry fermented fruit juice and probiotic fruit powder through sequential fermentation and deacidification of saccharomycetes and lactic acid bacteria

Abstract

The invention discloses a method for preparing sour cherry fermented fruit juice and probiotic fruit powder through sequential fermentation and deacidification of saccharomycetes and lactic acid bacteria. The method comprises the following steps: S1, inoculating saccharomycetes into sour cherry juice for primary fermentation to obtain fermentation liquor; and S2, inoculating lactic acid bacteria into the fermentation liquid obtained in the step S1, and carrying out secondary fermentation to obtain the sour cherry fermented fruit juice. Furthermore, a drying aid is added into the sour cherry fermented fruit juice, and spray drying is performed after homogenization treatment to obtain the sour cherry probiotic fruit powder. The content of total acid and malic acid in the sour cherry fruit juice can be effectively reduced, the product is endowed with special fermentation flavor, the fruit juice is dried through a spray drying method, the storage period of the sour cherry probiotic fruit powder is prolonged, and the survival rate of probiotics is increased. The method not only effectively maintains the nutritional quality of the sour cherries, but also widens the application field of the sour cherries.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a method for preparing sour cherry fermented juice and probiotic fruit powder by sequential fermentation and acid reduction of yeast and lactic acid bacteria. Background Art

[0002] Sour cherry, belonging to the genus Prunus in the Rosaceae family, is a perennial temperate economic tree of the drupe family. It has high yields, and its fruit is oblate or spherical, with a bright red or purple-red skin. Sour cherry is rich in sugars, organic acids, vitamins, and phytochemicals such as flavonoids, phenolic acids, anthocyanins, and melatonin. It can play an anti-inflammatory, analgesic, anti-oxidant, and prevent cardiovascular and cerebrovascular diseases, and is known as a "super fruit." The most abundant organic acid in sour cherry is malic acid, which is pleasant within a certain acid concentration range, but too much acid in sour cherry, especially malic acid, will make the taste too thorny and sour, resulting in poor fresh palatability and difficult to be accepted by consumers.

[0003] At present, the common methods of acid reduction include chemical acid reduction, physical acid reduction and biological acid reduction. Chemical and physical methods have adverse effects on the flavor and color of juice, while the two major biological acid reduction methods, malic acid-lactic acid fermentation (MLF) represented by lactic acid bacteria and malic acid-ethanol fermentation (MAF) represented by yeast, can not only reduce the impact of the acid reduction process on the quality and stability of juice, but also give the juice a unique ester aroma and lactic acid flavor.

[0004] pH is an important factor affecting the malic acid-lactic acid fermentation of lactic acid bacteria. When the pH is lower than 3.2, the growth of lactic acid bacteria is inhibited, thus affecting the acid reduction effect. The pH value of sour cherry juice is generally 3.0-3.2. The growth of lactic acid bacteria is inhibited in this high acid environment, making it difficult to successfully start the malic acid-lactic acid fermentation of sour cherry. The growth of acid-reducing yeast in sour cherry juice is not restricted and can start malic acid-ethanol fermentation, but the long fermentation cycle leads to a large accumulation of alcohol, affecting the flavor of the juice. At present, there are few reports on the research of sequential acid reduction fermentation of sour cherries by yeast and lactic acid bacteria. Combining sour cherries with lactic acid bacteria products has good use value and economic value.

[0005] The shelf life of live-bacteria fermented sour cherry juice is short and needs to be circulated under the cold chain. Making it into sour cherry probiotic powder can extend the shelf life, reduce transportation costs, and ensure the quality of sour cherry fermented powder. Therefore, it is very necessary to propose a method that can effectively reduce the acidity of sour cherry juice and extend the shelf life of sour cherry juice fermented products. Summary of the invention

[0006] The invention aims to provide a method for preparing sour cherry fermented juice and probiotic fruit powder by sequentially fermenting sour cherry with yeast and lactic acid bacteria to reduce acidity. The method uses sour cherry as a raw material and sequentially ferments with yeast and Lactobacillus rhamnosus, which can improve the degradation rate of organic acid, retain sour cherry functional components, increase the flavor of sour cherry products, enrich the types of sour cherry products, provide a theoretical basis for the production of probiotic fermented fruit and vegetable powder related products, and provide a sour cherry acid reduction process idea.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing sour cherry fermented juice by sequential fermentation with yeast and lactic acid bacteria to reduce acidity, comprising the following steps:

[0009] S1. Inoculating yeast into sour cherry juice for the first fermentation to obtain fermentation liquid;

[0010] S2, inoculating lactic acid bacteria into the fermentation liquid obtained in step S1 for a second fermentation to obtain the sour cherry fermented juice.

[0011] Based on the above technical scheme, the present invention adopts yeast and lactic acid bacteria to sequentially ferment and reduce acid to prepare sour cherry fermented juice: on the one hand, compared with the acid reduction by inoculating yeast alone, the sequential fermentation technology can greatly shorten the fermentation cycle and reduce the production of alcohol during the fermentation process; on the other hand, it overcomes the technical difficulty that lactic acid bacteria are difficult to start malic acid-lactic acid fermentation. The method of the present invention can improve the degradation rate of organic acid in sour cherry juice, reduce the overall acidity of the juice, and increase the flavor of the juice.

[0012] In the above method, the sour cherry juice is sour cherry puree, preferably prepared by removing pits from sour cherries, crushing and beating them, and then hydrolyzing them with pectinase. As an example, after removing pits from sour cherries, crushing and beating them, 0.05% pectinase is added for hydrolysis for 2 hours and filtered to obtain sour cherry juice, and then 0.15% ascorbic acid is added to the sour cherry juice to obtain sour cherry juice.

[0013] In the above method, further, the yeast is 71B yeast. Further, the conditions of the first fermentation are as follows: the fermentation temperature is 30°C, the yeast inoculation amount is 2×10 6 CFU / mL~8×10 6 CFU / mL (as an example, the yeast inoculation amount is 2-8% of the volume of the sour cherry juice), and the fermentation time is 36-48 hours. Preferably, the yeast inoculation amount is 4×10 6 CFU / mL (i.e., yeast inoculation amount is 4% of the fermentation liquid volume), and fermentation time is 40h.

[0014] In the above method, before inoculating 71B yeast in sour cherry juice, 71B yeast activation is required. After adding sterile water at a ratio of 1:10, the mixture is placed in an incubator at 30°C for activation for 30 minutes, and the number of viable bacteria is 1×10 8 CFU / mL. In the above method, further, the lactic acid bacteria is Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei or Oenococcus oeni, more preferably Lactobacillus such as Lactobacillus rhamnosus or Lactobacillus plantarum, and further preferably Lactobacillus rhamnosus. Further, the lactic acid bacteria is Lactobacillus rhamnosus, and the conditions of the second fermentation are as follows: the fermentation temperature is 31-37°C, and the inoculation amount of Lactobacillus rhamnosus is 2×10 based on the initial viable count in the fermentation broth obtained in step S1. 6 CFU / mL~6×10 6 CFU / mL (as an example, the lactobacillus inoculation amount is 2% to 6% of the fermentation broth volume), and the fermentation time is 24 to 48 hours. Preferably, the fermentation temperature is 34°C, and the inoculation amount of the Lactobacillus rhamnosus is 5×10 6 CFU / mL (i.e., the lactobacillus inoculation amount is 5% of the fermentation liquid volume), and the fermentation time is 42h. As an example, the plant lactobacillus is plant lactobacillus H87; the casei lactobacillus is casei lactobacillus SB27; the paracasei lactobacillus is paracasei lactobacillus ML446; and the rhamnosus lactobacillus is rhamnosus lactobacillus FN518.

[0015] In the above method, further, before inoculating Lactobacillus rhamnosus into the fermentation liquid obtained in step S1, Lactobacillus rhamnosus needs to be activated, and Lactobacillus rhamnosus is inoculated into MRS liquid culture medium and cultured at 37°C for 16-24h, and the number of viable bacteria is 1×10 8 CFU / mL.

[0016] In a second aspect, the present invention provides fermented sour cherry juice prepared by any of the methods described above.

[0017] In a third aspect, the present invention provides a method for preparing sour cherry probiotic fruit powder by sequential fermentation and acid reduction of yeast and lactic acid bacteria, comprising the following steps:

[0018] A method for preparing fermented sour cherry juice by sequential fermentation and acid reduction using yeast and lactic acid bacteria as described in any one of the above items; and,

[0019] A drying aid is added to the sour cherry fermented juice, and the sour cherry probiotic fruit powder is obtained by spray drying after homogenization.

[0020] In the above method, the mass of the drying aid is 10-15% of the sour cherry fermented juice, and the drying aid is inulin and skim milk powder; preferably, the spray drying inlet air temperature is 120°C, the amount of drying aid added is 15%, and the mass ratio of inulin to skim milk powder is 1:3. As an example, other conditions of the spray drying are as follows: outlet air temperature is 70°C, and air flow meter is 20l / min.

[0021] In a fourth aspect, the present invention provides sour cherry probiotic fruit powder prepared by any of the methods described above.

[0022] The present invention has the following beneficial effects:

[0023] The present invention is the first to apply brewer's yeast and lactic acid bacteria to the acid reduction of sour cherry juice by sequential inoculation and fermentation. The 71B yeast can maintain a good growth state in the high acidity of sour cherry juice, start malic acid-alcohol fermentation, consume and utilize malic acid as a carbon source, reduce the acidity of the juice, and provide a suitable fermentation pH value for lactic acid bacteria fermentation, thereby solving the problem that lactic acid bacteria are difficult to directly carry out biological acid reduction of sour cherry juice in a high-acid environment. Then, lactic acid bacteria are inoculated to start malic acid-lactic acid fermentation, continue to consume the malic acid content of sour cherries, further reduce the overall acidity of the juice, and increase the flavor of the juice. By combining it with spray drying technology, it can be made into low-acid sour cherry probiotic fruit powder, which can improve the survival rate of probiotics, extend the shelf life, reduce transportation costs, ensure the quality of sour cherry fermentation powder, enrich the product form of sour cherries, and improve competitiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the effect of the initial pH on the total acid degradation rate of sour cherry juice fermented by different strains in Example 1 of the present invention.

[0025] Figure 2 The pH changes of sour cherry juice during the shaking fermentation of different yeast species in Example 1 of the present invention.

[0026] Figure 3 The pH changes of sour cherry juice at different inoculation ratios under oscillating fermentation in Example 1 of the present invention are shown.

[0027] Figure 4 This is the growth curve of five lactic acid bacteria in sour cherry juice in Example 1 of the present invention.

[0028] Figure 5 It is the total acid degradation rate of sour cherry juice fermented by five lactic acid bacteria in Example 1 of the present invention.

[0029] Figure 6 The figure shows the influence of different inoculum amounts on the total acid reduction rate and the malic acid reduction rate in Example 2 of the present invention.

[0030] Figure 7This is the effect of fermentation temperature on the total acid reduction rate and malic acid reduction rate in Example 2 of the present invention.

[0031] Figure 8 This is the effect of fermentation time on the total acid reduction rate and malic acid reduction rate in Example 2 of the present invention.

[0032] Fig. 9 It is the interaction surface of the two factors in Example 2 of the present invention.

[0033] Fig.10 The figure shows the influence of different air inlet temperatures on the number of viable bacteria and powder yield in Example 3 of the present invention.

[0034] Fig.11 The figure shows the effect of different drying agent addition amounts on the viable bacteria count and flour yield in Example 3 of the present invention.

[0035] Fig.12 The figure shows the effect of the compounding ratio of inulin to skim milk powder on the number of viable bacteria and the powder yield in Example 3 of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0037] The methods used in the following examples, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0038] The sources of the raw materials in the following examples are as follows:

[0039] Sour cherries come from Yingguo Biotechnology (Qingdao) Co., Ltd.

[0040] Pectinase was purchased from Henan Wanbang Chemical Technology Co., Ltd. with an enzyme activity of 30,000 U / g.

[0041] Yeast 71B was purchased from Han Jiujiang Brewing House and produced by Lallemand Inc.; the production company of Oenococcus oeni is Chr. Hansen, and the trade name is Viniflora CH11 (low-temperature lactic acid bacteria CH11).

[0042] The acid-reducing yeast strain A3 and the acid-reducing yeast strain B5 were donated by the Agricultural Products Storage and Processing Laboratory of Jilin Agricultural University and are recorded in the document "Lu Siyan. Screening, identification and organic acid degradation characteristics of acid-reducing yeast strains in wild berries [D]. Changchun: Jilin Agricultural University, 2021." The public can obtain them from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0043] Lactobacillus plantarum H87, Lactobacillus casei SB27, Lactobacillus paracasei ML446, and Lactobacillus rhamnosus FN518 were donated by the Functional Dairy and Probiotics Engineering Laboratory, School of Food Science and Engineering, Ocean University of China. Lactobacillus plantarum H87 is recorded in the literature "Liang, Cong, et al. "Lactiplantibacillus plantarum H-87 prevents high-fat diet-induced obesity by regulating bile acid metabolism in C57BL / 6J mice." Food & function 12.10 (2021): 4315-4324.", Lactobacillus casei SB27 is recorded in the literature "Wang Shumei et al. Study on the effect of Lactobacillus casei SB27 on reducing the toxicity of N-nitrosamines on IEC-6 cells [J]. China Brewing, Vol. 41, No. 4, 2022", ... 446 is recorded in the document "Yang Wenjun et al. Effect of 2'-fucosyllactose on the colonization and anti-inflammatory ability of probiotics [J]", Food Industry Science and Technology, Volume 42, Issue 20, October 2021, and Lactobacillus rhamnosus FN 518 is recorded in the document "Yang Wenjun et al. Effect of 2'-fucosyllactose on the colonization and anti-inflammatory ability of probiotics [J]". The public can obtain it from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0044] The concentration of all bacterial suspensions before inoculation was 1×10 8 CFU / mL, based on an inoculation volume of 1% of the cherry juice volume, the initial concentration in the cherry juice after inoculation is 1×10 6 CFU / mL.

[0045] The composition of the culture medium in the following examples is as follows: YPD medium: 10 g yeast extract, 20 g peptone, 20 g anhydrous glucose dissolved in 1 L distilled water, sterilized at 121° C., 0.1 MPa for 20 min.

[0046] MRS medium: 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g dipotassium hydrogen phosphate, 2 g diammonium citrate, 5 g sodium acetate, 20 g anhydrous glucose, 1 mL Tween 80, 0.5 g magnesium sulfate, and 0.25 g manganese sulfate were dissolved in 1 L distilled water and sterilized at 121°C, 0.1 MPa for 20 min.

[0047] Example 1: Screening of yeast and lactic acid bacteria for sour cherry acid reduction fermentation

[0048] This example explores the optimal pH value for acid reduction fermentation of five lactic acid bacteria, uses this pH value as a standard to screen the yeast with the fastest pH increase rate to improve the juice fermentation environment to adapt to the normal growth of lactic acid bacteria, and determines the lactic acid bacteria fermentation strain with good growth condition and good acid reduction effect based on yeast fermentation. For specific determination experiments and result data, see Figure 1-4 .

[0049] 1. Determination of initial pH of sour cherry juice fermentation

[0050] Follow these steps to determine the initial pH of your tart cherry juice fermentation:

[0051] (1) Raw material pretreatment: whole, insect-free sour cherry fruits were washed, blanched in boiling water for 60 seconds, pitted, crushed and pulped, and 0.05% pectinase was added for enzymatic hydrolysis for 2 hours and filtered to obtain sour cherry juice. 0.15% ascorbic acid was then added to the sour cherry juice, and the sour cherry juice was allowed to stand at room temperature to obtain a sour cherry juice sample;

[0052] (2) Activation of strains: Yeast A3 and yeast B5 were activated for two generations and inoculated into YPD liquid medium at 2% inoculation volume. They were cultured at 30°C for 22 h, centrifuged at 5500×g for 8 min at 4°C, and the supernatant was discarded. The suspension was washed twice with sterile saline and then an equal volume of saline was added and fully shaken to obtain a uniform bacterial suspension. The suspension was diluted to OD 600nm = 1.0 for later use. Commercial 71B acid-reduced yeast powder was added with sterile water at a ratio of 1:10 and then activated in a 30°C incubator for 30 min for later use. At this time, the number of viable bacteria of the three yeasts was 1×10 8 CFU / mL. Lactobacillus plantarum H, Lactobacillus casei SB, Lactobacillus paracasei 446, and Lactobacillus rhamnosus FN were inoculated into MRS liquid culture medium at a 1% inoculation rate, cultured at 37°C for 24 hours, activated for two generations, and prepared bacterial suspensions in the same manner as above, diluted to OD 600nm = 0.5 for later use; commercial Oenococcus oeni powder was added to sterile water at a ratio of 1:1000 and then activated in a 28°C incubator for 20 minutes for later use. At this time, the viable counts of the five lactic acid bacteria were all 1×10 8 CFU / mL;

[0053] (3) Adjusting the pH value of the juice: Prepare a baking soda solution with a concentration of 1 mol / L, and adjust the pH values ​​of the sour cherry juice before inoculation to 3.2 (original pH value of the juice), 3.4, 3.6, 3.8 and 4.0, respectively. Sterilize at 90°C for 15 min and set aside.

[0054] (4) Inoculation of lactic acid bacteria: Five lactic acid bacteria (Lactobacillus plantarum H, Lactobacillus casei SB, Lactobacillus paracasei 446, Lactobacillus rhamnosus FN, and Oenococcus oenii) were inoculated into the above five samples respectively and fermented for 3 days with an inoculation amount of 4%. The fermentation temperature of Oenococcus oenii was 28° C. and the fermentation temperature of the other four lactic acid bacteria was 37° C. After the fermentation, the live bacteria of the sour cherry juice were preserved.

[0055] The specific experimental method is to use a pH meter to detect the pH of the juice, and to detect the total acid content according to GB / T 12456-2008 "Determination of total acid in food", and the acid is converted to malic acid equivalent. The acid reduction rate is calculated according to the following formula:

[0056] Acid reduction rate (%) = (1-total acid content after fermentation / total acid content before fermentation) × 100;

[0057] like Figure 1 As shown in the figure, when the pH of the sample is ≤3.8, the acid-reducing ability of Lactobacillus rhamnosus and Lactobacillus casei does not change significantly with the increase of pH; when the pH of the sample is 4.0, the acid-reducing ability of both lactic acid bacteria is greatly improved. The acid-reducing ability of Lactobacillus plantarum and Lactobacillus casei also shows an upward trend with the increase of the pH of the juice; Oenococcus oeni maintains a good acid-reducing ability throughout the process. On the whole, the pH value of the juice is selected to be 4.0 for the next step of yeast screening. In this environment, the five lactic acid bacteria have good fermentation acid-reducing ability.

[0058] 2. Determination of yeast species and culture conditions for acid reduction fermentation of sour cherry juice

[0059] Follow these steps to determine the type of yeast used in the sour cherry juice deacidification fermentation:

[0060] (1) Raw material pretreatment: whole, insect-free sour cherry fruits were washed, blanched in boiling water for 60 seconds, pitted, crushed and pulped, and 0.05% pectinase was added for enzymatic hydrolysis for 2 hours and filtered to obtain sour cherry juice. 0.15% ascorbic acid was then added to the sour cherry juice, and the sour cherry juice was allowed to stand at room temperature to obtain a sour cherry juice sample;

[0061] (2) Inoculating yeast: Yeast 71B, A3, and B5 were inoculated into sour cherry juice sterilized at 90° C. for 15 min, respectively, and fermented at 30° C. with shaking for 0-72 h. The inoculation amount was 4% of the volume of the sour cherry juice. The pH value of the fermentation liquid was detected during the fermentation process.

[0062] (3) Determination of 71B yeast fermentation conditions: inoculation of 71B yeast, shaking culture fermentation at 30°C for 0-48h, the inoculation amount was 2%, 4%, 6%, and 8% of the volume of the sour cherry juice, and the pH value of the fermentation liquid was detected during the fermentation process.

[0063] Depend on Figure 2 The results showed that 71B yeast oscillation fermentation 72h ( Figure 2 The pH of the fruit pulp increased significantly from 3.32 to 4.64, and it increased the pH of the sour cherry juice the fastest, so it was determined to be the fermentation yeast. The fermentation conditions of 71B yeast were further optimized. Figure 3 The results show that the pH of the juice environment first reaches 4.0 under the conditions of 40h fermentation and 4% inoculation. Therefore, the first fermentation process of 71B yeast is: inoculation 4%, fermentation time 40h, and the first fermentation liquid is centrifuged to remove yeast for later use.

[0064] 3. Screening of lactic acid bacteria for acid-reducing fermentation of sour cherry juice

[0065] Screening of lactic acid bacteria for acid reduction fermentation of sour cherry juice is carried out as follows:

[0066] (1) Raw material pretreatment: whole, insect-free sour cherry fruits were washed, blanched in boiling water for 60 seconds, pitted, crushed and pulped, and 0.05% pectinase was added for enzymatic hydrolysis for 2 hours and filtered to obtain sour cherry juice. 0.15% ascorbic acid was then added to the sour cherry juice, and the sour cherry juice was allowed to stand at room temperature to obtain a sour cherry juice sample;

[0067] (2) Inoculating yeast: inoculating yeast 71B into sour cherry juice sterilized at 90° C. for 15 min, and fermenting with shaking at 30° C. for 40 h. The inoculation amount is 4% of the volume of the sour cherry juice. After the fermentation is completed, centrifugation is performed to sterilize the juice, thereby obtaining a first fermentation liquid.

[0068] (3) Inoculation of lactic acid bacteria: Five lactic acid bacteria (Lactobacillus plantarum H, Lactobacillus casei SB, Lactobacillus paracasei 446, Lactobacillus rhamnosus FN, and Oenococcus oeni) were inoculated into the first fermentation broth for 0-96 h, respectively, with an inoculation amount of 4%. The fermentation temperature of Oenococcus oeni was 28° C., and the fermentation temperature of the other four lactic acid bacteria was 37° C. During the fermentation process, the number of live bacteria in the fermentation broth was detected and the total acid reduction rate of the juice at the 24th hour of fermentation was calculated. The fermentation broth was collected after the fermentation was completed.

[0069] The total colony count was determined by the plate count method, referring to GB 4789.2-2016.

[0070] Depend on Figure 4The results showed that in the first fermentation broth, the live counts of the five lactic acid bacteria (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Oenococcus oeni, and Lactobacillus rhamnosus) showed a gradual upward trend within the first 24 hours of fermentation; after 24 hours, the growth and adaptability of Oenococcus oeni and Lactobacillus rhamnosus were strong, and they kept rising to a stable state, while the other three showed a downward trend. The total acid reduction rate of the juice lactic acid bacteria after 24 hours of fermentation was further tested. Figure 5 The results showed that Lactobacillus rhamnosus had the highest total acid degradation rate, followed by Lactobacillus plantarum. Taking comprehensive considerations, Lactobacillus rhamnosus with good growth and acid reduction ability was selected for subsequent fermentation optimization experiments.

[0071] Example 2: Optimization of acid reduction conditions for Lactobacillus rhamnosus

[0072] 1. Single Factor Experiment

[0073] Taking the total acid reduction rate and malic acid reduction rate as indicators, the fermentation time (12, 24, 36, 48, 60h), fermentation temperature (28, 31, 34, 37, 40℃), and inoculation size (2, 4, 6, 8, 10%) were optimized to explore the effects of the above conditions on acid reduction fermentation. The initial culture conditions before optimization were 24h, the fermentation temperature was 37℃, and the inoculation size was 4%. When a single factor was optimized, the levels of other factors were kept unchanged, and the results of the single factor experiment after optimization were applied to the subsequent single factor experiment. The specific steps are as follows:

[0074] (1) Raw material pretreatment: whole, insect-free sour cherry fruits were washed, blanched in boiling water for 60 seconds, pitted, crushed and pulped, and 0.05% pectinase was added for enzymatic hydrolysis for 2 hours and filtered to obtain sour cherry juice. 0.15% ascorbic acid was then added to the sour cherry juice, and the sour cherry juice was allowed to stand at room temperature to obtain a sour cherry juice sample;

[0075] (2) Inoculating yeast: inoculating yeast 71B into sour cherry juice sterilized at 90° C. for 15 min, and fermenting with shaking at 30° C. for 40 h. The inoculation amount is 4% of the volume of the sour cherry juice. After the fermentation is completed, centrifugation is performed to sterilize the juice, thereby obtaining a first fermentation liquid.

[0076] (3) Inoculation of lactic acid bacteria: Lactobacillus rhamnosus FN was inoculated into the first fermentation broth and fermented at 37° C. for 24 h. The inoculation amount was 4%. After the fermentation was completed, the fermentation broth was collected.

[0077] Adjust the inoculum volume, and keep the other conditions the same as above to obtain Figure 6 The effects of different inoculation amounts on the total acid reduction rate and malic acid reduction rate are shown.

[0078] Adjust the fermentation temperature, and keep the other conditions the same as above to obtain Figure 7 The effects of different fermentation temperatures on the total acid reduction rate and malic acid reduction rate are shown.

[0079] Adjust the fermentation time, and keep the other conditions the same as above to obtain Figure 8 The effects of different fermentation times on the total acid reduction rate and malic acid reduction rate are shown.

[0080] Determination method: The organic acid content was determined by HPLC. The sour cherry fermentation broth sample was centrifuged at 8000rpm and 4℃ for 10 minutes to remove the precipitate. The supernatant was diluted 10 times with ultrapure water, filtered through a 0.22μm pore size filter membrane, and the filtrate was analyzed by the external standard method. Chromatographic conditions: Detector: K-2600 UV-visible detector, chromatographic column: Agilent ZORBAX-SBC 18 Column (4.6 mm × 250 mm, 5 μm), mobile phase: 55% 0.05 M NaH 2 PO 4 (pH 3.0), 40% ultrapure water and 5% methanol, flow rate: 0.2 mL / min, column temperature 20°C, detection wavelength 210 nm, injection volume: 22 μL; the total acid reduction rate determination method is the same as in Example 1.

[0081] Depend on Figure 6 It can be seen that when the inoculation amount of Lactobacillus rhamnosus is 4%, the total acid degradation rate reaches the maximum, and as the inoculation amount continues to increase, the total acid degradation rate shows a downward trend, and the malic acid degradation rate does not change significantly. Figure 7 The results showed that when the fermentation temperature reached 34℃, the total acid degradation rate was the highest, and the malic acid degradation rate was also good at this time; Figure 8 It can be seen that the total acid reduction rate shows a trend of increasing first and then decreasing. When the fermentation time is 36h, the total acid degradation rate is the highest; when the fermentation time is further extended, the accumulation of rhamnose fermentation acid leads to a decrease in the total acid reduction rate. Since the yeast fermentation consumes part of the malic acid in the early stage, the malic acid is completely consumed after 36h of fermentation. If the fermentation time is further extended, the malic acid reduction rate does not change significantly.

[0082] 2. Response surface optimization experiment

[0083] Based on the results of the single factor experiment, the total acid reduction rate with the greatest influence was selected as the corresponding indicator. The Box-Behnken central composite design principle was used to conduct a three-factor three-level response surface analysis experiment to investigate the effects of inoculation amount (A), fermentation temperature (B), and fermentation time (C) on the total acid reduction rate (Y). The scheme and results are shown in Table 1.

[0084] Inoculation amount (2%, 4%, 6%)

[0085] Fermentation temperature (31°C, 34°C, 37°C)

[0086] Fermentation time (24h, 36h, 48h)

[0087] Table 1 Response surface experiment results

[0088]

[0089] Table 2 Response surface variance analysis

[0090]

[0091] Note: *Significant difference (P<0.05); **Extremely significant difference (P<0.01).

[0092] As shown in Table 2, the P value of the quadratic polynomial regression model is less than 0.01, which is extremely significant. The P value of the lack-of-fit term is greater than 0.05, indicating that the lack-of-fit term is not significant. The model has a good fit to the experiment, and the determination coefficient R 2 The value is 0.9933, indicating that the model equation can accurately reflect the relationship between each factor and the response value. From the results of the significance analysis, it can be seen that the order of influence of each factor on the total acid reduction rate is: A>C>B. 2 , B 2 , C 2 The interaction terms AC had a very significant effect on the total acid reduction rate of sour cherry fermentation (P<0.01).

[0093] The Design-Expert 11.0 software was used to perform multiple regression fitting on the test results in Table 1, and the quadratic polynomial regression equation of each factor level and total acid reduction rate was obtained: Y = 32.94 + 2.47*A + 0.3750*B + 2.41*C-0.215*AB-1.36*AC-0.0.32*BC-0.27*A 2 -1.45*B 2 -2.09*C 2 . Fig. 9 The surface diagram of the interaction of two factors is shown in Figure 2. Through the analysis of the quadratic polynomial regression model, the optimal fermentation process of Lactobacillus rhamnosus is determined to be 4.68% inoculation, 34.16°C fermentation temperature, and 41.57h fermentation time, at which the acid reduction rate is 33.93%. In order to facilitate actual preparation, the optimal process is proposed as: 5% inoculation, 34°C fermentation temperature, and 42h fermentation time. The experiment was repeated 5 times under the optimized fermentation conditions, and the acid reduction rate was 33.62±0.01%, which was not significantly different from the theoretical value, indicating that the model has good predictive power.

[0094] Example 3: Study on spray drying of fermented sour cherry powder

[0095] 1. Spray drying single factor test

[0096] Taking the number of viable bacteria and powder yield as indicators, the inlet air temperature (120, 130, 140, 150℃), the amount of drying aid added (7.5%, 10%, 12.5%, 15%), and the compound ratio of drying aid, i.e., inulin: skim milk powder (2:1, 1:1, 1:2, 1:3) were optimized to explore the effects of the above conditions on sour cherry fermented fruit powder. Before optimization, the initial inlet air temperature was 130℃, the amount of drying aid added was 10%, and the ratio of inulin: skim milk powder was 1:1. When a single factor was optimized, the levels of other factors were kept unchanged, and the results of the single factor experiment after optimization were applied to the subsequent single factor experiments. The specific steps are as follows:

[0097] (1) Raw material pretreatment: whole, insect-free sour cherry fruits were washed, blanched in boiling water for 60 seconds, pitted, crushed and pulped, and 0.05% pectinase was added for enzymatic hydrolysis for 2 hours and filtered to obtain sour cherry juice, and then 0.15% ascorbic acid was added to the sour cherry juice, and the sour cherry juice was allowed to stand at room temperature to obtain sour cherry juice;

[0098] (2) Inoculating yeast: inoculating yeast 71B into sour cherry juice sterilized at 90° C. for 15 min, and fermenting with shaking at 30° C. for 40 h. The inoculation amount is 4% of the volume of the sour cherry juice. After the fermentation is completed, centrifugation is performed to sterilize the juice, thereby obtaining a first fermentation liquid.

[0099] (3) Inoculating lactic acid bacteria: Lactobacillus rhamnosus FN was inoculated into the first fermentation broth and fermented at 34° C. for 42 h. The inoculation amount was 5%. After the fermentation was completed, a low-acid sour cherry fermentation broth was obtained.

[0100] (4) Spray drying: Add a drying aid to the low-acid sour cherry fermented liquid. The drying aid addition amount is 10%. The drying aid is inulin: skim milk powder with a mass ratio of 1:1. After homogenization, spray drying is performed. The initial inlet air temperature is 130°C, the outlet air temperature is 70°C, and the air flow meter is 20l / min to obtain sour cherry probiotic powder.

[0101] Adjust the inlet air temperature, and keep the other conditions the same as above to obtain Fig.10 The effect of different air inlet temperatures on the number of viable bacteria and powder output rate is shown.

[0102] Adjust the amount of drying agent added, and keep the other conditions the same as above to obtain Fig.11 The effects of different drying agent addition amounts on viable bacterial count and powder yield are shown.

[0103] Adjust the ratio of inulin to skim milk powder, and keep the other conditions the same as above to obtain Fig.12 The effects of different inulin and skim milk powder mixing ratios on viable bacterial count and powder extraction rate are shown.

[0104] The method for determining the number of viable bacteria is the same as in Example 1, and the powder yield is the ratio of the mass of the powder obtained by spray drying to the mass of the total solids contained in the material before spray drying. The specific formula is as follows:

[0105] Powder extraction rate (%) = fermented fruit powder mass / (juice solid content + drying agent mass) × 100

[0106] Depend on Fig.10 It can be seen that with the increase of inlet air temperature, the number of live bacteria and powder extraction rate first increased and then decreased. When the inlet air temperature was 130℃, the number of live bacteria was the largest, which was 7.20Log CFU / g, and the powder extraction rate was 32.21%. The effect of different drying agent addition amounts on the number of live bacteria and powder extraction rate is shown in Figure 2. Fig.11 When the amount of drying agent added is 10%, the number of live bacteria is the largest. When the amount added is higher than 10%, due to the excessive content of drying agent, the powder extraction rate increases, but a large amount of drying agent dilutes the sour cherry fermentation powder, so the number of live bacteria begins to decrease. Fig.12 The results showed that the survival rate of lactic acid bacteria could be improved by a suitable compounding ratio. With the decrease of the inulin / skim milk powder ratio, the number of live bacteria and the powder extraction rate both increased first and then decreased. When the compounding ratio was 1:1, the number of live bacteria was 7.21Log CFU / g. Continuing to reduce the compounding ratio, the powder extraction rate increased significantly, but the number of live bacteria decreased significantly. Increasing the compounding ratio increased the content of inulin, a sugar, which caused the wall adhesion phenomenon during spray drying, resulting in a decrease in the number of live bacteria and the powder extraction rate.

[0107] 2. Spray drying orthogonal test

[0108] The entropy weight TOPSIS method in comprehensive evaluation was used to conduct a comprehensive analysis of the orthogonal test data, and the standardized index values ​​were recalculated. The results are shown in Tables 3 and 4. The range R of the compounding ratio of the drying aid is the largest, which is the main influencing factor, followed by the addition amount and the inlet air temperature. From the results of variance and range analysis, it can be concluded that the influence of each factor on the content of the active ingredient is in the following order: compounding ratio of the drying aid > addition amount > inlet air temperature, and the compounding ratio of the drying aid has a significant effect on the comprehensive evaluation. The optimal process condition is selected as A 1 B 3 C 3 , that is, the air inlet temperature is 120℃, the addition amount is 15%, and the ratio is 1:3. The spray drying test of sour cherry fermented powder was repeated 3 times under this process, and the number of viable bacteria and the powder extraction rate were 7.84±0.02Log CFU / g and 40.65±1.05 respectively, and the number of viable bacteria reached 10 6 CFU / g requirement and good powder extraction rate, it can be used as sour cherry fermentation powder spray drying process.

[0109] Table 3 Results of orthogonal test for spray drying of sour cherry fermented powder

[0110]

[0111] Table 4 Analysis of variance of comprehensive scores

[0112]

[0113] 3. Physical and chemical indicators of sour cherry probiotic fermented fruit powder

[0114] According to the process of 71B yeast inoculation 4% fermentation 40h → Lactobacillus rhamnosus inoculation 5% fermentation at 34℃ for 42h → spray drying with an inlet air temperature of 120℃, an addition amount of 15%, and inulin: skim milk powder 1:3, the sour cherry probiotic fermented fruit powder was prepared, and the relevant properties of the fruit powder were tested. Rehydration time determination: take 2g of sample and add it to a beaker filled with 50mL of distilled water, stir at 800rpm until the powder is completely dissolved, and record the time (s) from the beginning of addition to complete dissolution. Solubility determination: take 1g of sample and add it to a beaker filled with 100mL of distilled water, stir at 600rpm for 5min, and then centrifuge at 3000×g for 5 minutes. Take 25mL of supernatant in a culture dish, dry it at 100℃ to constant weight, and record the weight change of the culture dish. Moisture content determination: weigh 2g of powder sample and place it in a weighing bottle dried to constant weight, bake at 102℃ for 4h, and cool the dryer to constant weight. The ratio of the weight change to the weight of the sour cherry fermented powder before drying is the moisture content in the powder sample. pH determination: dissolve the sample in water at a dilution ratio of 1:10 and measure it with a pH meter. Bulk density determination: weigh 5g of the sample, put it into a 10mL measuring cylinder, and then shake it for 1min to make the powder particles closely contact each other. Record the volume of the powder when it is flattened in the measuring cylinder, and then divide the powder mass by the powder volume. The unit is g / mL. The solid powder sample after spray drying is subjected to sensory scoring of the tissue state and color. Take 5g of the powder sample and add 40mL of drinking water at about 40℃ to prepare it. Select 10 food professionals who are experienced in beverage sensory evaluation and have received professional training to conduct sensory scoring of the prepared sour cherry probiotic beverage. The full score is 100 points. The sensory scoring standard is shown in Table 5.

[0115] Table 5 Sensory evaluation standards for fermented sour cherry powder

[0116]

[0117] Table 6 Quality inspection of sour cherry baking powder

[0118]

[0119] Table 6 shows the test results of various quality indicators of sour cherry fermented powder. As can be seen from Table 6, the sour cherry fermented powder prepared by the spray drying process is relatively loose, with a bulk density of 0.26±0.02g / mL, a viable bacterial count of 7.84±0.02Log CFU / g, and a powder yield of 40.65±1.05%; the fermented powder rehydration time is 105.50s, the pH of the solution after dissolution is 5.55±0.01, the solubility is 89.92%, and the moisture content of the fruit powder is 4.45±0.18%, which meets the requirement that the moisture content of fruit and vegetable powder must be less than 6%.

[0120] The fermented sour cherry powder is in the form of a fine powder, evenly distributed, yellow in color, with a rich fermented aroma, sour cherry flavor, and a moderately sweet and sour taste.

[0121] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for preparing sour cherry fermented juice by sequential fermentation and acid reduction with yeast and lactic acid bacteria, characterized in that: The steps include: S1. Inoculating yeast into sour cherry juice for the first fermentation to obtain fermentation liquid; S2, inoculating lactic acid bacteria into the fermentation liquid obtained in step S1 for a second fermentation to obtain the sour cherry fermented juice.

2. The method for preparing sour cherry fermented juice by sequential fermentation and acid reduction with yeast and lactic acid bacteria according to claim 1, characterized in that: The sour cherry juice is sour cherry puree, preferably prepared by removing pits from the sour cherries, crushing and pulping the cherries, and then enzymolyzing them with pectinase.

3. The method for preparing sour cherry fermented juice by sequential fermentation and acid reduction with yeast and lactic acid bacteria according to any one of claims 1-2, characterized in that: The yeast is 71B yeast.

4. The method for preparing sour cherry fermented juice by sequential fermentation and acid reduction with yeast and lactic acid bacteria according to claim 3, characterized in that: The conditions of the first fermentation are as follows: the fermentation temperature is 30°C, the yeast inoculation amount is 2×10 6 CFU / mL~8×10 6 CFU / mL, fermentation time 36-48h; preferably, the yeast inoculation amount is 4×10 6 CFU / mL, fermentation time 40h.

5. The method for preparing sour cherry fermented juice by sequential fermentation and acid reduction with yeast and lactic acid bacteria according to any one of claims 1 to 4, characterized in that: The lactic acid bacteria are Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei or Oenococcus oeni.

6. The method for preparing sour cherry fermented juice by sequential fermentation and acid reduction with yeast and lactic acid bacteria according to claim 5, characterized in that: The lactic acid bacteria is Lactobacillus rhamnosus, and the conditions of the second fermentation are as follows: the fermentation temperature is 31-37°C, and the inoculation amount of Lactobacillus rhamnosus is 2×10 6 CFU / mL~6×10 6 CFU / mL, fermentation time 24 to 48 h; preferably, the fermentation temperature is 34°C, and the inoculation amount of Lactobacillus rhamnosus is 5×10 6 CFU / mL, fermentation time 42h.

7. Fermented sour cherry juice prepared by the method according to any one of claims 1 to 6.

8. A method for preparing sour cherry probiotic fruit powder by sequential fermentation and acid reduction with yeast and lactic acid bacteria, characterized in that: The steps include: A method for preparing sour cherry fermented juice by sequential fermentation and acid reduction using yeast and lactic acid bacteria as described in any one of claims 1 to 6; and, A drying aid is added to the sour cherry fermented juice, and the sour cherry probiotic fruit powder is obtained by spray drying after homogenization.

9. The method for preparing sour cherry probiotic fruit powder by sequential fermentation and acid reduction with yeast and lactic acid bacteria according to claim 8, characterized in that: The mass of the drying aid is 10-15% of the sour cherry fermented juice, and the drying aid is inulin and skim milk powder; preferably, the spray drying inlet air temperature is 120°C, the drying aid addition amount is 15%, and the mass ratio of inulin to skim milk powder is 1:

3.

10. Sour cherry probiotic fruit powder prepared by the method according to any one of claims 8-9.

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