Preparation method of fermented fruit juice

Abstract

The invention discloses a preparation method of fermented fruit juice, and belongs to the field of food fermentation processing. The method comprises the following steps: inoculating a mixed bacterium of activated zygosaccharomyces bayer, gluconobacter oxydans and foal foal xylose into tea sugar water, and culturing to obtain a domesticated culture solution; and adding the domesticated culture solution into a fruit juice raw material for fermentation to prepare the fermented fruit juice. The functional component content, especially the total phenol content and the protocatechuic acid content, of the fermented fruit juice is increased, meanwhile, the variety of organic acids is increased, the taste of the kombucha fermented fruit juice is improved, and meanwhile the oxidation resistance of the product is improved.

Description

Technical Field

[0001] The invention belongs to the field of food fermentation processing, and particularly relates to a method for fermenting apple juice by utilizing compound kombucha. Background Art

[0002] As a natural fermentation agent (mixed system), kombucha, which is used to ferment kombucha beverages, has not yet achieved standardized mass production and commercialization in my country. Factors such as the diversity of kombucha fermentation raw materials, the complexity of strain sources, and differences in fermentation conditions lead to significant differences in the composition of microorganisms during the fermentation process, which further affects the quality, safety, and health benefits of the product.

[0003] By rationally compounding existing strains and using compound kombucha for fermentation, the fermentation stability of kombucha can be effectively improved and the fermentation cycle can be reduced. Developing a simple, controllable, and stable synthetic microbial community through artificial means for the development and preparation of multifunctional active fermented beverages based on apple juice is of great significance for clarifying its microbial effects, softening its taste, and enhancing its efficacy. Summary of the invention

[0004] In view of the defects or shortcomings of the prior art, the present invention provides a method for preparing fermented juice.

[0005] To this end, the present invention provides a method for preparing a fermented juice comprising:

[0006] Strain domestication: inoculating a mixed bacteria of activated Zygosaccharomyces bailii, Gluconobacter oxydans and Bacillus xylosus into tea sugar water to obtain a domesticated culture solution; the preservation number of Zygosaccharomyces bailii is CCTCC NO: M2022204, the preservation number of Gluconobacter oxydans is CGMCC NO: 18500, and the preservation number of Bacillus xylosus is GDMCC NO: 62237; the tea sugar water is prepared by brewing tea leaves with hot water, filtering out the tea leaves, and adding glucose, sucrose or white sugar to the tea water;

[0007] Fruit juice fermentation: using glucose, sucrose or white sugar to adjust the soluble solid content in the fruit juice to 15-17° Brix to obtain a fruit juice raw material; adding the domesticated culture solution to the fruit juice raw material for fermentation to prepare the fermented fruit juice.

[0008] An optional scheme is that the strain domestication is to inoculate activated Bayer's yeast, Gluconobacter oxydans and Xylella fastidiosa into tea sugar water and culture for 2 to 3 generations to obtain a domestication culture solution.

[0009] The optional scheme is that the amount of tea leaves and hot water used is 9-11g tea leaves / L water; the amount of glucose, sucrose or white sugar added is 70-90g / L.

[0010] Optionally, the tea leaves are black tea.

[0011] Optionally, the bacterial mixture contains equal amounts of Zygosaccharomyces bailii, Gluconobacter oxydans and Bacillus xylosus.

[0012] An optional solution is that the fruit juice is apple juice, peach juice, pear juice, kiwi juice, grape juice or a compound fruit juice.

[0013] The present invention increases the functional component content of the fermented juice, especially the total phenol content and the protocatechuic acid content, and increases the types of organic acids, thereby improving the taste of the fermented juice of kombucha fungus and increasing the antioxidant capacity of the product. The strains used in the scheme of the present invention are all purchased and separated from food, and are all preserved in a microbial preservation center, and are safe and harmless for food fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The content of monomeric phenols in the kombucha fermented apple beverages prepared in different embodiments.

[0015] Figure 2 Organic acid content in the kombucha fermented apple beverage prepared in different embodiments.

[0016] Figure 3 Sensory scores of the kombucha fermented apple beverages prepared in different embodiments. DETAILED DESCRIPTION

[0017] Unless otherwise specified, the scientific and technical terms used herein are understood according to the knowledge of ordinary technicians in the relevant fields.

[0018] The deposit number of Zygosaccharomyces bailii used in the present invention is CCTCCNO:M2022204; the deposit number of Gluconobacter oxydans used is CGMCC NO:18500; the deposit number of Komagataeibacter xylinus used is GDMCC NO:62237. The above strains are all isolated from food and do not pose any food safety risk. Various bacteria are sequentially activated and domesticated or only activated and then used as fruit juice fermentation strains in the following examples and comparative examples. The following examples use apple juice as an example to further explain the present invention.

[0019] The activation method of the bacteria is as follows: Bayer yeast (Zygosaccharomyces Bailii) is inoculated on a yeast culture medium (YPD), activated at 28°C for 24 hours, and cultured continuously for two generations; wherein the preparation method of the yeast culture medium (YPD) is specifically as follows: 10.0g of yeast powder, 20.0g of peptone, 20.0g of glucose, distilled water is diluted to 1000mL, and high-pressure sterilization is performed at 121°C for 20min.

[0020] Gluconobacter oxydans (or Komagataeibacterxylinus) was inoculated on acetic acid bacteria medium (YPM), activated at 30°C for 48 hours, and cultured for two generations to obtain activated strains of Gluconobacter oxydans (or Komagataeibacterxylinus); the preparation method of the acetic acid bacteria medium (YPM) was specifically as follows: 5.0 g of yeast powder, 3.0 g of peptone, 25.0 g of mannitol, distilled water was used to make the volume to 1000 mL, and high-pressure sterilization was performed at 121°C for 20 minutes.

[0021] The strain acclimation method is as follows: (1) using a centrifuge to collect activated strain cells at 6000 rpm for 10 min at 4°C, and then washing the collected cells with sterile water; (2) resuspending a single strain cell, two strain cells, or three strain cells (i.e., one, two combinations, or three combinations of Zygosaccharomyces bailii, Gluconobacter oxydans, and Bacillus xylosus; when two strains are combined, the concentration ratio of the two strains is 1:1; when three strains are combined, the concentration ratio of the three strains is 1:1:1) to a final concentration of 10 8 CFU / ml, and a bacterial suspension was prepared; (3) the bacterial suspension was added to tea sugar water for culturing and domestication, with a total inoculation amount of 6% (i.e., 6 ml of bacterial suspension was inoculated in 100 ml of tea sugar water), and cultured at about 30°C for 7 days. After continuous culture for 2 generations, the domesticated culture solution was obtained for use;

[0022] The preparation method of tea sugar water is as follows: add 10g / L of black tea to 80-100°C hot water and keep it for 10 minutes, then add 80g / L of white sugar and stir until it is completely dissolved.

[0023] Apple juice fermentation (culture fluid after acclimation): adjust the soluble solid content of apple juice to 16°Brix with white sugar, pasteurize at 75°C for 10 minutes, and then transfer it to a fermentation container; inoculate the acclimation culture fluid into apple juice for fermentation, the inoculation amount is 10% (i.e., 100ml of strain acclimation culture fluid is inoculated into 100ml of apple juice), fermentation is carried out in a constant temperature incubator at 30°C, and fermentation is allowed to stand for 5 days.

[0024] Apple juice fermentation (activated strains / commercially available kombucha for fermented juice): the soluble solids content of the apple juice was adjusted to 16° Brix with white sugar, pasteurized at 75°C for 10 min, and then transferred to a fermentation container; the acclimated culture solution was inoculated into the apple juice for fermentation, with an inoculation amount of 10% (i.e., 100 ml of the above-mentioned single strain cell, two strain cell or three strain cell suspension, or commercially available kombucha for fermented juice activated bacterial suspension was inoculated into 100 ml of apple juice), and the fermentation was carried out in a constant temperature incubator at 30°C and allowed to ferment for 5 days.

[0025] The preparation method of apple juice is specifically as follows: apples without obvious diseases and insect pests are selected, the apples are cut into pieces, and the juice is squeezed by a juicer, and the clarified apple juice is obtained after filtering.

[0026] The activation method of commercially available kombucha for fermented juice (Zhanjiang Mingte Trading Co., Ltd.) was carried out according to the strain instruction manual.

[0027] The following methods for determining the indicators of the products in the examples are as follows:

[0028] Total phenol determination: The total phenol content was determined using the Folin phenol method. Gallic acid was used as the standard. 0.01 g of gallic acid standard was accurately weighed into a 100 mL volumetric flask, and the volume was adjusted with distilled water to prepare a 0.1 mg / mL gallic acid standard solution. Distilled water was used to prepare 0 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L and 0.5 mg / L gallic acid solutions. 0.2 mL of each solution was mixed with 1.0 mL of 10% Folin phenol, and the mixture was reacted for 10 min. Subsequently, 0.8 mL of 7.5% Na2CO3 was added and mixed, and the mixture was placed at 30°C for 1 h. The OD value at 760 was determined. A standard curve was drawn with the concentration of gallic acid as the horizontal axis and the OD value at 760 nm as the vertical axis.

[0029] The samples were diluted 50 times and measured in the same manner as the standards, and the results were expressed as gallic acid equivalents (gGAE / L).

[0030] Antioxidant capacity determination: DPPH free radical scavenging ability: The fermented apple juice samples prepared in different embodiments were diluted 300 times, and then an equal amount of 0.2 mM DPPH solution was added. After mixing, the mixture was reacted in the dark for 30 minutes, and the absorbance at 517 nm was measured. The group without sample was used as blank control, and the DPPH free radical scavenging effect (%) of the fermented apple juice was calculated.

[0031] ABTS free radical scavenging ability: The fermented apple juice samples prepared in different embodiments were diluted 200 times, and then an equal amount of ABTS working solution (absorbance at 734 nm was 0.7±0.02) was added, mixed and reacted in the dark for 10 minutes, and the absorbance at 734 nm was measured. The group without sample was used as blank control, and the ABTS free radical scavenging effect (%) of the fermented apple juice was calculated.

[0032] Monomeric phenol determination: The kombucha fermented apple juice samples prepared in different embodiments were first pretreated. The pH of 10 mL of fermented apple juice sample was adjusted to 7.0 with 1 mol / L NaOH solution, and then extracted 3 times with ethyl acetate (10 mL) to obtain a neutral phenol solution. The remaining aqueous phase was adjusted to pH 2.0 with 1 mol / L HCl solution, and then extracted 3 times with ethyl acetate (10 mL) to obtain an acidic phenol solution. The neutral and acidic solutions were then dried together by rotary evaporation at 35°C, and the residue was redissolved with 5 mL of methanol. The re-solution was filtered with a 0.22 μm filter membrane to obtain the pretreated fermented apple juice sample, which was then subjected to chromatographic detection;

[0033] The instrument and chromatographic conditions are as follows: a C18 chromatographic column is used. Mobile phase A is an aqueous solution containing 1% acetic acid, and mobile phase B is acetonitrile. The gradient elution procedure is as follows: the volume fraction of mobile phase B increases from 10% to 55% (within 50 min), then increases to 100% (within 10 min), then decreases to 10% (within 5 min, and finally equilibrates at this volume fraction for 10 min to facilitate the next injection. The flow rate of the mobile phase is 0.8 mL / min, and the sample injection volume is 10 μL. The column temperature of the chromatographic column is 30°C. The monomer phenol standard solutions used for the determination include quercetin, chlorogenic acid, ellagic acid, ECG, EGCG, epicatechin, catechin, EGC, protocatechuic acid, and gallic acid.

[0034] Organic acid determination: Determine the content and type of organic acids in fermented juice according to the method described in GB 5009.157-2016 "Determination of organic acids in food". Mobile phase A is an aqueous solution containing 0.1% phosphoric acid, and mobile phase B is methanol. The external standard method is used to determine the organic acid components, with retention time as the qualitative method and peak area as the quantitative method. The organic acid standard solution used for the determination contains 7 common organic acids, including citric acid, malic acid, succinic acid, oxalic acid, acetic acid, pyruvic acid, and glucuronic acid.

[0035] Sensory evaluation and analysis: 20 people with sensitive taste and smell were invited to form a sensory evaluation team to evaluate the samples of kombucha fermented apple juice prepared in different implementation methods in terms of color, state, taste, flavor, etc. The scoring criteria are shown in Table 1 below.

[0036] Table 1 Sensory evaluation standards

[0037]

[0038]

[0039] Example:

[0040] On the basis of the above scheme, different fermentation bacteria were used to ferment apple juice. The fermentation bacteria used in each embodiment and comparative example are shown in Table 2. Afterwards, the apple juice prepared in each embodiment and comparative example after fermentation was sampled and centrifuged to determine its total phenol content, antioxidant capacity and sensory evaluation. The total phenol content in the fermented apple juice beverage prepared in different embodiments is shown in Table 2;

[0041] Table 2 Total phenol content of different samples

[0042]

[0043]

[0044] Note: Different letters in the same column indicate that there are significant differences (P < 0.05) between the fermented apple juices prepared in different embodiments.

[0045] As shown in Table 2, the total phenol content of Examples 1 and 2 is significantly higher than that of the control example, indicating that the fermentation of Bayer's Zygosaccharomyces and Gluconobacter oxidans can increase the phenol content. Among them, the total phenol content of Example 4 is the highest, reaching 2.44gGAE / L, indicating that the combined fermentation of Bayer's Zygosaccharomyces and Gluconobacter oxidans can achieve better results and significantly increase the total phenol content. Compared with the control example, the total phenol content of the fermented apple juice sample of Example 7 has been significantly improved, which is 2.32g GAE / L, which is lower than that of Example 4, but also significantly higher than that of the control example, indicating that the combined fermentation of red tea fungi enhances the nutritional components of apple juice and is more beneficial to the human body.

[0046] The results of the antioxidant test are shown in Table 3. The free radical scavenging ability of Example 2 is higher than that of Example 1, indicating that Gluconobacter oxydans has a significant effect on enhancing antioxidant performance. Examples 4 and 5 further verify this conclusion. The free radical scavenging ability of apple juice fermented with compound kombucha is significantly improved. The DPPH free radical scavenging ability of Example 7 is significantly higher than that of the control example, increasing from 71.61% to 75.36%, and the ABTS free radical scavenging ability increases from 73.74% to 80.31%.

[0047] Table 3 Comparison of antioxidant properties of fermented apple juice

[0048] Sample name DPPH free radical scavenging ability % ABTS free radical scavenging ability % Example 1 63.15±0.50d 71.05±0.27e Example 2 69.81±1.65b,c 76.91±0.89c Example 3 54.82±0.50e 63.80±0.33f Example 4 74.32±1.39a 85.66±0.64a Example 5 68.46±1.50c 71.28±0.43e Example 6 63.57±0.92d 64.42±0.32f Example 7 75.36±0.76a 80.31±2.30b Comparative Example 1 70.10±1.51b,c 74.28±0.64d Comparative Example 2 71.61±1.86b 73.74±1.99d Comparative Example 3 71.14±1.50b 78.58±1.15b,c

[0049] Note: Different letters in the same column indicate significant differences (P < 0.05) among the fermented apple juices prepared by different methods.

[0050] The sensory evaluation results are shown in Table 4, which shows that there is no obvious difference in color of the fermented apple juices prepared in different embodiments, all of which are brown-yellow, a unique color of kombucha fermented beverages, and the fermented liquid is clear and transparent, but there are great differences in taste and flavor. Compared with other examples and control examples, the fermented apple juice of Example 7 has a mild sour taste, no sour feeling, and the tea aroma and fruity flavor of the product are more prominent, the smell is coordinated, and the taste is refreshing.

[0051] Table 4 Sensory analysis of fermented apple juice

[0052]

[0053]

[0054] Based on the results shown in Tables 2-4 above, the antioxidant capacity of Examples 4 and 7 has been significantly improved, but the fermented apple juice of Example 4 has a slightly unpleasant sour and rancid taste, which is not as refreshing as that of Example 7.

[0055] The product obtained in Example 7 was further subjected to liquid phase analysis to determine its monomer phenol content and organic acid content as well as sensory analysis. The results are as follows: Figure 1 , Figure 2 and Figure 3 shown.

[0056] The results of the determination of monomer phenols in apple juice (AJ), commercially available kombucha fermented juice (FAJ1, comparative example 2), and the fermented juice of the present invention (FAJ2, example 7) are as follows: Figure 1 The results show that the fermented juice of the present invention has a higher phenol content and a stronger antioxidant property.

[0057] The results of organic acid content determination are as follows Figure 2 It is shown that compared with apple juice, the organic acid types of fermented apple juice are richer and the contents are also increased to varying degrees, especially the glucuronic acid and acetic acid contents, which are significantly increased after fermentation. In addition, the succinic acid content of the fermented juice of the present invention is significantly increased from the original 0.65g / L to 35.96g / L, giving the fermented juice a soft sour taste, making the taste more refreshing, and enhancing the overall flavor complexity of the product; the malic acid content is increased from the original 0.81g / L to 3.91g / L.

[0058] The sensory evaluation results are as follows Figure 3The results show that apple juice is bright golden yellow, commercial kombucha fermented juice is unique brown yellow, and there is no significant difference in color between the fermented juice of the present invention and the commercial kombucha fermented juice. In terms of taste, apple juice has a relatively simple taste; commercial kombucha fermented juice has a relatively heavy sour taste; the fermented juice of the present invention has a refreshing taste with appropriate sweetness and sourness.

Claims

1. A method for preparing fermented fruit juice, characterized in that: Methods include: Strain domestication: inoculating a mixed bacteria of activated Zygosaccharomyces bailii, Gluconobacter oxydans and Bacillus xylosus into tea sugar water to obtain a domesticated culture solution; the preservation number of Zygosaccharomyces bailii is CCTCC NO: M2022204, the preservation number of Gluconobacter oxydans is CGMCC NO: 18500, and the preservation number of Bacillus xylosus is GDMCC NO: 62237; the tea sugar water is prepared by brewing tea leaves with hot water, filtering out the tea leaves, and adding glucose, sucrose or white sugar to the tea water; Fruit juice fermentation: using glucose, sucrose or white sugar to adjust the soluble solid content in the fruit juice to 15-17° Brix to obtain a fruit juice raw material; adding the domesticated culture solution to the fruit juice raw material for fermentation to prepare the fermented fruit juice.

2. The method for preparing fermented juice according to claim 1, characterized in that: The strain domestication is to inoculate activated Zygosaccharomyces bailii, Gluconobacter oxydans and Xylella fastidiosa into tea sugar water and culture for 2 to 3 generations to obtain domestication culture fluid.

3. The method for preparing fermented juice according to claim 1, characterized in that: The amount of tea leaves and hot water used is 9-11g tea leaves / L water; the amount of glucose, sucrose or white sugar added is 70-90g / L.

4. The method for preparing fermented fruit juice according to claim 3, characterized in that: The tea leaves are black tea.

5. The method for preparing fermented juice according to claim 1, characterized in that: The contents of Zygosaccharomyces bailii, Gluconobacter oxydans and Bacillus xylosus in the mixed bacteria were the same.

6. The method for preparing fermented juice according to claim 1, characterized in that: The fruit juice is apple juice, peach juice, pear juice, kiwi juice, grape juice or compound fruit juice.

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