A method for preparing fermented juice

Abstract

The application discloses a preparation method of fermented fruit juice and belongs to the field of food fermentation processing. The disclosed method comprises the following steps: inoculating activated mixed bacteria of bayer conjugated yeast, oxidized glucose bacillus and xylose hippodrome bacillus into tea sugar water to culture domesticated culture solution; and adding the domesticated culture solution into fruit juice raw materials to ferment and prepare fermented fruit juice. The application improves the functional component content of the fermented fruit juice, especially the total phenol content and the content of protocatechuic acid, simultaneously increases the types of organic acids, improves the taste of black tea fungus fermented fruit juice, and improves the antioxidant capacity of the product.

Description

Technical Field

[0001] This invention belongs to the field of food fermentation and processing, and specifically relates to a method for fermenting apple juice using a compound kombucha. Background Technology

[0002] Kombucha, a fermented beverage, is a natural starter culture (mixed system) that has not yet achieved standardized mass production and commercialization in my country. The diversity of raw materials, the complexity of strain sources, and the differences in fermentation conditions lead to significant variations in the microbial composition during the fermentation process, further affecting the product's quality, safety, and health benefits.

[0003] By rationally combining existing microbial strains and using blended kombucha for fermentation, the fermentation stability of kombucha can be effectively improved and the fermentation cycle shortened. Developing simple, controllable, and stable synthetic microbial communities through artificial means for the preparation of multifunctional active fermented beverages based on apple juice is of great significance for clarifying their microbial roles, smoothing their taste, and enhancing their efficacy. Summary of the Invention

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

[0005] Therefore, the present invention provides a method for preparing fermented fruit juice, comprising:

[0006] Strain domestication: A mixture of activated Bayer zygosacchariformis, Glucosamine oxidase, and Xylostella spp. was inoculated into tea sugar water and cultured to obtain a domestication culture solution; the preservation number of Bayer zygosacchariformis is CCTCC NO:M2022204, the preservation number of Glucosamine oxidase is CGMCC NO:18500, and the preservation number of Xylostella spp. is GDMCC NO:62237; the tea sugar water is prepared by steeping tea leaves in hot water, filtering out the tea leaves, and then adding glucose, sucrose, or white sugar to the tea water;

[0007] Fruit juice fermentation: The soluble solids content in the fruit juice is adjusted to 15-17°Brix using glucose, sucrose or white sugar to obtain fruit juice raw material; the domestication culture medium is added to the fruit juice raw material for fermentation to prepare fermented fruit juice.

[0008] An alternative approach is to inoculate activated Bayer zygosaccharizoides, Glucosamine oxytocinoides, and Xylose-1,000 bacteria into a tea-sugar solution and culture for 2-3 generations to obtain an acclimatization culture medium.

[0009] An alternative is to use 9-11g of tea leaves per liter of water, and add 70-90g of glucose, sucrose, or white sugar per liter.

[0010] Alternatively, the tea leaves can be black tea.

[0011] An alternative is to use the same amount of Bayer zygosaccharomyces, Glucosamine oxidans, and Xylostella spp. in the mixed cultures.

[0012] Alternatively, the juice may be apple juice, peach juice, pear juice, kiwi juice, grape juice, or a mixed juice.

[0013] This invention increases the content of functional components in fermented fruit juice, especially the total phenolic content and protocatechuic acid content, while also increasing the variety of organic acids, improving the taste of kombucha-fermented fruit juice, and enhancing the product's antioxidant capacity. The strains used in this invention are all purchased and isolated from food products and are preserved in microbial preservation centers, making them safe and harmless for food fermentation. Attached Figure Description

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

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

[0016] Figure 3 Sensory scores of kombucha-fermented apple beverages prepared in different embodiments. Detailed Implementation

[0017] Unless otherwise specified, the scientific and technical terms used in this article are intended for understanding by those skilled in the art.

[0018] The *Zygosaccharomyces bailii* used in this invention has the accession number CCTCC NO: M2022204; the *Gluconobacter oxydans* used has the accession number CGMCC NO: 18500; and the *Komagataeibacter xylinus* used has the accession number GDMCC NO: 62237. All of the above strains were isolated from food and pose no food safety risk. The various bacteria were sequentially activated and domesticated, or activated only, and used as the fruit juice fermentation strains in the following examples and comparative examples. Apple juice is used as an example in the following examples to further explain and illustrate this invention.

[0019] The activation method of the bacteria is as follows: Bayer zygosaccharomyces Bailii is inoculated onto yeast culture medium (YPD), activated at 28℃ for 24h, and cultured for two generations; the yeast culture medium (YPD) is prepared by taking 10.0g of yeast powder, 20.0g of peptone, 20.0g of glucose, and distilled water to a final volume of 1000mL, and autoclaving at 121℃ for 20min.

[0020] Gluconobacter oxydans (or Komagataeibacter xylinus) was inoculated onto acetic acid bacteria medium (YPM), activated at 30℃ for 48 h, and cultured for two generations to obtain activated strains of Gluconobacter oxydans (or Komagataeibacter xylinus). The acetic acid bacteria medium (YPM) was prepared as follows: 5.0 g yeast powder, 3.0 g peptone, 25.0 g mannitol were added, and the volume was adjusted to 1000 mL with distilled water. The mixture was then autoclaved at 121℃ for 20 min.

[0021] The strain domestication method is as follows: (1) Centrifuge at 6000 rpm for 10 min at 4℃ to collect the activated strain cells, and wash the collected cells with sterile water; (2) Resuspend single strain cells, two strain cells, or three strain cells (i.e., one, two, or three combinations of Bayer Zygomycin, Glucosamine oxytocin, and Xylostella xylostella; 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) until the final concentration is 10. 8 CFU / ml, to prepare bacterial suspension; (3) Add bacterial suspension to tea sugar water for culture and acclimatization, with a total inoculation amount of 6% (i.e., 6ml bacterial suspension inoculated in 100ml tea sugar water), and culture at about 30℃ for 7 days. After two generations of continuous culture, the acclimatization culture solution is obtained for use.

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

[0023] Apple juice fermentation (acclimated culture medium): Adjust the soluble solids content of apple juice to 16°Brix with white sugar, pasteurize at 75℃ for 10 min, and then transfer to a fermentation container; inoculate the acclimated culture medium into the apple juice for fermentation, with an inoculation amount of 10% (i.e., inoculate 10 ml of the bacterial strain acclimated culture medium into 100 ml of apple juice), and ferment in a constant temperature incubator at 30℃ for 5 days.

[0024] Apple juice fermentation (activated strain / commercially available kombucha for fermented fruit juice): Adjust the soluble solids content of apple juice to 16°Brix with white sugar, pasteurize at 75℃ for 10 minutes, and then transfer to a fermentation container; inoculate the acclimatized culture into the apple juice for fermentation, with an inoculation amount of 10% (i.e., inoculate 10 ml of the above single strain cell, two strain cell, or three strain cell suspension in 100 ml of apple juice, or activated kombucha suspension for commercially available kombucha for fermented fruit juice), and ferment in a constant temperature incubator at 30℃ for 5 days.

[0025] The specific method for preparing apple juice is as follows: select apples without obvious diseases or pests, cut them into pieces, juice them using a juicer, and filter the juice to obtain clear apple juice.

[0026] The activation method for commercially available kombucha for fermented fruit juice (Zhanjiang Mingte Trading Co., Ltd.) should be carried out according to the strain's instructions for use.

[0027] The following are the methods for measuring the various indicators of the products in the examples:

[0028] Total phenol determination: The total phenol content was determined using the Folin-Ciocalteu method, with gallic acid as the standard. Accurately weigh 0.01 g of gallic acid standard into a 100 mL volumetric flask, and dilute to volume with distilled water to prepare a 0.1 mg / mL gallic acid standard solution. Gallic acid solutions of 0 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, and 0.5 mg / L were prepared using distilled water. 0.2 mL of each solution was mixed thoroughly with 1.0 mL of 10% Folin-Ciocalteu, reacted for 10 min, and then 0.8 mL of 7.5% Na₂CO₃ was added and mixed thoroughly. The mixture was then reacted at 30 °C for 1 h, and the OD value at 760 nm was measured. A standard curve was plotted with gallic acid concentration on the x-axis and the OD value at 760 nm on the y-axis.

[0029] Dilute the sample 50 times and determine it using the same method as the standard. The result is expressed as gallic equivalent (gGAE / L).

[0030] Antioxidant capacity determination: DPPH free radical scavenging capacity: Fermented apple juice samples prepared by different methods 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 the sample was used as a blank control, and the DPPH free radical scavenging effect (%) of the fermented apple juice was calculated.

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

[0032] Determination of monomeric phenols: Kombucha fermented apple juice samples prepared using different methods were pretreated. First, the pH of 10 mL of fermented apple juice sample was adjusted to 7.0 with 1 mol / L NaOH solution, then extracted three 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 three 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. Finally, the residue was redissolved with 5 mL of methanol, and the reconstituted solution was filtered through a 0.22 μm filter membrane to obtain the pretreated fermented apple juice sample, which was then subjected to chromatographic analysis.

[0033] The instrumentation and chromatographic conditions were as follows: a C18 column was used. Mobile phase A was an aqueous solution containing 1% acetic acid, and mobile phase B was acetonitrile. The gradient elution program was as follows: the volume fraction of mobile phase B was increased from 10% to 55% (within 50 min), then increased to 100% (within 10 min), then decreased to 10% (within 5 min), and finally equilibrated at this volume fraction for 10 min to facilitate the next injection. The flow rate of the mobile phase was 0.8 mL / min, and the sample injection volume was 10 μL. The column temperature was 30℃. The monomeric phenol standard solutions used for determination included quercetin, chlorogenic acid, ellagic acid, ECG, EGCG, epicatechin, catechin, EGC, protocatechuic acid, and gallic acid.

[0034] Organic acid determination: The content and types of organic acids in fermented fruit juice were determined according to the method described in GB 5009.157-2016 "Determination of Organic Acids in Food". Mobile phase A was an aqueous solution containing 0.1% phosphoric acid, and mobile phase B was methanol. The external standard method was used to determine the organic acid components, with qualitative analysis based on retention time and quantitative analysis based on peak area. The standard solutions of organic acids used for determination contained seven common organic acids: citric acid, malic acid, succinic acid, oxalic acid, acetic acid, pyruvic acid, and glucuronic acid.

[0035] Sensory evaluation analysis: A sensory evaluation group composed of 20 people with sensitive taste and smell was invited to evaluate the color, state, taste and flavor of the kombucha fermented apple juice prepared by different implementation methods. The scoring criteria are shown in Table 1 below.

[0036] Table 1 Sensory Evaluation Criteria

[0037]

[0038]

[0039] Example:

[0040] Based on the above scheme, different fermentation bacteria were used for apple juice fermentation. The fermentation bacteria used in each embodiment and comparative example are shown in Table 2. After fermentation, the apple juice samples prepared in each embodiment and comparative example were centrifuged to determine their total phenol content, antioxidant capacity, and sensory evaluation. The total phenol content in the fermented apple juice beverages prepared by different implementation methods 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 significant differences between fermented apple juice prepared using different implementation methods (P < 0.05).

[0045] As shown in Table 2, the total phenolic content of Examples 1 and 2 was significantly higher than that of the control, indicating that fermentation with Bayer's kombucha and *Glucosium oxyphylla* can increase the phenolic content. Among them, Example 4 had the highest total phenolic content, reaching 2.44 g GAE / L, indicating that the combined fermentation of Bayer's kombucha and *Glucosium oxyphylla* achieved better results and significantly increased the total phenolic content. Compared to the control, the total phenolic content of the fermented apple juice sample in Example 7 was significantly increased to 2.32 g GAE / L. Although lower than that of Example 4, it was still significantly higher than that of the control, indicating that the combined kombucha fermentation enhanced the nutritional components of the apple juice and was more beneficial to the human body.

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

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

[0048] Sample Name DPPH free radical scavenging capacity % 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 Compare with Example 1 70.10±1.51b,c 74.28±0.64d Compare with Example 2 71.61±1.86b 73.74±1.99d Compare with Example 3 71.14±1.50b 78.58±1.15b,c

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

[0050] The sensory evaluation results are shown in Table 4. The table shows that the fermented apple juice prepared using different methods had no significant difference in color, all exhibiting the characteristic brownish-yellow color of kombucha fermented beverages, and the fermentation liquid was clear and transparent. However, there were significant differences in taste and flavor. Compared to other examples and the control example, the fermented apple juice of Example 7 had a milder acidity, without any astringency, and the tea and fruit aromas were more prominent, with a harmonious aroma and refreshing taste.

[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 both Example 4 and Example 7 was significantly improved. However, the fermented apple juice of Example 4 had a slightly unpleasant sour smell, which was not as refreshing as that of Example 7.

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

[0056] The monomeric phenol determination results of apple juice (AJ), commercially available kombucha fermented juice (FAJ1, control example 2), and the fermented juice of the present invention (FAJ2, example 7) are as follows: Figure 1 As shown in the figure. The results indicate that the fermented fruit juice of the present invention has a higher phenol content and stronger antioxidant properties.

[0057] The results were obtained by determining the organic acid content. Figure 2 As shown, compared with apple juice, fermented apple juice has a richer variety of organic acids, and the content of all of them is increased to varying degrees, especially the content of glucuronic acid and acetic acid, which are significantly increased after fermentation. In addition, the succinic acid content of the fermented juice of this invention is significantly increased, from the original 0.65g / L to 35.96g / L, giving the fermented juice a mellow sour taste, making the taste fresher, 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] Sensory evaluation results as follows Figure 3As shown, apple juice is a clear golden yellow, while commercially available kombucha fermented juice is a distinctive brownish-yellow. The fermented juice of this invention shows no significant difference in color from commercially available kombucha fermented juice. In terms of taste, apple juice has a relatively simple flavor; commercially available kombucha fermented juice has a stronger sour and astringent taste; the fermented juice of this invention has a refreshing taste with a balanced sweet and sour flavor.

Claims

1. A method for preparing fermented fruit juice, characterized in that, include: Strain domestication: A mixed culture of activated Bayer zygosaccharomyces, glucosamine oxidase, and xylose-rich bacteria was inoculated into tea sugar water to obtain a domestication culture solution; the preservation number of Bayer zygosaccharomyces is CCTCC NO:M2022204, the preservation number of glucosamine oxidase is CGMCC NO:18500, and the preservation number of xylose-rich bacteria is GDMCC NO:62237; the tea sugar water is prepared by brewing tea with hot water, filtering out the tea leaves, and then adding glucose, sucrose, or white sugar to the tea water; Fruit juice fermentation: The soluble solids content in the fruit juice is adjusted to 15-17 °Brix using glucose, sucrose, or white sugar to obtain the fruit juice raw material; the domestication culture solution is added to the fruit juice raw material for fermentation to prepare fermented fruit juice; the mixed bacteria contain the same amount of Bayer zygosaccharomyces, Glucosobacterium oxysporum, and Xylostella spp.; the tea is black tea; the fruit juice is apple juice.

2. The method for preparing fermented fruit juice according to claim 1, characterized in that, Strain domestication involves inoculating activated Bayer zygosaccharizoides, Glucosamine oxyphylla, and Xylose-1,000 bacteria into tea-sugar water and culturing for 2-3 generations to obtain a domestication culture medium.

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

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