Syzygium samarangense fermented beverage and preparation method thereof

Abstract

The invention provides a syzygium samarangense fermented beverage and a preparation method thereof, and belongs to the technical field of fermented beverages. Lactobacillus plantarum CGMCC1.16089 is adopted to ferment the syzygium samarangense juice, the lactobacillus plantarum strain CGMCC1.16089 can stably grow in the syzygium samarangense juice, the variety and the content of total flavonoids, polyphenols, amino acids and volatile compounds are remarkably increased after fermentation, the nutritional value and the quality of the syzygium samarangense juice are remarkably improved, and the syzygium samarangense juice is suitable for being eaten by people. Therefore, the wax apple juice has better edible value and wide market prospect.

Description

Technical Field

[0001] This invention belongs to the field of fermented beverage technology, specifically relating to a wax apple fermented beverage and its preparation method. Background Technology

[0002] Lactobacillus plantarum, a Gram-positive probiotic widely distributed in nature, has garnered significant attention for its multiple health benefits, including boosting immunity, regulating blood lipids and cholesterol, and maintaining intestinal microecological balance. Currently, commercially available lactic acid bacteria fermented products are primarily dairy-based. While some products enrich their product offerings by adding fruit and vegetable juices, these often require the addition of large amounts of sucrose, sweeteners, and preservatives to improve taste. This not only diminishes the product's health benefits but also fails to meet the demands of modern consumers for healthy eating.

[0003] Wax apples, a characteristic fruit of the Myrtaceae family, are rich in polysaccharides, minerals, organic acids, and flavonoids, among other bioactive components, possessing both nutritional and medicinal value. However, this fruit has significant limitations: firstly, the concentrated harvesting period and short shelf life, coupled with difficulties in storage and transportation, lead to unstable market supply; secondly, the fresh fruit has a limited flavor, failing to satisfy the diverse taste preferences of the public.

[0004] To address the aforementioned issues, domestic and international research has confirmed that lactic acid bacteria fermentation technology is an ideal solution for effectively improving fruit and vegetable products. This technology not only extends product shelf life but also simultaneously enhances flavor and quality, preserves nutrients, and ensures food safety. Fermenting wax apples with *Lactobacillus plantarum* holds promise for developing novel fermented beverages that combine health benefits with distinctive flavor. Summary of the Invention

[0005] This invention provides a fermented wax apple beverage and its preparation method. The fermented wax apple juice is rich in nutrients and has a strong aroma.

[0006] This invention provides a method for preparing a microbial agent that can ferment wax apple juice, comprising the following steps: inoculating activated Lactobacillus plantarum CGMCC1.16089 onto an MRS slant culture medium and placing it at 4°C to obtain the microbial agent.

[0007] In a preferred embodiment of the present invention, the activation includes thawing frozen Lactobacillus plantarum CGMCC1.16089, inoculating it at a 2% inoculum into MRS liquid medium, and anaerobically culturing it at 37°C until OD. 600 The value reaches 0.8~1.2, and the activated strain is obtained by continuous subculturing 2~3 times.

[0008] The present invention also provides a microbial agent prepared using the above-described preparation method.

[0009] The present invention also provides a method for fermenting wax apple juice, comprising activating Lactobacillus plantarum in the above-mentioned bacterial agent, inoculating it into wax apple juice for constant temperature anaerobic static fermentation, and obtaining wax apple fermented juice; wherein the pH value of the wax apple juice is 7.0±0.1.

[0010] In a preferred embodiment of the present invention, the wax apple juice comprises washing the wax apple pulp, pulping it, filtering it through a sieve, diluting it with deionized water, and pasteurizing the diluted wax apple juice.

[0011] In a preferred embodiment of the present invention, the mass ratio of fruit pulp to water during dilution is 1:(1~1.5).

[0012] In a preferred embodiment of the present invention, the pasteurization temperature is 85°C and the time is 15 minutes.

[0013] In a preferred embodiment of the present invention, the inoculum amount of the activated Lactobacillus plantarum in the wax apple juice is 2%.

[0014] In a preferred embodiment of the present invention, the temperature of the constant-temperature anaerobic static fermentation is 37°C, and the fermentation time is 8~24h.

[0015] The present invention also provides a wax apple fermented juice prepared using the above-described fermentation method.

[0016] The present invention also provides a fermented wax apple beverage prepared using the above-mentioned wax apple fermentation juice.

[0017] Beneficial effects: The *Lactobacillus plantarum* CGMCC1.16089 described in this invention can grow stably in wax apple juice, especially reaching a maximum viable count of 7.3483 × 10⁻⁶ cells after 8 hours of fermentation. 8 With a concentration of CFU / mL, it has good commercial application value and can be used to prepare fermentation agents for wax apple juice.

[0018] In this embodiment of the invention, the *Lactobacillus plantarum* CGMCC1.16089 is used to ferment wax apple juice. The fermented wax apple juice prepared can significantly increase the content of total flavonoids and polyphenols, especially the content of flavonols, flavones, dihydroflavones, flavanols, isoflavones, chalcones, dihydroflavonols, and anthocyanins, thus exhibiting good antioxidant capacity.

[0019] Meanwhile, the content of amino acids such as asparagine, L-aspartic acid, L-glutamic acid, L-arginine, L-isoleucine, L-glutamine, L-homoserine, L-tryptophan, L-lysine, citrulline, tyrosine, L-valine, L-proline, L-phenylalanine, L-ornithine, L-histidine, L-threonine, and L-tyrosine in the fermented wax apple juice prepared by this invention is significantly increased, thus significantly enhancing the nutritional value of the fermented wax apple juice.

[0020] Moreover, the types and contents of volatile compounds in the fermented wax apple juice of the present invention are significantly increased. For example, the contents of aroma substances such as (S)-2-methylbutanal, trans-3-hexenol acetate, 2-octenal, β-sesquiphlene, linalool propionate, geraniol, 3-octen-2-one, isoeugenol, (Z)-5-octenal, 2,3,5-trimethylfuran, sennatriene, 5-methyl-2-furfural, hexanal, decanal, methyl jasmonate, anethole, diallyl trisulfide, dihydrojasmone, 3-hexenal, eugenol, 4-ethyl-2-methoxyphenol, and butyl butyrate are increased, which greatly enhances the aroma of the fermented wax apple juice.

[0021] In summary, after fermentation with Lactobacillus plantarum CGMCC1.16089, the total flavonoid, polyphenol, and amino acid content in wax apple juice was significantly increased, enhancing its nutritional value; the types and contents of volatile aroma components were also significantly increased, greatly improving the aroma of wax apple juice, which can significantly improve the quality of wax apple juice, giving it better edible value and a broad market prospect. Attached Figure Description

[0022] Figure 1 A graph showing the changes in the viable count of Lactobacillus plantarum CGMCC1.16089; Figure 2 The graph shows the determination of the total antioxidant capacity of fermented wax apple juice; Figure 3 The graph shows the determination of total flavonoid content in fermented wax apple juice; Figure 4 The graph shows the determination of amino acid content in fermented wax apple juice; Figure 5 The content of various aroma compounds in fermented wax apple juice. Detailed Implementation

[0023] This invention provides a method for preparing a microbial agent that can ferment wax apple juice, comprising the following steps: inoculating activated Lactobacillus plantarum CGMCC1.16089 onto an MRS slant culture medium and placing it at 4°C to obtain the microbial agent.

[0024] The activation described in this invention includes thawing frozen Lactobacillus plantarum CGMCC1.16089, inoculating it at a 2% inoculum size into MRS liquid medium, and anaerobically culturing it at 37°C for 18-24 hours until OD (Organic Demand). 600 The concentration reaches 0.8~1.2, and activated strains are obtained by continuous subculturing 2~3 times. The composition of the MRS liquid culture medium of the present invention includes: 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract and 20.0 g / L glucose, which are sterilized at 121°C for 15 minutes before use.

[0025] The present invention also provides a microbial agent prepared using the above-described preparation method.

[0026] The present invention also provides a method for fermenting wax apple juice, comprising activating Lactobacillus plantarum in the above-mentioned bacterial agent, inoculating it into wax apple juice for constant temperature anaerobic static fermentation, and obtaining wax apple fermented juice; wherein the pH value of the wax apple juice is 7.0±0.1.

[0027] The *Lactobacillus plantarum* CGMCC1.16089 strain described in this invention can grow normally in wax apple juice. In one embodiment, the *Lactobacillus plantarum* CGMCC1.16089 strain was inoculated into wax apple juice for fermentation. The *Lactobacillus plantarum* CGMCC1.16089 strain started at 0.8267 × 10⁻⁶. 8 CFU / mL increased to 7.0626 × 10⁻⁶ h at 32 h. 8 The CFU / mL concentration initially increased and then decreased, indicating that the viable count of the fermentation strain in the juice needed to be ≥10. 8 Based on the CFU / mL, it is believed that Lactobacillus plantarum CGMCC1.16089 can grow stably in wax apple juice and has the potential to be developed into a commercial product.

[0028] This invention activates *Lactobacillus plantarum* CGMCC1.16089 using MRS liquid medium. The strain, thawed from its -80℃ glycerol storage, is inoculated at a 2% inoculum volume into MRS liquid medium and anaerobically cultured at 37℃ for 18-24 hours until OD (Organic Discharge) is reached. 600 The concentration of the bacteria reached 0.8~1.2. After 2~3 consecutive subcultures, the activated strain was obtained and inoculated into MRS slant medium and stored at 4℃.

[0029] The wax apple juice described in this invention is obtained by washing and pulping the wax apple pulp, filtering it, and then diluting it with deionized water. In one embodiment, the filtration process uses a 400-mesh filter to obtain the wax apple juice. The juice is then diluted, and the mass ratio of pulp to deionized water during dilution is any value within the range of 1:(1~1.5), such as 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5.

[0030] This invention adjusts the pH value of diluted wax apple juice. For example, NaHCO3 solution can be added dropwise to the juice to stabilize the pH value of the diluted wax apple juice at 7.0±0.1. The acidified juice is immediately dispensed into brown glass bottles, sealed, and frozen in a -20°C refrigerator away from light for later use.

[0031] Before fermentation, this invention also includes sterilizing the wax apple juice, such as through pasteurization at 85°C for 15 minutes. The activated bacterial strain is inoculated into the prepared diluted wax apple juice at a 2% inoculation rate, mixed thoroughly, and then placed in a 37°C constant temperature anaerobic incubator for static fermentation. The fermentation time can be 8–24 hours.

[0032] The present invention also provides a wax apple fermented juice prepared using the above-described fermentation method.

[0033] The fermented wax apple juice of this invention can significantly increase the total phenols and total flavonoids in wax apple juice, such as increasing the content of flavonols, flavones, dihydroflavones, flavanols, isoflavones, chalcones, dihydroflavonols, and anthocyanins. The fermented wax apple juice of this invention can also increase the content of amino acids in wax apple juice, such as asparagine, L-aspartic acid, L-glutamic acid, L-arginine, L-isoleucine, L-glutamine, L-homoserine, L-tryptophan, L-lysine, citrulline, tyrosine, L-valine, L-proline, L-phenylalanine, L-ornithine, L-histidine, L-threonine, and L-tyrosine. The fermented wax apple juice of the present invention can also enhance the aroma components, especially the content of volatile compounds: (S)-2-methylbutanal, trans-3-hexene, acetate, 2-octenal, β-sesquicaprene, o-cresol, linalool propionate, geraniol, 3-octen-2-one, isoeugenol, 2-ethyl-2-hexenal, (Z)-5-octenal, 2,3,5-trimethylfuran, sennatriene, 5-methyl-2-furfural, hexanal, decanal, methyl jasmonate, anethole, diallyl trisulfide, dihydrojasmone, 3-hexenal, eugenol, 4-ethyl-2-methoxyphenol, butyl butyrate, and 2-butylfuran.

[0034] The present invention also provides a fermented wax apple beverage prepared using the above-mentioned wax apple fermentation juice.

[0035] The fermented wax apple juice described in this invention is rich in nutrients and aroma components. It can be consumed directly or used as a raw material to prepare fermented wax apple beverages.

[0036] To further illustrate the present invention, the following detailed description of a wax apple fermented beverage and its preparation method provided by the present invention is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1 1. Activation and preservation of bacterial strains Lactobacillus plantarum CGMCC1.16089 strain was activated using MRS liquid medium. The strain, preserved at -80℃ with glycerol, was thawed and inoculated at a 2% inoculation rate into the medium. It was then anaerobically cultured at 37℃ for 18–24 hours until OD (Organic Depth) reached. 600The concentration of the bacteria reached 0.8~1.2. After 2~3 consecutive subcultures, the activated strain was obtained and inoculated into MRS slant medium and stored at 4℃.

[0038] 2. Detection of bacterial growth in wax apple juice (1) Preparation of wax apple juice The wax apples were selected from Wanning, Hainan. The wax apple pulp was washed, pulped, and the juice was filtered through a 400-mesh sieve. The juice was then diluted with deionized water at a 1:1 ratio to obtain wax apple juice. Under stirring conditions, 94% NaHCO3 solution was added dropwise to the wax apple juice at a rate of 1-2 drops per second using a 1 mL pipette, while simultaneously monitoring the pH with a calibrated pH meter. The addition was stopped when the pH stabilized at 7.0 ± 0.1. The acidified juice was immediately dispensed into brown glass bottles, sealed, and frozen at -20°C in the dark for later use.

[0039] (2) Inoculation and fermentation of microbial strains The activated strain was inoculated into the wax apple juice prepared in step (1) at a 2% inoculation rate, mixed well, and then placed in a 37℃ constant temperature anaerobic incubator for static fermentation.

[0040] (3) Growth detection of microbial strains Since inoculation, samples were aseptically taken every 4 hours. The bacterial culture at each time point was serially diluted with 0.85% physiological saline, and 100 μL was evenly spread on MRS solid plates. After anaerobic incubation at 37°C for 48 hours, viable bacteria were counted.

[0041] The growth changes of the strain are as follows Figure 1 As shown, the *Lactobacillus plantarum* CGMCC1.16089 strain started at 0.8267 × 10⁻⁶. 8 CFU / mL increased to 7.0626 × 10⁻⁶ h at 32 h. 8 The CFU / mL concentration initially increased and then decreased. Based on current research and patent practices in fermented fruit juice products both domestically and internationally, the viable count of the fermentation strain in the fruit juice needs to reach ≥10⁻⁶ CFU / mL. 8 Only when the concentration of CFU / mL is it considered valuable for development into a commercial product. The viable count of this strain reached its maximum value of 7.3483 × 10⁻⁶ cells / mL after 8 hours of fermentation in wax apple. 8 CFU / mL. This indicates that *Lactobacillus plantarum* CGMCC1.16089 can grow stably in wax apple juice.

[0042] Example 2 This example provides a fermented wax apple juice, and the specific preparation steps are as follows: (1) Wash the fresh wax apple pulp, pulp the pulp, filter the juice through a 400-mesh filter screen, and dilute it with deionized water at a ratio of 1:1. Under stirring conditions, add 94% NaHCO3 solution dropwise at a rate of 1-2 drops per second using a 1 mL pipette, while monitoring the pH in real time with a calibrated pH meter. Stop adding the solution when the pH stabilizes at 7.0±0.1 to obtain the original wax apple juice.

[0043] (2) Heat the wax apple juice to 85°C and pasteurize it for 15 minutes. After the juice cools down, inoculate the activated Lactobacillus plantarum CGMCC1.16089 strain from Example 1 into the wax apple juice prepared in step (1) at a 2% inoculation rate. After mixing, place it in a 37°C constant temperature anaerobic incubator and let it ferment for 8 hours to obtain wax apple fermented juice.

[0044] Example 3 This embodiment provides a fermented wax apple juice, and the specific preparation steps are as follows: (1) Wash the fresh wax apple pulp, pulp the pulp, filter the juice through a 400-mesh filter screen, and dilute it with deionized water at a ratio of 1:1.5. Under stirring conditions, add 94% NaHCO3 solution to the juice dropwise at a rate of 1-2 drops per second using a 1 mL pipette, while monitoring the pH in real time with a calibrated pH meter. Stop adding the solution when the pH stabilizes at 7.0±0.1 to obtain the original wax apple juice.

[0045] (2) Heat the wax apple juice to 85°C and pasteurize it for 15 minutes. After the juice cools down, inoculate the activated Lactobacillus plantarum CGMCC1.16089 strain from Example 1 into the wax apple juice prepared in step (1) at a 2% inoculation rate. After mixing, place it in a 37°C constant temperature anaerobic incubator and let it ferment for 24 hours to obtain wax apple fermented juice.

[0046] Comparative Example 1 This comparative example provides a fermented wax apple juice, and the specific preparation steps are as follows: (1) Inoculate the commercially available fruit juice fermentation bacteria into MRS liquid medium, centrifuge the bacterial solution to remove the supernatant, wash with sterile water, and resuspend in sterile water to achieve a bacterial density of ≥10. 8 CFU / mL.

[0047] (2) Wash the fresh wax apple pulp, pulp the pulp, filter the juice through a 400-mesh filter screen, and dilute it with deionized water at a ratio of 1:1. Under stirring conditions, add 94% NaHCO3 solution to the juice dropwise at a rate of 1-2 drops per second using a 1 mL pipette, while monitoring the pH in real time with a calibrated pH meter. Stop adding the solution when the pH stabilizes at 7.0±0.1 to obtain the original wax apple juice.

[0048] (3) Heat the wax apple juice to 85°C and pasteurize it for 15 minutes. After the juice cools down, inoculate the bacterial solution obtained in step (1) into the wax apple juice prepared in step (2) at a 2% inoculation rate, mix well, and place it in a 37°C constant temperature anaerobic incubator for static fermentation for 8 hours to obtain wax apple fermented juice.

[0049] Comparative Example 2 This comparative example provides a fermented wax apple juice, and the specific preparation steps are as follows: (1) Wash the fresh wax apple pulp, pulp the pulp, filter the juice through a 400-mesh filter screen, and dilute it with deionized water at a ratio of 1:1. Under stirring conditions, add 94% NaHCO3 solution to the juice dropwise at a rate of 1-2 drops per second using a 1 mL pipette, while monitoring the pH in real time with a calibrated pH meter. Stop adding the solution when the pH stabilizes at 7.0±0.1 to obtain the original wax apple juice.

[0050] (2) Heat the wax apple juice to 85°C and pasteurize it for 15 minutes. After the juice cools down, add sterile water at a volume ratio of 2%, mix well, and place it in a 37°C constant temperature anaerobic incubator for static fermentation for 8 hours to obtain wax apple fermented juice.

[0051] Comparative Example 3 This comparative example provides a fermented wax apple juice, and the specific preparation steps are as follows: (1) Inoculate the commercially available fruit juice fermentation bacteria into MRS liquid medium, centrifuge the bacterial solution to remove the supernatant, wash with sterile water, and resuspend in sterile water to achieve a bacterial density of ≥10. 8 CFU / mL.

[0052] (2) Wash the fresh wax apple pulp, pulp the pulp, filter the juice through a 400-mesh filter screen, and dilute it with deionized water at a ratio of 1:1.5. Under stirring conditions, add 94% NaHCO3 solution to the juice dropwise at a rate of 1-2 drops per second using a 1 mL pipette, while monitoring the pH in real time with a calibrated pH meter. Stop adding the solution when the pH stabilizes at 7.0±0.1 to obtain the original wax apple juice.

[0053] (3) Heat the wax apple juice to 85°C and pasteurize it for 15 minutes. After the juice cools down, inoculate the bacterial solution obtained in step (1) into the wax apple juice prepared in step (2) at a 2% inoculation rate, mix well, and place it in a 37°C constant temperature anaerobic incubator for static fermentation for 24 hours to obtain wax apple fermented juice.

[0054] Comparative Example 4 (1) Wash the fresh wax apple pulp, pulp the pulp, filter the juice through a 400-mesh filter screen, and dilute it with deionized water at a ratio of 1:1.5. Under stirring conditions, add 94% NaHCO3 solution to the jujube juice at a rate of 1-2 drops per second using a 1 mL pipette, and monitor the pH in real time with a calibrated pH meter. Stop adding the solution when the pH stabilizes at 7.0±0.1 to obtain the original wax apple juice.

[0055] (2) Heat the wax apple juice to 85°C and pasteurize it for 15 minutes. After the juice cools down, add sterile water at a volume ratio of 2%, mix well, and place it in a 37°C constant temperature anaerobic incubator for static fermentation for 24 hours to obtain wax apple fermented juice.

[0056] The quality of the wax apple fermented juice provided in Examples 2-3 and Comparative Examples 1-4 was measured, and the details are as follows: 1. Analysis of total antioxidant capacity (1) Determination of total antioxidant capacity of ABTS Mix 7.4 mmol / L ABTS solution and 2.6 mmol / L potassium persulfate solution at a volume ratio of 1:1, and let stand at room temperature in the dark for 12-16 hours to prepare the ABTS working solution. Before use, dilute with PBS buffer (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. Add the reagents sequentially to a 96-well plate: Experimental group: 10 μL sample solution and 190 μL ABTS working solution; Blank group: 10 μL anhydrous ethanol and 190 μL LABTS working solution; Control group: 10 μL sample solution and 190 μL anhydrous ethanol.

[0057] Immediately after reacting at room temperature in the dark for 6 minutes, the absorbance was measured at a wavelength of 734 nm. A concentration gradient standard curve was prepared using Trolox as a standard. The total antioxidant capacity of the wax apple juice was calculated according to the equation of the standard curve. The results are expressed in μmol Trolox / mL. The calculation formula is as follows: Where: C—concentration (μM) calculated based on the Trolox standard curve; D: sample dilution factor (D=1 in this experiment); V: original sample volume (ml).

[0058] (2) Determination of total antioxidant capacity of DPPH Weigh 3.94 mg of DPPH reagent, dissolve it in anhydrous ethanol, and dilute to 100 mL to prepare a 0.1 mmol / L DPPH stock solution. Store in the dark for later use. Add the reagents sequentially to a 96-well plate: Experimental group: 10 μL sample solution + 190 μL LPPH working solution; Blank group: 10 μL anhydrous ethanol + 190 μL DPPH working solution; Control group: 10 μL sample solution + 190 μL anhydrous ethanol.

[0059] After mixing, incubate at room temperature in the dark for 30 minutes. Immediately measure the absorbance at 517 nm. Construct a concentration gradient standard curve using Trolox as a standard. Calculate the total antioxidant capacity of the wax apple juice according to the standard curve equation. The result is expressed as μmol Trolox / mL. The calculation formula is as follows: Where: C—concentration (μM) calculated based on the Trolox standard curve; D: sample dilution factor (D=1 in this experiment); V: original sample volume (ml).

[0060] The total antioxidant capacity of fermented wax apple juice is as follows: Figure 2 As shown, regardless of whether it is ABTS or DPPH, the total antioxidant capacity of Example 2 is significantly higher than that of Example 3 and the various comparative examples.

[0061] 2. Analysis of total flavonoid content This experiment used the aluminum reagent colorimetric method to quantitatively analyze flavonoids in the original juice and fermented juice of wax apple. The specific experimental methods and results are as follows: Weigh 10.0 mg of rutin standard, dissolve it in 70% ethanol, and dilute to a volumetric flask of 100 mL to obtain a 0.1 mg / mL rutin standard solution. Store at 4°C protected from light. Pipette 0, 0.5, 1.0, 2.0, 3.0, and 4.0 mL of the rutin standard solution into 10 mL volumetric flasks, and add 70% ethanol to each flask to a final volume of 5 mL. Add 0.3 mL of 5% NaNO₂ solution sequentially, shake well, and let stand for 6 min; add 0.3 mL of 10% Al(NO₃)₃ solution, shake well, and let stand for 6 min; then add 4 mL of 4% NaOH solution, and dilute to a final volume of 10 mL with 70% ethanol. Shake well and let stand for 15 min. Take 1 mL of fermented wax apple juice, centrifuge at 12000 rpm for 10 min, and dilute the supernatant 10 times with 70% ethanol. Take 1 mL of the diluted solution and develop the color using the standard curve method described above, with 70% ethanol as a blank control, and measure the absorbance at a wavelength of 510 nm. Then, the total flavonoid content was calculated based on the sample absorbance and the standard curve.

[0062] Depend on Figure 3 It can be seen that, compared with the comparative example, the wax apple juice prepared in Example 2 and Example 3 has a higher content of flavonoids. When the fermentation time is 8 hours, the total flavonoid content in the wax apple juice is the highest.

[0063] 3. Amino acid content analysis This experiment analyzed the amino acids in the original juice and fermented juice of wax apple using LC-MS. The specific experimental conditions and results are as follows: Chromatographic conditions: Ultra-high performance liquid chromatography system, using ACQUITYUPLC®HSST3 (2.1×100mm, 1.8µm) column (Waters, Milford, MA, USA), flow rate of 0.3mL / min, column temperature of 40℃, injection volume of 2μL.

[0064] In positive ion mode, the mobile phase consisted of 0.1% formic acid acetonitrile (B2) and 0.1% formic acid water (A2), with the gradient elution program as follows: 0–1 min, 10% B2; 1–5 min, 10%–98% B2; 5–6.5 min, 98% B2; 6.5–6.6 min, 98%–10% B2; 6.6–8 min, 10% B2.

[0065] In negative ion mode, the mobile phase consisted of acetonitrile (B3) and 5 mM ammonium formate water (A3), with the gradient elution program as follows: 0–1 min, 10% B3; 1–5 min, 10%–98% B3; 5–6.5 min, 98% B3; 6.5–6.6 min, 98%–10% B3; 6.6–8 min, 10% B3.

[0066] Mass spectrometry conditions: ThermoQ Exactive Focus mass spectrometer (ThermoFisher Scientific, USA), electrospray ionization (ESI) source, and data acquisition in both positive and negative ion modes. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 40 alb, and the auxiliary gas was 10 alb. The capillary temperature was 325 °C. A first-stage full scan was performed at a resolution of 70,000 m / z, with a first-stage ion scan range of 100–1000 m / z. Second-stage fragmentation was performed using an HCD with a collision energy of 30 eV and a second-stage resolution of 17,500 m / z. The first three ions acquired were fragmented, and unnecessary MS / MS information was removed using dynamic exclusion.

[0067] The content of various amino acids in fermented wax apple juice, such as Figure 4 As shown, the content includes 9 essential amino acids (of which arginine is essential for infants) and 9 non-essential amino acids. In Example 2, when fermented with *Lactobacillus plantarum* CGMCC1.16089 for 8 hours, the amino acid content was the highest, with the most significant differences observed in arginine and aspartic acid content. This demonstrates that the nutritional value of wax apple juice is enhanced after 8 hours of fermentation with *Lactobacillus plantarum* CGMCC1.16089, especially for infants and young children.

[0068] 4. Aroma Analysis Tests revealed 37 volatile compounds in the fermented wax apple juice. Classified by compound structure, these included 7 terpenes, 8 aldehydes, 6 esters, 5 ketones, 6 aromatics, 3 furans, and 2 others (methyl jasmonate and diallyl trisulfide).

[0069] Changes in the content of various aroma compounds after wax apple fermentation are as follows: Figure 5 As shown: Compared with the comparative example, the examples have a higher content of volatile aroma compounds, with significant differences in the content of aldehydes and lipids. In particular, Example 2 has the highest content of aroma compounds, including (S)-2-methylbutanal, 3-hexenal, 2-octenal, decanal, hexanal, 4-methylbenzaldehyde, (Z)-5-octenal, 2-ethyl-2-hexenal, linalool propionate, geraniol acetate, trans-3-hexenol acetate, (E)-3-heptenol acetate, butyl butyrate, allyl nonanoate, etc. These volatile aroma compounds can significantly enhance the aroma of wax apple juice. For example, (S)-2-methylbutanal has… It has a chocolate aroma; hexenal has a fresh aroma of green leaves and vegetable roots; octenal has a fatty aroma; decanal has a citrus aroma; hexanal has a fruity aroma; 4-methylbenzaldehyde has a floral and almond aroma; (Z)-5-octenal has a plant leaf aroma with woody and apple notes; 2-ethyl-2-hexenal has a baked aroma; linalool propionate has a sweet bergamot and floral aroma; geraniol acetate has a sweet bergamot fruit aroma with rose and lavender notes; trans-3-hexenol acetate has a grassy and green leaf aroma; (E)-3-heptenol acetate has a green and fruity aroma; butyl butyrate has a pineapple and banana-like fruity aroma; allyl nonanoate has a peach aroma.

[0070] In summary, the *Lactobacillus plantarum* CGMCC1.16089 strain identified in this application significantly optimizes the nutritional and flavor characteristics of wax apple juice through fermentation. After 8 hours of fermentation, the levels of flavonoids and polyphenols in the wax apple juice increased, resulting in a marked enhancement of its antioxidant activity. Simultaneously, the amino acid content increased significantly, improving its nutritional value. Furthermore, the fermentation process promoted the formation of aromatic volatile compounds, effectively improving the aroma characteristics of the wax apple juice. These quality enhancements give fermented wax apple juice superior edible value and market competitiveness.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a microbial agent capable of fermenting wax apple juice, characterized in that, The process includes the following steps: Inoculating activated Lactobacillus plantarum CGMCC1.16089 onto MRS slant agar medium and placing it at 4°C to obtain the bacterial agent.

2. The preparation method according to claim 1, characterized in that, The activation process involves thawing frozen Lactobacillus plantarum CGMCC1.16089, inoculating it at a 2% inoculum size into MRS liquid medium, and anaerobically culturing it at 37°C until OD500. 600 The value reaches 0.8~1.2, and the activated strain is obtained by continuous subculturing 2~3 times.

3. The microbial agent prepared using the preparation method described in claim 1 or 2.

4. A method for fermenting wax apple juice, characterized in that, The method involves activating the *Lactobacillus plantarum* in the bacterial agent described in claim 3, then inoculating it into wax apple juice for constant-temperature anaerobic static fermentation to obtain wax apple fermented juice; the pH value of the wax apple juice is 7.0±0.

1.

5. The fermentation method according to claim 4, characterized in that, The wax apple juice is made by washing the wax apple pulp, pulping it, filtering it through a sieve, diluting it with deionized water, and then pasteurizing the diluted wax apple juice.

6. The fermentation method according to claim 5, characterized in that, The pasteurization temperature is 85°C and the time is 15 minutes.

7. The fermentation method according to claim 4, characterized in that, After activation, the amount of *Lactobacillus plantarum* inoculated into the wax apple juice is 2%.

8. The fermentation method according to claim 4, characterized in that, The temperature for the constant-temperature anaerobic static fermentation is 37℃, and the fermentation time is 8~24h.

9. The fermented wax apple juice prepared by the fermentation method according to any one of claims 4 to 8.

10. A fermented wax apple beverage prepared using the wax apple fermentation juice according to claim 9.

Images