Passion fruit and black tea low-sugar beverage

Abstract

The invention relates to the technical field of food processing, and discloses a passion fruit and black tea low-sugar beverage which comprises the following components in parts by weight: 25-35 parts of passion fruit pulp; 65-75 parts of black tea soup; the sweetening agent is prepared from the following components in parts by weight: 0.01 to 0.03 part of acetoacetic acid, 0.005 to 0.015 part of erythritol and 0.002 to 0.006 part of stevioside; the nanometer embedded particles are nanometer particles formed by cross-linking zein and chitosan according to the mass ratio of 3: 1, the particle size is 50-80 nm, and 0.1-0.3 part of volatile aroma components are embedded in the nanometer embedded particles; the endogenous stabilizer is prepared from 0.05 to 0.1 part of extracellular polysaccharide generated by lactobacillus acidophilus and 0.8 to 1.0 part of tea polyphenol. Through the synergistic interaction between natural sweet metabolites (such as acetoacetic acid) generated by microbial fermentation and sugar substitute molecules, on the premise that the sugar content is not increased, the sweetness intensity is multiplied, and through self-assembly of exopolysaccharides generated in situ in the fermentation process and tea polyphenol, a three-dimensional stable network is formed. Long-acting stability of suspended particles can be realized without depending on exogenous colloids such as pectin and carrageenan.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and specifically to a low-sugar passion fruit black tea drink. Background Art

[0002] With the upgrade of the healthy consumption trend, the market demand for low-sugar drinks continues to grow, but the industry still faces multiple technical bottlenecks. In traditional processes, to maintain the perception of sweetness, a high-dose compounding scheme of sugar substitutes is generally adopted, resulting in problems such as metallic bitterness and an abrupt artificial sweet taste in the drink, and it is difficult to meet consumers' pursuit of natural flavors.

[0003] In terms of physical stability, the existing technologies rely on exogenous colloids such as pectin and carrageenan to maintain the suspension system. The introduction of such chemical additives not only goes against the trend of clean labels, but is more likely to cause molecular chain breakage under high-temperature or long-term storage conditions, leading to precipitation, delamination, and turbidity deterioration. There are inherent defects in the control of the release of flavor components. Aroma substances and sweet molecules escape or are oxidized and inactivated in advance during storage, resulting in a lagging and single taste experience when drinking. These systematic defects restrict the commercialization process of low-sugar drinks, and there is an urgent need to achieve a coordinated improvement in sensory quality, stability performance, and health attributes through technological innovation. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a low-sugar passion fruit black tea drink, which solves the problems of insufficient sweetness perception, strong dependence on chemical additives, and poor physical stability in the development of existing low-sugar drinks.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A low-sugar passion fruit black tea drink, comprising the following components in parts by weight: Passion fruit pulp: 25 - 35 parts; Black tea soup: 65 - 75 parts; Sweetener: composed of 0.01 - 0.03 parts of acetoacetic acid, 0.005 - 0.015 parts of erythritol, and 0.002 - 0.006 parts of stevioside; Nano-encapsulation particles: nano-particles formed by cross-linking zein and chitosan in a mass ratio of 3:1, with a particle size of 50 - 80 nm, encapsulating 0.1 - 0.3 parts of volatile aroma components; Endogenous stabilizer: composed of 0.05 - 0.1 parts of exopolysaccharide produced by Lactobacillus acidophilus and 0.8 - 1.0 parts of tea polyphenols.

[0006] Preferably, the mass ratio of erythritol to stevioside in the sweetener is 1:0.5.

[0007] Preferably, the volatile aroma components in the nano-encapsulation particles are ethyl hexanoate and β-ionone, and their mass ratio to the nano-particles is 1:10 - 1:12.

[0008] Preferably, the proportion of hydrogen bond interaction between exopolysaccharide and tea polyphenols in the endogenous stabilizer is ≥60%.

[0009] Preferably, the pH of the beverage is 3.8 - 4.0, and the soluble solids content is ≤5 °Brix.

[0010] A preparation method of a low - sugar passion fruit black tea beverage includes the following steps: S1. Raw material pretreatment: Break the shell of passion fruit to obtain pulp and filter it, then mix it with a color - protecting agent; Crush black tea and pre - soak it to obtain tea soup; S2. Co - fermentation of two strains: Inoculate Lactobacillus acidophilus and Saccharomycopsis fibuligera into the mixed solution, and control the oxygen content and pH in stages for fermentation; S3. Preparation of nanoparticles: Cross - link zein and chitosan to form nanoparticles; S4. Aroma encapsulation: Mix the volatile components in the fermentation broth with the nanoparticles, and adjust the pH to complete encapsulation; S5. Construction of a stable network: Promote the self - assembly of exopolysaccharide and tea polyphenols through inactivation treatment, and finally carry out sterilization and filling.

[0011] Preferably, the staged control in step S2 includes: In the first stage, control the dissolved oxygen ≤0.2 mg / L, the temperature is 32 ± 1 °C, and the pH is 4.0 - 4.2; In the second stage, the dissolved oxygen rises to 0.5 - 1.0 mg / L, and the pH naturally drops to 3.8 - 4.0.

[0012] Preferably, the inactivation conditions in step S5 are treatment at 50 ± 2 °C for 20 - 30 minutes, and add 0.05 - 0.1% of sodium citrate to adjust the ionic strength; The sterilization conditions are treatment at 80 ± 2 °C for 15 - 20 seconds, and the light transmittance of the packaging material is <5%.

[0013] Preferably, in S3, the cross - linking reaction uses transglutaminase, and the addition amount is 0.05 - 0.1% of the total mass of zein and chitosan.

[0014] Preferably, in S4, the pH is adjusted to 4.5 ± 0.1.

[0015] The present invention provides a low - sugar passion fruit black tea beverage. It has the following beneficial effects: 1. Through the synergistic effect between the natural sweet metabolic products (such as acetoacetic acid) produced by microbial fermentation and the sugar substitute molecules, the intensity of the sweet taste is doubled without increasing the sugar content. This mechanism breaks through the technical bottleneck of traditional formulations that rely on high doses of sugar substitutes to compensate for sweetness, effectively avoiding the metallic bitterness or off-flavor residues caused by excessive sugar substitutes, and making the sweetness of the beverage closer to natural fruit aroma.

[0016] 2. The present invention utilizes the extracellular polysaccharides generated in situ during the fermentation process and tea polyphenols to self-assemble into a three-dimensional stable network, enabling long-term stability of suspended particles without relying on exogenous colloids such as pectin and carrageenan.

[0017] 3. Based on the pH-responsive encapsulation system and the complexation structure of sweet molecules, the present invention precisely controls the release timing of aroma components and sweet substances in different sections of the oral cavity. When consumed, a strong flavor is instantly released, while the components remain stable during storage, overcoming the problems of taste lag or premature dissipation caused by slow-release technologies in traditional processes.

[0018] 4. The present invention avoids the addition of synthetic sweeteners, preservatives, and artificial stabilizers, and only achieves the simultaneous optimization of sweetness, stability, and safety through strain selection and process control, meeting consumers' core demands for "low-sugar, zero-additive" healthy beverages and possessing significant market competitiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a flowchart of the method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] Example 1: Please refer to the attached Figure 1 , the embodiment of the present invention provides a method for preparing a low-sugar passion fruit black tea beverage, including the following steps: S1: Raw material pretreatment Passion fruit pulp: 30 parts (by volume), mixed with 0.03 parts of vitamin C for color protection; Black tea soup: 70 parts (by volume), prepared by pre-soaking Dianhong broken tea crushed to 70 mesh at a ratio of tea: water = 1:18 (by mass) at 48 °C for 22 minutes.

[0022] S2: Co-fermentation of two strains Strain inoculation: Lactobacillus acidophilus (1×10 8CFU / mL) and Saccharomycopsis fibuligera (2×10 8 CFU / mL) were mixed at a volume ratio of 1:2.5, and the total inoculation amount was 4%; Stage 1 (0 - 6 h): Dissolved oxygen was 0.1 mg / L, temperature was 32 °C, pH was 4.1, and stirring rate was 75 rpm; Stage 2 (6 - 12 h): Dissolved oxygen was 0.75 mg / L, and pH naturally dropped to 3.9.

[0023] S3: Nanoparticle preparation Zein:Chitosan = 3:1 (mass ratio), 0.075% transglutaminase was added, and crosslinking was carried out at 55 °C for 35 minutes to obtain particles with a particle size of 65 nm.

[0024] S4: Aroma encapsulation Volatile components (ethyl hexanoate:β-ionone = 1:1) and nanoparticles were mixed at a mass ratio of 1:11, the pH was adjusted to 4.5, and stirring was carried out at 550 rpm for 2.5 hours.

[0025] S5: Stable network construction Inactivation conditions: Treatment at 50 °C for 25 minutes, and 0.075% sodium citrate was added; The extracellular polysaccharide concentration after self-assembly was 1.8 g / L, and the tea polyphenol concentration was 900 mg / L.

[0026] S6: Sterilization and filling Pasteurization: Treatment at 80 °C for 17.5 seconds, and filled into UV-blocking PET bottles with a light transmittance of 3%.

[0027] Example 2: Please refer to the appendix Figure 1 , The embodiment of the present invention provides a preparation method of a low-sugar passion fruit black tea drink, including the following steps: S1: Raw material pretreatment Passion fruit pulp: 25 parts (by volume), mixed with 0.025 parts of vitamin C; Black tea soup: 75 parts (by volume), tea leaves:water = 1:15 (mass ratio), pre-soaked at 45 °C for 20 minutes.

[0028] S2: Co-fermentation of two bacteria Strain inoculation: The volume ratio of the two bacteria was 1:2, and the total inoculation amount was 3%; Stage 1 (0 - 6 h): Dissolved oxygen was 0.05 mg / L, temperature was 31 °C, pH was 4.0, and stirring was 50 rpm; Stage 2 (6 - 12 h): Dissolved oxygen was 0.5 mg / L, and pH dropped to 3.8.

[0029] S3: Nanoparticle preparation Zein:chitosan = 3:1, adding 0.05% transglutaminase, crosslinking at 53°C for 30 minutes, particle size 50nm.

[0030] S4: Aroma encapsulation Aroma:nanoparticle = 1:10, pH 4.4, stirring at 500 rpm for 2 hours.

[0031] S5: Stable network construction Inactivation conditions: treating at 48°C for 20 minutes, adding 0.05% sodium citrate; Concentration of exopolysaccharide 1.5 g / L, concentration of tea polyphenols 800 mg / L.

[0032] S6: Sterilization and filling Pasteurization: treating at 78°C for 15 seconds, light transmittance of the package 4.5%.

[0033] Example 3: Please refer to the appendix Figure 1 , the embodiment of the present invention provides a preparation method of a low-sugar passion fruit black tea drink, including the following steps: S1: Raw material pretreatment Passion fruit pulp: 35 parts (by volume), mixed with 0.035 part of vitamin C; Black tea soup: 65 parts (by volume), tea leaves:water = 1:20 (by mass ratio), pre-soaking at 50°C for 25 minutes.

[0034] S2: Co-fermentation of two strains Strain inoculation: volume ratio of the two strains 1:3, total inoculation amount 5%; Stage 1 (0 - 6h): dissolved oxygen 0.2 mg / L, temperature 33°C, pH 4.2, stirring at 100 rpm; Stage 2 (6 - 12h): dissolved oxygen 1.0 mg / L, pH drops to 4.0.

[0035] S3: Nanoparticle preparation Zein:chitosan = 3:1, adding 0.1% transglutaminase, crosslinking at 57°C for 40 minutes, particle size 80nm.

[0036] S4: Aroma encapsulation Aroma:nanoparticle = 1:12, pH 4.6, stirring at 600 rpm for 3 hours.

[0037] S5: Stable network construction Inactivation conditions: treating at 52°C for 30 minutes, adding 0.1% sodium citrate; Concentration of exopolysaccharide 2.0 g / L, concentration of tea polyphenols 1000 mg / L.

[0038] S6: Sterilization and filling Pasteurization: Treat at 82 °C for 20 seconds, and the light transmittance of the packaging is 4.8%.

[0039] Comparative Example 1: Compared with Example 1, the difference lies in that only single-strain fermentation with Lactobacillus acidophilus is used (Saccharomycopsis fibuligera is not inoculated), and the remaining steps and parameters are the same.

[0040] Comparative Example 2: Compared with Example 1, the difference lies in that the nanoparticles are only prepared from zein (chitosan and the cross-linking agent transglutaminase are not added), and the remaining steps and parameters are the same.

[0041] Comparative Example 3: Compared with Example 1, the difference lies in that no staged fermentation control is carried out (dissolved oxygen ≤ 0.2 mg / L and pH 4.0 - 4.2 are maintained throughout the process), and the remaining steps and parameters are the same.

[0042] Comparative Example 4: Compared with Example 1, the difference lies in that exogenous pectin is used to replace the endogenous stable network (0.1% pectin is added, and the self-assembly of extracellular polysaccharide and tea polyphenol is not retained), and the remaining steps and parameters are the same.

[0043] Comparative Example 5: Compared with Example 1, the difference lies in that the sweeteners only contain erythritol and stevioside (acetoacetic acid is not retained), and the mass ratio of the two is 1:1, and the remaining steps and parameters are the same.

[0044] Comparative Example 6: Compared with Example 1, the difference lies in that the pasteurization conditions are 95 °C / 30 seconds (replacing the original 80 °C / 17.5 seconds), and the remaining steps and parameters are the same.

[0045] Test Example 1: Verification experiment on the metabolic complementarity of dual-strain co-fermentation Experimental purpose Verify the metabolic complementarity of the dual-strain co-fermentation of Lactobacillus acidophilus and Saccharomycopsis fibuligera, and compare the effects of single-strain fermentation (Comparative Example 1) on the extraction rate of tea polyphenols, the production amount of acetoacetic acid, and the cellulase activity.

[0046] Experimental steps Fermentation process Example group (Examples 1 - 3): Conduct dual-strain co-fermentation according to the claim parameters, and control the dissolved oxygen and pH in stages.

[0047] Control group (Comparative Example 1): Only inoculate Lactobacillus acidophilus, and the fermentation conditions are the same as those in Example 1 (dissolved oxygen ≤ 0.2 mg / L and pH 4.0 - 4.2 are maintained throughout the process).

[0048] Sample collection Time points: end of stage I (6 h), end of stage II (12 h).

[0049] Sampling location: sample evenly from the middle of the fermenter, take 50 mL each time, and immediately terminate the reaction in an ice bath.

[0050] Index detection Tea polyphenol extraction rate: Take 5 mL of the fermentation broth, centrifuge (8000×g, 10 min), and determine the supernatant by the Folin-Ciocalteu method (at a wavelength of 760 nm).

[0051] Calculation formula: Tea polyphenol extraction rate ; Acetoacetic acid content: Take 1 mL of the fermentation broth, filter it through a 0.22 μm filter membrane, and detect it by HPLC (C18 column, mobile phase methanol: 0.1% phosphoric acid = 15:85, flow rate 1 mL / min, detection wavelength 210 nm).

[0052] Cellulase activity: Take 10 mL of the fermentation broth, centrifuge and take the supernatant, and determine the cellulase activity by the DNS method (calculated in terms of glucose equivalent, unit U / mL).

[0053] Data processing Each experiment in each group was repeated 3 times, and the average value ± standard deviation was taken.

[0054] The test results are shown in Table 1: Table 1 Experimental data for verifying the metabolic complementarity of dual-bacteria co-fermentation Experimental summary: Through the comparison between co-fermentation of two strains and single-strain fermentation, this experiment revealed the crucial role of the metabolic complementarity between Lactobacillus acidophilus and Saccharomycopsis fibuligera in the extraction rate of tea polyphenols and the synthesis of sweet precursors. Under the synergistic effect of staged oxygen control and pH regulation, the anaerobic environment in the first stage (dissolved oxygen ≤ 0.2 mg / L, pH 4.0 - 4.2) preferentially activated the metabolic pathway of Lactobacillus acidophilus, efficiently converting citric acid in passion fruit into acetoacetic acid, with its content reaching 131.4 - 142.6 mg / L, significantly higher than 120.5 mg / L in single-strain fermentation. This process benefited from the selective utilization of citric acid by lactic acid bacteria under anaerobic conditions. Yeast secreted cellulase in the microaerobic environment (dissolved oxygen 0.5 - 1.0 mg / L) in the second stage, releasing bound tea polyphenols by enzymatic hydrolysis of the tea cell wall, increasing the extraction rate to 88.5 - 92.3%, far exceeding 65.8% in single-strain fermentation. Experimental data showed that the sequential division of labor in the metabolism of the two strains avoided substrate competition. Meanwhile, the metabolic by-products of yeast (such as reducing sugars) provided additional carbon sources for Lactobacillus acidophilus, forming a positive feedback loop.

[0055] The core of the synergistic effect of the two strains lies in the coupled design of the cross-species metabolic network. The acidic environment (pH naturally dropped to 3.8 - 4.0) generated by Lactobacillus acidophilus in the first stage not only inhibited the growth of miscellaneous bacteria but also created suitable conditions for the enzymatic hydrolysis of yeast in the second stage. The cellulase secreted by yeast (activity 25.3 - 28.7 U / mL) specifically degraded the tea fiber structure, increasing the release efficiency of tea polyphenols by more than 35%. Meanwhile, acetoacetic acid produced by the metabolism of Lactobacillus acidophilus, as a natural sweet precursor, effectively neutralized the aftertaste when compounded with exogenous sweeteners. In single-strain fermentation, the lack of this metabolite led to a significant decline in sweet coordination. The integration of this metabolic-enzymatic double path broke through the efficiency bottleneck of traditional single-strain or chemical extraction methods.

[0056] From the perspective of technical advantages, staged dynamic regulation achieved precise orientation of the metabolic activities of the strains. The efficient and synchronous production of tea polyphenols and acetoacetic acid in the examples verified the guiding role of oxygen control and pH sequential regulation on the functions of the microbial community. In contrast, in Comparative Example 1, due to the lack of the enzymatic hydrolysis contribution of yeast, the extraction rate of tea polyphenols decreased significantly, and no cellulase activity was detected, directly resulting in a weak flavor and insufficient functional components in the beverage. In addition, the interaction of the metabolic products of the two strains (such as the self-assembly of EPS and tea polyphenols) laid the foundation for the construction of a subsequent stable network, forming an all-chain innovation from fermentation to finished products. Through the cross-integration of microbial metabolic regulation and materials science, the problem of synergistic optimization of flavor, stability, and health attributes in the development of low-sugar beverages was solved.

[0057] Test Example 2: Verification Experiment on the Functional Embedding Performance of Nanoparticles Verify the encapsulation efficiency of zein-chitosan cross-linked nanoparticles for aroma components and their pH-responsive release characteristics, and compare the performance differences with Comparative Example 2 without chitosan cross-linking.

[0058] Experimental procedures Sample preparation Example group (Examples 1-3): Prepare cross-linked nanoparticles (containing chitosan and transglutaminase) according to the claim parameters.

[0059] Control group (Comparative Example 2): Prepare particles only with zein (without chitosan cross-linking).

[0060] Encapsulation efficiency test Take 10 mL of the nanoparticle suspension and mix it with the volatile aroma components (ethyl hexanoate: β-ionone = 1:1), and stir magnetically (500 rpm) for 2 hours.

[0061] Centrifuge (12,000×g, 15 min) to separate the unencapsulated free aroma, and measure the residual aroma concentration in the supernatant by GC-MS.

[0062] Calculation formula for encapsulation rate: ; Accelerated stability test Place the encapsulated nanoparticle suspension in an incubator at 40 °C, sample and detect the aroma residual rate weekly for 4 weeks.

[0063] pH-responsive release test Simulate the oral environment: Take 2 mL of the encapsulated particle suspension, adjust the pH to 6.8 with PBS buffer, and oscillate (100 rpm) at 37 °C for 30 minutes.

[0064] After centrifugation, measure the aroma concentration released into the solution and calculate the release rate.

[0065] Particle size detection Dilute the nanoparticle suspension to 0.1 mg / mL and measure the particle size distribution with a dynamic light scattering instrument The test results are shown in Table 2: Table 2 Comparative data on the encapsulation performance of nanoparticles Experimental summary: This experiment revealed the protection and controlled release mechanism of the zein-chitosan composite structure on aroma components by comparing the embedding performance of cross-linked and non-cross-linked nanoparticles. The amino groups of chitosan and the carboxyl groups of zein form a cross-linked network under the catalysis of glutamine transaminase, constructing nanoparticles with both a hydrophobic core and a positively charged surface. During the embedding stage, the positive charge on the particle surface (Zeta potential +25mV) adsorbs negatively charged aroma molecules (such as ethyl hexanoate) through electrostatic action, while the hydrophobic core embeds non-polar components (such as β-ionone) through van der Waals forces, so that the embedding rate of the embodiment group reaches 88.6-92.3%, far exceeding the 65.8% of non-cross-linked particles. The cross-linked structure also significantly reduces the average particle size of the particles (51.8-78.9nm vs. 132.6nm), reducing the physical escape of aroma due to interfacial tension.

[0066] The pH response characteristics of the nanoparticles are derived from the protonation-deprotonation behavior of chitosan. In the acidic environment of the beverage (pH3.8-4.0), the chitosan amino groups are protonated and positively charged, which enhances the electrostatic bonding with the aroma molecules. At the same time, the hydrophobic core is tightened to form a double aroma-locking barrier. Under the simulated neutral pH of the oral cavity (6.8), chitosan is deprotonated, the electrostatic effect is weakened, and the hydrophilicity of the particle surface is increased, triggering the rapid release of aroma (release rate 78.4-83.7%). The dynamic release characteristics enable the beverage to maintain a stable aroma during the storage period, and instantly release a rich fruity aroma when drinking, overcoming the taste lag problem caused by the sustained release of traditional microcapsules.

[0067] The long-term protection ability of the cross-linked structure was further verified in the accelerated experiment. The dense cross-linked network of the particles in the embodiment group effectively blocked the penetration of oxygen and water molecules at high temperature (40°C). After 4 weeks, the aroma residual rate was still over 85%, while the non-cross-linked particles had a loose structure and a residual rate of only 53.2%. In addition, the narrow particle size distribution of the cross-linked particles (standard deviation ≤ 5.2nm) ensures the uniformity of encapsulation and avoids sudden release of aroma caused by local overload. Precise regulation of the entire chain from encapsulation to release is achieved, providing an innovative solution for low-sugar beverages that takes into account both flavor stability and sensory experience.

[0068] Test Example 3: Experimental description of the necessity of phased oxygen control and pH adjustment The effects of phased oxygen control and pH adjustment on acetoacetate production, tea polyphenols release and yeast activity were verified, and the metabolic efficiency differences when anaerobic conditions were maintained throughout the process (Comparative Example 3) were compared.

[0069] Experimental procedures Fermentation process Example group (Examples 1 - 3): Control the dissolved oxygen and pH in stages according to the claims (Stage 1: 0 - 6 h, dissolved oxygen ≤ 0.2 mg / L, pH 4.0 - 4.2; Stage 2: 6 - 12 h, dissolved oxygen 0.5 - 1.0 mg / L, pH decreases naturally).

[0070] Control group (Comparative Example 3): Maintain the dissolved oxygen ≤ 0.2 mg / L and pH 4.0 - 4.2 throughout the process, and the other parameters are the same as those in Example 1.

[0071] Sample collection Time points: End of Stage 1 (6 h), End of Stage 2 (12 h).

[0072] Sampling method: Take 50 mL of the fermentation broth under sterile conditions and immediately centrifuge (8000×g, 10 min) to separate the cells and the supernatant.

[0073] Index detection Acetoacetic acid production: Take 1 mL of the supernatant, filter it through a 0.22 μm filter membrane, and detect it by HPLC (C18 column, mobile phase methanol: 0.1% phosphoric acid = 15:85, detection wavelength 210 nm).

[0074] Tea polyphenol release: Determine the tea polyphenol concentration in the supernatant by the Folin - Ciocalteu method and calculate the release rate based on the unfermented tea broth as the benchmark.

[0075] Yeast survival rate: Resuspend the cell precipitate in PBS, dilute it serially, spread it on YPD plates, culture it at 30 °C for 48 h, and count the number of viable cells.

[0076] Data processing Repeat each experiment 3 times and calculate the mean ± standard deviation.

[0077] The test results are shown in Table 3: Table 3 Effects of staged oxygen control and pH adjustment on metabolic efficiency Experiment summary: Stage - by - stage oxygen control and pH regulation achieved sequential functional complementarity between Lactobacillus acidophilus and yeast by dynamically regulating the metabolic activity of the microbial community. In the anaerobic environment (dissolved oxygen ≤ 0.2 mg / L) of stage one, Lactobacillus acidophilus preferentially utilized citric acid to generate acetoacetic acid (content reached 130.5 - 142.6 mg / L at 12 h), and its metabolites caused the pH of the fermentation broth to naturally drop to 3.8 - 4.0, providing acidic activation conditions for the enzymatic hydrolysis of yeast. In stage two, the micro - aerobic environment (dissolved oxygen 0.5 - 1.0 mg / L) relieved the oxygen restriction on yeast and activated the cellulase secreted by it (activity 25.3 - 28.7 U / mL), efficiently degrading the lignin - cellulose complex in the tea cell wall, and the release amount of tea polyphenols increased to 53.8 - 58.3 mg / g, significantly higher than 41.2 mg / g under full - process anaerobic conditions. The stage - by - stage preparation, through the coordinated changes of oxygen control and pH, directed the metabolic pathways of the two strains, avoiding efficiency losses caused by substrate competition.

[0078] The sequential switching of oxygen control plays a key regulatory role in the activity of the microbial community. The strict anaerobiosis in stage one inhibited the early proliferation of yeast (survival rate ≤ 1.8×10 8 CFU / mL), ensuring that Lactobacillus acidophilus exclusively utilized carbon sources to generate sweet precursors; while the micro - aerobic environment in stage two promoted the secretion of enzyme systems related to the release of tea polyphenols (such as polyphenol oxidase and cellulase) by activating the oxidative phosphorylation metabolism of yeast. At the same time, the natural drop in pH (4.2 → 3.8) enhanced the proton motive force of Lactobacillus acidophilus and accelerated the synthesis of acetoacetic acid, while in Comparative Example 3, maintaining a high pH (4.0 - 4.2) throughout the process led to a more than 15% reduction in metabolic efficiency. The division of labor among the microbial community based on the dynamic response of environmental parameters broke through the metabolic bottleneck of traditional single - stage fermentation.

[0079] Experimental data further verified the contribution of stage - by - stage regulation to product stability. The yeast survival rate in the experimental example group (1.8 - 2.1×10 8 CFU / mL) was about 3 times higher than that in Comparative Example 3 (6.7×10 7 CFU / mL), indicating that the micro - aerobic environment significantly improved the physiological activity of yeast. The continuous proliferation of yeast in stage two not only promoted the release of tea polyphenols, but the extracellular polysaccharide (EPS) produced by its metabolism also self - assembled with tea polyphenols through hydrogen bonds, providing a structural basis for the formation of subsequent stable networks. In Comparative Example 3, due to the inhibited yeast activity and insufficient EPS production, the suspension stability of the beverage directly decreased. The cross - stage metabolic - structural coordination achieved the full - process optimization from the preparation of flavor precursors to the construction of physical stability, providing a process path with both high efficiency and stability for low - sugar beverages.

[0080] Test Example 4: Explanation of the Experiment for Verifying the Performance of the Endogenous Stable Network Experimental Purpose Verify the performance of the endogenous stable network formed by the self-assembly of exopolysaccharide (EPS) and tea polyphenols, and compare the differences in suspension stability, thermal stability and antioxidant activity with exogenous pectin (Comparative Example 4).

[0081] Experimental procedures Sample preparation Example group (Examples 1-3): Retain the self-assembly network of EPS and tea polyphenols in the fermentation broth.

[0082] Control group (Comparative Example 4): Add 0.1% pectin (replacing the endogenous network), and the other process parameters are the same as those in Example 1.

[0083] Suspension stability test Take 50 mL of the beverage, centrifuge at 3000×g for 15 minutes, and measure the proportion of the sediment volume (sedimentation rate).

[0084] Calculation formula: Sedimentation rate (%) = Thermal stability test Place the sample in a water bath at 80 °C and heat for 30 minutes. After cooling to 25 °C, measure the viscosity change (Brookfield viscometer, rotor LV-62, rotation speed 60 rpm).

[0085] Viscosity retention rate: Retention rate (%) = Antioxidant activity test DPPH radical scavenging rate: Take 1 mL of the sample and mix it with 2 mL of 0.1 mM DPPH ethanol solution, and react in the dark for 30 minutes.

[0086] After centrifugation, measure the absorbance at 517 nm and calculate the scavenging rate: Scavenging rate (%) = The test results are shown in Table 4: Table 4 Comparison of the performance between the endogenous stable network and exogenous pectin Experimental summary: The endogenous stable network forms a three-dimensional hydrogen bond network through the molecular self-assembly of EPS and tea polyphenols, and its structural stability is significantly better than that of traditional exogenous pectin. The β-glucan chain in EPS binds to the phenolic hydroxyl group of tea polyphenols through hydrogen bonds (accounting for ≥60%) to form a dense and dynamically reversible network structure, reducing the centrifugal sedimentation rate to 3.2-4.8%, much lower than 15.6% of exogenous pectin. In the thermal stability test, the hydrogen bond network remains intact at 80 °C (viscosity retention rate ≥89.5%), while pectin shows a viscosity loss of 31.7% due to high-temperature degradation, demonstrating the advantage of the endogenous network in tolerating thermal stress.

[0087] The difference in antioxidant performance stems from the retention of tea polyphenols and their synergistic effect. The endogenous network entraps tea polyphenols through hydrogen bonding and hydrophobic interactions, preventing their oxidative inactivation and maintaining the DPPH scavenging rate at 77.6 - 82.3%; while the exogenous pectin in Comparative Example 4 lacks the tea polyphenol protection mechanism, with a scavenging rate of only 54.2%. In addition, the polysaccharide component in EPS can synergistically scavenge free radicals with tea polyphenols, forming a dual antioxidant pathway of "physical entrapment - chemical synergy".

[0088] The self - assembly mechanism of the endogenous network further reduces the additive dosage. The natural ratio of EPS to tea polyphenols in the examples (1:0.3 - 0.5) is generated in situ during the fermentation process without the need for additional stabilizers, while in Comparative Example 4, even with the exogenous addition of 0.1% pectin, the same level of stability cannot be achieved. Based on the synergistic design of microbial metabolism and molecular self - assembly, the simultaneous improvement of the stability and health attributes of low - sugar beverages has been realized.

[0089] Test Example 5: Explanation of Sweetness Synergy and Dynamic Release Verification Experiment Experimental Purpose Verify the sweetness synergy effect between acetoacetic acid and sugar substitutes and the dynamic release characteristics triggered by oral pH, and compare the effects of the absence of acetoacetic acid (Comparative Example 5) on sweetness intensity, bitterness residue, and sugar substitute dosage.

[0090] Experimental Procedures Sample Preparation Example Group (Examples 1 - 3): Retain the acetoacetic acid in the fermentation broth and compound it with erythritol (0.3%) and stevioside (0.01%).

[0091] Control Group (Comparative Example 5): Remove acetoacetic acid (activated carbon adsorption), and the sugar substitute ratio is the same as that in Example 1.

[0092] Determination of Sweetness Intensity - Time Curve Use an electronic tongue to simulate the pH value change from the oral cavity to the throat (pH6.8 → 4.5 → 3.8), and record the sweetness signal intensity within 0 - 30 seconds (with a 10% sucrose solution as the reference value of 100%).

[0093] Detection of Bitterness Residue Sensory Evaluation: Blind - test by 10 trained reviewers and evaluate the post - bitterness intensity on a scale of 0 - 5 (0 = no bitterness, 5 = strong bitterness).

[0094] Instrument Detection: Use the bitterness sensor (SAP8S) of the electronic tongue to measure the bitterness signal value.

[0095] Analysis of Sugar Substitute Components Take 1 mL of the sample, filter it through a 0.22 μm filter membrane, and determine the proportion of erythritol and steviol glycosides by HPLC (Aminex HPX-87H column, mobile phase 5 mM H2SO4, flow rate 0.6 mL / min).

[0096] The test results are shown in Table 5 as follows: Table 5 Sweetness Synergy and Dynamic Release Performance Data Experimental Summary: The sweetness synergy effect between acetoacetic acid and sugar substitutes stems from the complementary coupling of intermolecular hydrogen bonds and hydrophobic interactions. The β-ketoacid group of acetoacetic acid binds to the adjacent hydroxyl group of erythritol through a hydrogen bond (bond length ~1.8 Å) to form a stable complex structure, which can simultaneously activate multiple binding domains of the human tongue sweet receptor T1R2 / T1R3, increasing the peak sweetness intensity to 138.7 - 148.3% (40 - 50% higher than that of Comparative Example 5). In addition, the hydrophobic methyl group of acetoacetic acid and the terpene skeleton of steviol glycosides enhance the intermolecular adsorption through van der Waals forces (interaction energy ~3 kcal / mol), further amplifying the efficiency of sweet signal transmission, so that the sugar substitute dosage can be reduced by 30% while still maintaining equivalent sweetness.

[0097] The dynamic release characteristics and bitter taste neutralization mechanism depend on the pH-responsive behavior of acetoacetic acid. In the oral neutral environment (pH 6.8), the acetoacetic acid-sugar substitute complex dissociates due to the reduced degree of carboxyl protonation, rapidly releasing sweet molecules to form an "instantaneous sweet taste burst"; while in the acidic region of the throat (pH ≤ 4.5), acetoacetic acid is reprotonated, and its ketoacid group undergoes a nucleophilic addition reaction with the bitter aldehydes (such as phenylacetaldehyde) of the oxidation products of tea polyphenols to form odorless condensates, reducing the bitter taste sensory score to below 1.6 points (3.8 points for Comparative Example 5). This not only optimizes the sweet taste time sequence but also blocks the sensory exposure of bitter substances.

[0098] From the perspective of technical advantages, the introduction of acetoacetic acid has achieved a double breakthrough in sweetness enhancement and sugar substitute reduction. The proportion of sugar substitutes in the example group (0.28 - 0.31%) is significantly lower than that of Comparative Example 5 (0.42%), and the metallic bitter aftertaste of steviol glycosides is completely masked by acetoacetic acid, enabling the beverage to still present a natural fruit sweet flavor under low-sugar conditions. Based on the design of the synergy between metabolites and sugar substitute molecules, it has broken through the problems of off-flavors and health hazards caused by excessive sugar substitutes in traditional formulations, providing an innovative solution for the sensory and nutritional balance of low-sugar beverages.

[0099] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A low-sugar passion fruit black tea drink, characterized in that, Comprising the following components in parts by weight: Passion fruit pulp: 25 - 35 parts; Black tea soup: 65 - 75 parts; Sweetener: composed of 0.01 - 0.03 parts of acetoacetic acid, 0.005 - 0.015 parts of erythritol, and 0.002 - 0.006 parts of stevioside; Nano - encapsulated particles: nano - particles formed by cross - linking zein and chitosan in a mass ratio of 3:1, with a particle size of 50 - 80 nm, encapsulating 0.1 - 0.3 parts of volatile aroma components; Endogenous stabilizer: composed of 0.05 - 0.1 parts of exopolysaccharide produced by Lactobacillus acidophilus and 0.8 - 1.0 parts of tea polyphenols.

2. The low-sugar passion fruit black tea drink according to claim 1, wherein In the sweetener, the mass ratio of erythritol to stevioside is 1:0.

5.

3. A low-sugar passion fruit black tea drink according to claim 1, wherein In the nano - encapsulated particles, the volatile aroma components are ethyl hexanoate and β - ionone, and their mass ratio to the nano - particles is 1:10 - 1:

12.

4. A low-sugar passion fruit black tea drink according to claim 1, characterized in that, In the endogenous stabilizer, the proportion of hydrogen - bond interaction between exopolysaccharide and tea polyphenols is ≥60%.

5. A low-sugar passion fruit black tea drink according to claim 1, characterized in that, The pH of the beverage is 3.8 - 4.0, and the soluble solids ≤5°Brix.

6. A preparation method of a low-sugar passion fruit black tea drink. For the low-sugar passion fruit black tea drink according to any one of claims 1-5, it is characterized in that, Including the following steps: S1. Raw material pretreatment: Break the shell of passion fruit to obtain pulp and filter it, then mix it with a color - protecting agent; Crush black tea and pre - soak it to obtain tea soup; S2. Co - fermentation of two bacteria: Inoculate Lactobacillus acidophilus and Saccharomycopsis fibuligera into the mixed solution, and control the oxygen content and pH in stages for fermentation; S3. Preparation of nano - particles: Cross - link zein and chitosan to form nano - particles; S4. Aroma encapsulation: Mix the volatile components in the fermentation broth with the nano - particles, and adjust the pH to complete encapsulation; S5. Construction of a stable network: Promote the self - assembly of exopolysaccharide and tea polyphenols through inactivation treatment, and finally carry out sterilization and filling.

7. The preparation method of a low-sugar passion fruit black tea beverage according to claim 6, wherein The staged control in step S2 includes: In stage one, control the dissolved oxygen ≤0.2 mg / L, temperature 32±1℃, pH 4.0 - 4.2; In stage two, the dissolved oxygen rises to 0.5 - 1.0 mg / L, and the pH naturally drops to 3.8 - 4.

0.

8. The preparation method of a passion fruit black tea low-sugar drink according to claim 6, characterized in that The inactivation conditions in step S5 are treatment at 50±2℃ for 20 - 30 minutes, and add 0.05 - 0.1% of sodium citrate to adjust the ionic strength; The sterilization conditions are treatment at 80±2℃ for 15 - 20 seconds, and the light transmittance of the packaging material <5%.

9. The preparation method of a passion fruit black tea low-sugar drink according to claim 6, characterized in that, In S3, the cross - linking reaction uses transglutaminase, and the addition amount is 0.05 - 0.1% of the total mass of zein and chitosan.

10. The preparation method of a low-sugar passion fruit black tea drink according to claim 6, characterized in that, In S4, adjust the pH to 4.5±0.1.

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