Method for preparing jingang honey pomelo enzyme beverage through synergistic fermentation of lactic acid bacteria and yeast

Abstract

The application discloses a method for preparing Jinggang honey pomelo enzyme beverage through synergistic fermentation of lactic acid bacteria and yeast. The method comprises the following steps: mixing D-(+)-trehalose dihydrate, L-proline, manganese sulfate monohydrate and Jinggang honey pomelo base juice; introducing Saccharomyces cerevisiae, passing sterile air to perform normal pressure fermentation and monitoring oxidation-reduction potential; when the potential drops to-100 mV to-150 mV, cutting off the air inlet, introducing Lactobacillus plantarum and adding L-malic acid solution; then, closing the exhaust valve to maintain micro-pressure to perform static anaerobic fermentation, and finally, centrifuging and sterilizing to obtain the finished product. Through dynamic control of oxidation-reduction potential and material supplement, the method induces lactic acid bacteria to start specific metabolism and generate transmembrane proton motive force, drives manganese ions to enrich in cells against the concentration gradient, so as to activate endogenous enzymes to realize in-situ debittering, and at the same time, the method uses trehalose hydration layer and micro-pressure fermentation environment to improve the survival rate of bacterial cells and the stability of enzyme conformation.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation and food processing technology, specifically a method for preparing Jinggang pomelo enzyme beverage through co-fermentation of lactic acid bacteria and yeast. Background Technology

[0002] Jinggang pomelo is rich in vitamin C and flavonoids, making it valuable for fermented fruit and vegetable beverages. However, pomelo tissue contains substances like naringin, which can cause a noticeable bitter taste in the final product during processing and fermentation. Current fruit and vegetable enzyme fermentation processes typically use a mixture of yeast and lactic acid bacteria. To address the bitterness, a common method is to add exogenous commercial naringinase to the fermentation system for degradation. However, this exogenously added commercial enzyme is prone to conformational changes and inactivation in the low pH environment of the later stages of fermentation, increasing production costs and resulting in unstable degradation effects.

[0003] To avoid adding exogenous enzymes, existing technologies attempt to utilize endogenous β-glucosidases from lactic acid bacteria such as *Lactobacillus plantarum* for spontaneous debittering. The catalytic active site of this endogenous enzyme requires specific metal ions as cofactors. In conventional fermentation broth systems, metal ions mainly enter the cells via passive diffusion due to concentration gradients. This diffusion method is inefficient, making it difficult for sufficient cofactors to cross the membrane and enter the lactic acid bacteria cells, resulting in low catalytic activity of the endogenous enzyme and failing to meet the sensory requirements for debittering.

[0004] In conventional yeast and lactic acid bacteria mixed fermentation systems, the ethanol produced by yeast metabolism and the organic acids produced by lactic acid bacteria metabolism cause osmotic pressure stress and toxic damage to the cell membranes. The accumulation of these fermentation products disrupts the fluidity and integrity of the cell membranes, leading to a decrease in the survival rate of the mixed microbial community in the later stages of fermentation. The reduction in the number of viable cells not only lowers the overall metabolic rate of fermentation but also further limits the ability of the microorganisms to degrade bitter substances. Summary of the Invention

[0005] The technical problem solved by this invention is that in the existing Jinggang pomelo enzyme, during the mixed fermentation of lactic acid bacteria and yeast, the metabolic products of the strains inhibit each other, resulting in low cell survival rate, and the activity of endogenous β-glucosidase of Lactobacillus plantarum is limited under conventional fermentation conditions, resulting in low biodegradation efficiency of bitter substances such as naringin.

[0006] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a composition for preparing Jinggang pomelo enzyme beverage, employing the following technical solution: A composition for preparing Jinggang pomelo enzyme beverage is made from the following raw materials in parts by weight: 100 parts of Jinggang pomelo base juice; 1.5-2.0 parts of D-(+)-trehalose dihydrate; 0.3-0.5 parts of L-proline; 0.01-0.02 parts of manganese sulfate monohydrate; and 0.1-0.15 parts of L-malic acid.

[0007] By employing the above technical solution, a fermentation system with physicochemical protection and metabolic induction functions was constructed using a specific ratio of trehalose, L-proline, manganese sulfate, and L-malic acid, achieving the following technical effects: D-(+)-trehalose dihydrate, as a non-specifically compatible solute, can replace water molecules through hydrogen bonding, forming a stable amorphous hydrated layer on the outer side of the phospholipid bilayer of the microbial cell membrane, maintaining the liquid crystal fluidity of the cell membrane under conditions of ethanol and organic acid accumulation; L-proline provides a slow-release nitrogen source and is converted into ester aroma precursors in the yeast metabolic pathway; manganese sulfate monohydrate provides free divalent manganese ions, serving as an essential metal cofactor for the core enzyme in subsequent enzymatic debittering; L-malic acid, as a specific carbon source substrate, provides the material basis for triggering specific metabolic pathways and establishing transmembrane potential in the mid-to-late stages of fermentation. The above components do not exhibit unexpected chemical antagonism in the fermentation system, jointly constructing a fermentation environment that supports high survival rates of both microorganisms and efficient debittering.

[0008] Preferably, the raw materials further include fermentation strains, which are composed of brewer's yeast seed liquid and Lactobacillus plantarum seed liquid; the effective viable cell concentration of the brewer's yeast seed liquid is 3.0×10^6 CFU / mL, and the effective viable cell concentration of the Lactobacillus plantarum seed liquid is 3.0×10^7 CFU / mL.

[0009] By adopting the above technical solution and setting a specific initial concentration of live bacteria, it is ensured that the brewing yeast has a sufficient aerobic proliferation rate during the aerobic stage, while ensuring that Lactobacillus plantarum can quickly become the dominant bacterial group and start organic acid metabolism after inoculation, thus avoiding contamination and proliferation of wild bacteria.

[0010] Preferably, it is made from the following raw materials in parts by weight: 100 parts of Jinggang pomelo base juice, 1.8 parts of D-(+)-trehalose dihydrate, 0.4 parts of L-proline, 0.015 parts of manganese sulfate monohydrate, and 0.12 parts of L-malic acid.

[0011] By adopting the above technical solution, this ratio achieves a reaction kinetic equilibrium between the concentration of free manganese ions in the system and the proton kinetic potential triggered by L-malic acid, resulting in the highest transmembrane transport efficiency of the metal cofactor, while avoiding osmotic pressure imbalance caused by excessive addition.

[0012] Secondly, the present invention provides a method for preparing Jinggang pomelo enzyme beverage by co-fermentation of lactic acid bacteria and yeast, using the following technical solution: A method for preparing Jinggang pomelo enzyme beverage through co-fermentation of lactic acid bacteria and yeast, using the above-mentioned composition for preparing Jinggang pomelo enzyme beverage, includes the following steps: (1) Matrix mixing: Take the original juice of Jinggang pomelo base, add D-(+)-trehalose dihydrate, L-proline and manganese sulfate monohydrate to it, stir until completely dissolved, and place the mixture in the fermentation equipment; (2) Microenvironment remodeling fermentation: Open the vent valve of the fermentation equipment to introduce sterile air, adjust the initial oxidation-reduction potential (ORP) of the fermentation system to +130mV to +160mV, add brewing yeast, carry out fermentation under normal pressure, and continuously monitor the ORP data of the fermentation system; (3) ORP-triggered coupled fermentation: When the ORP of the fermentation system naturally drops to -100mV to -150mV, immediately close the air inlet valve to cut off the supply of sterile air, simultaneously inoculate with Lactobacillus plantarum, and add L-malic acid solution to the fermentation system; (4) Micro-pressure static fermentation: After inoculation and feeding, completely close the exhaust valve of the fermentation equipment, use the system’s spontaneous gas generation to maintain a constant micro-pressure in the tank for static anaerobic fermentation until the pH value of the fermentation liquid drops to 3.5-3.8, and then terminate the fermentation. (5) Post-processing: After depressurization, the fermented mash is centrifuged to separate solid and liquid. The clear supernatant is sterilized, cooled and then bottled to obtain Jinggang pomelo enzyme beverage.

[0013] By adopting the above technical solution, this invention utilizes the coupling of biophysical parameters and metabolic substrates to achieve targeted enrichment and in-situ enzymatic debittering of metal cofactors. The specific mechanism of action is as follows: 1. Dynamic Remodeling of Oxidation-Reduction Potential: A positive initial ORP is set by introducing sterile air in the first stage, utilizing the facultative anaerobic nature of Saccharomyces cerevisiae to consume dissolved oxygen in the system. The proliferation and oxygen-consuming metabolism of yeast cause the system's ORP to continuously decrease, transforming the conventional time-based inoculation control process into a physical parameter control process based on the thermodynamic state of the fermentation broth.

[0014] 2. Proton Dynamic Potential Coupling and Active Transmembrane Transport of Cofactors: When the ORP of the fermentation system drops to a micro-hypoxic state of -100mV to -150mV, the energy metabolism of *Lactobacillus plantarum* is limited. Simultaneous inoculation of *Lactobacillus plantarum* under this specific physical environment, along with the supplementation of L-malic acid, induces the *Lactobacillus plantarum* to initiate the malic acid-lactic acid fermentation metabolic pathway. This substrate transformation process is accompanied by transmembrane proton transfer, generating an electrochemical gradient across the lactic acid bacteria cell membrane and establishing a proton dynamic potential. This proton dynamic potential acts as the driving force for active transport, prompting the transport of divalent manganese ions free in the environment in the first stage against the concentration gradient and their accumulation within the lactic acid bacteria cell. This transport efficiency is higher than that of passive diffusion dominated by conventional concentration gradients.

[0015] 3. Enzymatic Conformation Activation and In-situ Debittering: *Lactobacillus plantarum* contains β-glucosidase, an enzyme capable of degrading naringin, but this enzyme is inactive. The high concentration of manganese ions enriched intracellularly through proton kinetic potential directly acts as a metal cofactor for this endogenous enzyme, binding to the enzyme's active site to form a holoenzyme conformation with catalytic activity. The activated enzyme specifically cleaves naringin molecules in Jinggang pomelo juice, achieving in-situ enzymatic debittering within the system without the need for external commercial enzyme preparations.

[0016] 4. Micropressure Steady-State and Conformation Maintenance: During the closed fermentation stage, micropressure is spontaneously established using carbon dioxide produced by microbial metabolism. This micropressure increases the solubility of carbon dioxide in the liquid phase, altering solvent activity. Under the combined physicochemical effects of low ORP, micropositive pressure, and the trehalose hydration layer, the spatial conformational stability of β-glucosidase increases, mitigating acid-induced inactivation of the enzyme due to pH decrease in the later stages of fermentation.

[0017] Preferably, before step (1), the main raw materials are pre-treated as follows to obtain the base juice of Jinggang pomelo: fresh and whole Jinggang pomelo fruits are peeled and seeded, and the juice is collected by cold pressing. The juice is filtered through a 100-mesh filter to remove the pulp fibers and obtain a clear filtrate. The filtrate is heated to 85°C and maintained for 15 minutes for pasteurization, and then cooled to 25°C for later use.

[0018] By adopting the above technical solutions, cold pressing and mechanical filtration remove the coarse fibers that produce sediment, ensuring the clarity of the fermentation liquid; specific pasteurization parameters inactivate polyphenol oxidase to prevent browning while preserving heat-sensitive flavor substances in the juice system.

[0019] Preferably, in step (1), the mass fractions of each component in the base juice of Jinggang pomelo are as follows: D-(+)-trehalose dihydrate 1.5%-2.0%, L-proline 0.3%-0.5%, and manganese sulfate monohydrate 0.01%-0.02%; in step (3), the mass fraction of L-malic acid solute in the L-malic acid solution is 0.1%-0.15% in the base juice of Jinggang pomelo.

[0020] By adopting the above technical solution, the specific mass fraction range of each substance is defined, ensuring that the system will not cause osmotic shock of lactic acid bacteria due to excessive concentration, nor will it cause failure to establish proton kinetic potential or insufficient cofactor concentration due to excessively low concentration.

[0021] Preferably, in step (2), the amount of brewing yeast added is 3%-5% of the fermentation liquid volume; the temperature of microenvironment remodeling fermentation is constant at 25℃-28℃, and the exhaust valve is kept open during fermentation.

[0022] By adopting the above technical solution, the temperature and inoculum range are matched to the optimal aerobic proliferation conditions of brewer's yeast. The normally open exhaust valve prevents the ORP from dropping too quickly due to early carbon dioxide accumulation, and provides the conditions required for yeast to complete proline metabolism.

[0023] Preferably, in step (3), the amount of *Lactobacillus plantarum* inoculated is 5%-8% of the fermentation liquid volume; the specific preparation method of L-malic acid solution is as follows: L-malic acid solid powder is dissolved in purified water to prepare an aqueous solution with a mass fraction of 20%, and then filtered and sterilized through a microporous membrane with a pore size of 0.22 μm.

[0024] By adopting the above technical solution, lactic acid bacteria can be introduced at a high inoculum level and dominate the fermentation process after obtaining proton kinetic potential; L-malic acid is sterilized by cold sterilization filtration, which avoids the degradation of organic acids caused by heat sterilization and ensures the effective concentration of trigger substrate.

[0025] Preferably, in step (4), the pressure range of the tank surface maintained by the spontaneous gas generation of the control system is 0.05MPa-0.08MPa, the temperature of static anaerobic fermentation is controlled at 28°C-30°C, and the fermentation time is 48-72 hours.

[0026] By adopting the above technical solution, the set micro-pressure range is sufficient to change the solvation effect of the microenvironment to stabilize the enzyme conformation, and it is also within the low-pressure safety range of conventional fermentation equipment; the fermentation temperature matches the optimal acid production and catalytic temperature of Lactobacillus plantarum, and the fermentation time is sufficient to degrade naringin to below the sensory threshold.

[0027] Preferably, in step (5), the centrifugation parameters are centrifugation at 4000-5000 r / min for 10-15 minutes; sterilization is performed by ultra-high temperature instantaneous sterilization at a temperature of 135°C-140°C for 3-5 seconds.

[0028] By adopting the above technical solution, the centrifugation parameters remove bacterial cells and macromolecular flocculants, ensuring the light transmittance and colloidal stability of the enzyme beverage system; while achieving commercial sterility requirements, the instantaneous high temperature reduces the volatilization and loss of aromatic esters.

[0029] This invention provides a method for preparing Jinggang pomelo enzyme beverage through co-fermentation of lactic acid bacteria and yeast. It has the following beneficial effects: First, this invention achieves enzymatic degradation of bitter substances in a fermentation system. By adding L-malic acid to the fermentation system at a specific redox potential, Lactobacillus plantarum undergoes malic acid-lactic acid fermentation. This process consumes protons across the membrane and establishes a proton kinetic potential across the cell membrane, driving free divalent manganese ions in the fermentation broth to enter the cell against the concentration gradient. The manganese ions enriched in the cell meet the cofactor requirements for the activation of endogenous β-glucosidase, enabling the enzyme to cleave naringin. The degradation process is completed without the addition of exogenous commercial debittering enzymes.

[0030] Secondly, this invention improves the survival rate of mixed microbial communities in the later stage of fermentation. By adding D-(+)-trehalose dihydrate and L-proline, this invention enables trehalose to form a hydrated layer on the outside of the microbial cell membrane through hydrogen bonding, maintaining the liquid crystal fluidity of the cell membrane under stress. This material basis buffers the osmotic pressure and toxicity effects caused by the accumulation of ethanol and organic acids in the fermentation system, maintaining the number of viable bacteria required for the fermentation process.

[0031] Third, this invention increases the spatial conformational stability of endogenous enzymes. This invention utilizes carbon dioxide produced by microbial metabolism during the closed fermentation stage to spontaneously establish and maintain a micro-positive pressure within the fermentation equipment. The micro-positive pressure increases the solubility of carbon dioxide in the liquid phase and changes the solvent activity. Combined with the low redox potential and the physicochemical effects of the trehalose hydration layer, this increases the spatial conformational stability of β-glucosidase and slows down the inactivation of the enzyme as the environmental pH decreases. Attached Figure Description

[0032] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Please see Figure 1 This invention provides a method for preparing Jinggang pomelo enzyme beverage through co-fermentation of lactic acid bacteria and yeast, comprising: raw material: Jinggang pomelos are selected from fresh, intact fruits with yellow-green to light yellow peels, weighing between 1,000 and 1,500 grams each, free from mechanical damage and pests.

[0035] The trehalose used is commercially available food-grade D-(+)-trehalose dihydrate with a purity greater than or equal to 99.0%, chemical formula C12H22O11·2H2O, and CAS registration number 6138-23-4. This substance is a conventional compound with a known chemical structure, and its specific molecular stereochemistry will not be described here.

[0036] L-proline is made from commercially available food-grade L-pyrrolidine-2-carboxylic acid with a purity greater than or equal to 99.0%, chemical formula C5H9NO2, and CAS registration number 147-85-3. This substance is a common natural amino acid with a known chemical structure.

[0037] The manganese sulfate used is commercially available food-grade manganese sulfate monohydrate with a purity greater than or equal to 99.0%, chemical formula MnSO4·H2O, and CAS registration number 10034-96-5. This substance is a conventional inorganic salt with a known chemical structure.

[0038] L-malic acid is commercially available food-grade L-hydroxysuccinic acid with a purity greater than or equal to 99.0%, chemical formula C4H6O5, and CAS registration number 97-67-6. This substance is a common organic acid with a known chemical structure.

[0039] The brewing yeast used is brewing yeast preserved by the China Industrial Microbial Culture Collection Center (CICC), with the preservation number CICC1046.

[0040] The Lactobacillus plantarum strain used was the Lactobacillus plantarum strain preserved by the China Industrial Microbial Culture Collection Center (CICC), with the accession number CICC20022.

[0041] YPD liquid culture medium, MRS liquid culture medium, and sterile physiological saline with a mass fraction of 0.9% are all commercially available, ready-to-use biochemical reagents.

[0042] Preparation Example 1: This preparation example provides yeast and lactic acid bacteria seed cultures, including the following steps: Saccharomyces cerevisiae was inoculated into YPD liquid medium for activation and expansion culture. After collecting the cells, they were washed and resuspended with sterile physiological saline (0.9% by mass). The concentration of the yeast suspension was adjusted to 3.0 × 10^6 CFU / mL to obtain Saccharomyces cerevisiae seed culture for later use. Lactobacillus plantarum was inoculated into MRS liquid medium for activation and expansion culture. After collecting the cells, they were washed and resuspended with sterile physiological saline (0.9% by mass). The concentration of the lactic acid bacteria suspension was adjusted to 3.0 × 10^7 CFU / mL to obtain Lactobacillus plantarum seed culture for later use.

[0043] Preparation Example 2: This preparation example provides a base juice for Jinggang pomelo, including the following steps: Fresh, whole Jinggang pomelo fruits are peeled and seeded. The pulp is then cold-pressed using a screw juicer to collect the juice. The juice is then filtered through a 100-mesh stainless steel filter to remove large pulp fibers and obtain a clear filtrate. The filtrate is placed in a heating device and heated to 85°C. It is then kept at this temperature for 15 minutes for pasteurization. Subsequently, it is rapidly cooled to 25°C using a heat exchanger to obtain standardized Jinggang pomelo base juice for later use.

[0044] Preparation Example 3: This preparation example provides an L-malic acid solution, including the following steps: Weigh a certain amount of L-malic acid solid powder and dissolve it in sterile purified water to prepare an L-malic acid aqueous solution with a mass fraction of 20%. Stir until completely dissolved, then filter the solution through a sterile microporous membrane with a pore size of 0.22 μm to remove bacteria. Collect the filtrate to obtain a sterile L-malic acid solution for later use.

[0045] Example 1: This embodiment provides a method for preparing Jinggang pomelo enzyme beverage through co-fermentation of lactic acid bacteria and yeast, including the following steps: Take the Jinggang pomelo base juice obtained in Preparation Example 2, add 1.8% D-(+)-trehalose dihydrate, 0.4% L-proline, and 0.015% manganese sulfate monohydrate by mass, stir until completely dissolved, pump the mixture into a sterile fermenter equipped with an online ORP electrode, pH electrode, and pressure sensor, open the fermenter's vent valve, introduce sterile air, and start mechanical stirring. Adjust the initial ORP of the fermentation system to +145mV, inoculate with the brewing yeast seed liquid prepared in Preparation Example 1 at an inoculation rate of 4% of the fermentation liquid volume, and ferment at 26°C. Keep the vent valve open and continuously monitor the ORP data of the fermentation system. When the ORP naturally drops to -120mV, the air inlet valve is immediately and completely closed to cut off the supply of sterile air. At the same time, the *Lactobacillus plantarum* seed liquid prepared in Preparation Example 1 is inoculated at an inoculation rate of 6% of the fermentation liquid volume, and the L-malic acid solution prepared in Preparation Example 3 is pumped in through the feed pump to achieve a final mass fraction of 0.12% in the fermentation liquid. After inoculation and feeding, the exhaust valve of the fermenter is completely closed, and the gauge pressure inside the tank is maintained at 0.06MPa by the system's spontaneous gas generation. The jacket temperature of the fermenter is kept constant at 29℃, and static anaerobic fermentation is carried out for 60 hours. When the pH value of the fermentation liquid drops to 3.6, the exhaust valve is opened to release pressure and terminate the fermentation. The fermentation mash is centrifuged at 4500r / min for 12 minutes, and the clear supernatant is taken and subjected to ultra-high temperature instantaneous sterilization at 138℃ for 4 seconds. After aseptic cooling, it is bottled to obtain the Jinggang pomelo enzyme beverage.

[0046] Example 2: This embodiment provides a method for preparing Jinggang pomelo enzyme beverage by co-fermentation of lactic acid bacteria and yeast, including the following steps: take the Jinggang pomelo base juice obtained in Preparation Example 2, add 1.5% D-(+)-trehalose dihydrate, 0.3% L-proline, and 0.01% manganese sulfate monohydrate by mass to it, stir until completely dissolved, pump the mixture into a sterile fermenter equipped with an online ORP electrode, pH electrode, and pressure sensor, open the fermenter vent valve, introduce sterile air and start mechanical stirring, and adjust the initial ORP of the fermentation system to +130mV. Inoculate the *Saccharomyces cerevisiae* seed culture prepared in Preparation Example 1 at an inoculation rate of 3% of the fermentation broth volume, and ferment at 25°C. Keep the exhaust valve open and continuously monitor the ORP data of the fermentation system. When the ORP naturally drops to -100mV, immediately and completely close the air inlet valve to cut off the supply of sterile air. At the same time, inoculate the *Lactobacillus plantarum* seed culture prepared in Preparation Example 1 at an inoculation rate of 5% of the fermentation broth volume, and pump the L-malic acid solution prepared in Preparation Example 3 into the fermentation broth through a feed pump to achieve a final mass fraction of 0.1% in the fermentation broth. After inoculation and feeding are completed, the exhaust valve of the fermenter is completely closed. The system uses its own gas generation to maintain the gauge pressure inside the tank at 0.05 MPa and the jacket temperature of the fermenter at a constant 28°C. Static anaerobic fermentation is carried out for 48 hours. When the pH value of the fermentation liquid drops to 3.8, the exhaust valve is opened to release pressure and terminate the fermentation. The fermentation mash is centrifuged at 4000 r / min for 15 minutes. The clear supernatant is taken and sterilized at 135°C for 5 seconds. After aseptic cooling, it is bottled to obtain Jinggang pomelo enzyme beverage.

[0047] Example 3: This embodiment provides a method for preparing Jinggang pomelo enzyme beverage through co-fermentation of lactic acid bacteria and yeast, including the following steps: Take the Jinggang pomelo base juice obtained in Preparation Example 2, add 2.0% D-(+)-trehalose dihydrate, 0.5% L-proline, and 0.02% manganese sulfate monohydrate by mass, stir until completely dissolved, pump the mixture into a sterile fermenter equipped with an online ORP electrode, pH electrode, and pressure sensor, open the fermenter's vent valve, introduce sterile air, and start mechanical stirring, adjust the initial ORP of the fermentation system to +160mV, inoculate with the brewing yeast seed liquid prepared in Preparation Example 1 at an inoculation rate of 5% of the fermentation liquid volume, ferment at 28°C, keep the vent valve open, continuously monitor the ORP data of the fermentation system, and when O When the RP naturally drops to -150mV, the air inlet valve is immediately and completely closed to cut off the supply of sterile air. At the same time, the *Lactobacillus plantarum* seed liquid prepared in Preparation Example 1 is inoculated at an inoculation rate of 8% of the fermentation liquid volume, and the L-malic acid solution prepared in Preparation Example 3 is pumped in through the feed pump to achieve a final mass fraction of 0.15% in the fermentation liquid. After inoculation and feeding, the exhaust valve of the fermenter is completely closed. The system uses its own gas generation to maintain the gauge pressure inside the tank at 0.08MPa, and the jacket temperature of the fermenter is kept constant at 30℃. Static anaerobic fermentation is carried out for 72 hours. When the pH value of the fermentation liquid drops to 3.5, the exhaust valve is opened to release pressure and terminate the fermentation. The fermentation mash is centrifuged at 5000r / min for 10 minutes, and the clear supernatant is taken and sterilized at 140℃ for 3 seconds. After aseptic cooling, it is bottled to obtain the Jinggang pomelo enzyme beverage.

[0048] Comparative Example 1: Compared with Example 1, the difference is that D-(+)-trehalose dihydrate, L-proline, manganese sulfate monohydrate and L-malic acid were not added, and the fermentation process was not monitored or triggered by ORP. After fermentation with Saccharomyces cerevisiae for 24 hours, Lactobacillus plantarum was directly inoculated. During the resting fermentation stage after inoculation, the exhaust valve was kept open to maintain normal pressure fermentation. All other aspects were the same.

[0049] Comparative Example 2: Compared with Example 1, the difference is that when the ORP of the fermentation system naturally dropped to -120mV, only Lactobacillus plantarum was introduced, and L-malic acid solution was not pumped in through the feed pump. All other aspects were the same.

[0050] Comparative Example 3: Compared with Example 1, the difference is that no manganese sulfate monohydrate was added to the initial fermentation base juice, otherwise it is the same.

[0051] Comparative Example 4: Compared with Example 1, the difference is that after inoculation with Lactobacillus plantarum and feeding, the exhaust valve of the fermenter was kept open, and the system's spontaneous gas generation was not used to establish micro-pressure. The entire anaerobic fermentation stage was maintained at atmospheric pressure. All other aspects were the same.

[0052] Comparative Example 5: Compared with Example 1, the difference is that D-(+)-trehalose dihydrate and L-proline were not added to the initial fermentation base juice, but all other aspects were the same.

[0053] Test Example 1: Dynamic Determination of Intracellular Manganese Ion Accumulation Concentration in Lactic Acid Bacteria This test example uses inductively coupled plasma mass spectrometry to quantitatively analyze the intracellular manganese ion concentration of Lactobacillus plantarum during the fermentation process of each embodiment and comparative example.

[0054] Take 50 mL of fermentation broth samples from each example and comparative example at 0, 12, 24, 36, and 48 hours after inoculation with *Lactobacillus plantarum*. Centrifuge at 8000 rpm at 4°C to remove the supernatant and collect the bacterial precipitate. Wash the bacterial cells twice with a 20 mmol / L disodium ethylenediaminetetraacetate solution to elute metal ions attached to the cell surface. Resuspend the cells in ultrapure water and wash them three times. Dry the cells to constant weight in a vacuum freeze dryer and record the dry weight. Place 50 mg of the lyophilized bacterial powder in a digestion vessel, add a mixture of nitric acid and hydrogen peroxide, and microwave digest until the solution is clear. After removing the acid from the digestion solution and making up to volume with ultrapure water, determine the manganese concentration in the sample using inductively coupled plasma mass spectrometry (ICP-MS) and calculate the micrograms of manganese per gram of dry cell weight.

[0055] Table 1. Results of intracellular manganese ion enrichment concentration in lactic acid bacteria at different fermentation times (unit: μg / g)

[0056] In the systems of Examples 1 to 3, the intracellular manganese ion concentration of *Lactobacillus plantarum* increased from hour 12 to hour 24 of fermentation, reaching a peak at hour 36. Examples 1 and 3 were conducted by introducing a micro-hypoxic state at a specific redox potential and supplementing with L-malic acid to initiate the malic-lactic fermentation pathway of *Lactobacillus plantarum*. The malic-lactic fermentation process consumes protons across the cell membrane, establishing a proton kinetic potential across the cell membrane. This transmembrane potential promotes the accumulation of free manganese ions against their concentration gradient within the cell, satisfying the cofactor requirements for endogenous β-glucosidase activation.

[0057] Comparative Example 1, without the addition of exogenous manganese salt, showed values ​​at the baseline level. Comparative Example 2, without the addition of L-malic acid, did not establish a proton kinetic potential in the cell membrane; manganese ions passively diffused based on their concentration gradient, resulting in lower intracellular accumulation than in the example. Comparative Example 3, without the addition of exogenous manganese salt, showed intracellular manganese ion accumulation limited by the baseline concentration. Comparative Example 4, without micropressure control, showed a manganese ion transport trajectory similar to the example in the early stages, indicating that the construction of ion transport channels was not affected by micropressure control. Comparative Example 5, without the addition of trehalose and L-proline, resulted in decreased transmembrane protein transport activity and lower active transport efficiency in the lactic acid bacteria due to ethanol stress from yeast metabolism, compared to the example. The results indicate that the parameter control and added substances in the examples work together to influence the formation of transmembrane potential and the targeted transport of manganese ions.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A composition for preparing Jinggang pomelo enzyme beverage, characterized in that, Made from the following parts by weight of raw materials: 100 servings of Jinggang pomelo base juice; D-(+)-trehalose dihydrate 1.5-2.0 parts; L-proline 0.3-0.5 parts; 0.01-0.02 parts of manganese sulfate monohydrate; L-malic acid 0.1-0.15 parts.

2. The composition according to claim 1, characterized in that, The raw materials also include fermentation strains, which are composed of brewer's yeast seed liquid and Lactobacillus plantarum seed liquid; Preferably, the effective viable cell concentration of the Saccharomyces cerevisiae seed liquid is 3.0 × 10^6 CFU / mL, and the effective viable cell concentration of the Lactobacillus plantarum seed liquid is 3.0 × 10^7 CFU / mL.

3. The composition according to claim 1, characterized in that, It is made from the following ingredients in parts by weight: 100 parts of Jinggang pomelo base juice, 1.8 parts of D-(+)-trehalose dihydrate, 0.4 parts of L-proline, 0.015 parts of manganese sulfate monohydrate, and 0.12 parts of L-malic acid.

4. A method for preparing Jinggang pomelo enzyme beverage through co-fermentation of lactic acid bacteria and yeast, characterized in that, Using the composition as described in any one of claims 1 to 3 includes the following steps: (1) Matrix mixing: Take the original juice of Jinggang pomelo base, add D-(+)-trehalose dihydrate, L-proline and manganese sulfate monohydrate to it, stir until completely dissolved, and place the mixture in the fermentation equipment; (2) Microenvironment remodeling fermentation: Open the vent valve of the fermentation equipment to introduce sterile air, adjust the initial oxidation-reduction potential (ORP) of the fermentation system to +130mV to +160mV, add brewing yeast, carry out fermentation under normal pressure, and continuously monitor the ORP data of the fermentation system; (3) ORP-triggered coupled fermentation: When the ORP of the fermentation system naturally drops to -100mV to -150mV, immediately close the air inlet valve to cut off the supply of sterile air, simultaneously inoculate with Lactobacillus plantarum, and add L-malic acid solution to the fermentation system; (4) Micro-pressure static fermentation: After inoculation and feeding, completely close the exhaust valve of the fermentation equipment, use the system’s spontaneous gas generation to maintain a constant micro-pressure in the tank for static anaerobic fermentation until the pH value of the fermentation liquid drops to 3.5-3.8, and then terminate the fermentation. (5) Post-processing: After depressurization, the fermented mash is centrifuged to separate solid and liquid. The clear supernatant is sterilized, cooled and then bottled to obtain Jinggang pomelo enzyme beverage.

5. The method according to claim 4, characterized in that, Before step (1), the main raw materials are pre-treated as follows to obtain the Jinggang pomelo base juice: Fresh, whole Jinggang pomelo fruits are peeled and seeded. The juice is collected by cold pressing and then filtered through a 100-mesh filter to remove pulp fibers and obtain a clear filtrate. The filtrate is then heated to 85°C and maintained for 15 minutes for pasteurization, and then cooled to 25°C for later use.

6. The method according to claim 4, characterized in that, In step (1), the mass fractions of each component in the base juice of Jinggang pomelo are as follows: D-(+)-trehalose dihydrate 1.5%-2.0%, L-proline 0.3%-0.5%, and manganese sulfate monohydrate 0.01%-0.02%; In step (3), the L-malic acid solute in the L-malic acid solution accounts for 0.1%-0.15% of the mass fraction of the Jinggang pomelo base juice.

7. The method according to claim 4, characterized in that, In step (2), the amount of brewing yeast added is 3%-5% of the fermentation liquid volume; the temperature of the microenvironment remodeling fermentation is constant at 25℃-28℃, and the exhaust valve is kept open during fermentation.

8. The method according to claim 4, characterized in that, In step (3), the amount of *Lactobacillus plantarum* introduced is 5%-8% of the fermentation broth volume; The L-malic acid solution is prepared by dissolving L-malic acid solid powder in purified water to prepare an aqueous solution with a mass fraction of 20%, which is then filtered and sterilized through a microporous membrane with a pore size of 0.22 μm.

9. The method according to claim 4, characterized in that, In step (4), the pressure range of the tank maintained by the spontaneous gas production of the system is 0.05MPa-0.08MPa, the temperature of the static anaerobic fermentation is controlled at 28°C-30°C, and the fermentation time is 48-72 hours.

10. The method according to claim 4, characterized in that, In step (5), the centrifugation parameters are centrifugation at 4000-5000 r / min for 10-15 minutes; the sterilization is carried out by ultra-high temperature instantaneous sterilization, with a sterilization temperature of 135°C-140°C and a sterilization time of 3-5 seconds.

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