A yeast for reducing caffeine content and its application in decaffeinated beverage

Abstract

The present application relates to the technical field of plant beverages, and particularly relates to a yeast for reducing caffeine content and application of the yeast in caffeine-reduced beverages. Zygosaccharomyces rouxii ) HX001, the preservation number of which is CCTCC NO: M 2026714, and the preservation unit is China Center for Type Culture Collection, and the preservation date is April 17, 2026. The Zygosaccharomyces rouxii HX001 screened in the present application can be used for reducing caffeine in real low-sugar or non-high-sugar tea and caffeine product beverage systems, and has a potential for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of plant-based beverage technology, specifically to a yeast strain that reduces caffeine content and its application in decaffeinated beverages. Background Technology

[0002] Caffeine is a naturally occurring xanthine alkaloid that is widely found in plants such as coffee beans, tea leaves, and cocoa, and is an important functional component of coffee and tea.

[0003] Currently, caffeine removal technology is mainly applied to the pre-treatment stage of coffee beans and tea processing, relying on physical or chemical methods to remove caffeine. There are three common processes: The first is organic solvent extraction, which selectively dissolves and removes caffeine using solvents, but carries the risk of solvent residue and can also remove some substances, damaging the original flavor. The second is water extraction, which utilizes the property of caffeine dissolving in hot water for repeated extraction, but this method requires high temperature and long processing time, easily leading to the loss of flavor components and also suffers from low production efficiency and high waste generation, increasing the environmental burden. The third is supercritical carbon dioxide extraction, which offers high selectivity and good product quality, and is currently a relatively advanced technology. However, this process relies on high-pressure equipment, is complex, still suffers from flavor substance loss, and can only be applied to the raw material processing stage, unable to flexibly control the caffeine content during beverage preparation. In summary, existing caffeine removal technologies generally have three shortcomings: first, they only apply to the raw material pre-treatment stage, making it difficult to accurately control the caffeine content of the finished beverage; second, the removal process easily causes the loss of aromatic substances and functional components, affecting the flavor of the beverage; and third, some technologies have complex processes or solvent safety issues, hindering large-scale promotion.

[0004] In recent years, with the development of food biotechnology, some studies have found that some environmental microorganisms can metabolize and degrade caffeine, making microbial fermentation for caffeine reduction a new research direction. However, existing research still has many limitations: First, most studies use artificially prepared pure caffeine culture systems, relying on high-concentration carbon source culture media. However, actual coffee and tea drinks have complex compositions and are mostly low in sugar. Various components in the system can inhibit microbial growth and metabolism, so laboratory results cannot be directly applied to actual production. Second, the traditional coffee bean fermentation process for reducing caffeine mainly involves mixing coffee beans with water and then allowing them to ferment naturally. Whether it is natural fermentation or artificial inoculation fermentation, the mechanism of action is not clear, and it cannot be confirmed that the reduction in caffeine content is due to microbial degradation. Moreover, the principle of this process is similar to water treatment. Soaking in water itself causes caffeine to dissolve, while also removing flavor substances. This process cannot prove that it has a real caffeine degradation ability, nor can it achieve targeted and controllable removal of caffeine. In addition, most of the reported caffeine-degrading strains are environmental strains or non-food grade strains, and their safety is unclear, so they cannot be directly applied to food production. In addition, most current decaffeination technologies focus on raw material pretreatment, making it difficult to accurately reduce caffeine while preserving the original flavor. Technical solutions for controlling caffeine content in finished beverages remain very limited.

[0005] Therefore, developing a technical solution that can regulate caffeine content in real coffee or tea beverage systems through food-grade microbial fermentation, while taking into account flavor stability and industrial feasibility, is of great significance for meeting the demand for low-caffeine healthy beverages. Summary of the Invention

[0006] In view of this, this application proposes a yeast strain that reduces caffeine content and its application in decaffeinated beverages.

[0007] The specific technical solution is as follows: In one aspect, the present invention provides a strain of *L. rouxii* (…). Zygosaccharomyces rouxii HX001, accession number CCTCC NO: M 2026714, deposited at China Center for Type Culture Collection, on April 17, 2026.

[0008] Furthermore, the described *Zygosacchariformis* exhibits high osmotic pressure tolerance, acid tolerance, and aerobic properties, as well as significant salt and high sugar tolerance.

[0009] Furthermore, the *Gnaphalium rouxiense* possesses caffeine tolerance and / or caffeine degradation capabilities.

[0010] Furthermore, the *Zygosacchariformis* can tolerate caffeine concentrations not exceeding 600 mg / L.

[0011] In one aspect, the present invention provides a microbial inoculant containing the above-mentioned *Zygosacchariformis* as an active ingredient.

[0012] In one aspect, the present invention provides the use of the above-mentioned *Gnaphalium rouxiense* or the above-mentioned microbial agent in the preparation of low-caffeine beverages.

[0013] In one aspect, the present invention provides a fermentation process for reducing the caffeine content in caffeinated beverages, comprising the following steps: The above-mentioned *Zygosacchariformis* or the above-mentioned microbial inoculum was activated and cultured, and the bacterial cells were collected. The bacterial cells were inoculated into a sugary beverage with an initial caffeine concentration of 50-1500 mg / L at a mass ratio of bacterial cells to beverage of 1:2 to 1:15; fermentation was carried out at 15℃ to 35℃, and after filtering the bacterial cells, a beverage with reduced caffeine was obtained.

[0014] The amount of sugar added is 0-10% of the total mass of the beverage, and the sugar is selected from at least one of glucose, D-fructose, sucrose, brown sugar, lactose, D-allulose, D-tagatose, xylose, and xylitol.

[0015] Furthermore, the initial caffeine concentration is 200-400 mg / L, preferably 300 mg / L.

[0016] Furthermore, the sugar is brown sugar, and its addition amount is 1-5% of the beverage mass, preferably 2%.

[0017] Furthermore, the mass ratio of the bacteria to the beverage is 1:2 to 1:10, preferably 1:5.

[0018] Furthermore, the fermentation temperature is 28~32℃, preferably 30℃.

[0019] Furthermore, the fermentation time is 36 to 60 hours, preferably 48 hours.

[0020] Furthermore, the caffeinated beverage is coffee liquid, tea beverage, or coffee-tea combination beverage.

[0021] In one aspect, the present invention provides a low-caffeine beverage prepared using the above-described fermentation process, wherein the caffeine content of the low-caffeine beverage is reduced by more than 10% compared to before fermentation.

[0022] Compared with the prior art, the present invention has the following advantages: This invention screened and preserved a strain of *Gnaphalium rouxiense* (R. rouxiense). Zygosaccharomyces rouxiiHX001. This strain not only exhibits excellent caffeine tolerance (able to grow and reproduce normally in a medium with a caffeine concentration of 600 mg / L), but also demonstrates highly efficient caffeine degradation activity. This strain provides the food industry with a safe and controllable biological caffeine-degrading resource. Under optimal fermentation conditions, for beverages with an initial caffeine concentration of 300 mg / L, the caffeine degradation rate can reach 21% after 48 hours of fermentation, which is comparable to or even better than the highest degradation rate achieved by existing technologies in pure tea systems. Attached Figure Description

[0023] Figure 1 This is a plate photograph showing the colony morphology of *Zygosacchariformis*.

[0024] Figure 2 Microscopic image of *Gnaphalium rouxiense*.

[0025] Figure 3 The degradation rate of caffeine by different microorganisms.

[0026] Figure 4 The caffeine degradation rate of *Gnaphalium roximatee* obtained by screening in this invention at different temperatures.

[0027] Figure 5 The caffeine degradation rate of *Zygomyces rouxii* obtained by screening in this invention under different carbon source conditions.

[0028] Figure 6 The caffeine degradation rate of *Zygomyces rouxii* obtained by screening in this invention under different proportions of brown sugar is shown.

[0029] Figure 7 The caffeine degradation rate of *Zygosacchariformis* obtained in this invention under different cell-to-coffee ratios is shown.

[0030] Figure 8 The caffeine degradation rate of *Gnaphalium roximatee* obtained by screening in this invention at different reaction times is shown. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0032] In the following examples, the high-performance liquid chromatograph is a Shimadzu LC-20A from Japan.

[0033] In the following examples, the tannin-based Italian coffee concentrate was purchased from Nongfu Spring Co., Ltd.

[0034] In the following examples, the formulation of the MRS medium is as follows (per 1 L): 10.0g peptone; 10.0g beef extract; 5.0g yeast extract; 20.0g glucose; 2.0g dipotassium hydrogen phosphate; 2.0g triammonium citrate; 5.0g sodium acetate; 0.2g magnesium sulfate; 0.05g manganese sulfate; 1.0mL Tween 80; distilled water to 1000mL; pH adjusted to 6.2±0.2 (25℃).

[0035] In the following examples, the YPD culture medium formulation is as follows (per 1 L): Yeast extract 10g; peptone 20g; glucose 20g; distilled water added to 1000 mL; pH adjusted to 5.4±0.2 (25℃) before sterilization; penicillin 0.1g (added after cooling to 50℃ after sterilization).

[0036] In the following examples, the formulation of GYC medium is as follows (per 1 L): Yeast extract 10g; glucose 50g; calcium carbonate 5g; distilled water to 1000mL; pH adjusted to 6.8±0.2 (25℃) before sterilization; anhydrous ethanol 10mL (added after sterilization and cooling to 60℃ to avoid volatilization).

[0037] Example 1: Screening of caffeine-resistant strains Sample sources: Pickled vegetables from Yanbian region, handmade yogurt from Inner Mongolian herders, sauerkraut, salted vegetables, as well as traditional fermented soy products (such as fermented black beans) and traditional fermented teas (such as Pu'er tea and Kang brick tea) were collected.

[0038] Isolation and purification: Appropriate amounts of each sample were serially diluted and plated onto MRS solid medium (containing 0.2% CaCO3), YPD medium, and GYC medium (containing 0.2% CaCO3), and anaerobically cultured at 30℃ for 48 h. Single colonies of different morphologies were picked and purified by repeated streak plating, resulting in a total of 89 strains.

[0039] Caffeine tolerance screening: The above 89 purified strains were inoculated into MRS, YPD, and GYC liquid media containing different concentrations of caffeine (50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg / L) and incubated at 30℃ for 48–72 h. The growth of each strain under different caffeine concentrations was observed (expressed as OD). 600 (A decrease of ≤50% from the control is considered tolerable), and the highest tolerable concentration for each strain is recorded.

[0040] Results: From 89 bacterial strains, 12 strains capable of tolerating caffeine concentrations ≥500 mg / L were screened. Bacterial strains were preliminarily identified using 16S rDNA, while fungal (yeast) strains were molecularly identified using 18S rDNA and ITS sequences, respectively. The strains mainly belonged to the genus *Zygosaccharomyces*. Zygosaccharomyces Leuconostoc ( ) Leuconostoc Lactobacillus ( ) Lactobacillus ), Weissella spp. Weissella ) and yeast ( Saccharomyces ).

[0041] Example 2: Screening of caffeine-degrading strains The 12 caffeine-tolerant bacterial strains (caffeine tolerance concentration ≥500 mg / L) obtained in Example 1 were activated and cultured, and the bacterial cells were collected by centrifugation. The bacterial cells were added to unsweetened coffee solution (natural pH) containing 300 mg / L caffeine at a ratio of bacterial cells to coffee solution of 1:5 (w / w), and the reaction was carried out at 30°C and 200 rpm for 48 h with shaking. Samples were taken at 0, 6, 12, 24, 36, 48, and 72 h, and the caffeine content was determined by HPLC. Inactivated bacterial cells (boiled at 100°C for 10 min) were used as a negative control, and the caffeine degradation rate at each time point was calculated.

[0042] Of the 12 caffeine-tolerant strains, only 3 showed detectable caffeine degradation activity (degradation rate ≥5% after 48 hours). Among them, *Zygosacchariformis* (… Zygosaccharomyces rouxii HX001 exhibited the highest caffeine degradation rate of 21.22% within 48 hours, significantly higher than the other two strains, a lactobacillus strain and a yeast strain (degradation rates of 9.59% and 7.89%, respectively). Figure 3 Furthermore, no decrease in caffeine content was observed in the negative control. Most microorganisms (including most probiotics), even if they can tolerate high concentrations of caffeine, generally lack the ability to degrade caffeine; however, the *Zygosacchariformis* strain obtained in this invention possesses both high caffeine tolerance (≥500 mg / L) and caffeine degradation activity, a characteristic extremely rare among yeasts, providing a crucial foundation for its direct decaffeination application in caffeinated plant beverages.

[0043] Example 3: Molecular identification and preservation information of *Zygosacchariformis* Genomic DNA was extracted from *Zygosaccharomyces rouxii*, and the full-length ITS sequence was amplified (as shown in SEQ ID NO.1). Sequencing was performed, and the sequence was compared with the NCBI database using BLAST. A phylogenetic tree was constructed, and the results showed that strain HX001 is related to *Zygosaccharomyces rouxii* (…). Zygosaccharomyces rouxiiThe homology with the type strain reached 100.0%. Combined with physiological and biochemical characteristics (tolerance to high osmotic pressure, acidity, and aerobic activity, with significant salt and high sugar tolerance, etc.), this strain was identified as *Zygosacchariformis* var. *roux*. Zygosaccharomyces rouxii The strain was deposited with the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2026714.

[0044] SEQ ID NO.1: .

[0045] Example 4: Effect of fermentation temperature on caffeine degradation rate The *Zygosacchariformis* cells were prepared according to the method in Example 2. A commercially available caffeinated plant-based beverage (Tanbing Italian coffee concentrate, initially diluted to approximately 300 mg / L, with total sugar adjusted to 2%) was used as the reaction matrix. Cells were added at a ratio of 1:5 (w / w) to beverage, and the reaction was carried out at 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C with shaking at 200 rpm for 48 h. The caffeine content at each temperature for 48 h was determined by HPLC, and the degradation rate was calculated.

[0046] The HPLC method for caffeine detection is as follows: Chromatographic column: Agilent 5 TC-C18 column (5µm particle size, 250mm length × 4.6mm diameter) or equivalent column.

[0047] Mobile phase: Acetonitrile:water = 1:9.

[0048] Flow rate: 1.0 mL / min.

[0049] Detection wavelength: 273nm.

[0050] Column temperature: 30℃.

[0051] Injection volume: 10µL.

[0052] Within the temperature range of 30-35℃, *Zygosaccharomyces rouxii* exhibited the highest caffeine degradation rate in beverages, reaching 18.4%. Figure 4 When the temperature is below 25℃ or above 40℃, the degradation rate decreases significantly (<5%). Therefore, the optimal fermentation temperature is determined to be 30~35℃ (this temperature range is used in subsequent examples unless otherwise specified, with 30℃ being preferred).

[0053] Example 5: Effect of carbon source type and dosage on caffeine degradation rate Under the optimal temperature conditions (30°C) of Example 4, 0.5%, 1%, 2%, 4%, and 8% (w / w) of different carbon sources (glucose, D-fructose, sucrose, brown sugar, lactose, D-allulose, D-tagatose, xylose, and xylitol) were added to caffeinated plant beverages, respectively, with no added carbon source and bacterial cells as blank controls. The caffeine degradation rate was detected by HPLC after 48 h.

[0054] Adding a carbon source can significantly improve the rate of caffeine degradation. Brown sugar is the most effective among these sources. Figure 5 When the addition amount was 2%, the degradation rate reached 16.3% after 48 hours; when the addition amount was further increased to 8%, the degradation rate did not increase significantly. Figure 6 The degradation rate of the blank control (without added carbon source) was only 4.2%. Therefore, brown sugar was determined to be the optimal carbon source, and the optimal addition amount was 2%.

[0055] Example 6: Effect of the ratio of bacterial cells to coffee liquid on caffeine degradation rate With a fixed mass of caffeinated plant-based beverage, *Zygosacchariformis* cells were added at ratios of 1:2, 1:3, 1:5, 1:8, 1:10, and 1:15 (w / w) of cell-to-beverage mixture. The mixture was reacted for 48 hours under the optimal conditions (30°C, 2% brown sugar) determined in Examples 4-5. The caffeine degradation rate at each ratio was determined by HPLC.

[0056] The higher the bacterial count ratio, the higher the degradation rate. When the bacterial count to beverage ratio is 1:5, the degradation rate reaches 15.1%; further increasing the bacterial count to 1:2 only increases the degradation rate to 16.3%, but the cost increases significantly. When the ratio drops below 1:10, the degradation rate drops below 8%. Figure 7 Taking into account both degradation efficiency and economics, the optimal ratio was determined to be 1:5 for microbial cells to beverage.

[0057] Example 7: Effect of fermentation time on caffeine degradation rate Fermentation was carried out under optimal conditions (30℃, 2% brown sugar, cell count:beverage ratio = 1:5). Samples were taken at 0, 6, 12, 18, 24, 30, 36, 42, 48, 60, and 72 hours. Caffeine content was determined by HPLC. The pH value, total bacterial count, and reducing sugar content of the system were also measured.

[0058] During fermentation, the caffeine degradation rate slowly increased to approximately 6% from 0 to 12 hours; a rapid degradation period occurred from 24 to 48 hours, with the degradation rate increasing from 10.3% to 19.5%; after 48 hours, the degradation rate leveled off, reaching 19.8% at 72 hours. Figure 8 The pH value gradually decreased from an initial 6.2 to 4.1 after 48 hours, and the total bacterial count reached its peak at 48 hours (approximately 10). 9 The reducing sugar content decreased from 2% to 0.5% (CFU / mL). Based on these factors, the optimal fermentation time was determined to be 48 hours.

[0059] Example 8: Degradation effect at different initial caffeine concentrations Plant-based beverage substrates with caffeine concentrations of 50, 100, 200, 300, 400, and 500 mg / L were prepared (all with 2% brown sugar added). Fermentation was carried out under optimal conditions (30℃, cell:beverage = 1:5, 48 h). The caffeine residue at each initial concentration was determined by HPLC after 48 h, and the degradation rate and absolute degradation amount were calculated.

[0060] Caffeine degradation rate (%) = (C0) C t ) / C0×100%; Absolute degradation amount (mg) = (C0) C t )×V; C0: Initial concentration of caffeine (mg / mL or mg / L) in the experimental group (0h). C t : Residual concentration of caffeine in the experimental group (after treatment) (mg / mL or mg / L). V: Volume of the sample solution (mL or L).

[0061] When the initial caffeine concentration is 50–300 mg / L, the degradation rate stabilizes at 16–20%, with an absolute degradation amount of approximately 10–30 mg / L. When the initial concentration is ≥400 mg / L, the degradation rate gradually decreases to below 10%, but the absolute degradation amount still increases with increasing concentration. This indicates that *Zygosacchariformis* is suitable for decaffeination treatment of plant beverages with low to medium caffeine concentrations (≤300 mg / L).

[0062] Example 9: Fermentation of a fruit juice-coffee composite system Based on the single-factor optimization results of Examples 4-8, the optimal process conditions for fermenting caffeinated plant beverages with *Zygosacchariformis* were determined to be: fermentation temperature 30℃, addition of 2% brown sugar as an external carbon source, a cell-to-beverage ratio of 1:5, and fermentation time 48 hours. Under these conditions, a degradation rate of approximately 20-22% can be achieved for beverages with an initial caffeine concentration ≤300 mg / L.

[0063] Based on the above optimal conditions, the application of *Zygosacchariformis* in compound plant-based beverages was further explored. Caffeinated plant-based beverages (cold brew coffee or tea) were mixed with natural fruit juices (apple juice, orange juice, grape juice, etc.) at volume ratios of 9:1, 7:3, 1:1, and 3:7. The initial caffeine concentration of the mixture was adjusted to 80–120 mg / L, and the initial total sugar concentration to 2–3% (supplemented with fructose). *Zygosacchariformis* was inoculated at the optimal ratio (cell volume: mixture = 1:5) and fermented at 30°C for 48 hours.

[0064] The optimal ratio of coffee (or tea) to apple juice is 7:3. After fermentation, the caffeine degradation rate reaches 15.8%, and the fructose and glucose content decreases slightly (approximately 30% is consumed).

[0065] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A strain of *Leucobryonia roux* ( Zygosaccharomyces rouxii HX001, characterized in that, The accession number is CCTCC NO: M 2026714, the depositary institution is the China Center for Type Culture Collection, and the deposit date is April 17, 2026.

2. The *Leucobryonius zygosacchari* according to claim 1 (… Zygosaccharomyces rouxii HX001, characterized in that, The *L. rouxae* yeast possesses caffeine tolerance and / or caffeine degradation capabilities.

3. A microbial inoculant, characterized in that, The microbial agent contains the *Zootralzyme roux* as described in claim 1 or 2 as an active ingredient.

4. The *L. rouxae* as described in claim 1 or 2 (… Zygosaccharomyces rouxii HX001, or the application of the microbial agent as described in claim 3 in the preparation of low-caffeine beverages.

5. A fermentation process for reducing the caffeine content in caffeinated beverages, characterized in that, Includes the following steps: The Lurni conjugated yeast as described in claim 1 or 2 ( Zygosaccharomyces rouxii HX001, or the microbial agent described in claim 3, is activated and cultured, and the bacterial cells are collected; the bacterial cells are inoculated into a sugary beverage with an initial caffeine concentration of 50-1500 mg / L at a mass ratio of bacterial cells to beverage of 1:2 to 1:15; fermentation is carried out at a fermentation temperature of 15℃ to 35℃, and after filtering the bacterial cells, a beverage with reduced caffeine is obtained. The amount of sugar added is 0.5-10% of the total mass of the beverage, and the sugar is selected from at least one of glucose, D-fructose, sucrose, brown sugar, lactose, D-allulose, D-tagatose, xylose, and xylitol.

6. The fermentation process according to claim 5, characterized in that, The initial concentration of caffeine is 200~400 mg / L.

7. The fermentation process according to claim 5, characterized in that, The sugar is brown sugar, and its addition amount is 1-5% of the beverage weight.

8. The fermentation process according to claim 5, characterized in that, The mass-to-volume ratio of the microorganisms to the beverage is 1:2 to 1:10; The fermentation temperature is 28℃~32℃.

9. The fermentation process according to any one of claims 5 to 8, characterized in that, The caffeinated beverage is coffee liquid, tea beverage, or coffee-tea combination beverage.

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