A method for low-temperature micro-aerobic segmental fermentation of apple fruit wine based on complex yeast synergistic metabolism

Abstract

The application discloses a kind of based on composite yeast synergistic metabolism-low temperature microaerophilic section fermentation apple fruit wine method, method, comprising: S0: apple lock fresh pretreatment, obtain bagged lock fresh apple;S1: wall breaking treatment;S2: vacuum flash sterilization;S3 three-stage fermentation, divided into four steps: S3.0 inoculant online activation;S4 online clarification-energy recovery;S5 cold flavoring-isobaric filling, obtain apple fruit wine finished product.The application adopts non-brewing yeast, brewing yeast and rhizopus three fungus coupling strategy, in the preparation of apple fruit wine early stage, add non-brewing yeast to produce fruity ester precursor, in the preparation of apple fruit wine late stage, add brewing yeast and rhizopus fermentation.The application uses apple as raw material, through "lock fresh-three temperature zone-three fungus coupling" integrated process, successfully solved the traditional fruit wine low juice yield, polyphenol loss is big, aroma single, batch fluctuation big and other pain points.

Description

Technical Field

[0001] This invention relates to the field of fruit wine bio-manufacturing technology, and in particular to a method for preparing apple wine that simultaneously improves the retention rate of monomeric phenols, the complexity of ester aromas, and shortens the fermentation cycle by using brewing yeast CR1 in synergistic metabolism with non-brewing yeast OM and Rhizopus, and by multi-stage fermentation under low temperature and micro-oxygen conditions. Background Technology

[0002] China's apple cider processing industry holds a significant position within the fruit wine industry, being one of the core categories besides wine, with a market share significantly higher than many specialty small berry wines. Its growth rate (≈8-12%) is synchronized with the trend of consumption upgrading, and the market foundation is solid and continues to expand. As a staple fruit, apples can be stably supplied year-round through cold storage and processing raw materials. The mature and resilient supply chain provides a solid foundation for the large-scale and standardized development of the apple cider industry. At the same time, apples are rich in various bioactive substances such as apple polyphenols, quercetin, and dietary fiber, and their health attributes are increasingly valued by consumers. Combined with its deep-rooted culinary culture and diverse flavor versatility, the apple cider industry presents a steady and broad development prospect.

[0003] In summary, under current technology, polyphenols suffer significant oxidation losses, resulting in reduced functionality; flavor homogenization and insufficient floral and fruity aromas; long single-stage fermentation at room temperature (8–12 days) poses a high risk of microbial contamination; and existing equipment cannot precisely control the coupling of dissolved oxygen and temperature, leading to large batch-to-batch variations.

[0004] Therefore, developing a new process and supporting equipment for apple wine with high polyphenol content, high aroma, and short production cycle has become an urgent need for the industry. Summary of the Invention

[0005] This invention provides a method for apple wine fermentation based on the synergistic metabolism of compound yeast and low-temperature micro-aerobic staged fermentation.

[0006] This invention employs a three-strain coupling strategy involving non-sacchariculture yeast, sacchariculture yeast, and Rhizopus. In the early stage of apple wine preparation, non-sacchariculture yeast is added to produce fruit aroma ester precursors, while in the later stage of apple wine preparation, sacchariculture yeast and Rhizopus are added for fermentation.

[0007] The ratio of non-Saccharomyces cerevisiae, Saccharomyces cerevisiae, and Rhizopus is 3~7:8~12:0.5~1.5.

[0008] The non-brewing yeast mentioned is OM, a non-brewing yeast from Yantai Diboshi Brewing Machine Co., Ltd. The brewing yeast used is CR1 brewing yeast from Yantai Diboshi Brewing Machine Co., Ltd. The Rhizopus mentioned is Rhizopus from Angel Yeast Co., Ltd.

[0009] The method is performed according to the following steps: The "CR1+OM+Rhizopus" three-fungus coupling strategy was adopted: H0–24: Non-Saccharibril OM proliferates independently, producing fruit flavor esters (isoamyl acetate, ethyl lactate) precursors; Between 24 and 72 hours: Saccharomyces cerevisiae CR1 and Rhizopus suspensions were added online, with the OM:CR1:Rhizopus ratio automatically adjusted to 5:10:1 using a pH-linked peristaltic pump. Rhizopus was used solely as a flavor enhancer, with an inoculum of 0.05–0.10% (w / w apple pulp), added 6 hours into the early fermentation stage, and the viable count decreased to 10 within 24 hours. 3 Below CFU / mL.

[0010] From 72 h to the fermentation endpoint: the temperature was slowly increased from 22 ℃ to 26 ℃, which activated the CR1 higher alcohol-ester synthesis pathway and inhibited the excessive accumulation of ethyl acetate in OM.

[0011] The ratio of bacterial agent compound and the timing of replenishment are controlled by a closed loop of two parameters: the ethanol generation rate (dE / dt) and the polyphenol degradation rate (dA / dt) fed back by the fiber optic probe.

[0012] A method for apple wine based on complex yeast synergistic metabolism-low temperature microaerobic staged fermentation includes the following steps: S0: Apple pretreatment for freshness locking, resulting in bagged fresh-locked apples; S1: Cell wall breaking process; S2: Vacuum flash sterilization; S3 three-stage fermentation consists of four steps: S3.0 microbial agent online activation: Brewing yeast CR1: Rehydrate at 35~39 ℃ for 10~20 min; Non-brewing yeast OM: Rehydrate at 26~30 ℃ for 10~30 min. S3.1 Low-temperature ester aroma stage, with a temperature of 17~19 ℃ and a duration of 18–30 h, using only non-Saccharibrew yeast OM; S3.2 During the microbial equilibrium period, the temperature is 21–23 ℃, and the duration is 42–54 h. Saccharomyces cerevisiae CR1 and Rhizopus are added in a specific ratio to achieve an OM:CR1:Rhizopus count of 3–7:8–12:0.5–1.5. S3.3 High-temperature aroma enhancement period, with a temperature of 25~27 ℃, lasting until the end.

[0013] S4 Online Clarification-Energy Recovery; S5 cold flavoring-isobaric bottling yields the finished apple wine.

[0014] In step S0, the apple freshness-preserving pretreatment specifically includes: S0.1 Apple picking and initial selection; S0.2 Ultrasonic-bubble combined washing: The apples after initial selection are washed with a combination of microbubbles and ultrasound to remove surface dirt and pesticide residues. The water temperature is controlled at 0℃~8℃. After washing, the apples are drained. S0.3 Tea polyphenols-chitosan biphasic freshness locking; S0.4 Dynamic precooling; S0.5 Modified atmosphere packaging.

[0015] In step S0.2, the pre-selected apples are cleaned using a combination of 0.7~0.9 MPa microbubbles and 26~30 kHz ultrasound for 60 s~120 s; In step S0.3, the tea polyphenol-chitosan biphasic freshness locking process specifically includes: a) Soak the drained apples in a tea polyphenol solution; b) Immediately after removal, spray with chitosan solution to form an edible semi-permeable membrane; In step S0.4, dynamic precooling specifically includes: keeping the apples after dual-phase freshness locking in a cold air tunnel at -2 to -0.5 ℃ for 2 to 5 minutes; In step S0.5, modified atmosphere packaging specifically includes: After modified atmosphere packaging, apples are filled with a mixture of O2, CO2 and N2 gases, and then heat-sealed using barrier bags. The relative humidity inside the bag is maintained at 90%–92%, resulting in bagged apples that are kept fresh.

[0016] In step S1, the cell wall breaking process specifically includes: S1.1 Automatic bag breaking and weighing: The magnetic elevator feeds the freshness-locking bags of bagged apples into the bag breaking screw and weighs them by batch; S1.2 Microjet cell disruption: 70~90 MPa instantaneous jet breaks down the waxy layer of fruit. S1.3 Vacuum degassing: Vacuum degassing at -0.09 to -0.07 MPa for 20~40 s to remove dissolved oxygen.

[0017] In step S2, vacuum flash sterilization specifically includes: S2.1: Flash-steam the apples after cell wall breaking treatment at 40~50℃ for 20~40 seconds. S2.2 Instantaneous cooling: The ice water plate drops to 12~22 ℃.

[0018] In step S5, the cold-mixed fragrance-isobaric filling specifically includes: S5.1 Cold Aroma: The apple wine semi-finished product obtained from the three-stage fermentation is left to stand at 2~8℃ for 20~28 hours, and then acacia honey is added; S5.2 Microfiltration sterilization: ceramic membrane filtration; S5.3 Isobaric filling-nitrogen sealing yields the finished apple wine.

[0019] This invention pioneers a "three-temperature zone + three-microbe coupling" model to solve the problems of single aroma and easy degradation of polyphenols in cider. An online closed-loop fiber optic probe correlates polyphenol and ethanol curves with microbial metabolism, enabling intelligent determination of the fermentation endpoint with an error of less than 1 hour. Through residual pressure centrifugation and heat pump recovery, steam consumption is reduced by more than 30%, achieving "negative carbon" brewing and realizing the energy utilization of CO2 exhaust gas. The combination of microjet and vacuum flash evaporation achieves commercial sterilization at a low temperature of 45°C, reducing the loss of heat-sensitive polyphenols by 20% compared to traditional pasteurization. The highly compact equipment reduces the footprint by 50%, avoids batch transfer contamination, and meets the FSSC22000 aseptic requirements.

[0020] Compared with the prior art, the beneficial effects of the present invention are reflected in: 1. Significantly improved polyphenol retention rate: The entire process involves low-temperature preservation and 45℃ vacuum flash evaporation, resulting in PPO passivation of ≥90% and an average retention rate of 92% for key polyphenols such as chlorogenic acid and rutin, which is 15–20% higher than the traditional 95℃ pasteurization process, and the wine has a more stable color.

[0021] 2. The aroma has been upgraded from a "single fruity aroma" to a "three-dimensional complex aroma": The three temperature zones of 18℃–22℃–26℃, combined with the three bacteria CR1+OM+Rhizopus, amplify the fruity esters, floral esters, and mellow esters in sequence, with a total ester content of 4.7 g / L, which is 2.3 times that of traditional single-strain fermentation, and improves the sensory score by 12–15 points.

[0022] 3. Batch stability CV < 5% The fiber optic probe provides real-time closed-loop control of bacterial community ratio and temperature gradient, with key indicator fluctuation coefficients below 5%, completely solving the industry pain points of large batch differences and difficult quality control in traditional methods.

[0023] 4. Energy consumption reduced by >30%, achieving "negative carbon" utilization of exhaust gas: The residual pressure of CO2 directly drives the centrifuge, and the latent heat of the exhaust gas is recovered by a heat pump to supply CIP and preheat the next batch of flash evaporation. This saves 0.38 tons of steam per kiloliter of wine, reduces workshop humidity by 25%, and reduces equipment corrosion rate by 40%. Attached Figure Description

[0024] Figure 1 This is a schematic flowchart of the method for making apple wine based on the synergistic metabolism of compound yeast and low-temperature micro-aerobic segmented fermentation according to the present invention. Detailed Implementation

[0025] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the scope of the present invention.

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0027] The present invention will now be described in detail with reference to the accompanying drawings and exemplary embodiments.

[0028] strain source Rhizopus: Angel Yeast Co., Ltd. CR1 (Brewing Yeast): Yantai Diboshi Craft Brewing Machine Co., Ltd. OM (Non-Saccharomyces cerevisiae): Yantai Diboshi Craft Brewing Machine Co., Ltd. Example 1 (Freshness Preservation - Three-Temperature Zone Three-Bacterial Coupling): like Figure 1 As shown, a method for processing apple wine is carried out according to the following steps: S0 apple freshness-locking pretreatment consists of six steps: S0.1 Harvesting and Preliminary Selection: Apples that are 90% ripe and have a soluble solids content of ≥14 °Brix are picked and delivered to the processing workshop within 3 hours. Moldy or mechanically damaged apples are removed.

[0029] S0.2 Ultrasonic-bubble combined washing: The surface was cleaned with a combination of 0.8 MPa microbubbles and 28 kHz ultrasound for 90 seconds to remove mud, sand and pesticide residues, while the water temperature was controlled below 8 ℃.

[0030] S0.3 Tea polyphenols-chitosan biphasic freshness locking: a) Immerse the drained apples in a 0.35% (w / v) tea polyphenol solution (pH 4.2) for 5 min to inhibit surface microorganisms and preliminarily passivate PPO; b) Immediately after removal, spray with 1% low molecular weight chitosan solution (<50 kDa) for 30 seconds to form an edible semi-permeable membrane that blocks oxygen.

[0031] S0.4 Dynamic precooling: The fruit is kept in a -1 ℃ cold air tunnel for 3 minutes to allow the core temperature to drop to 2 ℃ within 5 minutes, thus preventing ice crystal formation.

[0032] S0.5 Modified atmosphere packaging: The bag is filled with a mixture of 8% O2 + 10% CO2 + 82% N2 and heat-sealed using a 40 µm PE / PET high-barrier bag, with the relative humidity inside the bag maintained at 90%–92%.

[0033] S0.6 Cold chain transportation: The entire process is carried out in a 2–4 ​​℃ cold chain, and the fruit enters the subsequent micro-jet cell disruption process within 48 hours to ensure that the polyphenol oxidase (PPO) activity is deactivated by ≥90% and that the fruit retains its elasticity without the "sponge-like" defects caused by quick-freezing.

[0034] S1 micro-jet cell disruption consists of three steps: S1.1 Automatic bag breaking and weighing: The magnetic lift feeds the fresh-locking bags into the bag-breaking screw, and the bags are weighed in batches of 500 kg ± 2 kg.

[0035] S1.2 Microjet cell disruption: An instantaneous jet of 80 MPa breaks down 100% of the fruit wax layer, resulting in a juice yield of 83% to 92%.

[0036] S1.3 Vacuum degassing: -0.08 MPa for 30 s removes 70% of dissolved oxygen and prevents early browning.

[0037] S2 vacuum flash sterilization consists of two steps: S2.1 Flash evaporation at 45℃ for 30 s: PPO (polyphenol oxidase) was completely inactivated, and the total bacterial count decreased by 3.2 log; the aroma condensate was returned to the fragrance jar.

[0038] S2.2 Instantaneous Cooling: The temperature was lowered to 18°C ​​in 3 seconds by ice water plate replacement, in preparation for subsequent low-temperature fermentation.

[0039] S3 three-stage fermentation consists of four steps: S3.0 microbial agent online activation: CR1: Rehydrate at 37℃ for 15 min; OM: Rehydrate at 28℃ for 20 min; Peristaltic pump on standby.

[0040] S3.1 Low-temperature ester aroma period (18 ℃, 0–24 h): If only OM is used and the fiber optic probe monitors ethyl lactate levels >1.5 g / L, the system will automatically proceed to the next stage.

[0041] S3.2 Microbial community equilibrium period (22 ℃, 24–72 h): The peristaltic pump replenishes CR1 and Rhizopus suspension in proportion, so that OM:CR1:Rhizopus number = 5:10:1; microporous circulation aeration 0.2 vvm.

[0042] S3.3 High-temperature aroma enhancement period (26 ℃, 72 h – endpoint): If the fiber is determined to have dE / dt < 0.3 g / (L·h) and dA / dt < 0.05 AU / h, the automatic cooling to 5 ℃ will terminate.

[0043] S4 online clarification-energy recovery, consisting of two steps: S4.1 CO2 residual pressure centrifugation: The exhaust gas is directly driven by a disc centrifuge at 0.12 MPa, resulting in a wine turbidity of <5 NTU and a fruit pomace water content of ≤50%.

[0044] S4.2 Heat pump regeneration: The latent heat of the exhaust gas is heated to 75 ℃ by the heat pump evaporator, which is used for preheating of CIP / SIP and the next batch of flash evaporation.

[0045] S5 Cold-Blend Fragrance - Isobaric Filling, consisting of three steps: S5.1 Cool-toned fragrance: After standing at 5℃ for 24 hours, adding 0.05% acacia honey improved the aroma blending by 18%.

[0046] S5.2 Microfiltration sterilization: 0.45 µm ceramic membrane filtration, microorganisms ≤10 CFU / mL.

[0047] S5.3 Isobaric Filling - Nitrogen Sealing: The isobaric filling machine operates at 0.08 MPa, with nitrogen replacement of O2 <1%; the CO2 content inside the bottle is 2.0 g / L, resulting in a slightly effervescent taste.

[0048] This invention employs a two-step process of "freshness locking-relay fermentation". Front-end freshness locking: Apples are sliced ​​after ultrasonic-bubble cleaning, treated with 0.35% tea polyphenols to inhibit PPO, 1% chitosan to form a film, pre-cooled from -1℃ to 2℃ with cold air, and sealed with 8% O2 + 10% CO2 modified atmosphere packaging, maintaining elasticity for 48 hours. Back-end relay: Microjet cell wall disruption achieves a juice yield of 92%, followed by vacuum flash sterilization and a three-stage fermentation process—rapidly producing fruit flavor esters within 6 hours using OM single-strain fermentation at 18℃; primary fermentation is completed at 22℃ with the addition of CR1 and Rhizopus at a ratio of 5:10:1; the aroma enhancement stage is determined by optical fiber, ensuring a total ester content of 4.7 g / L and 92% retention of chlorogenic acid. The exhaust heat is used to preheat the next batch of juice. During the cold flavoring stage, the juice is left to stand at 5 ℃ for 24 h and 0.05% acacia honey is added. The final volatile acid is <0.4 g / L. The aroma is distinctly divided into three stages: fruity, floral and mellow. The batch CV is <5%.

[0049] Example 2 (Freshness Locking - Single Brewing Yeast CR1): The preservation and pretreatment were the same as in Example 1, except that the fermentation stage was carried out at 26 ℃ with a single CR1. The final polyphenol retention rate was 76%, the total ester was 1.9 g / L, the sensory score was 74.6, and the color was slightly dark.

[0050] Example 3 (Freshness Locking - Double-Strain CR1 + OM): The preservation and pretreatment were the same as in Example 1. The fermentation stage used a three-temperature zone of 18 ℃-22 ℃-26 ℃, CR1+OM dual bacteria, polyphenol retention rate of 89%, total ester of 4.7 g / L, sensory score of 82.8, and distinct layers of fruit aroma, floral aroma and alcohol aroma.

[0051] Example 4 (Freshness Locking - Three-Bacterium CR1 + OM + Rhizopus): The preservation and pretreatment were the same as in Example 1. Three bacteria were used in the fermentation stage, with Rhizopus accounting for 10%, polyphenol retention rate of 92%, and total ester of 4.9 g / L. The taste was more mellow, but the Rhizopus flavor was slightly stronger, and the sensory score was 80.1.

[0052] The results are shown in Table 1: The color, taste, texture, and aroma of the wine varied significantly with different fermentation conditions under different examples (P < 0.5). The sample treated with fresh-locked single brewer's yeast CR1 had the lowest sensory score of 63.4 points, while the sample treated with fresh-locked triple-strain CR1 + OM + Rhizopus had the highest total sensory score of 85.1 points.

[0053] Table 1. Sensory evaluation results of samples from different embodiments

[0054] Table 2. Sensory evaluation criteria for samples from different embodiments

[0055] In summary, this invention, using apples as raw material, successfully solves the pain points of traditional fruit wine, such as low juice yield, significant polyphenol loss, monotonous aroma, and large batch fluctuations, through an integrated process of "freshness locking - three-temperature zone - three-microbial coupling". The freshness locking pretreatment enables apples to complete surface enzyme inhibition, film formation, pre-cooling, and modified atmosphere sealing within 3 hours of harvesting, achieving polyphenol oxidase inactivation ≥90%; micro-jet cell disruption + vacuum flash evaporation achieves low-temperature sterilization and aroma recovery; three-stage fermentation at 18℃-22℃-26℃, combined with precise relay of CR1, OM, and a small amount of Rhizopus, increases total esters to 4.9 g / L, chlorogenic acid retention ≥92%, and batch CV <5%; residual pressure of CO2 exhaust gas drives centrifugation and recovers latent heat, reducing energy consumption by 30%.

Claims

1. A method for making apple wine based on synergistic metabolism of compound yeast and low-temperature microaerobic staged fermentation, characterized in that, Includes the following steps: S0: Apple pretreatment for freshness locking, resulting in bagged fresh-locked apples; S1: Cell wall breaking process; S2: Vacuum flash sterilization; S3 three-stage fermentation consists of four steps: S3.0 microbial agent online activation: Brewing yeast CR1: Rehydrate at 35~39 ℃ for 10~20 min; Non-brewing yeast OM: Rehydrate at 26~30 ℃ for 10~30 min; S3.1 Low-temperature ester aroma stage, with a temperature of 17~19 ℃ and a duration of 18–30 h, using only non-Saccharibrew yeast OM; S3.2 During the microbial equilibrium period, the temperature is 21–23 ℃, and the duration is 42–54 h. Saccharomyces cerevisiae CR1 and Rhizopus are added in a specific ratio to achieve an OM:CR1:Rhizopus count of 3–7:8–12:0.5–1.

5. S3.3 High-temperature aroma enhancement period, with a temperature of 25~27 ℃, lasting until the end; S4 Online Clarification-Energy Recovery; S5 cold flavoring - isobaric bottling yields the finished apple wine.

2. The method for apple wine based on compound yeast synergistic metabolism-low temperature microaerobic segmented fermentation according to claim 1, characterized in that, In step S0, the apple freshness-preserving pretreatment specifically includes: S0.1 Apple picking and initial selection; S0.2 Ultrasonic-bubble combined washing: The apples after initial selection are washed with a combination of microbubbles and ultrasound to remove surface mud and pesticide residues. The water temperature is controlled at 0℃~8℃. After washing, the apples are drained. S0.3 Tea polyphenols-chitosan biphasic freshness locking; S0.4 Dynamic precooling; S0.5 Modified atmosphere packaging.

3. The method for apple wine based on compound yeast synergistic metabolism-low temperature microaerobic segmented fermentation according to claim 2, characterized in that, In step S0.2, the pre-selected apples are cleaned by a combination of 0.7~0.9 MPa microbubbles and 26~30 kHz ultrasound for 60 s~120 s.

4. The method for apple wine based on compound yeast synergistic metabolism-low temperature microaerobic segmented fermentation according to claim 2, characterized in that, In step S0.3, the tea polyphenol-chitosan biphasic freshness locking process specifically includes: a) Soak the drained apples in a tea polyphenol solution; b) Immediately after removal, spray with chitosan solution to form an edible semi-permeable membrane.

5. The method for apple wine based on compound yeast synergistic metabolism-low temperature microaerobic segmented fermentation according to claim 2, characterized in that, In step S0.4, dynamic precooling specifically includes: keeping the apples after dual-phase freshness locking in a cold air tunnel at -2 to -0.5 ℃ for 2 to 5 minutes.

6. The method for apple wine based on compound yeast synergistic metabolism-low temperature microaerobic segmented fermentation according to claim 2, characterized in that, In step S0.5, modified atmosphere packaging specifically includes: After modified atmosphere packaging, apples are filled with a mixture of O2, CO2 and N2 gases, and then heat-sealed using barrier bags. The relative humidity inside the bag is maintained at 90%–92%, resulting in bagged apples that are kept fresh.

7. The method for apple wine based on compound yeast synergistic metabolism-low temperature microaerobic segmented fermentation according to claim 1, characterized in that, In step S1, the cell wall breaking process specifically includes: S1.1 Automatic bag breaking and weighing: The magnetic elevator feeds the freshness-locking bags of bagged apples into the bag breaking screw and weighs them by batch; S1.2 Microjet cell disruption: 70~90 MPa instantaneous jet breaks down the waxy layer of fruit. S1.3 Vacuum degassing: Vacuum degassing at -0.09 to -0.07 MPa for 20~40 s to remove dissolved oxygen.

8. The method for apple wine based on compound yeast synergistic metabolism-low temperature microaerobic segmented fermentation according to claim 1, characterized in that, In step S2, vacuum flash sterilization specifically includes: S2.1: Flash-steam the apples after cell wall breaking treatment at 40~50℃ for 20~40 seconds. S2.2 Instantaneous cooling: The ice water plate drops to 12~22 ℃.

9. The method for apple wine based on compound yeast synergistic metabolism-low temperature microaerobic segmented fermentation according to claim 1, characterized in that, In step S5, the cold-mixed fragrance-isobaric filling specifically includes: S5.1 Cold Aroma: The apple wine semi-finished product obtained from the three-stage fermentation is left to stand at 2~8℃ for 20~28 hours, and then acacia honey is added; S5.2 Microfiltration sterilization: ceramic membrane filtration; S5.3 Isobaric filling-nitrogen sealing yields the finished apple wine.

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