A pectinase complex with low pectin esterase activity, an enzymatic fermentation method for reducing the content of methanol and higher alcohols in tangerine fruit wine, application and tangerine fruit wine

Abstract

This invention provides a pectinase complex with low pectin esterase activity, an enzymatic fermentation method for reducing methanol and higher alcohol content in orange wine, its application, and the resulting orange wine, specifically belonging to the field of fruit wine brewing technology. The pectinase complex with low pectin esterase activity described in this invention is derived from Aspergillus niger (… Aspergillus niger The pectinase complex with low pectin esterase activity was prepared by fermentation using CICC 40273. The pectin esterase activity was less than 0.05 U / mg, the pectin lyase activity was ≥7000 U / g, and the polygalacturonase activity was ≥4500 U / g. This invention's pectinase complex with low pectin esterase activity can specifically degrade pectin, reduce the methanol content in orange wine, and exhibits high enzymatic hydrolysis efficiency, stable and significant effects, and a significant improvement in sensory quality.

Description

Technical Field

[0001] This invention belongs to the field of fruit wine brewing technology, specifically relating to a pectinase complex with low pectin esterase activity, an enzymatic fermentation method for reducing methanol and higher alcohol content in orange wine and its application, and orange wine. Background Technology

[0002] When brewing fruit wine from citrus fruits, the high pectin and protein content often leads to high methanol levels, affecting the wine's safety and palatability. Methanol formation is primarily related to the activity of pectin esterase (PE) within pectinase enzymes. Pectinase is a complex enzyme system, mainly including pectin esterase (PE), polygalacturonase (PG), and pectin lyase (PL). PE catalyzes the hydrolysis of methyl ester bonds in pectin, releasing methanol; while PG and PL mainly break down pectin molecular chains, promoting liquid clarification, but produce little or no methanol. Therefore, controlling the activity of PE in pectinase preparations is crucial for controlling methanol formation.

[0003] Currently, the mainstream methods for controlling methanol in the industry are to use pectinase preparations with low PE activity or to peel the raw materials. However, these methods have significant limitations: the enzymatic hydrolysis efficiency (in terms of juice yield and viscosity reduction) of commercially available low-methanol (i.e., low-PE activity) pectinases is sometimes lower than that of traditional pectinases with high PE activity. Therefore, developing a method to reduce methanol content in citrus wine with higher enzymatic hydrolysis efficiency has significant industrial application value. Summary of the Invention

[0004] The purpose of this invention is to provide a pectinase complex with low pectin esterase activity, an enzymatic fermentation method for reducing methanol and higher alcohol content in orange wine, its application, and the resulting orange wine. The pectinase complex with low pectin esterase activity described in this invention can specifically degrade pectin, reduce the methanol content in orange wine, exhibits high enzymatic hydrolysis efficiency, stable and significant effects, and significantly improves sensory quality.

[0005] This invention provides a pectinase complex with low pectin esterase activity, derived from Aspergillus niger (… Aspergillus niger The pectinase complex with low pectin esterase activity was prepared by fermentation using CICC 40273. The pectin esterase activity was less than 0.05 U / mg, the pectin lyase activity was ≥7000 U / g, and the polygalacturonase activity was ≥4500 U / g.

[0006] This invention also provides a method for preparing the pectinase complex described in the above technical solution, comprising the following steps: Liquid fermentation of Aspergillus niger CICC 40273 was carried out, the fermentation broth was collected, and crude enzyme solution was extracted. The crude enzyme solution was subjected to ion exchange chromatography to remove the pectin esterase component, and the flow-through was collected. The flow-through solution was concentrated and dried to obtain pectinase with low pectin esterase activity.

[0007] Preferably, the pH value of the liquid fermentation is 4.8~5.2; the aeration rate of the liquid fermentation is 0.8~1.2 vvm; and the temperature of the liquid fermentation is 31~34℃.

[0008] This invention also provides an enzymatic fermentation method for reducing the methanol and higher alcohol content in orange wine based on the pectinase complex described in the above technical solution, comprising the following steps: Remove the peel and pith of the citrus fruit, with a removal rate of ≥15%, then crush the fruit to obtain citrus pulp; The citrus pulp and the pectinase complex described in the above technical solution are mixed and subjected to the first enzymatic hydrolysis to obtain the first enzymatic hydrolysate. The first hydrolysate is mixed with acidic protease and subjected to a second hydrolysis to obtain the second hydrolysate. The second enzymatic hydrolysate is mixed with brewer's yeast and fermented to obtain orange wine.

[0009] Preferably, the temperature of the first enzymatic hydrolysis is 35~40℃; the pH value of the first enzymatic hydrolysis is 3.5~4.0; the time of the first enzymatic hydrolysis is 1~2h; and the amount of pectinase complex with low pectin esterase activity added to the citrus pulp in the first enzymatic hydrolysis is 30~50 mg / kg.

[0010] Preferably, the temperature of the second enzymatic hydrolysis is 47~52℃; the time of the second enzymatic hydrolysis is 40~60min; and the amount of acidic protease added to the citrus pulp in the second enzymatic hydrolysis is 10~20 mg / kg.

[0011] Preferably, the fermentation is a gradient temperature-controlled fermentation; the conditions for the gradient temperature-controlled fermentation are as follows: the first fermentation is carried out at 16~18℃ for 12~36h, the temperature is raised to 20~25℃ for the second fermentation, and when the residual sugar content is less than 5 g / L, the temperature is lowered to 12~14℃ for the third fermentation for 7~15d.

[0012] Preferably, the brewing yeast includes Angel Yeast RV171.

[0013] The present invention also provides the application of the enzymatic fermentation method described above in the preparation of orange wine with low methanol and higher alcohol content; wherein the methanol content in the orange wine is ≤100 mg / L and the total amount of higher alcohols is ≤300 mg / L.

[0014] The present invention also provides orange wine prepared by the enzymatic fermentation method described in the above technical solution.

[0015] This invention provides a pectinase complex with low pectin esterase activity. The pectinase complex of this invention can specifically degrade pectin, reduce the methanol content in orange wine, and exhibits high enzymatic hydrolysis efficiency, stable and significant effects, resulting in a significant improvement in sensory quality.

[0016] Furthermore, this invention provides an enzymatic fermentation method for reducing methanol and higher alcohol content in orange wine. This method reshapes the fermentation substrate through specific staged enzymatic hydrolysis (first targeting pectin degradation, then optimizing the nitrogen source sequence), combined with fermentation regulation. This significantly reduces methanol and higher alcohol content in orange wine, improves enzymatic hydrolysis efficiency, and results in a marked improvement in sensory quality. Experimental results show that, at the same addition amount, treatment with commercially available pectinase with low pectin esterase activity still resulted in a significantly higher methanol content than this invention, while the juice yield and viscosity reduction rate were lower. This invention overcomes the deficiency of insufficient enzymatic hydrolysis efficiency in commercially available pectinases with low pectin esterase activity, improving sensory quality while reducing methanol and higher alcohol content, and has significant industrial application value. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A process flow diagram of the enzymatic hydrolysis fermentation method provided by the present invention; Figure 2 A bar chart comparing the methanol and higher alcohol content in the final products of the embodiments and comparative examples provided by the present invention; Figure 3 A photograph of the orange wine provided in Embodiment 4 of the present invention; Figure 4 A photograph of orange wine provided for Comparative Example 10 of this invention. Detailed Implementation

[0019] This invention provides a pectinase complex with low pectin esterase activity, derived from Aspergillus niger (… Aspergillus nigerThe pectinase complex with low pectin esterase activity was prepared by fermentation with CICC 40273. The pectin esterase activity was less than 0.05 U / mg, the pectin lyase activity was ≥7000 U / g, and the polygalacturonase activity was ≥4500 U / g. In a specific embodiment, the pectin esterase activity was less than 0.01 U / mg. The pectinase complex with low pectin esterase activity of this invention was prepared by fermenting Aspergillus niger CICC 40273 strain and then removing the pectin esterase component. The pectinase complex with low pectin esterase activity of this invention has both pectin lyase (PL) and polygalacturonase (PG) activities. This invention does not have a specific limitation on the source of Aspergillus niger CICC 40273 strain; conventional commercially available products well known to those skilled in the art can be used.

[0020] This invention also provides a method for preparing the pectinase complex described in the above technical solution, comprising the following steps: Liquid fermentation of Aspergillus niger CICC 40273 was carried out, the fermentation broth was collected, and crude enzyme solution was extracted. The crude enzyme solution was subjected to ion exchange chromatography to remove the pectin esterase component, and the flow-through was collected. The flow-through solution was concentrated and dried to obtain pectinase with low pectin esterase activity.

[0021] This invention involves liquid fermentation of *Aspergillus niger* CICC 40273, collecting the fermentation broth, and extracting a crude enzyme solution. Prior to liquid fermentation, *Aspergillus niger* CICC 40273 is activated and cultured as a seed culture. In a specific embodiment, activation can be performed using potato dextrose agar (PDA) slant. In a specific embodiment, the activation temperature can be 28–32°C, specifically 28°C, 30°C, or 32°C. In a specific embodiment, the activation time can be 68–76 hours, specifically 70 hours, 72 hours, 74 hours, or 76 hours. Activation is performed until the spore layer is abundant. A spore suspension is prepared using sterile water for seed culture, and the spore concentration in the suspension can be 1 × 10⁻⁶. 7 ~1×10 9 CFU / mL, specifically 1×10 8CFU / mL. After activation culture, the present invention performs seed culture. In a specific embodiment, the seed culture uses a seed culture medium. In a specific embodiment, the composition of the seed culture medium may be: glucose 20-40 g / L, soybean meal 10-20 g / L, ammonium sulfate 3-7 g / L, potassium dihydrogen phosphate 1-3 g / L, and magnesium sulfate 0.3-0.7 g / L, natural pH. In a specific embodiment, the inoculum size of the seed culture may be 3-7% (v / v), specifically 5%. In a specific embodiment, the temperature of the seed culture may be 28-32℃, specifically 28℃, 30℃, or 32℃. In a specific embodiment, the seed culture is a shaking culture, and the shaking speed may be 150-200 rpm, specifically 180 rpm. In a specific embodiment, the seed culture time may be 20-28 h, specifically 24 h. The present invention uses a fermentation culture medium for liquid fermentation. In a specific embodiment, the fermentation medium may consist of: 30-50 g / L citrus peel powder (40-80 mesh), 15-25 g / L wheat bran, 8-12 g / L ammonium sulfate, 3-7 g / L yeast extract, 2-4 g / L dipotassium hydrogen phosphate, 0.3-0.7 g / L magnesium sulfate, and 0.05-0.15% (v / v) surfactant (which may be Tween-80), with an initial pH of natural (approximately 5.0-6.0). In a specific embodiment, seed culture is inoculated into the fermentation medium for liquid fermentation. In a specific embodiment, the inoculation amount for liquid fermentation may be 6-10%, specifically 8%. In a specific embodiment, the temperature for liquid fermentation may be 31-34°C, specifically 32°C, 33°C, or 34°C. In a specific embodiment, the liquid fermentation is stirred at a speed of 180-220 rpm, specifically 180 rpm, 200 rpm, or 220 rpm. In a specific embodiment, the aeration (sterile compressed air) rate of the liquid fermentation can be 0.8~1.2 vvm, specifically 0.8 vvm, 1 vvm, or 1.2 vvm. In a specific embodiment, the pH value of the liquid fermentation is 4.8~5.2, specifically 4.9, 5.0, or 5.1. The pH value control of this invention can be automatic, achieved through the automatic control of the addition of alkali solution. In a specific embodiment, the alkali solution can be a 2~10% sodium hydroxide aqueous solution. In a specific embodiment, the liquid fermentation time can be 65~80 hours, specifically 70 hours or 75 hours. After liquid fermentation, a fermentation broth is obtained. This invention can remove mycelia and insoluble residues from the fermentation broth through solid-liquid separation. In a specific embodiment, the solid-liquid separation method can be filtration or centrifugation. In a specific embodiment, the filtration can be a plate and frame filter.In a specific embodiment, the centrifugation speed can be 5000~10000 rpm, specifically 6000 rpm, 8000 rpm, or 10000 rpm; the centrifugation time is 10~30 min, specifically 15 min, 20 min, or 25 min; the centrifugation temperature is 4℃ to maintain enzyme activity. The filtrate or supernatant is collected to obtain the liquid fraction. After solid-liquid separation, the present invention can concentrate and decolorize the liquid fraction to obtain a crude enzyme solution (or crude enzyme concentrate). In a specific embodiment, the concentration can be performed using an ultrafiltration membrane system with a molecular weight cutoff of 8~12 kDa, specifically 10 kDa, which also has a decolorizing effect.

[0022] After obtaining the crude enzyme solution, the present invention performs ion exchange chromatography to remove the pectin esterase component and collects the flow-through. In a specific embodiment, ion exchange chromatography can be used to specifically remove the pectin esterase component from the crude enzyme solution, ultimately obtaining a pectinase complex mainly composed of PL and PG with extremely low PE activity. Before ion exchange chromatography, the present invention dialyzes or dilutes the crude enzyme solution to make its conductivity consistent with the equilibration buffer. In a specific embodiment, the equilibration buffer can be 10-50 mM, pH 4.8-5.2, specifically 20 mM, pH 5.0 acetate-sodium acetate buffer. In a specific embodiment, the packing material for the ion exchange chromatography can be a weak anion exchange medium, such as DEAE Sepharose FastFlow. In a specific embodiment, the ion exchange chromatography column can be fully equilibrated using the above-mentioned acetate-sodium acetate buffer. In a specific embodiment, the treated crude enzyme solution is loaded with a sample, the loading volume being 3-10% of the column volume, specifically 5%. In a specific embodiment, the flow-through can be washed with 3-5 column volumes of equilibration buffer and collected (this fraction is rich in PL and PG). The pectin esterase (PE) activity of this fraction of the complex has been significantly reduced. In another specific embodiment, after collecting the flow-through, a linear gradient elution with 0-0.6 M NaCl can be performed over 8-15 column volumes. Pectin esterase (PE) is mainly eluted in this gradient elution phase (typically in the 0.15-0.4 M NaCl range), and this fraction can be discarded or collected separately.

[0023] After obtaining the flow-through liquid, the present invention concentrates and dries the flow-through liquid to obtain pectinase with low pectin esterase activity. After collecting and combining the target flow-through liquids, the present invention can concentrate and desalt them using an ultrafiltration membrane with a molecular weight cutoff of 8-12 kDa, specifically 10 kDa. After concentration and desalting, the present invention adds a lyophilization protectant to the concentrate. In a specific embodiment, the lyophilization protectant can be trehalose or mannitol, and the amount of the lyophilization protectant added can be 3-10% of the total solids. In a specific embodiment, the drying can be freeze-drying to obtain a powdered pectinase complex with low pectin esterase activity. The pectinase complex with low pectin esterase activity prepared under the above method conditions of the present invention has a pectin lyase (PL) activity ≥7000 U / g, a polygalacturonase (PG) activity ≥4500 U / g, and a pectin esterase (PE) activity <0.05 U / mg protein, specifically <0.01 U / mg.

[0024] This invention also provides an enzymatic fermentation method for reducing the methanol and higher alcohol content in orange wine based on the pectinase complex described in the above technical solution, comprising the following steps: Remove the peel and pith of the citrus fruit, with a removal rate of ≥15%, then crush the fruit to obtain citrus pulp; The citrus pulp and the pectinase complex described in the above technical solution are mixed and subjected to the first enzymatic hydrolysis to obtain the first enzymatic hydrolysate. The first hydrolysate is mixed with acidic protease and subjected to a second hydrolysis to obtain the second hydrolysate. The second enzymatic hydrolysate is mixed with brewer's yeast and fermented to obtain orange wine.

[0025] Figure 1 This is a process flow diagram of the enzymatic fermentation method of the present invention. The peel and pith of the citrus fruit are removed, with a removal rate ≥15%, and the fruit is crushed to obtain citrus pulp. In this invention, the citrus fruit can be mature citrus fruit. In a specific embodiment, the citrus fruit can be mandarin oranges or Wogan tangerines. The present invention does not have specific limitations on the method for removing the peel and pith of the citrus fruit; conventional mechanical methods are sufficient. The present invention does not have specific limitations on the crushing conditions; conventional crushing methods are sufficient. This invention reduces methanol-forming substrates from the source by physically removing areas rich in pectin and bitter substances.

[0026] After obtaining citrus pulp, the present invention mixes the citrus pulp with a pectinase complex with low pectin esterase (PE) activity as described in the above technical solution, and performs a first enzymatic hydrolysis to obtain a first hydrolysate. In the citrus pulp treatment, compared with commercially available ordinary pectinase (such as Comparative Example 4), the pectinase of the present invention can significantly reduce the methanol content while achieving better juice yield and viscosity reduction effect. In a specific embodiment, the temperature of the first enzymatic hydrolysis is 35~40℃, which can be 38~39℃, specifically 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃; the pH value of the first enzymatic hydrolysis is 3.5~4.0, which can be 3.6~3.8, specifically 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0; the time of the first enzymatic hydrolysis is 1~2h, which can be 1.2~1.8h, specifically 1h, 1.2h, 1.4h, 1.5h, 1.6h, 1.8h or 2h; in the first enzymatic hydrolysis, the amount of pectinase complex with low pectin esterase activity added to the citrus pulp is 30~50 mg / kg, which can be 35~45 mg / kg, specifically 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg or 50 mg / kg. This invention utilizes a pectinase complex with low pectin esterase activity to target and degrade the pectin structure, which can efficiently decompose pectin and minimize methanol generation while improving juice yield and clarity.

[0027] After obtaining the first enzymatic hydrolysate, the present invention mixes the first enzymatic hydrolysate with an acidic protease for a second enzymatic hydrolysis to obtain the second enzymatic hydrolysate. After obtaining the first enzymatic hydrolysate, without changing the reaction system, the present invention raises the material temperature to perform a second enzymatic hydrolysis. In a specific embodiment, the acidic protease can be a commercially available acidic protease preparation, such as NovoCor® ABG acidic protease manufactured by Novonesis. In a specific embodiment, the temperature of the second enzymatic hydrolysis is 47~52℃, specifically 48~50℃, or more specifically 47℃, 48℃, 49℃, 50℃, 51℃, or 52℃; the time of the second enzymatic hydrolysis is 40~60 min, specifically 45~55 min, or more specifically 40 min, 45 min, 50 min, 55 min, or 60 min; in the second enzymatic hydrolysis, the amount of acidic protease added to the citrus pulp is 10~20 mg / kg, specifically 12~18 mg / kg, or more specifically 10 mg / kg, 12 mg / kg, 15 mg / kg, 16 mg / kg, 18 mg / kg, or 20 mg / kg. The second enzymatic hydrolysis of this invention utilizes acidic protease to optimize the nitrogen source form of the fruit juice. Specifically, it can hydrolyze large molecular proteins in the fruit juice that are difficult for yeast to directly utilize into easily absorbed short peptides and free amino acids, thereby reducing the excessive synthesis of higher alcohols by brewer's yeast due to nitrogen deficiency from the metabolic source.

[0028] After obtaining the second enzymatic hydrolysate, the present invention mixes the second enzymatic hydrolysate with brewer's yeast and ferments it to obtain orange wine. In a specific embodiment, after obtaining the second enzymatic hydrolysate, the present invention adjusts the sugar content and pH value of the second enzymatic hydrolysate before mixing it with brewer's yeast. In a specific embodiment, the sugar content can be 18~22°Brix, specifically 18°Brix, 19°Brix, 20°Brix, 21°Brix, or 22°Brix. In a specific embodiment, the pH value can be 3.2~3.6, specifically 3.2, 3.3, 3.4, 3.5, or 3.6. In a specific embodiment, the fermentation is a gradient temperature-controlled fermentation; the conditions for the gradient temperature-controlled fermentation are: first fermentation at 16~18℃ for 12~36h, second fermentation at 20~25℃, and when the residual sugar content is below 5 g / L, third fermentation at 12~14℃ for 7~15d. In a specific embodiment, the first fermentation is the adaptation period, the second fermentation is the logarithmic phase, and the third fermentation is the stationary phase. Starting fermentation at a lower temperature of 16-18°C allows the yeast to smoothly adapt to the nutrient environment of the second enzymatic hydrolysate. The temperature setting for the second fermentation ensures sufficient fermentation. During the third fermentation, when the residual sugar content drops below 5 g / L, the temperature is lowered to 12-14°C, which promotes yeast sedimentation and stabilizes the flavor of the wine. In a specific embodiment, the temperature of the first fermentation can be 16°C, 17°C, or 18°C. In a specific embodiment, the duration of the first fermentation can be 12-36 hours, specifically 24 hours. In a specific embodiment, the temperature of the second fermentation can be 20-25°C, specifically 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C. In a specific embodiment, the temperature of the third fermentation can be 12°C, 13°C, or 14°C. In a specific embodiment, the duration of the third fermentation is 7-15 days. In a specific embodiment, the residual sugar content of the third fermentation is ≤4 g / L and the wine is basically clarified, thus ending fermentation and allowing for subsequent conventional clarification and aging processes. In a specific embodiment, the brewing yeast includes Angel Fruit Wine Yeast RV171. The juice after the first and second enzymatic hydrolysis is then mixed with the brewing yeast for gradient temperature-controlled fermentation, which can systematically and efficiently reduce the content of methanol and higher alcohols simultaneously, and significantly improve sensory quality. The method described in this invention is stable and easy to industrialize.

[0029] The present invention also provides the application of the enzymatic fermentation method described above in the preparation of orange wine with low methanol and higher alcohol content; wherein the methanol content in the orange wine is ≤100 mg / L and the total amount of higher alcohols is ≤300 mg / L.

[0030] This invention also provides orange wine prepared by the enzymatic fermentation method described above. The orange wine of this invention has low methanol and higher alcohol content, and exhibits high and stable sensory quality.

[0031] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a pectinase complex with low pectin esterase activity, an enzymatic fermentation method for reducing methanol and higher alcohol content in orange wine, its application, and orange wine. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Unless otherwise specified, all percentages are weight-to-volume ratios (w / v). Unless otherwise specified, the test materials used in the examples are all commercially available products.

[0033] Example 1 Preparation of pectinase complex with low pectin esterase activity.

[0034] Aspergillus niger CICC 40273 was activated on a PDA slant at 30℃ for 72 hours until the spore layer was full. A spore suspension (concentration 1×10⁻⁶) was then prepared with sterile water. 8 (CFU / mL).

[0035] Prepare seed culture medium: glucose 30 g / L, soybean meal 15 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 2 g / L and magnesium sulfate 0.5 g / L, natural pH, inoculate with 5% spore suspension, and culture at 30℃ and 180 rpm for 24 h with shaking.

[0036] Fermentation medium was prepared as follows: 40 g / L orange peel powder (60 mesh), 20 g / L wheat bran, 10 g / L ammonium sulfate, 5 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, Tween-80 0.1%, and natural pH. The seed culture was inoculated into the fermenter at an 8% inoculum rate. Fermentation was carried out for 72 h at a fermentation temperature of 32℃, a stirring speed of 200 rpm, an aeration rate of 1.0 vvm, and an automatically controlled pH (5.0 via the addition of 5% NaOH solution).

[0037] After centrifuging the fermentation broth at 8000 rpm for 20 min, the supernatant was concentrated using a 10 kDa ultrafiltration membrane at 4℃ to obtain crude enzyme solution.

[0038] After dialyzing the crude enzyme solution against 20 mM, pH 5.0 acetate-sodium acetate buffer, it was loaded onto a DEAE Sepharose Fast Flow column (5% column volume loading). The column was washed with 5 column volumes of equilibration buffer (20 mM, pH 5.0 acetate-sodium acetate buffer), and the flow-through was collected.

[0039] The flow-through solution was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, 5% trehalose was added, and the solution was freeze-dried to obtain the target enzyme powder, namely a pectinase complex with low pectin esterase activity.

[0040] The enzyme powder was tested and found to have an activity of 8200 U / g for PL, 5200 U / g for PG, and 0.008 U / mg protein for PE.

[0041] Example 2 Preparation of pectinase complex with low pectin esterase activity.

[0042] Aspergillus niger CICC 40273 was activated on a PDA slant at 28℃ for 76 hours until the spore layer was full. A spore suspension (concentration 1×10⁻⁶) was then prepared with sterile water. 8 (CFU / mL).

[0043] Prepare seed culture medium: glucose 30 g / L, soybean meal 15 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 2 g / L and magnesium sulfate 0.5 g / L, natural pH, inoculate with 5% spore suspension, and culture at 30℃ and 180 rpm for 24 h with shaking.

[0044] Fermentation medium was prepared as follows: 40 g / L orange peel powder (60 mesh), 20 g / L wheat bran, 10 g / L ammonium sulfate, 5 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, Tween-80 0.1%, and natural pH. The seed culture was inoculated into the fermenter at an 8% inoculum rate. Fermentation was carried out for 72 hours at a fermentation temperature of 34℃, a stirring speed of 200 rpm, an aeration rate of 1.1 vvm, and an automatically controlled pH (5.2 via continuous addition of 5% NaOH solution).

[0045] After centrifuging the fermentation broth at 8000 rpm, the supernatant was concentrated using a 10 kDa ultrafiltration membrane at 4℃ to obtain crude enzyme solution.

[0046] After dialyzing the crude enzyme solution against 20 mM, pH 5.0 acetate-sodium acetate buffer, it was loaded onto a DEAE Sepharose Fast Flow column (5% column volume loading). The column was washed with 5 column volumes of equilibration buffer (20 mM, pH 5.0 acetate-sodium acetate buffer), and the flow-through was collected.

[0047] The flow-through solution was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, 5% mannitol was added, and the solution was freeze-dried to obtain the target enzyme powder, namely a pectinase complex with low pectin esterase activity.

[0048] The enzyme powder was tested and found to have an activity of 8120 U / g for PL, 5000 U / g for PG, and 0.009 U / mg protein for PE.

[0049] Example 3 Preparation of pectinase complex with low pectin esterase activity.

[0050] Aspergillus niger CICC 40273 was activated on a PDA slant at 29℃ for 74 hours until the spore layer was full. A spore suspension (concentration 1×10⁻⁶) was then prepared with sterile water. 8 (CFU / mL).

[0051] Prepare seed culture medium: glucose 30 g / L, soybean meal 15 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 2 g / L and magnesium sulfate 0.5 g / L, natural pH, inoculate with 5% spore suspension, and culture at 30℃ and 180 rpm for 24 h with shaking.

[0052] Fermentation medium was prepared as follows: 40 g / L orange peel powder (60 mesh), 20 g / L wheat bran, 10 g / L ammonium sulfate, 5 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, Tween-80 0.1%, and natural pH. The seed culture was inoculated into the fermenter at an 8% inoculum rate. Fermentation was carried out for 72 hours at a fermentation temperature of 33℃, a stirring speed of 200 rpm, an aeration rate of 1.1 vvm, and an automatically controlled pH (by adding 5% NaOH solution) of 4.9.

[0053] After centrifuging the fermentation broth at 8000 rpm, the supernatant was concentrated using a 10 kDa ultrafiltration membrane at 4℃ to obtain crude enzyme solution.

[0054] After dialyzing the crude enzyme solution against 20 mM, pH 5.0 acetate-sodium acetate buffer, it was loaded onto a DEAE Sepharose Fast Flow column (5% column volume loading). The column was washed with 5 column volumes of equilibration buffer (20 mM, pH 5.0 acetate-sodium acetate buffer), and the flow-through was collected.

[0055] The flow-through solution was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, 5% trehalose was added, and the solution was freeze-dried to obtain the target enzyme powder, namely a pectinase complex with low pectin esterase activity.

[0056] The enzyme powder was tested and found to have an activity of 7900 U / g for PL, 5100 U / g for PG, and 0.0075 U / mg protein for PE.

[0057] Comparative Example 1 The other preparation steps and conditions are the same as in Example 1, except that the pH value was not automatically controlled during the fermentation process and was allowed to decrease naturally (eventually dropping to around 3.2).

[0058] Tests showed that the enzyme powder obtained after ion exchange had an activity of 3500 U / g for PL and 2200 U / g for PG.

[0059] Following the method of Example 4, orange wine was brewed using the pectinase complex prepared in Comparative Example 1, resulting in a juice yield of 71.2%, a viscosity reduction rate of 48.5%, and a methanol content of 145 mg / L. This indicates that maintaining the pH in a slightly acidic range of 4.8–5.2 is crucial for maintaining high PL and PG activity expression.

[0060] Comparative experiments revealed that when the pH of liquid fermentation was allowed to decrease without control, the metabolic pathways for the synthesis of PL and PG by *Aspergillus niger* were inhibited. Despite subsequent ion-exchange chromatography, the low initial levels of the main functional enzymes (PL / PG) in the original crude enzyme solution significantly degraded the catalytic efficiency (juice extraction, viscosity reduction) of the final enzyme powder. During winemaking, this "insufficient enzyme activity" resulted in incomplete pectin degradation, preventing the rapid remodeling of the substrate and leading to methanol control failure and low juice yield. The results indicate that setting the pH value (4.8–5.2) is a key technical parameter for maintaining high PL and PG activity and ensuring the quality of fruit wine.

[0061] Comparative Example 2 The other preparation steps and conditions are the same as in Example 1, except that the ventilation rate is adjusted to 0.3 vvm (low dissolved oxygen).

[0062] Tests showed that the cell growth in the fermentation broth was reduced, with the enzyme powder's PL activity at only 3400 U / g and PG activity at 2050 U / g. The fermentation time was extended to 96 hours.

[0063] The results showed that too low aeration rate can also lead to insufficient enzyme activity. An aeration rate of 0.8~1.2 vvm is a necessary condition to ensure aerobic fermentation and efficient enzyme production of Aspergillus niger.

[0064] Comparative Example 3 The other preparation steps and conditions are the same as in Example 1, except that the fermentation temperature is set to 28°C (lower than the range of this invention), and the other conditions are the same as in Example 1.

[0065] Tests showed that the fermentation cycle was extended to 96 hours, with PL activity at 5600 U / g and PG activity at 3800 U / g.

[0066] Orange wine was brewed according to the method in Example 4, with a juice yield of 74.5% and a methanol content of 132 mg / L. The results indicate that 31–34°C is the optimal temperature range for enzyme production during liquid fermentation.

[0067] Example 4 1. Raw material processing: Take 10 kg of fresh, unrotten "Wogan" oranges, rinse off surface dirt with clean water, and then remove the outer peel (orange peel) manually or mechanically. Supplement this by manually peeling the white pith (white layer and network of fibers), ensuring a removal rate of over 15%. Specifically, the total weight of peel and pith removed should account for over 92% of the total weight of the peel and pith. Place the resulting pure pulp in a crusher and crush it into a uniform pulp for later use.

[0068] 2. Staged targeted enzymatic hydrolysis: Phase 1: Adjust the pH of the orange pulp to 3.8 and the temperature to 38°C. Add 40 mg / kg of the pectinase complex with low pectin esterase activity prepared in Example 1, and stir slowly for 1.5 h.

[0069] Second stage: Heat to 50℃, add 15 mg / kg of acidic protease (enzyme activity 50,000 U / g), and react for 50 min.

[0070] 3. Fermentation After enzymatic hydrolysis, the juice was pressed and adjusted to a sugar content of 20°Brix and a pH of 3.4. Activated commercial Angel Yeast (RV171) was inoculated at a rate of 0.25 g / L. The fermentation temperature program was as follows: fermentation at 17℃ for 24 hours, then the temperature was increased to 21℃ for continued fermentation. When the residual sugar was less than 5 g / L, the temperature was lowered to 13℃ for 12 days, at which point fermentation was complete.

[0071] Example 5 The raw material processing is the same as in Example 4.

[0072] First stage: Adjust the pH to 3.5, set the temperature to 40°C, add 50 mg / kg of the pectin lyase complex prepared in Example 1, and react for 1 hour.

[0073] Second stage: Heat to 47℃, add 20 mg / kg of acidic protease, and react for 40 min.

[0074] The fermentation conditions were the same as in Example 4.

[0075] Example 6 The raw material processing is the same as in Example 4.

[0076] First stage: Adjust the pH to 4.0, set the temperature to 35°C, add 30 mg / kg of the pectin lyase complex prepared in Example 1, and react for 2 hours.

[0077] Second stage: Heat to 52℃, add 10 mg / kg of acidic protease, and react for 60 min.

[0078] The fermentation conditions were the same as in Example 4.

[0079] Example 7 The raw material processing is the same as in Example 4.

[0080] First stage: Adjust the pH to 3.7, set the temperature to 37°C, add 40 mg / kg of the pectin lyase complex prepared in Example 1, and react for 1.2 h.

[0081] Second stage: Heat to 49℃, add 15 mg / kg of acidic protease, and react for 45 min.

[0082] The fermentation conditions were the same as in Example 4.

[0083] Comparative Example 4 The difference from Example 4 lies in the enzymatic hydrolysis process: only an equal amount (45 mg / kg) of commercially available common pectinase (Pectinex® Ultra SP-L, PE activity approximately 2.5 U / mg) was used for enzymatic hydrolysis at 38°C for 1.5 h, without the second-stage protease treatment. The fermentation conditions were exactly the same as in Example 4.

[0084] Comparative Example 5 Same as Example 4, but the raw material peeling and de-coring removal rate is only 11.5%. Specifically, the total mass of peel and de-coring removed accounts for 70% of the total mass of peel and de-coring.

[0085] Comparative Example 6 Same as Example 4, but in the first stage, an equal amount of commercially available Pectinex® Ultra SP-L (high PE activity) was used to replace the self-made enzyme, and the two-stage enzymatic hydrolysis was completed.

[0086] Comparative Example 7 Same as Example 4, but without the first stage of pectinase treatment, only the second stage of protease treatment is performed.

[0087] Comparative Example 8 Same as Example 4, but first perform the second stage protease treatment (50°C, 18 mg / kg, 50 min), and after completion, lower the system temperature to 38°C, and then perform the first stage pectinase treatment (pH 3.8, self-made enzyme 45 mg / kg, 1.5 h).

[0088] Comparative Example 9 The enzymatic hydrolysis process is the same as in Example 4, but the fermentation stage is kept at a constant temperature of 20°C throughout, without using gradient temperature control.

[0089] Comparative Example 10 The only difference from Example 4 is the enzymatic hydrolysis process: Phase 1: Using an equal amount (40 mg / kg) of commercially available low-methanol pectinase (Novonesis, Pectinex® XXL, characterized by effective inhibition of PE activity), enzymatic hydrolysis was performed for 1.5 h at pH 3.8 and a temperature of 38°C.

[0090] No second-stage protease treatment is performed.

[0091] The fermentation conditions were exactly the same as in Example 4.

[0092] Effect verification and statistical analysis Key indicators of the raw wines obtained in the above embodiments and comparative examples were tested. Three parallel samples were set up for each experimental group. Data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 26.0 software, and Duncan's multiple comparison method was used to test for significant differences between groups at the p < 0.05 level. The results are shown in Table 1. Figure 2 This is a bar chart comparing the methanol and higher alcohol content in the final products of the examples and comparative examples.

[0093] Sensory evaluation method: The orange fruit wines prepared in the examples and comparative examples were evaluated according to the relevant provisions of QB / T 8218-2026 Guidelines for Sensory Evaluation of Wines and Fruit Wines.

[0094] An evaluation team of 10 professionally trained sensory evaluators (half male and half female, aged 25-45) was formed. The evaluation was conducted in a qualified sensory analysis laboratory (temperature 20-22℃, free from odor interference). Samples were randomly numbered and presented in a blind sample format.

[0095] The evaluation indicators include four dimensions: appearance (20%), aroma (30%), taste (40%), and style (10%). A 100-point scale is used for scoring, with each indicator assigned a weighted score (appearance 0-20 points, aroma 0-30 points, taste 0-40 points, style 0-10 points), for a total score of 100 points. The final results are converted to a 10-point scale (total score ÷ 10) and presented in Table 1.

[0096] During the scoring process, evaluators work independently without interfering with each other. Each sample is evaluated twice, and the highest and lowest scores are removed before the average is taken. The results are expressed as "mean ± standard deviation".

[0097] Table 1. Analysis of Key Indicators for Orange Wine

[0098] Table 2. Analysis and statistical comparison of orange juice yield and viscosity-reducing effect.

[0099] The orange wines prepared in Examples 4 to 7 of this invention have consistently low methanol content (90-98 mg / L) and total higher alcohols (250-268 mg / L), and high sensory scores (≥8.3). This fully demonstrates that the method described in this invention can achieve excellent and stable results.

[0100] The comparative results demonstrate the necessity and synergy of each step in the technical solution of this invention: 1) Both stages of enzymatic hydrolysis are indispensable: Comparative Example 4 (using only ordinary pectinase) and Comparative Example 7 (using only protease) demonstrate that a single treatment cannot achieve a synergistic reduction in methanol and higher alcohols. Although the higher alcohol content in Comparative Example 7 was low, the methanol content was extremely high.

[0101] 2) The core role of low PE activity enzymes: Comparative Example 6 (using high PE commercial enzymes) had a higher methanol content than the Example Group, confirming that using pectinase with inhibited PE activity is the key to controlling methanol.

[0102] 3) Importance of processing order: All indicators of Comparative Example 8 (inverted order) were lower than those of Example 1, proving that the fixed order of pectinase first and then protease is crucial for simultaneous optimization of substrate.

[0103] 4) Threshold for pretreatment removal rate: The methanol and higher alcohol content in Comparative Example 5 (removal rate 11.5%) was higher than that in Example 4, confirming that a removal rate of more than 15% is a necessary condition for achieving source reduction.

[0104] 5) Overall optimization of the process: The content of higher alcohols in Comparative Example 9 (constant temperature fermentation) was higher than that in Example 1, indicating that the gradient temperature control process and the optimized matrix have a synergistic effect, which can further stabilize the quality.

[0105] 6) Comparative Example 10 was treated with a commercially available low-methanol pectinase (Pectinex® XXL) using a single enzyme. Although the methanol content of the final product was lower than that of the traditional high-PE pectinase (Comparative Example 4), it was still higher than that of the group in the present invention. More importantly, at the same addition amount, its juice yield and viscosity reduction rate of citrus pulp were lower than those of the pectinase complex with low pectin esterase activity of the present invention. This confirms that the pectinase complex with low pectin esterase activity prepared by the present invention achieves extremely low PE activity while overcoming the defect of insufficient enzymatic hydrolysis efficiency that may exist in similar commercial products.

[0106] In summary, this invention, through a specific sequence of processes—"high removal rate pretreatment - low PE pectin hydrolysis - protease-optimized nitrogen source"—coupled with a suitable fermentation process, produces a significant synergistic effect, systematically solving the technical challenge of high methanol and higher alcohol content in orange wine. The comparative examples, from the opposite perspective, demonstrate that each technical feature is indispensable for achieving this superior effect.

[0107] Figure 3 The image shows the orange wine prepared in Example 4. Figure 4 The image shows the orange wine prepared in Comparative Example 10. Comparative observation reveals: Regarding clarity: The fruit wine prepared in Example 4 ( Figure 3 The wine was clear and bright, with no obvious suspended matter or sediment, exhibiting the typical clear appearance of fruit wine; while the fruit wine prepared in Comparative Example 10 ( Figure 4 The wine appears slightly cloudy with low transparency, indicating that its clarification effect is inferior to that of Example 4.

[0108] In terms of color: the fruit wine of Example 4 is a uniform light yellow with good luster, and has the color characteristics that orange fruit wine should have; the fruit wine of Comparative Example 10 is darker in color, lacks luster, and its appearance quality is significantly inferior.

[0109] Overall appearance quality: The fruit wine of Example 4 meets the sensory requirements of high-quality fruit wine in terms of appearance, while the fruit wine of Comparative Example 10 has obvious sensory defects.

[0110] The above comparative results show that, through the synergistic effect of a specific staged enzymatic hydrolysis process (first low-PE pectin hydrolysis, then acidic proteolysis) and gradient temperature-controlled fermentation, this invention not only significantly reduces the content of methanol and higher alcohols (as shown in Table 1, Example 4: methanol 92 mg / L, higher alcohols 255 mg / L; Comparative Example 10: methanol 135 mg / L, higher alcohols 390 mg / L), but also significantly improves the clarity and color of the fruit wine, achieving a dual optimization of safety and sensory quality.

[0111] Comparative Example 11 Raw material processing: Same as in Example 4.

[0112] Simultaneous enzymatic fermentation: The pH of the fruit pulp was adjusted to 3.8, and the temperature was controlled at 20℃. At the same time, 40 mg / kg of pectinase complex with low pectin esterase activity prepared in Example 1, 15 mg / kg of acidic protease (such as NovoCor® ABG), and activated Angel yeast RV171 (inoculation amount 0.25 g / L) were added.

[0113] The above materials are mixed evenly at once, without staged enzymatic hydrolysis, and directly enter the fermentation stage.

[0114] Fermentation conditions: Same as the gradient temperature-controlled fermentation program in Example 4 (17℃ for 24h → 21℃ → after residual sugar <5 g / L, the temperature was lowered to 13℃ and fermentation continued for 12 days).

[0115] Indicators: methanol content 160 ±10 mg / L, higher alcohol content 380 ±20 mg / L, juice yield 71% ±1%, viscosity reduction rate 49% ±1.5%. The results show that the staged enzymatic hydrolysis followed by gradient temperature-controlled fermentation in this invention can ensure the quality of the fruit wine.

[0116] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A pectinase complex with low pectin esterase activity, characterized in that, By Aspergillus niger ( Aspergillus niger The pectinase complex with low pectin esterase activity was prepared by fermentation using CICC 40273. The pectin esterase activity was less than 0.05 U / mg, the pectin lyase activity was ≥7000 U / g, and the polygalacturonase activity was ≥4500 U / g.

2. The method for preparing the pectinase complex according to claim 1, characterized in that, Includes the following steps: Liquid fermentation of Aspergillus niger CICC 40273 was carried out, the fermentation broth was collected, and crude enzyme solution was extracted. The crude enzyme solution was subjected to ion exchange chromatography to remove the pectin esterase component, and the flow-through was collected. The flow-through solution was concentrated and dried to obtain pectinase with low pectin esterase activity.

3. The preparation method according to claim 2, characterized in that, The pH value of the liquid fermentation is 4.8~5.2; the aeration rate of the liquid fermentation is 0.8~1.2 vvm; and the temperature of the liquid fermentation is 31~34℃.

4. A method for enzymatic fermentation to reduce the methanol and higher alcohol content in orange wine based on the pectinase complex of claim 1 or the pectinase complex prepared by the preparation method of claim 2 or 3, characterized in that, Includes the following steps: Remove the peel and pith of the citrus fruit, with a removal rate of ≥15%, then crush the fruit to obtain citrus pulp; The citrus pulp and the pectinase complex according to claim 1 or the pectinase complex prepared by the preparation method according to claim 2 or 3 are mixed and subjected to the first enzymatic hydrolysis to obtain the first enzymatic hydrolysate. The first hydrolysate is mixed with acidic protease and subjected to a second hydrolysis to obtain the second hydrolysate. The second enzymatic hydrolysate is mixed with brewer's yeast and fermented to obtain orange wine.

5. The enzymatic fermentation method according to claim 4, characterized in that, The temperature of the first enzymatic hydrolysis is 35~40℃; the pH value of the first enzymatic hydrolysis is 3.5~4.0; the time of the first enzymatic hydrolysis is 1~2h; and the amount of pectinase complex with low pectin esterase activity added to the citrus pulp in the first enzymatic hydrolysis is 30~50 mg / kg.

6. The enzymatic fermentation method according to claim 4, characterized in that, The temperature of the second enzymatic hydrolysis is 47~52℃; the time of the second enzymatic hydrolysis is 40~60min; and the amount of acidic protease added to the citrus pulp in the second enzymatic hydrolysis is 10~20 mg / kg.

7. The enzymatic fermentation method according to claim 4, characterized in that, The fermentation is a gradient temperature-controlled fermentation; the conditions for the gradient temperature-controlled fermentation are as follows: the first fermentation is carried out at 16~18℃ for 12~36h, the temperature is raised to 20~25℃ for the second fermentation, and when the residual sugar content is lower than 5 g / L, the temperature is lowered to 12~14℃ for the third fermentation for 7~15d.

8. The enzymatic fermentation method according to claim 4, characterized in that, The brewing yeast includes Angel Yeast RV171.

9. The application of the enzymatic fermentation method according to any one of claims 4 to 8 in the preparation of orange wine with low methanol and higher alcohol content; wherein the methanol content in the orange wine is ≤100 mg / L and the total amount of higher alcohols is ≤300 mg / L.

10. Orange wine prepared by the enzymatic fermentation method according to any one of claims 4 to 8.

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