Article pubs.acs.org/JAFC
Enzymes Extracted from Apple Peels Have Activity in Reducing Higher Alcohols in Chinese Liquors Qi’an Han,† Junling Shi,*,§ Jing Zhu,† Hongliang Lv,† and Shuangkui Du† †
College of Food Science and Engineering, Northwest A&F University, 28 Xinong Road, Yangling, Shaanxi Province 712100, China Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an, Shaanxi Province 710072, China
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ABSTRACT: As the unavoidable byproducts of alcoholic fermentation, higher alcohols are unhealthy compounds widespread in alcoholic drinks. To investigate the activity of apple crude enzymes toward higher alcohols in liquors, five kinds of apple peels, namely, Fuji, Gala, Golden Delicious, Red Star, and Jonagold, were chosen to prepare enzymes, and three kinds of Chinese liquors, namely, Xifeng (containing 45% ethanol), Taibai (containing 50% ethanol), and Erguotou (containing 56% ethanol), were tested. Enzymes were prepared in the forms of liquid solution, powder, and immobilized enzymes using sodium alginate (SA) and chitosan. The treatment was carried out at 37 °C for 1 h. The relative amounts of different alcohols (including ethanol, 1-propanol, isobutanol, 1-butanol, isoamylol, and 1-hexanol) were measured using gas chromatography (GC). Conditions for preparing SA-immobilized Fuji enzymes (SA-IEP) were optimized, and the obtained SA-IEP (containing 0.3 g of enzyme) was continuously used to treat Xifeng liquor eight times, 20 mL per time. Significant degradation rates (DRs) of higher alcohols were observed at different degrees, and it also showed enzyme specificity according to the apple varieties and enzyme preparations. After five repeated treatments, the DRs of the optimized Fuji SA-IEP remained 70% for 1-hexanol and >15% for other higher alcohols. KEYWORDS: enzyme, higher alcohols, degradation, apple, Chinese liquor
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INTRODUCTION As a profound constituent of the diet, alcoholic drinks are pervasive in daily and social life. Chinese liquor is very popular around the world due to its rich and special flavor and taste. Among the aroma-producing and flavoring-producing substances of liquor, higher alcohols are the most prominent with a quantitatively large group, containing chiefly isoamylol, isobutanol (20%), 1-propanol (3−5%), and small amounts of other alcohols, esters, and aldehydes.1 The materials, microorganisms, and solid-state fermentation features determine a high alcohol content (38−65%) in Chinese liquors.2 Normally, the content of higher alcohols in Chinese liquors is at high level (244.47−1699.30 g/hL), especially in the rice-made liquor.3,4 Higher alcohols are unavoidable byproducts of alcoholic fermentation.5 It has been reported that higher alcohols can cause damage to the nervous system and have acute or chronic toxicity as well as liver toxicity. Furthermore, the toxicity of higher alcohols increases with molecular weight, boiling point, and chain length.6 They have destructive influence on liquor quality at high concentration.7 Too much higher alcohols (>400 mg/L) provokes an unpleasant flavor and a harsh taste in alcoholic drinks.8,9 Many efforts have been made to reduce higher alcohols in alcoholic drinks, such as using low higher-alcohol-producing yeast strains.10,11 Conventional methods and modern molecular biotechniques are used to breed low higher-alcohol-producing yeast strains.12−15 Nevertheless, the obtained yeast strains have a low ethanol production and thus inhibit the alcoholic fermentation. It also can be made by adjustments of fermentation conditions and materials. In the brewing process, the formation of higher alcohols is generally regulated by © 2014 American Chemical Society
controlling the fermentation temperature, sugar, oxygen and SO2 supply, and pH.16−19 Liquor-making raw materials have a decisive effect on the final formation of higher alcohol because of nitrogen concentration.20 Rapp and Versini21 have found that controlling α-amino nitrogen content can reduce higher alcohols, which in turn improves the liquor flavor. A low level of nitrogen content in materials normally results in a high content of higher alcohols in the end products, whereas appropriate and high concentrations of α-amino nitrogen inhibit the formation of higher alcohols.22−24 Adding some vitamins (thiamin, pantothenic acid) and micronutrients (zinc, boron, iron, and manganese) can control the production of higher alcohols owing to suppression of the metabolism process during alcoholic fermentation.25 However, it is difficult and impossible to manually control the production of higher alcohols during Chinese liquor making because the process involves multiple materials and many unidentified microorganisms, including bacteria, molds, and yeasts. Therefore, measures after alcoholic fermentation are also important to reduce higher alcohols in Chinese liquors. A nanomembrane filter is currently reported as an efficient implement to remove higher alcohols from liquors with the degradation rate above 44%. However, it is poor in specificity of alcohol varieties.26 In previous studies, we found some hexanol-degrading enzymes extracted from apple possessed high activity for the degradation of hexanol in acid condition. Hence, crude Received: Revised: Accepted: Published: 9529
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For all enzymes prepared from the five different apple varieties, samples of 0.125 g of EP, 2.5 mL of ES, SA-IEP prepared from 0.125 g of EP, SA-IES prepared from 2.5 mL of ES and, CTS-IES prepared from 2.5 mL of ES were added into 20 mL of Taibai liquor (50% ethanol by volume). The treatment was carried out at 37 °C for 1 h according to the method reported by Zhu et al.32 The corresponding inactive enzyme preparations were used at the same time as control. All treatments were carried out in triplicate. Optimization of the Conditions for Preparing Immobilized Fuji Enzyme Powder Using SA. The single-factor design was used to investigate the influence of enzyme dosage, sodium alginate concentration, calcium chloride concentration, and immobilizing time on higher alcohol degradation. In enzyme dosage effect tests, 10 mL of a 40 g/L sodium alginate solution was mixed with Fuji EP at different dosages of 0.1, 0.3, 0.5, and 0.8 g, dropped into 10 mL of a 20 g/L CaCl2 solution, and kept at 4 °C for 2 h to get the immobilized enzymes. In sodium alginate concentration effect tests, sodium alginate solutions with different concentrations (25, 30, 35, 40, and 45 g/L) were separately mixed with 0.3 g of Fuji EP, dropped into 10 mL of a 20 g/L CaCl2 solution, and kept at 4 °C for 2 h to get the immobilized enzymes. In CaCl2 solution concentration effect tests, 40 g/L sodium alginate solution was mixed with 0.3 g of Fuji EP and then dropped into 10 mL of a CaCl2 solution with different concentrations (15, 20, 25, 30, and 35 g/L) and kept at 4 °C for 2 h to get the immobilized enzymes. In immobilizing time effect tests, 40 g/L sodium alginate solution was mixed with 0.3 g of Fuji enzyme powder, dropped into 10 mL of a 20 g/L CaCl2 solution, and then kept 4 °C for different times (1.25, 2, 2.75, 3.5, and 4.25 h) to get immobilized enzymes. All of the obtained SA-IEPs were used to treat Taibai liquor (50% ethanol by volume) as described above, and their activities in degrading higher alcohols were measured using gas chromatography (GC) analysis. Response surface methodology was also used to test the comprehensive effect of different factors on the higher alcohol degradation by SA-IEP, and Box−Behnken central composite design was used to design the experiments. Enzyme dosage, sodium alginate concentration, and CaCl2 concentration were selected as independent variables, and their levels and codes are listed in Table 1. Immobilized
enzymes extracted from apple are suspected to have activity in reducing higher alcohols of alcoholic drinks in acid condition. In this study, we prepared crude enzymes from five different apple varieties and optimized the conditions of Fuji sodium alginate-immobilized enzyme powder (SA-IEP), then applied this new product to treat three Chinese liquors in which ethanol content was from 45 to 56% (v/v). The results are hoped to demonstrate an enzymatic method to reduce higher alcohols in high ethanol content liquors.
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MATERIALS AND METHODS
Apples and Chemicals. Fresh apple fruits of five different cultivars (Malus domestica Borkh. cv. Fuji, Gala, Golden Delicious, Red Star, and Jonagold) were bought from the local market of Yangling city, Shaanxi province, China. Three liquors with different ethanol contents (45% Xifeng, 50% Taibai, and 56% Erguotou) were bought from the local supermarket and used to test the effect of apple enzymes on reducing higher alcohols in these products. Acetone, 50% glutaraldehyde, citric acid, sodium citrate, and anhydrous calcium chloride were purchased from Bodi Chemical Ltd. (Tianjin, China). Ammonium sulfate was bought from Zonghengxing Industrial and Trading Co., Ltd., Chemical Reagent Branch (Tianjin, China). MD25 dialysis tubing was purchased from Sigma (St. Louis, MO, USA). Chitosan was obtained from Wolsen Biotechnology Co., Ltd. (Xi’an, China). Sodium alginate of food grade was obtained from Ming Yue Seaweed Group Co., Ltd. (Qingdao, China). All of these chemicals were of analytical grade. Preparation of Crude Enzymes. Crude enzymes were prepared using a previously developed method described by Zhu et al.27 In brief, apple peels (about 4 mm thick) were prepared as dry, fluffy, and white powders containing crude enzymes after removal of pigments and other ester-soluble impurities with cold acetone (−30 °C). A sample of 1 g of the prepared crude enzyme powder was extracted into 25 mL of citrate buffer (0.1 mol/L, pH 4.0) after extraction for 30 min at 4 °C with continuously gentle agitation. The liquid phase containing crude enzymes was obtained after a centrifugation at 4000 rpm and 4 °C for 30 min in an ALC PK121R refrigerated benchtop centrifuge (ALC International S.r.l., Cologno Honzese, Italy). The obtained supernatant was used as crude enzyme solution. Protein content in the prepared enzyme powder (EP) and enzyme solution (ES) was estimated using the method developed by Bradford.28 Preparation of Immobilized Enzymes. The prepared EP and ES were immobilized using the methods reported by Yen and Wang.29,30 In brief, sodium alginate solution was prepared by dissolving 0.4 g of sodium alginate in 10 mL of distilled water, and then 0.125 g of EP or 2.5 mL of ES was added, in which protein contents were equal. After thorough mixing, the mixture was dropped into 10 mL of 20 g/kg CaCl2 solution at a speed of 20 drops per minute using an injector to form beads containing enzymes. The enzyme beads were kept at 4 °C for 2 h to enhance the mechanical stability and then washed three times with distilled water, 20 mL per time. The obtained immobilized EP and ES were designated SA-IEP and SA-IES, respectively. Chitosan was also used to prepare immobilized enzymes according to the method described by Jin et al.31 Briefly, 0.565 g of chitosan polymer was dissolved in 3.75 mL of a 500 mL/L glutaraldehyde solution and then kept statically for 3 h to activate chitosan. After that, the treated chitosan powder was collected and thoroughly rinsed with sterilized water 10 times to remove the surplus glutaraldehyde and then mixed with 2.5 mL of ES for 1 h to get immobilized enzymes. The obtained powder was chitosan-immobilized enzyme solution and designated CTS-IES in the following text. For the treatments as control, EP and SA-IEP were replaced with equivalent inactive EP and SA-immobilized inactive EP, respectively. ES, SA-IES, and CTS-IES were replaced with equivalent citrate buffer (0.1 mol/L, pH 4.0), SA, and chitosan-immobilized citrate buffer (0.1 mol/L, pH 4.0), respectively. Inactive EP was obtained by heating EP in boiling water for 1 h. Effect of Enzymes on Higher Alcohol Content in Taibai Liquor. All prepared enzymes were preheated at 37 °C for 30 min.
Table 1. Factors and Their Levels of the Parameters Used in Box−Behnken Design factors level
sodium alginate A (g/L)
calcium chloride B (g/L)
enzyme powder C (g)
−1 0 1
35 40 45
15 20 25
0.2 0.3 0.4
time (2 h) and temperature (4 °C) were set as the fixed conditions. Design expert 8.0.5 software (Stat-Ease, Inc., Minneapolis, MN, USA) was used to perform the experimental design and data analysis. Effect of the Optimized SA-IEP Made from Fuji on Higher Alcohols of Different Liquors. Xifeng (45% ethanol v/v), Taibai (50% ethanol v/v), and Erguotou (56% ethanol v/v) were used here. To evaluate the effect of the optimized SA-IEP made from Fuji on liquor flavor, 20 mL of all liquors was sampled after treatment with SAIEP for 1 h and gas chromatography−mass spectrometry analysis (GCMS). The liquors without any treatment were used as control. All treatments were carried out in triplicate. Activity of the Optimized SA-IEP Made from Fuji in Continuous Treatments. The optimized SA-IEP was continuously used to treat Taibai liquor (containing 50% ethanol) eight times, 20 mL per time. For each time, the treatment was carried out at 37 °C for 1 h using preheated SA-IEP containing 0.3 g of enzyme powder. After each treatment, SA-IEP was collected by filtration, and 20 mL of new liquor was added again. This operation was repeated until a total of 9530
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Figure 1. Degradation rates of different alcohols in Taibai liquor (50% ethanol v/v) using crude enzyme preparations made from Fuji (A), Golden Delicious (B), Jonagold (C), Red Star (D), and Gala (E). SA-IEP, sodium alginate-immobilized enzyme powder; SA-IES, sodium alginateimmobilized enzyme solution; CTS-IES, chitosan-immobilized enzyme solution; EP, enzyme powder; ES, enzyme solution. The bars show standard deviation of triplicate. Different letters in the same group indicate significant difference at the P < 0.05 level, according to ANOVA and Tukey’s difference test. 9531
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Figure 2. Degradation rates of different alcohols in Taibai liquor (50% ethanol v/v) using Fuji SA-IEP prepared under different levels of enzyme powder (A), sodium alginate concentration (B), calcium chloride concentration (C), and immobilization time (D). Bars show standard deviation of triplicate. 160 mL of liquor was treated. Measurement of the residue higher alcohols in the liquor was carried out by GC after each treatment. Measurement of Higher Alcohol Degradation Rate. After each treatment, 3 mL of treated liquor was injected into an 8 mL GC vial together with 60 μL of internal standard (5% 4-methyl-2pentanol/acetone g/v) and sealed immediately for measurement. The measurement was carried out in a gas chromatograph (GC-17A/FID, Shimadzu, Japan) combined with a SupelcoWax 10 capillary column (30 m × 0.32 mm i.d. × 0.25 μm film thickness; Bellefonte, PA, USA). Nitrogen was used as carrier gas with a linear flow rate of 30 mL/min. The column temperature was programmed as follows: 55 °C for 3 min, increasing to 200 °C at a rate of 5 °C/min, held at 200 °C for 3 min. Before determination, all samples were kept at 60 °C for 30 min in a water bath and subsequently injected to the GC system at a sample volume of 300 μL. Volatile profiles of the liquors with and without treatment of Fuji SA-IEP were also tested using headspace solid phase microextraction coupled to gas chromatography spectrometry (HS-SPME-GC-MS) in a gas chromatograph−mass spectrometer equipped with a capillary column DB-WAX (3 m × 0.25 mm i.d. × 0.25 μm film thickness; Agilent, USA). Before measurement, the samples were kept at 60 °C for 30 min to release the volatiles from the liquid phase to the headspace. In measurements, 8 mL of the liquor sample was mixed together with 2.5 g of NaCl and 40 μL of an 8.00 mg/L 2-octanol solution (used as internal standard) and then was put into a 20 mL
SPME vial. The SPME procedure and chromatographic conditions were the same as those detailed by Rodrı ́guez Bencomo et al.33 The flow rate of carrier gas (He) was 1 mL/min. The oven temperature program was set from 40 °C (2.5 min) to 200 °C (5 min) at an increasing rate of 5 °C/min. Data acquisition was made in different segments with electronic impact mode. The transfer line was kept at 230 °C and fed into an electron ionization (EI) source with an emission current of 100 μA. The masses in the range of m/z 34−350 were tested. The volatile compounds were identified by comparing their mass spectra and retention indices with standards and from NIST 2002. Measurement of 3 mL of untreated liquors was also carried out as control. Each assay was performed in duplicate. The enzyme degradation rate (DR) of each higher alcohol by per milligram of crude enzyme was calculated according to eqs 1, 2, and 3:
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⎛ T × S0 ⎞ DR of treated sample (%) = ⎜1 − 1 ⎟ × 100 T0 × S1 ⎠ ⎝
(1)
⎛ C × S0 ⎞ DR of control (%) = ⎜1 − 1 ⎟ × 100 C0 × S1 ⎠ ⎝
(2)
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Table 2. Runs and Results of the Box−Behnken Design degradation rate (%) runa
sodium alginate (g/L)
calcium chloride (g/L)
enzyme powder (g)
ethanol
1-propanol
isobutanol
1-butanol
isoamylol
1-hexanol
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1 0 −1 1 0 0 −1 1 1 −1 0 −1 0 0 0
−1 0 1 1 1 0 0 0 0 −1 0 0 −1 −1 1
0 0 0 0 1 0 −1 1 −1 0 0 1 1 −1 −1
75.48 82.74 77.64 81.68 74.33 84.51 79.81 74.14 77.94 80.58 81.98 74.13 66.78 77.33 76.04
66.94 74.67 74.28 71.64 65.69 78.69 70.48 62.33 70.47 70.66 71.57 67.61 47.45 67.51 66.34
67.68 73.26 70.21 72.63 64.68 72.28 71.64 62.75 70.21 71.82 74.24 63.49 58.19 69.74 68.88
−6.11 19.24 0.62 10.41 −5.25 18.93 1.98 −13.67 2.62 5.79 19.53 −9.04 −16.3 0.42 0.46
28.16 54.87 36.40 42.99 26.56 51.46 41.26 24.17 39.67 41.72 58.28 25.29 4.07 35.52 30.68
74.98 88.53 80.38 87.49 74.09 89.80 82.82 59.39 81.37 83.33 87.27 70.83 49.53 78.67 76.27
a
Fifteen runs were carried out, including 12 factorial experiments and 3 central experiments to estimate error.
most alcohols. SA-IEP prepared from Golden Delicious had DR values >80% for 1-hexanol and DR values 50% for 1-hexanol, around 35% for isobutanol, but only 15% for ethanol (Figure 1C). SA-IEP made from Red Star had DR values about 28% for ethanol, 48% for 1-hexanol, and 15−28% for other higher alcohols (Figure 1D). SA-IEP made from Gala had DR values 33% for ethanol, 35% for 1-propanol, and 80% for ethanol and 1-hexanol, >70% for 1-propanol and isobutanol, around 52% for isoamylol, and only 20% for 1butanol, indicating high efficiency of Fuji SA-IEP in degrading 9533
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1800.43 4.65 62.94 309.26 117.61 6.94 30.31 233.83 272.95 922.09 3.61 3.43 0.18 1804.04
source
model A, sodium alginate B, calcium chloride C, enzyme powder AB AC BC A2 B2 C2 residual lack of fit pure error cor totel
a
df
sum of squares
0.7395
0.56
200.05 4.65 62.94 309.26 117.61 6.94 30.31 233.83 272.95 922.09 0.72 1.14 0.09
mean square
