- Email: [email protected]
Influence of pectinase treatment on fruit spirits from apple mash, juice and pomace
Process Biochemistry 46 (2011) 1909–1913
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Influence of pectinase treatment on fruit spirits from apple mash, juice and pomace Hui Zhang a , Edward E. Woodams b , Yong D. Hang b,∗ a b
Shenyang University of Chemical Technology, Department of Environmental & Bioengineering, Shenyang 110142, China Cornell University, Department of Food Science, 630 W. North Street, Geneva, NY 14456, USA
a r t i c l e
i n f o
Article history: Received 26 January 2011 Received in revised form 21 June 2011 Accepted 22 June 2011 Keywords: Apple Pectinase Spirits Alcohol Methanol
a b s t r a c t The current investigation was conducted to determine the influence of pectinase treatment on fruit spirits produced from apple mash, juice, and pomace. Crispin apples were processed into apple mash, juice, and pomace in our pilot-plant, and fermented with a commercial Red Star wine yeast (Sachharomyces cerevisiae Davis 904). After fermentation, the samples of fermented apple mash, juice, and pomace were distilled, and the distillates were analyzed by HPLC with a Bio-Rad Aminex HPX 87H column and a refractive index detector. Methanol, ethanol, n-propanol, iso-butanol, and iso-amyl alcohol were identified as the major alcohols in all the apple spirits. Student’s t-test results indicate that there are significant differences between the methanol concentrations of pectinase treated and non-pectinase treated apple spirits. Duncan’s multiple range tests show significant differences in the concentrations of methanol of the fruit spirits made from apple mash, juice, and pomace. Apple pomace yielded significantly higher methanol concentrations than apple mash and juice. Pectinase treatment had little effect on the concentrations of n-propanol, iso-butanol, and iso-amyl alcohol. It is concluded that fruit spirits made from the pectinase treated mash, juice, and pomace of Crispin apples had methanol concentrations significantly above the United States FDA guidance of 0.35% by volume or 280 mg/100 mL of fruit brandy containing 40% ethanol. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Fruit spirits are widely consumed in the United States, Canada and European countries. They are high-value alcoholic beverages produced commercially by the distillation of fermented fruit mash, juice, or pomace. Methanol and higher alcohols are major volatile compounds that influence the quality and safety of fruit spirits and can exert adverse effects on human health. Methanol, for example, has been reported to be associated with harmful health effects such as headache, fatigue, nausea, visual impairment, or complete blindness [1], and its presence in distilled fruit spirits is strictly controlled. The United States legal limit on methanol in distilled fruit spirits is 0.35% (v/v), or 280 mg/100 mL of 40% alcohol [2]. The EU general methanol limit for spirits made of fruits and marc Brandy is 400 mg/100 mL of 40% alcohol, but for spirits made of plums, apples, and pears, the methanol limit is 480 mg/100 mL of 40% alcohol [3]. Methanol is the product of the enzymatic hydrol-
∗ Corresponding author. Tel.: +1 315 787 2265; fax: +1 315 787 2284. E-mail addresses: [email protected], [email protected] (Y.D. Hang). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.06.020
ysis of fruit pectin by pectin methylesterase (EC 3.3.3.11) during alcoholic fermentation as follows [4]: Pectin methylesterase
Pectin + H2 O
−→
Pectic acid + Methanol
The commercial pectinase enzyme preparation derived from Aspergillus niger is widely used in the fruit juice processing industry to improve the juice yield [5,6]. However, the enzymatic treatment employed in fruit processing can cause a significant increase in the methanol concentrations of wine and brandy. Several investigators reported the influence of pectinase enzyme treatment on the methanol concentrations of wines from California and New York State in the United States [7–9]. Wilson et al. [10] reported the effect of processing treatments on the characteristics of juices and still ciders from apples grown in Ontario, Canada. Andraous et al. [11] reported that the methanol concentrations of fruit spirits made from apple and pear mashes with commercial pectinase enzyme preparations derived from A. niger were well above the United States FDA limits. Hang and Woodams [12] found the methanol content of grappa made from the sweet pomace of five New York grape varieties to be within the United States legal limit. Da porto et al. reported the influence of different enological techniques distillation processes on the concentrations of methanol and other volatile compounds in grape distillates [13]. Hernandez et al. found that
1910
H. Zhang et al. / Process Biochemistry 46 (2011) 1909–1913
Table 1 Chemical composition of apple mash, juice and pomace. Sample
◦
Brix
pH
wine yeast (1 g/kg), all the samples were incubated at room temperature (21–23 ◦ C) for 12 days. All the fermentation experiments were conducted in triplicate. Acidity% 2.3. Distillation
No pectinase treatment Mash 11.3 Juice 11.2 Pomace 11.5 Pectinase treatment Mash 11.2 Juice 11.0 Pomace 11.3
± 0.1 ±0 ± 0.7
3.2 ± 0 3.3 ± 0 3.6 ± 0
0.48 ± 0.01 0.46 ± 0 0.42 ± 0.03
±0 ±0 ± 0.4
3.1 ± 0 3.2 ± 0 3.1 ± 0
0.60 ± 0 0.58 ± 0 0.46 ± 0.02
the concentrations of methanol and the other volatile compounds varied considerably and were dependent on the fermentation conditions and the distillation methods. [14]. Glatthar et al. [15] found that the methanol concentrations of pear spirits could be significantly reduced by lowering the pH of pear mashes and shortening the fermentation time. Hang and Woodams [16] investigated the influence of apple variety and juice pasteurization on hard cider and eau-de-vie methanol content and found Crispin apples yielded more methanol in hard cider and eau-de-vie than other three varieties, and juice pasteurization prior to alcoholic fermentation significantly reduced the methanol content. The current study was undertaken to determine if there were any significant differences in the concentrations of methanol, ethanol, and higher alcohols (C3–C5) of the fruit spirits produced from apple mash, juice and pomace with and without pectinase enzyme treatments. 2. Materials and methods 2.1. Materials The Crispin apple variety (Malus domestica) is a cross between the Golden Delicious and the Indo apple varieties first grown in Japan and generally ripes in late October in Geneva, New York. Crispin apples grown in the Finger Lakes fruit region of New York State were processed into mashes with a comminuting machine (Model D, The W.J. Fitzpatrick Company, Chicago, USA) in our fruit and vegetable processing pilot plant at Cornell University’s Food Science Department in Geneva, NY. The apple mashes were divided into two lots. One lot of the apple mashes was mixed with a commercial pectinase preparation derived from A. niger, Rapidase ADEX-D and incubated at 50 ◦ C for 2 h. Rapidase ADEX-D was added at the rate of 100 g/ton as recommended by the enzyme supplier (DSM Food Specialties USA, Inc. Charlotte, NC, USA). Approximately 65% of the enzyme treated mashes was pressed into juice (CJE) and pomace (CPE) by a Hydraulic Rack and Frame Press (screen size No. 6, 34-in.). The remaining 35% of the enzyme treated mash (CME) was kept for direct fermentation. The other lot of apple mashes (CMNE), juice (CJNE), and pomace (CPNE) samples were prepared in the same manner except that there was no pectinase enzyme preparations added (Fig. 1). 2.2. Fermentation The fermentation experiments with apple mash and juice were conducted in 2000-mL fermenters equipped with U-tube airlocks, each containing 1000 g of apple mash or apple juice. Apple pomace was fermented in 1000-mL fermenters, each containing 300 g of apple pomace. After inoculation with commercial Red Star dry active
Approximately 250 mL of fermented apple mash, juice or pomace samples were distilled utilizing a laboratory distillation apparatus under controlled conditions [16]. Prior to distillation, samples of fermented apple mash were squeezed at 500–700 psi with Loomis Press (Loomis Engineering & MFG Company, Caldwell, NJ, USA). Samples of fermented apple pomace were prepared by blending with water in a Waring blender for 1 min and centrifuging at 16,270 rpm for 25 min. The supernatant fractions were used for distillation. Samples of fermented apple juice were distilled directly. 2.4. Analytical methods Methanol, ethanol, n-propanol, iso-butanol and iso-amyl alcohol were determined by HPLC (11) with the following conditions: Column, Bio-Rad Aminex HPX 87H; Eluent, 0.005 M sulfuric acid; flow rate, 0.6 mL/min; temperature, 50 ◦ C; detection, a refractive index detector Knauer K-2300 (Sonntek Inc., Upper Saddle River, NJ). ◦ Brix was measured with a Karl Zeiss refractometer (Jena, Germany). Fig. 2 shows the separation of volatile compounds by HPLC using a Bio-Rad Aminex HPX 87H column. Standards of methanol, ethanol, n-propanol, iso-butanol and iso-amyl alcohol were run first to determine the retention time of each alcohol, and then the samples were run. The titratable acidity, expressed as % malic acid, was determined by titrating the samples with 0.02 N NaOH to 8.2 [17]. pH value was measured with a Fisher pH meter Model 230. 2.5. Statistical analysis All experimental data were expressed as mean ± SD (standard deviation). Statistical analyses were conducted with the SAS statistical computer package (SAS Institute, Cary, NY, USA). The student’s t-test was used to determine if there was a significant difference in alcoholic compositions between apple spirits made from mash, juice, and pomace with and without enzyme treatment. The levels of confidence used in the statistical analysis of the data were 95%. Duncan’s multiple range test was used to evaluate if there were significant differences among spirits made from apple mash, juice, and pomace. The values that have no common superscript are significantly different according to Duncan’s multiple range test.
3. Results and discussion Crispin apples grown in the Finger Lakes region of New York State were found to have an average ◦ Brix value of 11.2, an average pH of 3.3, and an average titratable acidity of 0.42% expressed as malic acid (Table 1.). Student’s t-tests were used to determine if the pectinase enzyme treatment caused statistically significant differences in the concentrations of methanol and other alcoholic constituents of fruit spirits made from apple mash, juice and pomace. As shown in Table 2, the methanol concentrations of fruit spirits made from the nonpectinase treated apple mash and the pectinase treated apple mash are 87.7 mg and 533.2 mg/100 mL of 40% ethanol, respectively, The Student’s t-test results indicate that the difference in the methanol concentration was statistically significant (t = 55.29, p0.05 = 4.303), but there were no significant differences in the concentrations of n-propanol (t = −0.53), iso-butanol (t = 2.55), or iso-amyl alcohol
Table 2 Effect of pectinase treatment on the alcoholic constituents of apple spirits made from mash. Substrate
Pectinase treatment
Methanol in 40% ethanol (mg/100 mL)
n-Propanol in 40% ethanol (mg/100 mL)
Iso-Butanol in 40% ethanol (mg/100 mL)
Iso-Amyl alcohol in 40% ethanol (mg/100 mL)
Mash1 Mash 2 Mash 3
No No No Mean ± SD Yes Yes Yes Mean ± SD
91.3 92.3 79.4 87.7 ± 8.2B 526.8 532.0 540.9 533.2 ± 8.5A 55.29
19.1 15.6 13.9 16.2 ± 3.3A 16.6 15.1 15.1 156 ± 10A −0.53
58.7 52.2 51.5 54.1 ± 6.0A 60.5 65.5 62.9 63.0 ± 2.3A 2.55
179.3 173.5 179.6 177.5 ± 6.4A 181.6 182.2 173.4 179.1 ± 4.8A 0.37
Mash 1 Mash 2 Mash 3 t-value
The values that have no common superscript are significantly different (p < 0.05) according to Student’s t-test at the 95% confidence level.
H. Zhang et al. / Process Biochemistry 46 (2011) 1909–1913
1911
Apples Mashing
Mash (Enzyme treatment)
Mash (No enzyme treatment)
Treated mash (CME)
Untreated mash (CMNE)
Pressing
Fermentation
Fermentation
Treated juice(CJE) Treated pomace(CPE)
Fermentation
Fermentation
Pressing
Untreated juice(CJNE)
Fermentation
Untreated pomace(CPNE)
Fermentation
Fig. 1. Processing flow chart of Crispin apple mash, juice and pomace.
(t = 0.37) between the fruit spirits made from the pectinase treated apple mash and the non-pectinase treated apple mash. The Student’s t-test results in Table 3 indicate that there were significant differences in the concentrations of methanol (t = 27.28), n-propanol (t = −7.14), and iso-butanol (t = 34.64) between the fruit spirits made from the pectinase treated apple juice and the nonpectinase treated apple juice. The pectinase enzyme treatment, however, had no effect on the concentration of iso-amyl alcohol (t = 3.88). The effect of pectinase treatment on the methanol and other alcoholic constituents of the fruit spirits made from apple pomace is given in Table 4. According to the Student’s t-test results, there was a significant difference in the methanol concentrations of fruit spirits made from the pectinase treated apple pomace and the non-
pectinase treated apple pomace (t = 26.78). The fruit spirits made from the pectinase treated apple pomace had methanol concentrations (1000.1 ± 36.0 mg/100 mL 40% ethanol), nearly four times higher than the United States FDA legal limit of 280 mg/100 mL of 40% ethanol for fruit spirits. However, the differences in the concentrations of n-proponal (t = −1.36), iso-butanol (t = −0.64) and iso-amyl alcohol (t = −0.19) of fruit spirits between the pectinase treated apple pomace and the pectinase treated apple pomace were not statistically significant. Andraous et al. [10] reported that the methanol concentrations of the fruit spirits made from the pectinase treated fruit mashes of Gala, Red Delicious, Granny Smith apples, and Bartlett pears were over 423, 320, 464 and 607 mg/100 mL of 40% ethanol, respectively. However, they did not investigate the influence of
Fig. 2. Separation of volatile compounds by HPLC using a Bio-Rad Aminex HPX 87H column.
1912
H. Zhang et al. / Process Biochemistry 46 (2011) 1909–1913
Table 3 Effect of pectinase treatment on the alcoholic constituents of apple spirits made from juice. Substrate
Pectinase treatment
Methanol in 40% ethanol (mg/100 mL)
n-Propanol in 40% ethanol (mg/100 mL)
Iso-Butanol in 40% ethanol (mg/100 mL)
Iso-Amyl alcohol in 40% ethanol (mg/100 mL)
Juice 1 Juice 2 Juice 3
No No No Mean ± SD Yes Yes Yes Mean ± SD
81.2 38.1 36.5 51.9 ± 24.2B 402.7 400.3 392.9 398.7 ± 7.1A 27.28
14.1 14.7 15.0 14.6 ± 0.7A 12.9 12.8 13.1 12.9 ± 0.2B −7.14
38.5 42.7 43.7 41.6 ± 2.6B 51.1 54.7 55.1 53.6 ± 2.1A 34.64
139.7 146.8 146.1 144.2 ± 3.8A 185.1 192.7 164.4 180.7 ± 19.1A 3.88
Juice 1 Juice 2 Juice 3 t-value
The values that have no common superscript are significantly different (p < 0.05) according to Student’s t-test at the 95% confidence level.
Table 4 Student’s t-test of pectinase treatment on alcoholic constituents of apple spirits made from pomace. Substrate
Pectinase treatment
Methanol in 40% ethanol (mg/100 mL)
n-Propanol in 40% ethanol (mg/100 mL)
Iso-Butanol in 40% ethanol (mg/100 mL)
Iso-Amyl alcohol in 40% ethanol (mg/100 mL)
Pomace 1 Pomace 2 Pomace 3
No No No Mean ± SD Yes Yes Yes Mean ± SD
313.1 314.5 312.6 303.4 ± 22.6B 1025.4 966.8 1008.0 1000.1 ± 36.0A 26.78
16.9 21.5 23.2 20.5 ± 5.9A 19.5 13.0 13.0 15.2 ± 3.6A −1.36
44.6 48.0 44.7 45.8 ± 3.5A 46.9 44.0 43.9 45.0 ± 5.3A −0.46
185.1 176.1 177.6 179.6 ± 7.2A 185.9 181.5 169.1 178.9 ± 10.7A −0.19
Pomace 1 Pomace 2 Pomace 3 t-value
The values that have no common superscript are significantly different (p < 0.05) according to Student’s t-test at the 95% confidence level.
Table 5 Alcoholic constituents of fruit spirits made from apple mash, juice and pomace. Substrate
Pectinase treatment
Methanol in 40% ethanol (mg/100 mL)
Mash Juice Pomace Mash Juice Pomace
No No No Yes Yes Yes
87.7 51.9 303.4 533.2 398.7 1000.1
± ± ± ± ± ±
8.2B 24.2B 22.6A 8.5b 7.1c 36.0a
n-Propanol in 40% ethanol (mg/100 mL) 16.2 14.6 20.5 15.6 12.9 15.2
± ± ± ± ± ±
3.3BA 0.7B 5.9A 1.0a 0.2a 3.6a
Iso-butanol in 40% ethanol (mg/100 mL) 54.1 41.6 45.8 63.0 53.6 45.0
± ± ± ± ± ±
6.0A 2.6B 3.5B 2.3a 2.1b 5.3c
Iso-amyl alcohol in 40% ethanol (mg/100 mL) 177.5 144.2 179.6 179.1 180.7 178.9
± ± ± ± ± ±
6.4A 3.8B 7.2A 4.8a 19.1a 10.7a
The values (N = 3) that have no common superscript are significantly different at the 95% confidence level according to Duncan’s multiple range test.
pectinase treatment on the methanol concentrations of the fruit spirits produced from apple juice and pomace. Table 4 compares the concentrations of methanol and higher alcohols of the fruit spirits made from the apple mash, juice and pomace with and without pectinase enzyme treatment. The fruit spirits made from apple pomace, for example, had significantly higher amounts of methanol (303 mg/100 mL of 40% ethanol) than those made from apple mash (88 mg/100 mL of 40% ethanol) or apple juice (52 mg/100 mL of 40% ethanol). Only fruit spirits made from the non-pectinase treated apple mash or apple juice had methanol concentrations lower than the United States FDA limit of 280 mg/100 mL of 100 mL of 40% ethanol. The commercial A. niger pectinase enzyme preparation used in the processing of Crispin apples greatly affected the formation of methanol in the fruit spirits made from apple mash, juice, and pomace. For example, the fruit spirits made from the pectinase treated apple pomace contained the greatest amount of methanol (1000 mg/100 mL of 40% ethanol), whereas the methanol concentrations of fruit spirits produced from the pectinase apple mash and apple juice were 533 mg and 399 mg/100 mL of 40% ethanol, respectively. According to Duncan multiple range tests, the differences in the methanol concentrations of fruit spirits made from apple mash, juice and pomace were statistically significant. As shown in Table 5,
the methanol concentrations of all the fruit spirits made from the pectinase treated apple mash, juice, and pomace were significantly above the United States FDA legal limit of 280 mg/100 mL of 40% ethanol for fruit brandy. There were no significant differences in the concentrations of n-propanol and iso-amyl alcohol, but the fruit spirits made from apple mash had significantly higher iso-butanol than those made from apple juice and pomace.
4. Conclusion Fruit spirits have been successfully produced from the mash, juice, and pomace of Crispin apples. The use of an A. niger pectinase enzyme preparation in the production process was found to significantly increase the methanol concentrations of all the apple spirits. Fruit spirits made from apple pomace had the greatest amount of methanol, whereas those made from the non-pectinase treated apple mash and juice had methanol concentrations below the United States FDA legal limit of 280 mg/100 mL of 40%. However, the methanol concentrations of all the fruit spirits made from the pectinase treated apple mash, juice, and pomace are significantly above the government legal tolerance.
H. Zhang et al. / Process Biochemistry 46 (2011) 1909–1913
Acknowledgements This work was funded in part by Federal Formula Funds (Multistate project W1009). Dr. Hui Zhang was supported by the China Scholarship Council (Beijing) and Shenyang University of Chemical Technology. Shenyang, China. References [1] The Merck Index. An encyclopedia of chemicals and drugs. 13th ed. NJ: Whitehouse Station; 2001. [2] United States Code of Federal Regulations, Title 27-Alcohol, Tobacco Products and Firearms, Chapter 1, Subpart C. Standards of Identity for distilled spirits; 2003. [3] Regulation (EC) No 110/2008 of the European Parliament and of the Council of 15 January 2008 on the definition, description, presentation, labelling and the protection of geographical indications of spirit drinks and repealing Council Regulation (EEC) No 1576/89. Official Journal of the European Union. L39/16-50. 13.2.2008. [4] Dixon M, Webb E-C. The enzymes. London: Longmans, Green and Co. Ltd.; 1964. [5] Pilnik W, Voragen A-G-J. Pectic enzymes in fruit and vegetable juice manufacture. In: Nagodawithana T, Reed G, editors. Enzymes in food processing. 3rd ed. Academic Press; 1993. [6] Mantovani CF, Geimba MP, Brandeli A. Enzymatic clarification of fruit juices by fungal pectin lyase. Food Biotechnol 2005;19(173-181).
1913
[7] Lee C-Y, Robinson W-B, Van Buren J-P, Acree T-E, Stoewsand G-S. Methanol in wines in relation to processing and variety. Am J Enol Viticult 1975;26(4):184–7. [8] Gnekow B, Ough C-S. Methanol in wines and musts: source and amounts. Am J Enol Viticult 1976;27(1):1–6. [9] Bindler F, Voges E, Laugel P. The problem of methanol concentration admissible in distilled fruit spirits. Food Addit Contam 1988;5(3):343–51. [10] Wilson S-M, Le Maguer M, Duitschaever C-L, Buteau C, O Brien A. Effect of processing treatments on the characteristics of juices and still ciders from Ontario-grown apples. J Sci Food Agric 2003;83:215–24. [11] Andraous J-I, Claus M-J, Lindemann D-J, Berglung K-A. Effect of liquefaction enzymes on methanol concentration of distilled fruit spirits. Am J Enol Viticult 2004;55(2):199–202. [12] Hang Y-D, Woodams ED. Methanol content of grappa made from New York grape pomace. Bioresour Technol 2008;99:3923–5. [13] Da Porto C, Sensodoni A, Battistutta F. Composition and flavour of Muscat of canelli grape distillates obtained using different oenological techniques and unconventional distillation processes. Ital J Food Sci 1995;7(1):47–56. [14] Hernandez-Gomes L-F, Ubeda J, Briones A. Melon fruit distillates: comparison of different distillation methods. Food Chem 2003;82:539–43. [15] Glatthar J, Senn T, Pieper H-J. Investigations on reducing methanol content in distilled spirits made of Bartlett pears. Deut Lebensm Randsch 2001;97: 209–16. [16] Hang Y-D, Woodams EK. Influence of apple cultivar and juice pasteurization on hard cider and eau-de-vie methanol content. Bioresour Technol 2010;101:1396–8. [17] Sadler G-D, Murphy P-A. pH and titratable acidity. In: Niesen SS, editor. Food analysis. 3rd ed. New York: Kluwer Academic/Plenum Publishers; 2003.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Influence of pectinase treatment on fruit spirits from apple mash, juice and pomace Hui Zhang a , Edward E. Woodams b , Yong D. Hang b,∗ a b
Shenyang University of Chemical Technology, Department of Environmental & Bioengineering, Shenyang 110142, China Cornell University, Department of Food Science, 630 W. North Street, Geneva, NY 14456, USA
a r t i c l e
i n f o
Article history: Received 26 January 2011 Received in revised form 21 June 2011 Accepted 22 June 2011 Keywords: Apple Pectinase Spirits Alcohol Methanol
a b s t r a c t The current investigation was conducted to determine the influence of pectinase treatment on fruit spirits produced from apple mash, juice, and pomace. Crispin apples were processed into apple mash, juice, and pomace in our pilot-plant, and fermented with a commercial Red Star wine yeast (Sachharomyces cerevisiae Davis 904). After fermentation, the samples of fermented apple mash, juice, and pomace were distilled, and the distillates were analyzed by HPLC with a Bio-Rad Aminex HPX 87H column and a refractive index detector. Methanol, ethanol, n-propanol, iso-butanol, and iso-amyl alcohol were identified as the major alcohols in all the apple spirits. Student’s t-test results indicate that there are significant differences between the methanol concentrations of pectinase treated and non-pectinase treated apple spirits. Duncan’s multiple range tests show significant differences in the concentrations of methanol of the fruit spirits made from apple mash, juice, and pomace. Apple pomace yielded significantly higher methanol concentrations than apple mash and juice. Pectinase treatment had little effect on the concentrations of n-propanol, iso-butanol, and iso-amyl alcohol. It is concluded that fruit spirits made from the pectinase treated mash, juice, and pomace of Crispin apples had methanol concentrations significantly above the United States FDA guidance of 0.35% by volume or 280 mg/100 mL of fruit brandy containing 40% ethanol. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Fruit spirits are widely consumed in the United States, Canada and European countries. They are high-value alcoholic beverages produced commercially by the distillation of fermented fruit mash, juice, or pomace. Methanol and higher alcohols are major volatile compounds that influence the quality and safety of fruit spirits and can exert adverse effects on human health. Methanol, for example, has been reported to be associated with harmful health effects such as headache, fatigue, nausea, visual impairment, or complete blindness [1], and its presence in distilled fruit spirits is strictly controlled. The United States legal limit on methanol in distilled fruit spirits is 0.35% (v/v), or 280 mg/100 mL of 40% alcohol [2]. The EU general methanol limit for spirits made of fruits and marc Brandy is 400 mg/100 mL of 40% alcohol, but for spirits made of plums, apples, and pears, the methanol limit is 480 mg/100 mL of 40% alcohol [3]. Methanol is the product of the enzymatic hydrol-
∗ Corresponding author. Tel.: +1 315 787 2265; fax: +1 315 787 2284. E-mail addresses: [email protected], [email protected] (Y.D. Hang). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.06.020
ysis of fruit pectin by pectin methylesterase (EC 3.3.3.11) during alcoholic fermentation as follows [4]: Pectin methylesterase
Pectin + H2 O
−→
Pectic acid + Methanol
The commercial pectinase enzyme preparation derived from Aspergillus niger is widely used in the fruit juice processing industry to improve the juice yield [5,6]. However, the enzymatic treatment employed in fruit processing can cause a significant increase in the methanol concentrations of wine and brandy. Several investigators reported the influence of pectinase enzyme treatment on the methanol concentrations of wines from California and New York State in the United States [7–9]. Wilson et al. [10] reported the effect of processing treatments on the characteristics of juices and still ciders from apples grown in Ontario, Canada. Andraous et al. [11] reported that the methanol concentrations of fruit spirits made from apple and pear mashes with commercial pectinase enzyme preparations derived from A. niger were well above the United States FDA limits. Hang and Woodams [12] found the methanol content of grappa made from the sweet pomace of five New York grape varieties to be within the United States legal limit. Da porto et al. reported the influence of different enological techniques distillation processes on the concentrations of methanol and other volatile compounds in grape distillates [13]. Hernandez et al. found that
1910
H. Zhang et al. / Process Biochemistry 46 (2011) 1909–1913
Table 1 Chemical composition of apple mash, juice and pomace. Sample
◦
Brix
pH
wine yeast (1 g/kg), all the samples were incubated at room temperature (21–23 ◦ C) for 12 days. All the fermentation experiments were conducted in triplicate. Acidity% 2.3. Distillation
No pectinase treatment Mash 11.3 Juice 11.2 Pomace 11.5 Pectinase treatment Mash 11.2 Juice 11.0 Pomace 11.3
± 0.1 ±0 ± 0.7
3.2 ± 0 3.3 ± 0 3.6 ± 0
0.48 ± 0.01 0.46 ± 0 0.42 ± 0.03
±0 ±0 ± 0.4
3.1 ± 0 3.2 ± 0 3.1 ± 0
0.60 ± 0 0.58 ± 0 0.46 ± 0.02
the concentrations of methanol and the other volatile compounds varied considerably and were dependent on the fermentation conditions and the distillation methods. [14]. Glatthar et al. [15] found that the methanol concentrations of pear spirits could be significantly reduced by lowering the pH of pear mashes and shortening the fermentation time. Hang and Woodams [16] investigated the influence of apple variety and juice pasteurization on hard cider and eau-de-vie methanol content and found Crispin apples yielded more methanol in hard cider and eau-de-vie than other three varieties, and juice pasteurization prior to alcoholic fermentation significantly reduced the methanol content. The current study was undertaken to determine if there were any significant differences in the concentrations of methanol, ethanol, and higher alcohols (C3–C5) of the fruit spirits produced from apple mash, juice and pomace with and without pectinase enzyme treatments. 2. Materials and methods 2.1. Materials The Crispin apple variety (Malus domestica) is a cross between the Golden Delicious and the Indo apple varieties first grown in Japan and generally ripes in late October in Geneva, New York. Crispin apples grown in the Finger Lakes fruit region of New York State were processed into mashes with a comminuting machine (Model D, The W.J. Fitzpatrick Company, Chicago, USA) in our fruit and vegetable processing pilot plant at Cornell University’s Food Science Department in Geneva, NY. The apple mashes were divided into two lots. One lot of the apple mashes was mixed with a commercial pectinase preparation derived from A. niger, Rapidase ADEX-D and incubated at 50 ◦ C for 2 h. Rapidase ADEX-D was added at the rate of 100 g/ton as recommended by the enzyme supplier (DSM Food Specialties USA, Inc. Charlotte, NC, USA). Approximately 65% of the enzyme treated mashes was pressed into juice (CJE) and pomace (CPE) by a Hydraulic Rack and Frame Press (screen size No. 6, 34-in.). The remaining 35% of the enzyme treated mash (CME) was kept for direct fermentation. The other lot of apple mashes (CMNE), juice (CJNE), and pomace (CPNE) samples were prepared in the same manner except that there was no pectinase enzyme preparations added (Fig. 1). 2.2. Fermentation The fermentation experiments with apple mash and juice were conducted in 2000-mL fermenters equipped with U-tube airlocks, each containing 1000 g of apple mash or apple juice. Apple pomace was fermented in 1000-mL fermenters, each containing 300 g of apple pomace. After inoculation with commercial Red Star dry active
Approximately 250 mL of fermented apple mash, juice or pomace samples were distilled utilizing a laboratory distillation apparatus under controlled conditions [16]. Prior to distillation, samples of fermented apple mash were squeezed at 500–700 psi with Loomis Press (Loomis Engineering & MFG Company, Caldwell, NJ, USA). Samples of fermented apple pomace were prepared by blending with water in a Waring blender for 1 min and centrifuging at 16,270 rpm for 25 min. The supernatant fractions were used for distillation. Samples of fermented apple juice were distilled directly. 2.4. Analytical methods Methanol, ethanol, n-propanol, iso-butanol and iso-amyl alcohol were determined by HPLC (11) with the following conditions: Column, Bio-Rad Aminex HPX 87H; Eluent, 0.005 M sulfuric acid; flow rate, 0.6 mL/min; temperature, 50 ◦ C; detection, a refractive index detector Knauer K-2300 (Sonntek Inc., Upper Saddle River, NJ). ◦ Brix was measured with a Karl Zeiss refractometer (Jena, Germany). Fig. 2 shows the separation of volatile compounds by HPLC using a Bio-Rad Aminex HPX 87H column. Standards of methanol, ethanol, n-propanol, iso-butanol and iso-amyl alcohol were run first to determine the retention time of each alcohol, and then the samples were run. The titratable acidity, expressed as % malic acid, was determined by titrating the samples with 0.02 N NaOH to 8.2 [17]. pH value was measured with a Fisher pH meter Model 230. 2.5. Statistical analysis All experimental data were expressed as mean ± SD (standard deviation). Statistical analyses were conducted with the SAS statistical computer package (SAS Institute, Cary, NY, USA). The student’s t-test was used to determine if there was a significant difference in alcoholic compositions between apple spirits made from mash, juice, and pomace with and without enzyme treatment. The levels of confidence used in the statistical analysis of the data were 95%. Duncan’s multiple range test was used to evaluate if there were significant differences among spirits made from apple mash, juice, and pomace. The values that have no common superscript are significantly different according to Duncan’s multiple range test.
3. Results and discussion Crispin apples grown in the Finger Lakes region of New York State were found to have an average ◦ Brix value of 11.2, an average pH of 3.3, and an average titratable acidity of 0.42% expressed as malic acid (Table 1.). Student’s t-tests were used to determine if the pectinase enzyme treatment caused statistically significant differences in the concentrations of methanol and other alcoholic constituents of fruit spirits made from apple mash, juice and pomace. As shown in Table 2, the methanol concentrations of fruit spirits made from the nonpectinase treated apple mash and the pectinase treated apple mash are 87.7 mg and 533.2 mg/100 mL of 40% ethanol, respectively, The Student’s t-test results indicate that the difference in the methanol concentration was statistically significant (t = 55.29, p0.05 = 4.303), but there were no significant differences in the concentrations of n-propanol (t = −0.53), iso-butanol (t = 2.55), or iso-amyl alcohol
Table 2 Effect of pectinase treatment on the alcoholic constituents of apple spirits made from mash. Substrate
Pectinase treatment
Methanol in 40% ethanol (mg/100 mL)
n-Propanol in 40% ethanol (mg/100 mL)
Iso-Butanol in 40% ethanol (mg/100 mL)
Iso-Amyl alcohol in 40% ethanol (mg/100 mL)
Mash1 Mash 2 Mash 3
No No No Mean ± SD Yes Yes Yes Mean ± SD
91.3 92.3 79.4 87.7 ± 8.2B 526.8 532.0 540.9 533.2 ± 8.5A 55.29
19.1 15.6 13.9 16.2 ± 3.3A 16.6 15.1 15.1 156 ± 10A −0.53
58.7 52.2 51.5 54.1 ± 6.0A 60.5 65.5 62.9 63.0 ± 2.3A 2.55
179.3 173.5 179.6 177.5 ± 6.4A 181.6 182.2 173.4 179.1 ± 4.8A 0.37
Mash 1 Mash 2 Mash 3 t-value
The values that have no common superscript are significantly different (p < 0.05) according to Student’s t-test at the 95% confidence level.
H. Zhang et al. / Process Biochemistry 46 (2011) 1909–1913
1911
Apples Mashing
Mash (Enzyme treatment)
Mash (No enzyme treatment)
Treated mash (CME)
Untreated mash (CMNE)
Pressing
Fermentation
Fermentation
Treated juice(CJE) Treated pomace(CPE)
Fermentation
Fermentation
Pressing
Untreated juice(CJNE)
Fermentation
Untreated pomace(CPNE)
Fermentation
Fig. 1. Processing flow chart of Crispin apple mash, juice and pomace.
(t = 0.37) between the fruit spirits made from the pectinase treated apple mash and the non-pectinase treated apple mash. The Student’s t-test results in Table 3 indicate that there were significant differences in the concentrations of methanol (t = 27.28), n-propanol (t = −7.14), and iso-butanol (t = 34.64) between the fruit spirits made from the pectinase treated apple juice and the nonpectinase treated apple juice. The pectinase enzyme treatment, however, had no effect on the concentration of iso-amyl alcohol (t = 3.88). The effect of pectinase treatment on the methanol and other alcoholic constituents of the fruit spirits made from apple pomace is given in Table 4. According to the Student’s t-test results, there was a significant difference in the methanol concentrations of fruit spirits made from the pectinase treated apple pomace and the non-
pectinase treated apple pomace (t = 26.78). The fruit spirits made from the pectinase treated apple pomace had methanol concentrations (1000.1 ± 36.0 mg/100 mL 40% ethanol), nearly four times higher than the United States FDA legal limit of 280 mg/100 mL of 40% ethanol for fruit spirits. However, the differences in the concentrations of n-proponal (t = −1.36), iso-butanol (t = −0.64) and iso-amyl alcohol (t = −0.19) of fruit spirits between the pectinase treated apple pomace and the pectinase treated apple pomace were not statistically significant. Andraous et al. [10] reported that the methanol concentrations of the fruit spirits made from the pectinase treated fruit mashes of Gala, Red Delicious, Granny Smith apples, and Bartlett pears were over 423, 320, 464 and 607 mg/100 mL of 40% ethanol, respectively. However, they did not investigate the influence of
Fig. 2. Separation of volatile compounds by HPLC using a Bio-Rad Aminex HPX 87H column.
1912
H. Zhang et al. / Process Biochemistry 46 (2011) 1909–1913
Table 3 Effect of pectinase treatment on the alcoholic constituents of apple spirits made from juice. Substrate
Pectinase treatment
Methanol in 40% ethanol (mg/100 mL)
n-Propanol in 40% ethanol (mg/100 mL)
Iso-Butanol in 40% ethanol (mg/100 mL)
Iso-Amyl alcohol in 40% ethanol (mg/100 mL)
Juice 1 Juice 2 Juice 3
No No No Mean ± SD Yes Yes Yes Mean ± SD
81.2 38.1 36.5 51.9 ± 24.2B 402.7 400.3 392.9 398.7 ± 7.1A 27.28
14.1 14.7 15.0 14.6 ± 0.7A 12.9 12.8 13.1 12.9 ± 0.2B −7.14
38.5 42.7 43.7 41.6 ± 2.6B 51.1 54.7 55.1 53.6 ± 2.1A 34.64
139.7 146.8 146.1 144.2 ± 3.8A 185.1 192.7 164.4 180.7 ± 19.1A 3.88
Juice 1 Juice 2 Juice 3 t-value
The values that have no common superscript are significantly different (p < 0.05) according to Student’s t-test at the 95% confidence level.
Table 4 Student’s t-test of pectinase treatment on alcoholic constituents of apple spirits made from pomace. Substrate
Pectinase treatment
Methanol in 40% ethanol (mg/100 mL)
n-Propanol in 40% ethanol (mg/100 mL)
Iso-Butanol in 40% ethanol (mg/100 mL)
Iso-Amyl alcohol in 40% ethanol (mg/100 mL)
Pomace 1 Pomace 2 Pomace 3
No No No Mean ± SD Yes Yes Yes Mean ± SD
313.1 314.5 312.6 303.4 ± 22.6B 1025.4 966.8 1008.0 1000.1 ± 36.0A 26.78
16.9 21.5 23.2 20.5 ± 5.9A 19.5 13.0 13.0 15.2 ± 3.6A −1.36
44.6 48.0 44.7 45.8 ± 3.5A 46.9 44.0 43.9 45.0 ± 5.3A −0.46
185.1 176.1 177.6 179.6 ± 7.2A 185.9 181.5 169.1 178.9 ± 10.7A −0.19
Pomace 1 Pomace 2 Pomace 3 t-value
The values that have no common superscript are significantly different (p < 0.05) according to Student’s t-test at the 95% confidence level.
Table 5 Alcoholic constituents of fruit spirits made from apple mash, juice and pomace. Substrate
Pectinase treatment
Methanol in 40% ethanol (mg/100 mL)
Mash Juice Pomace Mash Juice Pomace
No No No Yes Yes Yes
87.7 51.9 303.4 533.2 398.7 1000.1
± ± ± ± ± ±
8.2B 24.2B 22.6A 8.5b 7.1c 36.0a
n-Propanol in 40% ethanol (mg/100 mL) 16.2 14.6 20.5 15.6 12.9 15.2
± ± ± ± ± ±
3.3BA 0.7B 5.9A 1.0a 0.2a 3.6a
Iso-butanol in 40% ethanol (mg/100 mL) 54.1 41.6 45.8 63.0 53.6 45.0
± ± ± ± ± ±
6.0A 2.6B 3.5B 2.3a 2.1b 5.3c
Iso-amyl alcohol in 40% ethanol (mg/100 mL) 177.5 144.2 179.6 179.1 180.7 178.9
± ± ± ± ± ±
6.4A 3.8B 7.2A 4.8a 19.1a 10.7a
The values (N = 3) that have no common superscript are significantly different at the 95% confidence level according to Duncan’s multiple range test.
pectinase treatment on the methanol concentrations of the fruit spirits produced from apple juice and pomace. Table 4 compares the concentrations of methanol and higher alcohols of the fruit spirits made from the apple mash, juice and pomace with and without pectinase enzyme treatment. The fruit spirits made from apple pomace, for example, had significantly higher amounts of methanol (303 mg/100 mL of 40% ethanol) than those made from apple mash (88 mg/100 mL of 40% ethanol) or apple juice (52 mg/100 mL of 40% ethanol). Only fruit spirits made from the non-pectinase treated apple mash or apple juice had methanol concentrations lower than the United States FDA limit of 280 mg/100 mL of 100 mL of 40% ethanol. The commercial A. niger pectinase enzyme preparation used in the processing of Crispin apples greatly affected the formation of methanol in the fruit spirits made from apple mash, juice, and pomace. For example, the fruit spirits made from the pectinase treated apple pomace contained the greatest amount of methanol (1000 mg/100 mL of 40% ethanol), whereas the methanol concentrations of fruit spirits produced from the pectinase apple mash and apple juice were 533 mg and 399 mg/100 mL of 40% ethanol, respectively. According to Duncan multiple range tests, the differences in the methanol concentrations of fruit spirits made from apple mash, juice and pomace were statistically significant. As shown in Table 5,
the methanol concentrations of all the fruit spirits made from the pectinase treated apple mash, juice, and pomace were significantly above the United States FDA legal limit of 280 mg/100 mL of 40% ethanol for fruit brandy. There were no significant differences in the concentrations of n-propanol and iso-amyl alcohol, but the fruit spirits made from apple mash had significantly higher iso-butanol than those made from apple juice and pomace.
4. Conclusion Fruit spirits have been successfully produced from the mash, juice, and pomace of Crispin apples. The use of an A. niger pectinase enzyme preparation in the production process was found to significantly increase the methanol concentrations of all the apple spirits. Fruit spirits made from apple pomace had the greatest amount of methanol, whereas those made from the non-pectinase treated apple mash and juice had methanol concentrations below the United States FDA legal limit of 280 mg/100 mL of 40%. However, the methanol concentrations of all the fruit spirits made from the pectinase treated apple mash, juice, and pomace are significantly above the government legal tolerance.
H. Zhang et al. / Process Biochemistry 46 (2011) 1909–1913
Acknowledgements This work was funded in part by Federal Formula Funds (Multistate project W1009). Dr. Hui Zhang was supported by the China Scholarship Council (Beijing) and Shenyang University of Chemical Technology. Shenyang, China. References [1] The Merck Index. An encyclopedia of chemicals and drugs. 13th ed. NJ: Whitehouse Station; 2001. [2] United States Code of Federal Regulations, Title 27-Alcohol, Tobacco Products and Firearms, Chapter 1, Subpart C. Standards of Identity for distilled spirits; 2003. [3] Regulation (EC) No 110/2008 of the European Parliament and of the Council of 15 January 2008 on the definition, description, presentation, labelling and the protection of geographical indications of spirit drinks and repealing Council Regulation (EEC) No 1576/89. Official Journal of the European Union. L39/16-50. 13.2.2008. [4] Dixon M, Webb E-C. The enzymes. London: Longmans, Green and Co. Ltd.; 1964. [5] Pilnik W, Voragen A-G-J. Pectic enzymes in fruit and vegetable juice manufacture. In: Nagodawithana T, Reed G, editors. Enzymes in food processing. 3rd ed. Academic Press; 1993. [6] Mantovani CF, Geimba MP, Brandeli A. Enzymatic clarification of fruit juices by fungal pectin lyase. Food Biotechnol 2005;19(173-181).
1913
[7] Lee C-Y, Robinson W-B, Van Buren J-P, Acree T-E, Stoewsand G-S. Methanol in wines in relation to processing and variety. Am J Enol Viticult 1975;26(4):184–7. [8] Gnekow B, Ough C-S. Methanol in wines and musts: source and amounts. Am J Enol Viticult 1976;27(1):1–6. [9] Bindler F, Voges E, Laugel P. The problem of methanol concentration admissible in distilled fruit spirits. Food Addit Contam 1988;5(3):343–51. [10] Wilson S-M, Le Maguer M, Duitschaever C-L, Buteau C, O Brien A. Effect of processing treatments on the characteristics of juices and still ciders from Ontario-grown apples. J Sci Food Agric 2003;83:215–24. [11] Andraous J-I, Claus M-J, Lindemann D-J, Berglung K-A. Effect of liquefaction enzymes on methanol concentration of distilled fruit spirits. Am J Enol Viticult 2004;55(2):199–202. [12] Hang Y-D, Woodams ED. Methanol content of grappa made from New York grape pomace. Bioresour Technol 2008;99:3923–5. [13] Da Porto C, Sensodoni A, Battistutta F. Composition and flavour of Muscat of canelli grape distillates obtained using different oenological techniques and unconventional distillation processes. Ital J Food Sci 1995;7(1):47–56. [14] Hernandez-Gomes L-F, Ubeda J, Briones A. Melon fruit distillates: comparison of different distillation methods. Food Chem 2003;82:539–43. [15] Glatthar J, Senn T, Pieper H-J. Investigations on reducing methanol content in distilled spirits made of Bartlett pears. Deut Lebensm Randsch 2001;97: 209–16. [16] Hang Y-D, Woodams EK. Influence of apple cultivar and juice pasteurization on hard cider and eau-de-vie methanol content. Bioresour Technol 2010;101:1396–8. [17] Sadler G-D, Murphy P-A. pH and titratable acidity. In: Niesen SS, editor. Food analysis. 3rd ed. New York: Kluwer Academic/Plenum Publishers; 2003.