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A publication of

CHEMICAL ENGINEERING TRANSACTIONS
VOL. 37, 2014
Guest Editors: Eliseo Ranzi, Katharina Kohse- Höinghaus Copyright © 2014, AIDIC Servizi S.r.l., ISBN 978-88-95608-28-0; ISSN 2283-9216 The Italian Association of Chemical Engineering www.aidic.it/cet DOI: 10.3303/CET1437102

Vinegar Production from Pineapple Wastes –Preliminary Saccharification Trials
Arianna Roda, Dante Marco De Faveri, Roberta Dordoni, Milena Lambri*
Istituto di Enologia e Ingegneria Agro-Alimentare, Università Cattolica del Sacro Cuore Via Emilia Parmense, 84, 29122 Piacenza, Italy milena.lambri@unicatt.it

This study is located in the within of a research devoted at processing wastes both in developing and in developed Countries, so reducing both environmental pollution and seasonal fruit losses. In particular, the full work intended to completely process pineapple wastes into vinegar which may be then used as dressing, food preservative, and disinfectant. The preliminary trials presented here deepened the first process step (i.e. the saccharification) and looked into the feasibility of producing the greatest yield of reducing sugars from peels and core of pineapples. Wastes were cut into thin strips, chopped in a mixer, and divided into samples of peel and core to which distilled water was added. For enhancing reducing sugar yield, physical treatments were arranged to disaggregate the fibrous structure followed by enzyme treatments to breakdown cellulose polymers and to hydrolyse sucrose. The optimal time-temperature conditions of each process step were searched for gaining the highest reducing sugars yield at the end of the saccharification. Cellulolytic enzymes were tested for 4-8-18-24 h at 30-40-50 °C, invertase addition was arranged, and amylolytic enzymes were evaluated. All determinations were done in duplicate and a factorial ANOVA with Tukey’s test at p ≤ 0.05 was used to measure the significance of the differences among treatments. The conditions allowing the greatest reducing sugar yield were: the addition to 100 g of waste fresh weight (fw) of 0.025 mL of thermostable α-amylase before a 10 min treatment at 143.27 kPa followed by 24 h-50 °C incubation with 0.05 g pectinase/kgfw, 6 g cellulase/kgfw, 1 g hemicellulase/kgfw, and 0.05 % glucoamylase and pullulanase (Venzyme/kgfw). Then, samples were incubated with 0.05 g invertase/kgfw for 3 h at 50 °C. Under these conditions, more than 100 g of reducing sugars per kg of fresh peels and about 330 g of reducing sugars per kg of fresh core were obtained.

1. Introduction
Most nations, whether economically advanced or at different stages of development are faced with the issue of disposal and treatment of wastes (Itelima et al., 2013). Agro-industrial wastes are generated in large amounts every year and their reuse in processes is of particular interest due to their availability, low cost, and characteristics that allow at obtaining different value-added compounds (De Freitas Borghi et al., 2009). Tropical and subtropical fruits processing have considerably higher ratios of by-products than the temperate fruits (Schieber et al., 2001). Pineapple (Ananas comosus) by-products are not exceptions. Indeed, several efforts have been made in order to utilize pineapple wastes, which have already been used as the substrate for the production of bromelain and organic acids (Dacera et al., 2009), fibre and phenolic anti-oxidants (Larrauri et al., 1997), ethanol and biogas (Nigam, 1999). Since pineapple has the second highest production volume of all tropical fruits in the world (FAO, 2009), the production of processed items results in massive waste generation, estimated about 40-50 % from fresh fruit as peels and core (Buckle, 1989). Even if pineapple residues are rich in sugars, especially in sucrose, glucose and fructose and other components like minerals and vitamins (Abdullah and Hanafi, 2008), they cannot be used in full. The pineapple peel contained an appreciable amount of insoluble fibre-rich fraction which primarily consisted of cellulose, pectin substances, hemicellulose, and notable proportions of lignin (Huang

Please cite this article as: Roda A., De Faveri D.M., Dordoni R., Lambri M., 2014, Vinegar production from pineapple wastes –preliminary saccharification trials, Chemical Engineering Transactions, 37, 607-612 DOI: 10.3303/CET1437102

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et al., 2011). A multitude of different pretreatment technologies have been suggested during the last decades (Alvira et al., 2010) in order to hydrolyze the cellulosic waste materials. The present study, devoted to turn pineapple wastes into vinegar, investigated throughout preliminary trials the first step (i.e. saccharification) of the process with the purpose of obtaining the greatest yield of reducing sugars from peel and core of pineapples.

2. Materials and methods
2.1 Materials Enzymes Cellulolytic and pectinolytic enzymes were: cellulase (from Aspergillus niger, 0.8 enzyme units/mg solid, Sigma C1184-25KU, Sigma-Aldrich, USA), hemicellulase (from Aspergillus niger, 1.5 enzyme units/mg solid, using a β-galactose dehydrogenase system and locust bean gum as a substrate, Sigma H2125150KU, Sigma-Aldrich, USA), and pectinase (>200 enzyme units PL/g, Enartis Zym Quick, Enartis, Novara, Italy). Amylolytic enzymes were: thermostable α-amylase (from Bacillus licheniformis, 135 KNU/g, Liquezyme-X), glucoamylase (from Aspergillus niger, 400 AGU/g, Dextrozyme GA), and pullulanase (from Bacillus acidopullulyticus, 400 AGU/g, Dextrozyme GX) and provided by M/s Novozymes A/S (Denmark). For sucrose hydrolysis, invertase (from bakery’s yeast Saccharomyces cerevisiae, >300 enzyme units/mg, Sigma-Aldrich, USA) was employed. Raw material Pineapples (average fresh weight of 1.73±0.49 kg) were purchased from the supermarket in Italy. They were kept at 22 °C before undergoing the saccharification process. The pineapples were washed and the wastes were separated from the edible pulp and the crown. The pineapple wastes used in the study, peel and core, were separately processed. The peels were manually cut in small pieces using knife and then chopped in an electric blender (La Moulinette, Moulinex, Groupe SEB, France) to obtain a homogeneous mixture. Similarly, pineapple core mash was prepared. Samples of 50 g and 20 g of peel and core respectively, were stored in freezer (-18 °C) prior to use. 2.2 Saccharification procedure Hydrolysis of cellulose polymers and of sucrose in peel and core samples (treatment 1 – T1) Each sample of pineapple peel (50 g) and core (20 g) was added in duplicate at 1:2 ratio to distilled water and put into 100 mL Pyrex bottle sealed with screw cap. Then, hydrolysis was performed at pH 4.00 adding 6 g/kgfw of cellulase, 1 g/kgfw of hemicellulase, and 0.05 g/kgfw of pectinase under different times and temperatures: 4-8-18-24 h and 30-40-50 °C, respectively. Then, the samples were brought down to 21±2 °C, filtrated using cheese cloth, and poured to the beaker. The filtered sample was added with 0.05 g/kgfw of invertase and left for 3 h at 50 °C before being centrifuged at 3000 rpm for 15 min at 24 °C. Physical pre-treatment and use of amylolytic enzymes on peel and core samples (treatment 2 – T2) To enhance reducing sugar yield, pineapple peel and core samples were subjected to 10 and 30 min 143.27 kPa treatment before enzymatic hydrolysis with and without the addition of 0.025 % thermostable α-amylase (Venzyme/kgfw). Then, 0.05 % glucoamylase and pullulanase (Venzyme/kgfw) were mixed with cellulolytic enzymes and incubated under the conditions tested in T1 which allowed at gaining the greatest sugar yield. The samples were filtrated and incubated with invertase under the condition reported in T1. 2.3 Chemical analyses Pineapple waste samples were analyzed in duplicate. The reducing sugar content was determined on liquid aliquots of peel and core juice before and after T1 and T2 according to Lane and Eynon (1923) method with Crison compact titrator (Crison Instruments SA, Alella, Spain). Carrez I and Carrez II reactive for sugar analisys were provided by Carlo Erba reagents (Milan, Italy). The acidity and pH were measured using the Crison TitroMatic 1S (Crison Instruments SA, Alella, Spain). 2.4 Data analysis and statistics. All determinations were done in duplicate and a factorial ANOVA with Tukey’s test at p ≤ 0.05 was used to measure the significance of the differences among the conditions tested within each treatment. The statistics package IBM SPSS Statistics 19 (IBM Corporation, New York, USA) was used.

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3. Results and discussion
Acidity and pH of pineapple wastes before and after T1 are reported in Table 1. The results were in agreement with Abdullah and Hanafi’s study (2008), and with Sasaki et al. (1991) who reported the pH of pineapple juice in the range value of 3.6-4.6. Table 1: Chemical analysis of pineapple waste samples before (control) and after T1. Values are means ± SD (n=15). Within each row, different letters indicate statistically different values according to post-hoc comparison (Tukey’s test) at p ≤ 0.05
Analysis Acidity pH Control peel 2.55±0.97 a 4.16±0.45 a Peel 3.54±1.05 a 3.89±0.10 a Control core 3.46±0.90 a 3.93±0.10 a Core 4.42±1.37 a 3.85±0.08 a

The results of control samples indicated that peel and core from pineapple are a substrate suitable for cultivation of bacteria, i.e. potentially used as a carbon source for organic acid fermentation (Abdullah and Hanafi, 2008). Indeed, the saccharification process can be performed without changing the pH value of substrate, as pH of the wastes is ideal for the activity of cellulolytic and pectinolytic enzymes (Romsayud et al., 2009). 3.1 Hydrolysis of cellulose polymers and of sucrose in peel and core samples (T1) Hydrolysis with cellulase, hemicellulase, and pectinase was tested at three temperatures (30-40-50 ºC), in a time range of 4-8-18-24 h, in order to gain the highest yield of reducing sugars (Table 2). The enzymes tested were more effective in the peel samples, probably because of the higher cellulosic content compared with the core (Abdullah and Hanafi, 2008). Moreover, according with Tengbord et al. (2001) more than 8 h hydrolysis at 30 °C improved the final sugar yield. As a consequence, peel and core were submitted to 18 and 24 h hydrolysis at 40 and 50 °C (Figure 1). In agreement with De Prados et al. (2010), high temperature and long time were found to be favourable for maximum sugar yield. Once determined the optimal time-temperature (24 h-50 °C) values for cellulolytic hydrolysis, the subsequent action of other enzymes was tested. From the data received from literature (Abdullah and Hanafi, 2008; Hemalatha and Anbuselvi, 2013) the amount of sucrose in the pineapple liquid waste is in the range of 16.75 to 40.10 g/L, in a significant percentage out of the total amount of reducing sugars (about 40 g/L) which primarily consists of glucose (>20 g/L) and fructose (