1. INTRODUCTION
⌅The evaluation of any economic activity may be performed based on the economic value added (EVA) model developed in 1982 by G.B. Stewart, which is still used successfully (Stewart, 2013)Stewart BG. 2013. Best-Practice EVA. The Definitive Guide to Measuring and Maximizing Shareholder Value. John Wiley & Sons, Inc., Hoboken.. In such context the valorization of the biomass waste from the industrial production of wine is important, as obtaining added value is a priority for any sustainable economy.
Romania is a known wine producer, being the 11th producer in the world and the 5th in Europe (European Commission, 2019European Commission. 2019. Evaluation of the CAP measures applicable to the wine sector. Case study report, Publications Office of the European Union, Luxembourg. https://ec.europa.eu/info/sites/info/files/food-farming-fisheries/key_policies/documents/case-study-wine-evaluation-romania_en.pdf ). A sustainable development for winery implies the adequate management of resources in the production chain and reduction of waste (Maicas and Mateo, 2020Maicas S, Mateo JJ. 2020. Sustainability of Wine Production. Sustainability 12, 559 https://doi.org/10.3390/su12020559 ). Waste minimization through technological innovation will generate an EVA equity indicator (Machová and Vrbka, 2018Machová V, Vrbka J. 2018. Value generators for businesses in agricultura, in Löster T, Pavelka T (Eds) International Days of Statistics and Economics Conference Proceedings. MELANDRIUM, Slany, Czech Republic, 1123-1132. https://msed.vse.cz/msed_2018/article/57-Machova-Veronika-paper.pdf ). A waste reduction policy is recommended for achieving a sustainable wine making process (Devesa-Rey et al., 2011Devesa-Rey R, Vecino X, Varela-Alende JL, Barral MT, Cruz JM, Moldes AB. 2011. Valorization of winery waste vs. the costs of not recycling. Waste Manage. 31 (11), 2327-2335. https://doi.org/10.1016/j.wasman.2011.06.001 ) and the implementation of the “zero waste” concept (Donner et al., 2020Donner M, Gohier R, de Vries H. 2020. A new circular business model typology for creating value from agrowaste. Sci. Total Environ. 716, 137065. https://doi.org/10.1016/j.scitotenv.2020.137065 ).
Winery wastes are an important part of wine production. Grape pomace is a waste which results as a by-product from the must production; it is composed of around 30% stems, 30% seeds and 40% skins and pulp. It is considered an agro-industrial waste, representing about 25% (w/w) of the weight of grapes processed and more than 9 million tons annually (Sirohia et al., 2020Sirohia R, Tarafdarb A, Singha S, Negic T, Kumar Gaurd V, Gnansounou, Bharathirajag B. 2020. Green processing and biotechnological potential of grape pomace: Current trends and opportunities for sustainable biorefinery. Bioresour. Technol. 314, 123771. https://doi.org/10.1016/j.biortech.2020.123771 , Cvejic Hogervorst et al., 2017Cvejic Hogervorst J, Miljić U, Puškaš V. 2017. Extraction of Bioactive Compounds from Grape Processing By-Products, in Galanakis C (Ed.) Handbook of Grape Processing By-Products explores. Sustainable solutions. AcademicPress, Cambridge Massachusetts, 105-132. https://doi.org/10.1016/B978-0-12-809870-7.00005-3 ).
There are several proposals for solving the problem of pomace. It can be used in different applications such as functional foods and supplements, pharmaceutical and cosmetic products (Galanakis, 2020Galanakis C. 2020. Life cycle assesment in food industry, in Galanakis C (Ed) The Interaction of Food Industry and Environment. Academic Press, Cambridge Massachusetts, 377-386. https://doi.org/10.1016/B978-0-12-816449-5.00011-4 ). The dispersal of it into landfill seems ecologically inappropriate (Dwyer et al., 2014Dwyer K, Hosseinian F, Rod M. 2014. The market potential of grape waste alternatives. J. Food Res. 3 (2), 91-106. https://doi.org/10.5539/jfr.v3n2p91 ). Viable solutions for winery waste valorization are: oil production (Al-Juhaimi and Ozcan, 2018Al-Juhaimi F, Ozcan MM. 2018. Effect of cold press and soxhlet extraction systems on fatty acid, tocopherol contents, and phenolic compounds of various grape seed oils. J. Food Process. Pres. 42 (1), e13417. https://doi.org/10.1111/jfpp.13417 ), manufacture of composite materials (Barbieri et al., 2013Barbieri L, Andreola F, Lancellotti I, Taurino R. 2013. Management of agricultural biomass wastes: Preliminary study on characterization and valorisation in clay matrix bricks. Waste Manage. 33 (11), 2307-2315. https://doi.org/10.1016/j.wasman.2013.03.014 ), extraction of the antioxidants for food supplements (Nowshehri et al., 2015Nowshehri JA, Bhat ZA, Shah MY. 2015. Blessings in disguise: Bio-functional benefits of grape seed extracts. Food Res. Int. 77 (3), 333-348. https://doi.org/10.1016/j.foodres.2015.08.026 ), bioconversion to valuable chemicals or biofuels (Rani et al., 2020Rani J, Yadav Indrajeet, Rautela A, Kumar S. 2020. Biovalorization of winery industry waste to produce value-added products, in Rathinam NK, Sani RK (Eds) Biovalorisation of Wastes to Renewable Chemicals and Biofuels, Elsevier, Amsterdam, 63-85. https://doi.org/10.1016/B978-0-12-817951-2.00004-3 ; Zacharof, 2017Zacharof MP. 2017. Grape Winery Waste as Feedstock for Bioconversions: Applying the Biorefinery Concept. Waste Biomass Valor. 8, 1011-1025.https://doi.org/10.1007/s12649-016-9674-2 ).
A better biomass valorization implies the separation of the pomace components (Toscano et al., 2013Toscano G, Riva G, Duca D, Foppa Pedretti E, Corinaldesi F, Rossini G. 2013. Analysis of the characteristics of the residues of the wine production chain finalized to their industrial and energy recovery. Biomass Bioenerg. 55, 260-267. https://doi.org/10.1016/j.biombioe.2013.02.015 ). The seeds represent a great part of these wastes, approximately 47% on dry base (Zhang et al., 2017Zhang N, Hoadley A, Patel J, Lim S, Li C. 2017. Sustainable options for the utilization of solid residues from wine production. Waste Manage. 60, 173-183. https://doi.org/10.1016/j.wasman.2017.01.006 ), making their valorization vital.
The paper investigates the valorization of grape seeds by oil extraction. Residual grape seeds from six Romanian cultivars were processed. In order to increase the outcome, a pre-treatment of the seeds with a commercial pectin lyase was experimented. The enzymatic treatment seems appropriate due to the progress in the industrial production of enzymes as well as their numerous industrial applications derived from their properties, namely: low toxicity, energy saving due to mild work conditions, biodegradability, etc. (Choi et al., 2015Choi J-M, Han S-S, Kim H-S. 2015. Industrial applications of enzyme biocatalysis: Current status and future aspects. Biotechnol. Adv. 33 (7) 1443-1454. https://doi.org/10.1016/j.biotechadv.2015.02.014 ). An improvement in oil quantity and/or quality was predicted.
2. MATERIALS AND METHODS
⌅2.1. Materials
⌅2.1.1. Sample preparation
⌅The grape seeds were isolated from the pomace resulting from the wine making process, washed, dried for 24 hours (h) at room temperature, and ground. Seeds from the grape varieties of the 2015 harvest were investigated, namely four red brands, including Cabernet Sauvignon, Feteasca Neagra, Merlot, Pinot Noir and two white brands Columna and Riesling Italian. The material was supplied by the winery of Murfatlar, situated in Dobrogea, a south-eastern region of Romania.
2.1.2. Commercial enzyme
⌅The pectin lyase solution (Pectinex XXL, activity 10.000 U mL-1 according to the supplier) was purchased from Novozymes A/SNovozymes A/S. 2012. Product Data Sheet Pectinex® XXL www.sinerji-as.com/files/0ec4f308-1c61-41b6-a897-725d0aba8a52.pdf (Bagsvaerd, Denmark).
2.1.3. Chemicals
⌅The 10-14% BF3 solution in methanol was supplied by Merck (Darmstadt, Germany). Petroleum ether (b.p. 40-60 ºC), analytical grade methylene chloride, methanol, and 96% ethanol solvents were purchased from Sigma Aldrich and were used as delivered. The citric acid and disodium phosphate dihydrate for the buffer solution were supplied by Sigma Aldrich.
2.2. Equipment and procedures
⌅UV-Vis spectra (200-800 nm) were acquired on a Helios Beta apparatus with Vision software (Thermo Electron Corporation, Waltham, MA, United States).
The 1H-NMR spectra were obtained on a Bruker Avance III 400 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany).
The GC analyses were performed on an Agilent Technologies 7890A instrument (2850 Centerville Road Wilmington, DE 19808-1610 USA), provided with a flame ionization detector.
2.2.1. Enzymatic treatment
⌅Portions of 10 g ground grape seeds were kept for certain amounts of time in a solution of 3 mL commercial enzyme and 37 mL buffer solution of pH 3.5 (0.1M citric acid and 0.2M Na2HPO4, 2.3/1 volume ratio), in 100 mL capped glass amber flasks, at room temperature (20 ºC), with gentle intermittent stirring. The optimal time for treatment was established based on the oil yield (see Figure 1). For each grape seed variety the experiments were conducted in triplicate. After treatment, the seeds were separated by filtration, washed and dried before extraction.
The residual solution was concentrated in a Heidolph rotary evaporator, in a water bath (50 ºC), and the residue was studied by NMR spectroscopy.
For checking the buffer effect on the grape seed a number of experiments were performed by keeping the seeds in the buffer solution (ratio w/v = 1/4) for 24 hours, at room temperature. The quantity of the oil from these samples was in the same range as the corresponding untreated oil.
2.2.2. Oil extraction
⌅The oil samples were obtained from the dried ground seeds (pre-treated or not with enzyme) by the Soxhlet extraction method with petroleum ether (b.p. 40-60 ºC), following the protocol ISO 659 (2009)ISO 659, 2009. Oilseeds - Determination of oil content (Reference method).. The solvent was partially recovered by evaporation under atmospheric pressure, heated on a water bath at 65 ºC, using a Heidolph rotary evaporator. The weight of the oil was measured with an analytical balance (accuracy of ± 0.0001 g) and the corresponding volume was calculated using the density value for grape seed oil of 0.92 g/mL (Ceriani et al., 2008Ceriani R, Paiva FR, Gonçalves CB, Batista EAC, Meirelles AJA. 2008. Densities and Viscosities of Vegetable Oils of Nutritional Value. J. Chem. Eng. 53 (8) 1846-1853. https://doi.org/10.1021/je800177e ). The value of the density was confirmed by checking on a number of experimental samples. The experiments were performed in triplicate, the average volumes of oil being presented in Figure 2. The initial quantity of seeds was the same for both types of experiments (with or without pre-treatment) as measured before pre-treatment.
2.2.3. GC analysis of the grape seed oil composition (FAP)
⌅The grape seed oil FAPs were determined following the ISO 12966 (2015)ISO 12966, 2015. Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 4: Determination by capillary gas chromatography. protocol. The free fatty acids were obtained by treatment of 0.5 mg oil with 0.9 mL NaOH 0.5 M solution in methanol, at reflux, for 10 min. Then, they were esterified by treatment with 0.1 mL BF3 solution and 0.8 mL methanol, at reflux, for another 10 min. After dilution with 4 mL distilled water, the fatty acid methyl esters (FAMEs) were extracted with methylene chloride and were submitted to a standard gas chromatographic (GC) analysis. GC analysis was performed on a highly polar capillary column Supelco SPTM 2560 (100 m × 0.25 mm, 0.20 μm film) with a biscyanopropyl stationary phase. A certified reference material (SupelcoTM 37 Component FAME Mix) was used for the identification of the FAPs (see table 1). For each oil sample, three GC analyses were performed. The composition was expressed as a percentage based on the ratio between each peak area and the total area.
| Cultivar | FN | FNE | CS | CSE | ME | MEE | PN | PNE | RI | RIE | CO | COE |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Fatty acid | FA Content (%) | |||||||||||
| Palmitic and Stearic | 10.58±0.02 | 10.71±0.01 | 11.11±0.02 | 11.29±0.01 | 9.02±0.02 | 9.55±0.03 | 10.34±0.01 | 10.42±0.01 | 9.88±0.02 | 9.99±0.03 | 8.78±0.01 | 8.83±0.02 |
| Oleic (18:1 cis-9) | 15.47±0.01 | 15.84±0.03 | 12.14±0.02 | 12.36±0.03 | 12.18±0.02 | 12.62±0.02 | 14.39±0.01 | 14.53±0.03 | 18.10±0.01 | 18.18±0.01 | 17.85±0.01 | 17.90±0.01 |
| Linoleic (18:2 cis,cis-9,12) | 73.76±0.02 | 73.28±0.01 | 76.48±0.02 | 76.12±0.01 | 78.34±0.01 | 77.60±0.01 | 75.05±0.02 | 74.76±0.01 | 71.79±0.02 | 71.54±0.03 | 73.13±0.01 | 73.00± 0.02 |
| Linolenic (18:3 cis,cis,cis 9,12,15) | 0.19±0.01 | 0.22±0.01 | 0.27±0.01 | 0.22±0.02 | 0.28±0.02 | 0.24±0.01 | 0.23±0.01 | 0.27±0.01 | 0.23±0.01 | 0.28±0.01 | 0.24±0.01 | 0.27±0.01 |
| TAC (μmol TE /100 g oil) | 72.84±1.14 | 176.75±1.31 | 62.43±1.49 | 131.64±1.45 | 109.26±1.32 | 216.18±1.19 | 134.19±1.28 | 234.48±1.54 | 33.45±1.31 | 58.73±1.40 | 50.14±1.33 | 85.12±1.49 |
Standard
deviation for n = 3
FAPs - fatty acid profiles, FA - fatty acids, TAC -
total antioxidative capacity, TE - trolox equivalent, FN - Feteasca
Neagra, FNE - Feteasca Neagra pre-treated with enzyme, CS - Cabernet
Sauvignon, CSE - Cabernet Sauvignon pre-treated with enzyme, ME -Merlot,
MEE -Merlot pre-treated with enzyme, PN - Pinot Noir, PNE - Pinot Noir
pre-treated with enzyme, CO - Columna, COE - Columna pre-treated with
enzyme, RI - Riesling Italian, RIE - Riesling Italian pre-treated with
enzyme.
2.2.4. Determination of the reductive power of the oil samples
⌅The CUPRAC assay (Apak et al., 2008Apak R, Guclu K, Ozyurek M, Celik SE. 2008. Mechanism of antioxidant capacity assays and the CUPRAC (cupric ion reducing antioxidant capacity) assay. Microchim. Acta 160, 413-419. https://doi.org/10.1007/s00604-007-0777-0 ) was applied to assess the total antioxidative capacity (TAC) of the oil. Samples of oil extracted by sonication in ethanol (1/10, v/v) were treated with 1 mL CuSO4 10-2 M aqueous solution, 1 mL of 7.5 × 10-3 M Neocuproine ethanolic solution, 1 mL CH3COONH4 buffer (pH 7) and 0.4 mL distilled water. After stirring for 30 min, at room temperature, the absorbance (Abs) at 450 nm was measured. TAC was expressed as a trolox equivalent (TE, μmol per 100 g oil) based on a previously drawn calibration curve (y = 0.058x; R2 = 0.99). The average values obtained from three experiments for each oil sample are presented in Table 1.
2.2.5. Determination of the total polyphenol content (TPPC) of the grape seeds
⌅The polyphenols (PPs) were quantified using the known Folin-Ciocalteu (FC) method (Ballus et al., 2015Ballus CA, Meinhart AD, de Souza Campos FA, Teixeira Godoy H. 2015. Total Phenolics of Virgin Olive Oils Highly Correlate with the Hydrogen Atom Transfer Mechanism of Antioxidant Capacity. J. Am. Oil. Chem. Soc. 92, 843-851. https://doi.org/10.1007/s11746-015-2629-0 ). Dried seeds (2 g), before and after the oil extraction, were extracted with 9 mL ethanol/water (2/1, v/v). Extract samples (0.1 mL) were mixed with the FC reagent (0.5 mL), distilled water (1.5 mL) and, after 5 min, with 20% Na2CO3 solution (1.5 mL). The mixture was incubated for 2 h, at room temperature, in the dark. The Abs at 750 nm was measured vs. a blank. The analyses were performed in triplicate, for seeds with or without enzymatic pre-treatment, before and after oil extraction. The TPPC was expressed as gallic acid equivalents (GAE, mg per 100 g dried seeds) derived from a previously obtained calibration curve (y = 0.061x; R2 = 0.98) (Table 2).
| Sample Variety | PN | ME | FN | CS | CO | RI |
|---|---|---|---|---|---|---|
| Analyzed sample | Polyphenols as GAE (mg/100 g seeds) | |||||
| Initial | 132.27±1.33 | 148.52±1.25 | 133.74±1.33 | 149.26±1.35 | 133.74±1.39 | 134.48±1.34 |
| After extraction | 127.09±1.53 | 128.57±1.34 | 130.05±1.44 | 144.09±1.59 | 128.57±1.35 | 127.09±1.33 |
| After pre-treatment and extraction | 124.14±1.49 | 125.62±1.43 | 115.27±1.48 | 135.22±1.54 | 127.83±1.58 | 124.14±1.34 |
Standard
deviation for n = 3
GAE - gallic acid equivalents, PN - Pinot Noir, ME
-Merlot, FN - Feteasca Neagra,
CS - Cabernet Sauvignon, CO - Columna, RI
- Riesling Italian.
2.3. Statistical analysis
⌅To identify sample clusters the principal component analysis (PCA) was applied to the grape seed oil compositions. The independent variables were selected by PCA. The ANOVA program was used for the comparison of the mean values. Data analysis was performed using the XLSTAT 2015 software (Addinsoft).
3. RESULTS AND DISCUSSION
⌅3.1. Effects of the pre-treatment with pectin lyase on the grape seed oil yields
⌅The research was focused on the valorization of grape seeds from the waste of a Romanian winery Murfatlar starting with the production of the grape oil. The oil obtained from grape seeds is a valuable material (Shinagawa et al., 2015Shinagawa FB, de Santana FC, Torres LRO, Mancini-Filho J. 2015. Grape seed oil: a potential functional food? Food Sci. Technol. (Campinas) 35, 399-406. https://doi.org/10.1590/1678-457X.6826 ). Recent clinical trials showed the beneficial effects of this oil on human health (Kaseb and Biregani 2016Kaseb F, Biregani AN. 2016. Effects of Olive Oil and Grape Seed Oil on Lipid Profile and Blood Pressure in Patients with Hyperlipidemia: A Randomized Clinical Trial. Food Nutr. Sci. 7 (8), 682-688. https://doi.org/10.4236/fns.2016.78069 ; Ismail et al., 2016Ismail AF, Salem AA, Eassawy MM. 2016. Hepatoprotective effect of grape seed oil against carbon tetrachloride induced oxidative stress in liver of γ-irradiated rat. J. Photochem. Photobiol B: Biology 160, 1-10.).
The solvent extraction standardized method used for the oil separation is simple and less energy demanding than other procedures (Castro-Lopez et al., 2016Castro-Lopez C, Rojas R, Sanchez-Alejo EJ, Nino-Medina G, Martínez-Avila GCG. 2016. Phenolic Compounds Recovery from Grape Fruit and By-Products: An Overview of Extraction Methods, in: Morata A and Loira I (Eds.) Grape and wine biotechnology, InTech, Rijeka, 103-123. https://doi.org/10.5772/64821 ). The yield is relatively high due to the permanent contact of ground seeds with clean solvent, which favors the extraction. The grinding of the seeds helps due to the generation of a larger contact area for seed-solvent. The solvent used, petroleum ether, is industrially accessible and not so toxic because of degrading rapidly in soil and water and having a half-life of 3-8 days in the air (Nalliah, 2014Nalliah RE. 2014. Petrolem ether, in Wexler Ph (Chief Ed.) Encyclopedia of Toxicology, 3rd edition, Academic Press, Cambridge Massachusetts, 834-837. https://doi.org/10.1016/B978-0-12-386454-3.00418-8 ).
Vegetable oils may be produced also by cold pressing the seeds but the yield is usually lower and it is difficult to obtain a constant quality of the product (Ustun-Argon et al., 2020Ustun Argon Z, Celenk VU, Gumus ZP. 2020 Cold pressed grape (Vitis vinifera) seed oil, in Ramadan MF (Ed.) Green Technology, Bioactive Compounds, Functionality, and Applications. Academic Press, Cambridge Massachusetts, 39-52. https://doi.org/10.1016/B978-0-12-818188-1.00005-0 ). The yield may be improved by using shockwaves but the process is complex and not always economically viable (Marousek, 2015aMarousek J. 2015a. Economic Analysis of the Pressure Shockwave Disintegration Process. IJSGE 12 (12) 1232-1235. https://doi.org/10.1080/15435075.2014.895740 ).
By comparison with other methods the solvent extraction of the grape seed oil has a low processing cost and is easy to handle (Galankis, 2020Galanakis C. 2020. Life cycle assesment in food industry, in Galanakis C (Ed) The Interaction of Food Industry and Environment. Academic Press, Cambridge Massachusetts, 377-386. https://doi.org/10.1016/B978-0-12-816449-5.00011-4 ).
For improving the extraction process of bioactive compounds a number of procedures have been experimented, among which can be found supercritical fluid extraction, pressurized liquid extraction, microwave and ultrasound assisted extraction, etc. (Azmir et al., 2013Azmir J, Zaidul ISM, Rahman MM, Sharif KM, Mohamed A, Sahena F, Jahurul MHA, Ghafoor K. 2013. Techniques for extraction of bioactive compounds from plant materials: A review. J. Food Eng. 117, 426-436. https://doi.org/10.1016/j.jfoodeng.2013.01.014 ; Kumar et al., 2017Kumar K, Yadav AN, Kumar V, Vyas P, Dhaliwal HS. 2017. Food waste: a potential bioresource for extraction of nutraceuticals and bioactive compounds. Bioresour. Bioprocess. 4, 18. https://doi.org/10.1186/s40643-017-0148-6 ). Some of these procedures require investments which are suitable only for a large scale production.
Improvements of the extraction process may be done by pre-treatment of seeds before extraction. The literature has reported the following procedures: heating (Ustun-Argon et al., 2020Ustun Argon Z, Celenk VU, Gumus ZP. 2020 Cold pressed grape (Vitis vinifera) seed oil, in Ramadan MF (Ed.) Green Technology, Bioactive Compounds, Functionality, and Applications. Academic Press, Cambridge Massachusetts, 39-52. https://doi.org/10.1016/B978-0-12-818188-1.00005-0 ), shockwave treatment (Marousek, 2015aMarousek J. 2015a. Economic Analysis of the Pressure Shockwave Disintegration Process. IJSGE 12 (12) 1232-1235. https://doi.org/10.1080/15435075.2014.895740 ), enzymatic hydrolysis (Passos et al., 2009Passos CP, Yilmaz S, Silva CM, Coimbra MA. 2009. Enhancement of grape seed oil extraction using a cell wall degrading enzyme cocktail. Food Chem. 115 (1), 48-53. https://doi.org/10.1016/j.foodchem.2008.11.064 ) or a combination of these methods (Marousek et al., 2015bMaroušek J, Hašková S, Maroušková A, Myšková K, Vaníčková R, Váchal J, Vochozka M, Zeman R, Žák J. 2015b. Financial and Biotechnological Assessment of New Oil Extraction Technology. Energy Sources, Part A: Recovery, Utilization, and Environmental Effects 37 (16) 1723-1728. https://doi.org/10.1080/15567036.2015.1048391 ).
According to the literature (González-Centeno et al., 2010Gonzalez-Centeno MR, Rossello C, Simal S, Garau MC, Lopez F, Femenia A. 2010. Physico-chemical properties of cell wall materials obtained from ten grape varieties and their byproducts: grape pomaces and stems. LWT - Food Sci. Technol. 43 (10), 1580-1586. https://doi.org/10.1016/j.lwt.2010.06.024 ), pectins are polymer constituents of the cell walls in fresh grapes or in grape pomace, which bind other constituents. The study of oil distribution inside the seed showed its presence near the external tegument (Pope et al., 1993Pope JM, Jonas D, Walker RR. 1993. Applications of NMR micro-imaging to the study of water, lipid, and carbohydrate distribution in grape berries. Protoplasma 173, 177-186. https://doi.org/10.1007/BF01379006 ). There is a relatively strong interaction between the oil and the seed walls leading to supramolecular structures (Scollary et al., 2012Scollary GR, Pasti G, Kallay M, Blackman J, Clark AC. 2012. Astringency response of red wines: Potential role of molecular assembly. Trends Food Sci. Technol. 27 (1), 25-36. https://doi.org/10.1016/j.tifs.2012.05.002 ) which block oil removal. Thus, to improve oil extraction, an enzymatic pre-treatment was carried out, with an enzyme specific for pectin breaking. The enzymatic treatment (see paragraph 2.2.1) was performed in batch mode, by maintaining the seeds in a buffered solution of Pectinex XXL, at room temperature and pH 3.5, parameters recommended for this type of enzymes (Najafian et al., 2009Najafian L, Ghodsvali A, Haddad Khodaparast MH, Diosady LL. 2009. Aqueous extraction of virgin olive oil using industrial enzymes. Food Res. Int. 42 (1), 171-175. https://doi.org/10.1016/j.foodres.2008.10.002 ). The pectin lyase cleaves the pectin (Yadav et al., 2009Yadav S, Yadav PK, Yadav D, Yadav KDS. 2009. Pectin lyase: A review. Process Biochem. 44 (1), 1-10. https://doi.org/10.1016/j.procbio.2008.09.012 ) giving water soluble compounds (saturated and unsaturated pectic-oligosaccharides). The fragmentation process is improved by the presence of citric acid, which acts both as buffer and ligand for calcium ions (Stanescu et al., 2010Stanescu MD, Dochia M, Radu D, Sirghie C. 2010. Green solution for cotton scouring. Fibres Text. East. Eur. 3 (80), 109-111. www.fibtex.lodz.pl ). Thus, these ions, which reinforce the pectin structure by making bridge bonds between chains (Ochoa-Villarreal et al., 2012Ochoa-Villarreal M, Aispuro-Hernandez E, Vargas-Arispuro I, Martínez-Tellez, MA. 2012. Plant Cell Wall Polymers: Function, Structure and Biological Activity of Their Derivatives, in De Souza Gomes A (Ed.) Polymerization, InTech, Rijeka, pp. 63-84. ), are removed, aiding in the elimination of pectins. The fracture of the cell walls facilitates the access of solvent and improves the oil extraction yield. The chosen pH was the optimal one for the commercial enzyme used. Room temperature was suitable for the enzyme and did not require added cost for heating. The only parameter to be established was the treatment time. An optimal time of 24 hours was established for the enzymatic treatment based on the volume of the resulted oils for different time ranges (see Figure 1).
The enzymatic pre-treatment of seeds, for 24 h with Pectinex XXL, led to more oil, the quantities obtained being 101.3-104.3% compared to untreated seeds (see Figure 2). Comparable results for the enzymatic pre-treatment of seeds have been found by other researchers (Passos et al., 2009Passos CP, Yilmaz S, Silva CM, Coimbra MA. 2009. Enhancement of grape seed oil extraction using a cell wall degrading enzyme cocktail. Food Chem. 115 (1), 48-53. https://doi.org/10.1016/j.foodchem.2008.11.064 ). The performance of the described procedure consisted of lower additional costs than those with enzyme cocktails (Passos et al., 2009Passos CP, Yilmaz S, Silva CM, Coimbra MA. 2009. Enhancement of grape seed oil extraction using a cell wall degrading enzyme cocktail. Food Chem. 115 (1), 48-53. https://doi.org/10.1016/j.foodchem.2008.11.064 ; Marousek et al., 2015Marousek J. 2015a. Economic Analysis of the Pressure Shockwave Disintegration Process. IJSGE 12 (12) 1232-1235. https://doi.org/10.1080/15435075.2014.895740 ). A preliminary calculation (Tociu, 2019aTociu M. 2019a. PhD Thesis, Study of natural fats from vegetable oils and milk: composition, factor of influence and authentication, Aspects of waste valorization, Bucharest) indicated a reduced cost (of over 100 times) for the pre-treatment with only Pectinex XXL compared to the cost of the treatment with a mixture of cellulase, xylanase and pectinase performed by Passos et al., (2009)Passos CP, Yilmaz S, Silva CM, Coimbra MA. 2009. Enhancement of grape seed oil extraction using a cell wall degrading enzyme cocktail. Food Chem. 115 (1), 48-53. https://doi.org/10.1016/j.foodchem.2008.11.064 , the differences in oil yield being insignificant.
Unfortunately, there are limitations to the application of enzymatic treatments such as the reproducibility of enzyme biosynthesis and the possible negative effects of the stabilizers on commercial products. These are impediments to the application of enzymatic treatments at large scale in processes where the enzyme properties (content, activity) are of great importance.
3.2. Effects of pre-treatment with pectin lyase on the oil properties
⌅3.2.1. FAPs of the grape seed oils
⌅One of the most important features of lipids is their fatty acid profile (FAP). The previous research involving enzymatic pre-treatment (Passos et al., 2009Passos CP, Yilmaz S, Silva CM, Coimbra MA. 2009. Enhancement of grape seed oil extraction using a cell wall degrading enzyme cocktail. Food Chem. 115 (1), 48-53. https://doi.org/10.1016/j.foodchem.2008.11.064 ; Marousek et al., 2015bMaroušek J, Hašková S, Maroušková A, Myšková K, Vaníčková R, Váchal J, Vochozka M, Zeman R, Žák J. 2015b. Financial and Biotechnological Assessment of New Oil Extraction Technology. Energy Sources, Part A: Recovery, Utilization, and Environmental Effects 37 (16) 1723-1728. https://doi.org/10.1080/15567036.2015.1048391 ) did not check this aspect for the oil applications. Thus, the extracted oils were analyzed by a GC standard method (see paragraph 2.2.3). The FAPs of the oil samples are presented in Table 1.
The main component of these oils was linoleic acid, a representative polyunsaturated fatty acid (PUFA). The saturated fatty acid (SFA) content was around 10%. Similar FAPs were revealed for Portuguese grape varieties (Fernandes et al., 2013Fernandes L, Casal S, Cruz R, Pereira JA, Ramalhosa E. 2013. Seed oils of ten traditional Portuguese grape varieties with interesting chemical and antioxidant properties. Food Res. Int. 50 (1), 161-166. https://doi.org/10.1016/j.foodres.2012.09.039 ) and Spanish wines with protected denomination of origin (Bada et al., 2015Bada JC, León-Camacho M, Copovi P, Alonso L. 2015. Characterization of grape seed oil from wines with protected denomination of origin (PDO) from Spain. Grasas Aceites 66 (3), e085. https://doi.org/10.3989/gya.1063142 ). Due to their low SFAs and high linoleic acid contents these oils present good nutritional qualities (Alsharari et al., 2017Alsharari ZD, Riserus U, Leander K, Sjogren P, Carlsson AC, Vikstrom M, Laguzzi F, Gigante B, Cederholm T, De Faire U, Hellenius ML, Marklund M. 2017. Serum Fatty Acids, Desaturase Activities and Abdominal Obesity - A Population-Based Study of 60-Year Old Men and Women. PloS one 12 (1), 0170684. https://doi.org/10.1371/journal.pone.0170684 ). According to the experimental results the pre-treatment changed the FAPs only slightly (≤ 0.4%).
As expected, the statistical analysis revealed a close correlation between PUFA and linoleic acid (y = 1.004x+0.1003; R2 = 0.99). The grape variety showed an impact on the content in fatty acids in oil. Principal component analysis (PCA) applied to FAPs disclosed the differences or similarities of the analyzed oils (Figure 3). The PCA analysis revealed the following eigenvalues: F1: 2.067, F2: 1.375, F3: 0.558, F4: 0.000. Thus, the variance values of the principal components (PCs) were 51.66% (F1), 34.39% (F2), 13.95% (F3) and 0.003% (F4). The cumulative percentage for PC1 and PC2 coordinates was over 86.00% so only these 2 PCs had to be considered.
The oils from Columna and Riesling (with a higher content in oleic acid) as well as Merlot and Cabernet Sauvignon (with higher content in linoleic acid) showed a good discrimination on the F1 (PC1) direction; whereas Feteasca Neagra and Cabernet Sauvignon (with high content in SFA) exhibited good discrimination on the F2 (PC2) direction.
A comparison with oils from other seeds seemed of interest. According to the literature the average values for PUFAs (%) (Chira et al., 2011Chira N, Todasca MC, Nicolescu A, Rosu A, Nicolae M, Rosca SI. 2011. Evaluation of the Computational Methods for Determining Vegetable Oils Composition using 1H-NMR Spectroscopy. Rev. Chim., 26 (1) 42-46. https://www.revistadechimie.ro/ ) for oils obtained from sunflower (59.77 ± 4.80), soybean (55.02 ± 2.88) and rapeseed (25.95 ± 1.70) were by far lower than those for the grape seed oil (75.00 ± 2.40%). Thus, this oil stands out for its high content in healthy PUFAs.
3.2.2. Total antioxidant capacity (TAC) of the grape seed oils
⌅Besides FAP, the antioxidant capacity of vegetable oils is essential for their applications (Chambre et al., 2019Chambre DR, Tociu M, Stanescu MD, Popescu C. 2019. Influence of composition on the thermal behavior of oils extracted from the seeds of some Romanian grapes. J. Sci. Food Agric. 99 (14), 6324-6332. https://doi.org/10.1002/jsfa.9909 ). The method used to establish the TAC was the CUPRAC assay. This assay is recommended due to its simplicity, reduced cost and reduced reaction time (Apak et al., 2008Apak R, Guclu K, Ozyurek M, Celik SE. 2008. Mechanism of antioxidant capacity assays and the CUPRAC (cupric ion reducing antioxidant capacity) assay. Microchim. Acta 160, 413-419. https://doi.org/10.1007/s00604-007-0777-0 ). It gives an accurate estimation of the TAC of the analyzed sample. The experimental results obtained for the studied oils are presented in Table 1. The oils obtained after the enzymatic treatments of seeds are richer in antioxidants, with the TAC values being 1.7-2.4 times higher compared to those of oils from untreated seeds (see Table 1). The enzymatic treatment destroyed not only the interactions of oil with the cell walls but also that of the antioxidants (Chamorro et al., 2012Chamorro S, Viveros A, Alvarez I, Vega E, Brenes A. 2012. Changes in polyphenol and polysaccharide content of grape seed extract and grape pomace after enzymatic treatment. Food Chem. 133 (2), 308-314. https://doi.org/10.1016/j.foodchem.2012.01.031 ), increasing the quantities of extracted antioxidants. As expected, the oils obtained from the red grape varieties had higher antioxidant contents and improved health benefits.
A comparison of the TAC of grape seed oils (see Table 1) with other vegetable oil TACs is of interest. Thus, from the literature data the TAC values, expressed as TE (mmol kg-1) are as follows: 1.79 for extra virgin olive oil, 2.20 (soybean), 1.29 (corn), 1.17 (sunflower) (Pellegrini et al., 2003Pellegrini N, Serafini M, Colombi B, Del Rio D, Salvatore S, Bianchi M, Brighenti F. 2003. Total Antioxidant Capacity of Plant Foods, Beverages and Oils Consumed in Italy Assessed by Three Different In Vitro Assays. J. Nutr. 133 (9), 2812-2819. https://doi.org/10.1093/jn/133.9.2812 ). Due to the enzymatic treatment the related grape seed oils are ranked in better positions by their TE values.
3.3. Complementary possible valorization of other wastes
⌅The processing of grape seeds for oil extraction generates new wastes. Solutions have to be found for further valorization.
An investigation by 1H-NMR (see Figure 4b) of the residue resulting from the concentration of the solutions obtained after enzymatic treatment using a rotavap showed the presence of valuable products.
Occurrence of pectic-oligosaccharides was suggested by the gel aspect of the residue, as well as the specific 1H-NMR peaks at 4.2-4.5 ppm and around the 5.1 ppm (Winning et al., 2007Winning H, Viereck N, Norgaard L, Larsen J, Engelsen SB. 2007. Quantification of the degree of blockiness in pectins using 1H NMR spectroscopy and chemometrics. Food Hydrocoll. 21 (2), 256-266. https://doi.org/10.1016/j.foodhyd.2006.03.017 ). Other specific peaks are covered by the concentrated commercial enzyme signal (see Figure 4a) and the signal of the citric acid (2.5-2.8 ppm) from the buffer. Small peaks at 6.6-7.5 ppm evidenced the presence of aromatic compounds which were most likely traces of polyphenols (PPs) (Franz et al., 2014Franz AH, Serebnitskaya I, Guidal G, Wallis C. 2014. Structure assignment and H/D-exchange behavior of several glycosylated polyphenols (14-8583QP). ARKIVOC V, 94-122. https://doi.org/10.3998/ark.5550190.p008.583 ). There was no peak at 5.29 ppm in the residue spectrum (Figure 4b), signal characteristic for -CH=CH- of fatty acid (Chambre et al., 2019Chambre DR, Tociu M, Stanescu MD, Popescu C. 2019. Influence of composition on the thermal behavior of oils extracted from the seeds of some Romanian grapes. J. Sci. Food Agric. 99 (14), 6324-6332. https://doi.org/10.1002/jsfa.9909 ) as one may see in Figure 5, proving that no oil was lost during pre-treatment.
The residual grape seeds from the extraction of oil may be another source for PPs (Maier et al., 2009Maier T, Schieber A, Kammerer DR, Carle R. 2009. Residues of grape (Vitis vinifera L.) seed oil production as a valuable source of phenolic antioxidants. Food Chem. 112 (3), 551-559. https://doi.org/10.1016/j.foodchem.2008.06.005 ) as proven by the performed Folin-Ciocalteu assay presented in Table 2.
After the extraction of the oil, the seeds hold over 86% of the initial quantity of PPs, most likely due to the reduced solubility of these compounds in petroleum ether. The literature data claim that vitamin E (tocopherols and tocotrienols) is the main antioxidant in grape seed oils (Wen et al., 2016Wen X, Zhu M, Hu R, Zhao J, Chen Z, Li J, Ni Y. 2016. Characterisation of seed oils from different grape cultivars grown in China. J. Food Sci. Technol. 53, 3129-3136. https://doi.org/10.1007/s13197-016-2286-9 ). This fact was also confirmed by the thermal behavior of grape oils (Chambre et al., 2019Chambre DR, Tociu M, Stanescu MD, Popescu C. 2019. Influence of composition on the thermal behavior of oils extracted from the seeds of some Romanian grapes. J. Sci. Food Agric. 99 (14), 6324-6332. https://doi.org/10.1002/jsfa.9909 ).
Further investigation into the valorization of residual grape seed after extraction were also performed by isolating the PPs and using the ligno-cellulosic material for dye adsorption (Tociu, 2019aTociu M. 2019a. PhD Thesis, Study of natural fats from vegetable oils and milk: composition, factor of influence and authentication, Aspects of waste valorization, Bucharest; Tociu et al., 2019bTociu M, Oprea O, Stanescu MD. 2019b. Preliminary investigations for the valorization of grape seeds after the oil extraction. U.P.B. Sci. Bull., Series B 81 (2), 47-56. https://www.scientificbulletin.upb.ro/rev_docs_arhiva/full828_896673.pdf ).
The economic impact of the enzymatic pre-treatment has to be analyzed. Therefore, an accurate EVA analysis should be performed, considering all the by-products from the grape seeds (oil, PPs, adsorbents, char) not only the oil. Thus, new investigations must be carried out.
4. CONCLUSIONS
⌅The paper presents the effect of a pectin lyase pre-treatment on grape seeds from six Romanian grape cultivars, raw materials for oil extraction. The following assertions resulted from the experimental work:
The effect of treatment on oil quantity is comparable to processes using cocktails of enzymes.
The statistical analysis indicates the influence of the seed origin on the FAP.
The enzymatic treatment does not affect the FAP of the extracted oils, which contain a high percentage of healthy unsaturated fatty acids.
The enzymatic pre-treatment increased significantly the antioxidant capacity of the extracted grape seed oils.
The antioxidant capacity of the oils is based mostly on vitamin E components as experimentally proven. Most of PPs remain in seeds.
The pre-treatment is performed in a batch system and in mild conditions not needing a complex production unit.
Further investigation into the residual solutions resulting from the enzymatic treatment to recover valuable products, e.g. oligopectins and PPs, are required.