A new fermented beverage from sugarcane (Saccharum officinarum L.) T molasses: Analysis of physicochemical properties and antioxidant capacity, and comparison with other industrial alcohol products

Elsevier Editorial System(tm) for LWT - Food Science and Technology Manuscript Draft Manuscript Number: Title: A new fermented beverage from sugarcane (Saccharum officinarum L.) molasses: analysis of physicochemical properties and antioxidant capacity, and comparison with other industrial alcohol products. Article Type: Research paper Keywords: Antioxidant capacity; Mead; Honey; Sugarcane molasses; Phenolics. Corresponding Author: Dr. José Javier Quesada-Granados, Ph.D. Corresponding Author's Institution: Universidad de Granada First Author: Cristina Samaniego-Sánchez, Ph.D. Order of Authors: Cristina Samaniego-Sánchez, Ph.D.; Gabino Marín-García; José Javier Quesada-Granados, Ph.D. Abstract: A new alcoholic beverage made from sugarcane (Saccharum officinarum L.) molasses was studied and compared with analogous products. Three honey meads and two beverages obtained from sugarcane molasses were analysed for alcohol content, acidity, pH and reducing sugars. Extracts were obtained using a rotary evaporator, and tested for antioxidant capacity and total content of phenols, tannins and flavonoids. Antioxidant capacity was measured by DPPH, ABTS, DMPD and FRAP assays. Total phenol content was measured by the Folin-Ciocalteu test. Total tannin and flavonoid contents were measured by colorimetric methods based on (+)-catechin equivalents. The correlation between antioxidant capacity and total phenols was determined. The results obtained showed that the physical and chemical characteristics of sugarcane molasses mead were similar to those of beer. The sugarcane molasses meads had a higher antioxidant capacity than the honey-based ones, from which we conclude that the sugarcane molasses product is a new and interesting alternative. Suggested Reviewers: José del Carmen Contreras Calderón PH.D., Universidad Nacional de Antioquia (Colombia) [email protected] Annalisa Maietti Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Ferrara (Italy) [email protected] Marta Mesías García Ph.D. Instituto de Ciencia y Tecnología de los Alimentos y la Nutrición (ICTAN), Consejo Superior de Investigaciones Científicas (Spain) [email protected] Highlights: 1. A new sugarcane molasses beverage was studied/compared with analogous products 2. The sugarcane molasses meads had a higher antioxidant capacity than the honey ones 3. The values of phenols are higher in the sugarcane meads than in the honey ones 4. The sugarcane mead is statistically different from similar alcoholic beverages 5. The sugarcane molasses product is a new and interesting alternative *Highlights (for review) 1 A new fermented beverage from sugarcane (Saccharum officinarum L.) molasses: analysis of 1 physicochemical properties and antioxidant capacity, and comparison with other industrial alco-2 hol products 3 C. Samaniego-Sánchez, G. Marín-García, J. J. Quesada-Granados* 4 Department of Nutrition and Food Science, Faculty of Pharmacy, University of Granada, Campus Cartu-5 ja s/n, 18071 Granada (Spain). 6 7 Corresponding Author 8 * Tel: +34 958240756. Email: [email protected] 9 *Manuscript Click here to view linked References 2 ABSTRACT: A new alcoholic beverage made from sugarcane (Saccharum officinarum L.) mo-10 lasses was studied and compared with analogous products. Three honey meads and two bever-11 ages obtained from sugarcane molasses were analysed for alcohol content, acidity, pH and reduc-12 ing sugars. Extracts were obtained using a rotary evaporator, and tested for antioxidant capacity 13 and total content of phenols, tannins and flavonoids. Antioxidant capacity was measured by 14 DPPH, ABTS, DMPD and FRAP assays. Total phenol content was measured by the Folin-Ciocalteu 15 test. Total tannin and flavonoid contents were measured by colorimetric methods based on (+)-16 catechin equivalents. The correlation between antioxidant capacity and total phenols was deter-17 mined. The results obtained showed that the physical and chemical characteristics of sugarcane 18 molasses mead were similar to those of beer. The sugarcane molasses meads had a higher antioxi-19 dant capacity than the honey-based ones, from which we conclude that the sugarcane molasses 20 product is a new and interesting alternative. 21 22 KEY WORDS: Antioxidant capacity, mead, honey, sugarcane molasses, phenolics. 23 24 25 3 1. Introduction 26 Mead is an ancient alcoholic beverage made from honey and water (Kahoun, Řezková, 27 Veškrnová, Královský, & Holčapek, 2008; Mendes-Ferreira et al., 2010). It contains at least 7% 28 ethanol and many other compounds, including sugars, acids, vitamins, antioxidants and miner-29 als. Reflecting its origin, the chemical composition of mead is similar to that of honey. The qual-30 ity of a mead depends on its parameters and on the content of certain compounds such as reduc-31 ing sugars, organic acids and phenolic compounds (Švecová, Bordovská, Kalvachová, & Hájek, 32 2015). The composition and phenol content of mead are influenced by many different factors, 33 including fermentation, storage and maturation (Wintersteen, Andrae, & Engeseth, 2005). 34 Although honey is the main ingredient of mead, another raw material, with similar characteris-35 tics, sugarcane molasses, can also be used to brew mead. 36 Sugarcane (Saccharum officinarum L.) is a major source of sugar, together with beet (Beta vul-37 garis L.) (Maurício Duarte-Almeida, Novoa, Linares, Lajolo, & Inés Genovese, 2006). Molasses, 38 the thick, dark syrup obtained as a byproduct from the processing of sugar cane and sugar beet 39 into sucrose, consists of fermentable carbohydrates (sucrose, glucose and fructose) and non-sugar 40 organic materials (betaine and other amino acids; minerals and trace elements; vitamins, espe-41 cially of the B-group, etc.). Although molasses is mainly used as a supplement for livestock feed 42 and as a source of carbon in fermentation processes, for example, for the production of ethanol, it 43 is also a traditional sweetener and colourant in cakes. Molasses is generally regarded as nutrition-44 ally safe (Valli et al., 2012). 45 In southern Spain, an important sugarcane industry became established in the provinces of 46 Malaga and Granada in the first decade of the 20th century. Sugarcane contains flavonoids and 47 other phenolic compounds, derived from naringenin, tricin, apigenin and luteolin (Smith & 48 Paton, 1985; Williams, Harborne, & Clifford, 1974), with antioxidant properties (McGhie, 1993). 49 4 Various studies have analysed the properties, antioxidant capacity and related properties of 50 honey mead (Kahoun et al., 2008; Mendes-Ferreira et al., 2010; Švecová et al., 2015), and those of 51 sugarcane molasses, considered as such (Asikin et al., 2018)but to our knowledge none have exam-52 ined the case of a fermented beverage obtained from sugarcane molasses as the main raw mate-53 rial. It should be taken into account that the composition and content of phenolic compounds in 54 honey meads and other fermented beverages are influenced by the ingredients used, the produc-55 tion process followed, the storage conditions, etc. and these factors are likely to affect sugarcane 56 molasses meads, too (Kahoun, Řezková, & Královský, 2017). 57 In the present study, we analyse the physical and chemical characteristics and the antioxidant 58 capacity of a new fermented beverage made from sugarcane molasses and from each of two yeast 59 strains (one typically used for beer and the other for wine). We then compare these findings with 60 those obtained for analogous alcoholic beverages. Thus, four different meads were crafted, using 61 sugarcane molasses and honey, and their antioxidant capacities compared, together with those 62 for other alcoholic beverages (beer and wine). 63 64 2. Materials and methods 65 2.1. Mead and sample preparation 66 Sugarcane molasses and honey for mead production were acquired in local commercial estab-67 lishments in Granada (Spain). The commercial meads used as controls (“semdry”) were also ac-68 quired from a company from Granada (Spain). Saccharomyces cerevisiae yeast, variety SafbrewTM 69 S-33, was acquired from Fermentis and Saccharomyces bayanus yeast, variety Bioferm Killer, was 70 acquired from Brouwland (www.brouwland.com). The meads were brewed following the method 71 described below. 72 73 5 Two batches of four meads were prepared, two made from honey and two from sugarcane mo-74 lasses. In each case, 500 mg of honey or sugarcane molasses were added to 1.5 L of water in a 75 stainless steel cooking pot. The mixture was heated to 80 ºC, and this temperature was main-76 tained for 10 minutes for pasteurisation to take place. The mixture was then cooled using indirect 77 cold water for nine minutes, until the temperature of the pasteurised mixture had fallen to 35 ºC. 78 The yeasts (S. cerevisiae and S. bayanus) were reconstituted following the manufacturer's recom-79 mendations, i.e. placing 7 g of each yeast in 0.3 L of water at 35 ºC for ten minutes. Then, the pas-80 teurised dilutions of honey and sugarcane molasses were each poured into two one-litre glass bot-81 tles. 0.15 L of the reconstituted yeasts were added, one type for each bottle, and the bottles were 82 then sealed with airlocks to keep them airtight. The fermentation process was controlled for 18 83 days, until the CO2 bubbling in the bottles ceased. When the fermentation had concluded, the 84 bottles were stored at 4 ºC until needed for analysis. Figure 1 shows a summary of the sample 85 preparation. 86 The mead samples were labelled according to the variety of yeast used, the raw material and the 87 batch number, as follows: SCH1 (S. cerevisiae Honey batch 1), SBH1 (S. bayanus Honey batch 1), 88 SCS1 (S. cerevisiae Sugarcane batch 1), SBS1 (S. bayanus Sugarcane batch 1), SEMDRY (Commer-89 cial honey mead), SCH2 (S. cerevisiae Honey batch 2), SBH2 (S. bayanus Honey batch 2), SCS2 (S. 90 cerevisiae Sugarcane batch 2) and SBS2(S. bayanus Sugarcane batch 2). 91 92 2.2. Equipment 93 Electronic weighing scale (Mettler AE 2000, precision 0.0001 g), mixer (Vortexer, Cleaver Scien-94 tific Ltd), high resolution spectrometer SYNAPT G2 HDMS Q-TOF. Waters, Lambda 25 UV/vis 95 spectrophotometer (Perkin-Elmer®, Madrid, Spain), Orion pH-meter. 96 97 6 2.3. Chemicals 98 All chemicals used were analytical reagent grade, unless otherwise stated. Folin–Ciocalteu phe-99 nol reagent was obtained from Merck (Darmstadt, Germany). Gallic acid, 6-hydroxy-2,5,7,8-100 tetramethyl-chroman-2-carboxylic acid (Trolox), 2,2-azinobis-(3-ethylbensothiazoline)-6-sulfonic 101 acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and N, N-dimethyl-p-phenylenediamine 102 (DMPD) were supplied by Sigma–Aldrich (Milan, Italy). Sodium nitrite, aluminium chloride, 103 catechin, vanillin, sodium acetate 3-hydrate, anhydrous sodium carbonate, ferric chloride 6-104 hydrate, orthophosphoric acid and Rebelein Vinikit were supplied by Panreac (Barcelona, Spain). 105 The 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) for the FRAP method was obtained from Fluka Chemi-106 cals (Buchs, Switzerland). 107 108 2.4. Alcohol content (% V/V), acidity, pH and reducing sugars 109 Alcohol content was determined using a pycnometer, as recommended by the European Brew-110 ery Convention (1975). Acidity and pH were determined following the methods described by the 111 American Society of Brewing Chemists (1942). 112 Reducing sugar content was measured using the Rebelein method described in European 113 Council regulation 1234/2007. Specifically, 2 mL of mead sample were mixed with 10 mL of cupric 114 solution and 5 mL of alkaline solution from the Rebelein kit. The mixture was heated and main-115 tained at boiling point for three minutes. The resulting solution was then cooled and mixed with 116 10 mL of potassium iodide, 10 mL of sulphuric acid and 10 mL of starch solution. The final solu-117 tion was titrated with thiosulphate solution until it turned yellow. 118 119 2.5. Extraction conditions 120 7 The extracts were obtained as follows (Socha, Gałkowska, Robak, Fortuna, & Buksa, 2015), 25 121 mL of mead sample were concentrated in a rotary evaporator in order to remove the alcohol. The 122 resulting solution was then diluted to the primary volume with distilled water. 123 The sample was adjusted to pH = 2 with HCl solution and then saturated with NaCl. The solu-124 tion obtained was extracted three times with ethyl acetate, using 25 mL of the solvent. The ethyl 125 acetate fraction was then collected and evaporated to dryness in a vacuum rotatory evaporator. 126 The dry residue after evaporation was dissolved in 5 mL of methanol. 127 128 2.6. Total content of phenols, flavonoids and tannins 129 Total phenol content was determined using a modified version of the Folin-Ciocalteu colori-130 metric method (Singleton & Rossi, 1965). 2.5 mL of deionised water and 500 µL of Folin-Ciocalteu 131 reagent were added to an appropriately diluted mead methanolic extract. The mixture was al-132 lowed to stand for five minutes, after which 2 mL of a 10% aqueous Na2CO3 solution were added. 133 The final volume was adjusted to 10 mL. The samples were allowed to stand for 90 minutes at 134 room temperature before measurement at 700 nm versus the blank, using a spectrophotometer. 135 The total phenol content is expressed as gallic acid equivalent (mg gallic acid/L mead), using the 136 gallic acid calibration curve. 137 Total flavonoid content was determined using a colorimetric method (Maietti et al., 2012). To 138 50, 100 and 200 μl of mead phenolic extract respectively, 2 mL of deionised water, 150 μL of 5% 139 NaNO2 solution, 300 μL of 10% AlCl3 solution, and 1 mL of NaOH 1N were added. The final vol-140 ume was adjusted to 5 mL with deionised water and the absorption was measured at 510 nm ver-141 sus the blank. The amount of total flavonoids is expressed as (+)-catechin equivalents (mg (+)-142 catechin/L) through the calibration curve of (+)-catechin. 143 14 In conclusion, the antioxidant capacity of sugarcane molasses meads is different from that of 287 honey meads, but similar to that of beer. 288 289 3.4. Correlation between antioxidant capacity (TEAC) and Total Phenols Content (TPC) 290 Table 9 summarises the correlation between total phenol contents and the different measure-291 ment methods used (Samaniego Sánchez et al., 2007). A very high positive correlation was ob-292 served between the total phenols in honey and sugarcane molasses meads and their antioxidant 293 capacity (Pearson’s correlation coefficients: 0-9 to 0.99) except for DMPD, in which case only a 294 moderate positive correlation was observed (Pearson’s correlation coefficients: 0.4 to 0.69). 295 The DPPH values also presented a very high positive correlation with ABTS and FRAP but there 296 was only a low positive correlation (Pearson’s correlation coefficients: 0.2 to 0.39) between the 297 DPPH and DMPD values. 298 The ABTS values obtained were almost perfectly correlated with the ABTS values (r=0.9912) but 299 there was only a low positive correlation (Pearson’s correlation coefficients: 0.2 to 0.39) with the 300 FRAP values. Finally, the Pearson’s correlation coefficients between FRAP and DMPD were mod-301 erate and positive. 302 In view of these results, it is confirmed that the DMPD method is not suitable for measuring the 303 antioxidant potential of mead. 304 305 3.5. Multivariate analysis 306 To determine whether the sugarcane molasses mead was different from the honey mead, based 307 on all data obtained, two multivariate analyses were conducted. The first was a cluster analysis 308 using the median method, which produced the dendrogram shown below (Figure 2): 309 15 310 This figure shows that the samples of sugarcane molasses mead, the new product (SCS and SBS) 311 closely resembled each other and constituted a well-defined cluster. Among all other beverages 312 compared, the most similar to this was the honey mead. It is interesting to note that the honey 313 mead obtained in our assay was similar to a commercial product marketed as SEMDRY. In conclu-314 sion, the sugarcane mead analysed in this study can be statistically differentiated from similar 315 alcoholic beverages. 316 317 To further corroborate the conclusion that the new product can be differentiated from existing 318 beverages, a discriminant analysis was also conducted. This method predicts whether a sample 319 belongs to one group or another, according to the overall data available. The results obtained from 320 this analysis are shown below (Figure 3). 321 322 The predictions made by the function achieve a 100% success rate. Thus, all the samples and 323 products differ sufficiently to be individually identifiable and the function correctly classified all 324 the samples from the data supplied. 325 326 327 4. Conclusion. 328 The sugarcane molasses meads had a higher antioxidant capacity than the honey-based ones 329 and values of phenols, flavonoids and tannins higher in the sugarcane molasses meads than in 330 the honey meads. The sugarcane mead analysed in this study can be statistically differentiated 331 from similar alcoholic beverages. In summary, by applying the method described in this paper we 332 16 obtained a new alcoholic beverage, made from sugarcane molasses, which differed significantly 333 both from traditional honey mead and from beer and red wine and it is a new and interesting 334 alternative. 335 336 Abbreviations 337 SCH1, Saccharomyces cerevisiae Honey batch 1; SBH1, Saccharomyces bayanus Honey batch 1; SCS1, 338 Saccharomyces Sugarcane batch 1; SBS1, Saccharomyces. bayanus Sugarcane batch 1; SEMDRY, 339 Commercial honey mead; SCH2, Saccharomyces cerevisiae Honey batch 2; SBH2, Saccharomyces ba-340 yanus Honey batch 2; SCS2, Saccharomyces cerevisiae Sugarcane batch 2; SBS2, Saccharomyces ba-341 yanus Sugarcane batch 2; Trolox, Gallic acid, 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic 342 acid; TPC, Total Phenols Content; ABTS, 2,2-azinobis-(3-ethylbensothiazoline)-6- sulfonic acid; DPPH, 343 2,2-diphenyl-1-picrylhydrazyl; DMPD, N, N-dimethyl-p-phenylenediamine; FRAP, Ferric Reducing 344 Antioxidant Power; TEAC, Trolox Equivalent Antioxidant Capacity; GAE, Gallic Acid Equivalent; Cat, 345 Catechin; TE, Trolox Equivalent. 346 347 Acknowledgements 348 We wish to express our gratitude to Glenn Harding for his assistance in improving the English language 349 version of the manuscript. 350 351 Funding 352 This study is part of Gabino Marín Garcia’s PhD thesis, from the Nutrition and Food Sciences Doctor-353 ate Program of the University of Granada and it was funded by PAIDI groups Junta de Andalucía 354 AGR141 and AGR279. 355 17 Notes 356 The authors have no competing financial interest to declare. 357 358 359 References 360 Asikin, Y., Wada, K., Imai, Y., Kawamoto, Y., Mizu, M., Mutsuura, M., & Takahashi, M. 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Discriminant function chart of all samples and beverages studied. 453 1 Table 1 1 Alcoholic content, acidity, pH and reducing sugars of the samplesa. 2 Sample % V/V Acidity (g tartaric acid/L) pH Reducing sugars (g/L) BATCH 1 SCH 8.5 ± 0.1 2.1 ± 0.0 4.04 ± 0.11 14.8 ± 0.25 SBH 8.5 ± 0.1 3.2 ± 0.1 3.85 ± 0.03 19.0 ± 0.75 SCS 11.0 ± 0.1 2.5 ± 0.1 4.49 ± 0.18 3.0 ± 0.30 SBS 11.0 ± 0.1 3.8 ± 0.2 4.31 ± 0.03 3.2 ± 0.21 Commercial honey mead SEMDRY 9.1 ± 0.1 2.1 ± 0.2 4.40 ± 0.21 25.47 ± 1.27 BATCH 2 SCH 7.5 ± 0.1 2.6 ± 0.4 3.75 ± 0.01 18.1 ± 0.10 SBH 6.9 ± 0.1 3.8 ± 0.4 3.58 ± 0.01 21.9 ± 0.12 SCS 9.5±0.1 2.9±0.4 4.42±0.01 2.6±0.31 SBS 10.0±0.1 3.8±0.0 4.17±0.02 2.9±0.20 a Values are the means ± SD (n=3). 3 4 Tables 8 Table 8 35 DPPH, ABTS, DMPD and FRAP comparison between beers, wines and meads in millimolars TE/L. 36 Beverage DPPH ABTS DMPD FRAP Beer a 0.35 ± 0.01 to 0.83 ± 0.01 0.14 ± 0.01 to 0.35 ± 0.01 - 22.99 ± 5.11 to 831.20 ± 3.83 Red wine b 9.2 ± 0.6 to 37.8 ± 2.8 7.9 ± 0.4 to 24.2 ± 0.8 5.8 ± 0.3 to 10.2 ± 0.5 22.195± 4.479 to 32.280 ± 4.479 Honey mead (SCH and SBH) 2.44 ± 0.03 to 5.89 ± 0.05 3.17 ± 0.11 to 7.73 ± 0.17 7.26 ± 0.18 to 20.91 ± 0.12 2.67 ± 0.06 to 6.67 ± 0.33 Sugarcane molasses mead (SCS and SBS) 11.50 ± 0.15 to 12.63 ± 0.10 58.37 ± 0.69 to 90.15 ± 1.29 8.29 ± 0.36 to 9,50 ± 0.63 60.82 ± 2.43 to 71.14 ± 0.25 a (Mitić et al., 2014; Zhao et al., 2010) 37 b (Busuricu, F.; Balaban, D.; Popescu, A.; Anghel, 2008; Katalinić et al., 2004; Ma et al., 2014; Šeruga 38 et al., 2011) 39 40 9 Table 9 41 Pearson’s correlation coefficients (r) between antioxidant capacity and total phenols. 42 Total phenols (Folin-Ciocalteu) DPPH ABTS FRAP DMPD Total phenols (Folin-Ciocalteu) 1 DPPH 0.9602 1 ABTS 0.9780 0.9669 1 FRAP 0.9853 0.9788 0.9912 1 DMPD 0.4674 0.3686 0.4871 0.4198 1 43 44 45 46 Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image CRediT author statement C. Samaniego-Sánchez: Conceptualization, Methodology, Investigation, Validation, Resources, Writing - Review & Editing. G. Marín-García: Conceptualization, Methodology, Investigation, Resources, Formal analysis, Writing - Original Draft, Visualization. J. J. Quesada-Granados: Conceptualization, Methodology, Formal analysis, Validation, Writing - Review & Editing, Visualization, Supervision, Project administration. Credit Author Statement