potentialapplicabilityofcocoapulp(theobromacacao l)asan...

Research ArticlePotential Applicability of Cocoa Pulp (Theobroma cacao L) as anAdjunct for Beer Production

Cassiane S O Nunes12 Marılia L C da Silva3 Geany P Camilloto3

Bruna A S Machado 4 Katharine V S Hodel4 Maria Gabriela B Koblitz5

Giovani B M Carvalho3 and Ana Paula T Uetanabaro 1

1Department of Biology and Biotechnology of Microorganisms State University of Santa Cruz Ilheus 45662900 Brazil2Bahian Federal Institute Catu Campus Catu 48110-000 Brazil3Department of Biotechnology State University of Feira de Santana Feira de Santana 44036-900 Brazil4University Center SENAI CIMATEC SENAI Institute of Innovation (ISI) in Advanced Health Systems (CIMATEC ISI SAS)National Service of Industrial LearningndashSENAI Salvador 41650-010 Bahia Brazil5Food and Nutrition Center Federal University of the State of Rio de Janeiro Rio de Janeiro 21941-901 Brazil

Correspondence should be addressed to Bruna A S Machado brunamachado17hotmailcom

Received 20 April 2020 Accepted 17 July 2020 Published 2 September 2020

Academic Editor Carin von Muhlen

Copyright copy 2020 Cassiane S O Nunes et al is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

e aim of this study was to evaluate the application of cocoa pulp as an adjunct for malt in beer production e cocoa pulp wasanalyzed for humidity proteins lipids sugars total soluble solids organic acids and minerals A study was carried out to reducethe cocoa pulp viscosity by enzymatic depectinization making its use viable in beer production e cocoa pulp showed relevantquantities of compounds important in fermentation such as sugars acids and minerals In fermentation using the adjunct theproportions of pulp used were 10 30 and 49 A significant difference was found between the adjunct and all-malt wortse 30cocoa pulp concentration as an adjunct for malt in the fermentation medium contributed the most to the fermentative per-formance of the yeasts at both 15 and 22degC based on the consumption of apparent extract (degPlato) ethanol production andcellular growth

1 Introduction

Cocoa (5eobroma cacao L) is known worldwide for the useof its beans in chocolate production [1ndash4] and is an im-portant commodity for some countries [5ndash8] Brazil is acountry with a large production of fruits and a high volumeof exports and in the global production of cocoa Braziloccupied the 6th ranked position in the 20142015 harvest[9 10] For a long period the production and commer-cialization of cocoa were the basis of the economy in someBrazilian states especially Bahia Brazil was the secondlargest producer of cocoa in the world until the appearanceof ldquowitchrsquos broomrdquo a disease caused by the fungus Mon-iliophthora perniciosa after which the agroindustry of cocoadeclined quickly causing a decrease in the production and

exportation of cocoa beans [11ndash13] Cocoa pulp is a richsubstrate which can be used in industrial processes toproduce by-products [10 14 15] e pulp is made up of agroup of mucilaginous spongy cells containing waterfructose glucose sucrose citric acid and several inorganicsalts [16]

Cocoa pulp shows a complex and variable microbiotaand spontaneous fermentation is a prevalent process in thefood industry [14 16ndash18] In studies performed with cocoapulp it was observed that it can be used as a substrate for thedevelopment of different microorganisms with a higherprevalence of fungus than bacteria and that within thisdiversity there is a trend for Saccharomyces cerevisiae tostand out [17] e use of cocoa as an industrial raw materialis advantageous compared to other potentially suitable

Hindawie Scientific World JournalVolume 2020 Article ID 3192585 14 pageshttpsdoiorg10115520203192585

tropical fruits as it is an abundant product derived from anestablished culture Research aims at obtaining pulp toproduce juice jam compotes fermented beverages andother processed products thereby offering special and at-tractive flavors [19]

Cocoa pulp can be easily fermented by yeasts such asS cerevisiae to produce an alcoholic beverage is yeast hasbeen used in fermentation processes for a long timeaccording to the first historical reports of beer and wineproduction [20ndash27]e wort composition is very importantfor beer production because it ensures the quality of the finalproduct and is a growth medium for the yeasts Severalenvironmental factors influence the production of metab-olites and the survival of the yeasts during industrial fer-mentation e main factors are temperature pH and sugarconcentration as well as pulp characteristics in the case ofbeer production with the use of the adjunct [28ndash31] Similarto cocoa pulp many other fruits can be used in the pro-duction of alcoholic beverages [32] Various experimentshave been carried out with fruits such as mango [33]pineapple [34] star fruit [35] banana [36ndash38] orange[39ndash41] grape [42] apple [43 44] cocoa [19] gabiroba [45]kiwi [46] and caja [47] for the production of fermentedbeverages However due to the different chemical compo-sitions of the fruits further studies are necessary includingthe type of fermented beverage to be produced the idealtemperature of fermentation and the type of treatment thatmust be applied to the fruit pulp or the wort in the pre-fermentative phase andor during fermentation Cocoa pulpconstitutes a very favorable raw material for the productionof beverages by alcoholic fermentation because it is rich incarbohydrates has low acidity and contains importantorganic acids [19 48ndash51]

A relevant aspect in the current beer market is the use ofadjuncts with a high concentration of fermentable sugarsthat contribute to higher production of ethanol from eachdegPlato of extract [52ndash55] e use of certain types of adjunctsonly contributes to the supply of carbohydrates and no othernutrients consequently its addition to the wort at the be-ginning of beer fermentation results in a similar balance ofnutrients to the fermentations without the addition of ad-junct [56ndash58] e main objective of this work was toevaluate the performance of fermentation in the productionof beer using cocoa pulp from the south region of Bahia(Brazil) as an adjunct for malt after the determination of itschemical composition

2 Materials and Methods

21 Obtaining and Storing the Cocoa Pulp Cocoa fruits wereobtained from clone VP 1151 which was supplied by theMars Center for Cocoa Science located in the south region ofthe state of Bahia (Ilheus Brazil) the cocoa fruits wereharvested at their optimum ripeness for consumption eselection step consisted of the rejection of defective fruits toavoid compromising the quality of the final pulp and size-and color-based selection from the commercially availablefruitse selected fruits were washed with clean tap water toremove impurities sanitized for 30min in a 100mgmiddotmLminus 1

free chlorine solution and rinsed with clean tap water toremove excess free chlorine en the cocoa fruits werebroken pulped using a bench depulper (model DM-Ji-05Macanuda) fractionated and stored in flexible polyethylenebags e pulp fractions were then frozen and stored atminus 18degC until chemical analysis

22 Wort Production e wort was produced according tothe conventional techniques of beer production at theLaboratory of Fermentation of the State University of Feirade Santana (UEFS) Bahia Brazil During this process 88 kgof malt (Chateau Pilsen 325 EBC from Belgium) wasground in a bench grinder to decrease the grain size andfacilitate hydrolysis catalyzed by enzymes during wortproduction e wort production ramp used was adaptedfrom Carvalho and Zambiazi [59]e initial pH value of thewort was adjusted to 53 by adding food-grade lactic acide wort was hot-packed (gt90degC) and stored at 0degC until use

23 Chemical Analysis of the Pulp and Wort e chemicalanalysis of the cocoa pulp and wort was performed at theLaboratory of Physical-Chemical Analysis of the Depart-ment of Technology State University of Feira de SantanaBahia e humidity was determined by direct drying in anoven at 105degC and ashes were obtained by incinerating thesamples in a muffle oven at 550degC Protein was determinedby the Kjeldahl method and lipids by gravimetry after ex-traction with petroleum ether using a Soxhlet extractor andstarch Pectin and titratable acidity were determinedaccording to the methodology recommended by Adolfo LutzInstitute [60] e sugars were determined by spectropho-tometry with the reducing sugars established by the DNSmethod (35-dinitrosalicylic acid) [61] and the total sugarsby the phenol-sulfuric method [62] e pH was determinedby the potentiometric method using a digital InstruthermpH meter (model PH-1700) and titratable acidity by neu-tralization with the results of the titration expressed in g ofmalic acid per 100 g of pulp according to the methodologydescribed by Carvalho et al [63]

e total phenolic content of the cocoa pulp was de-termined according to Singleton and Rossi [64] by the re-action between the pulp extract and FolinndashCiocalteu reagentin the presence of sodic carbonate based on the appearanceof blue due to the oxidation of phenols in the basic mediume absorbance was measured in a spectrophotometer(Shimadzu model UV mini 1240) at 765 nme quantity oftotal phenols was expressed in gallic acid equivalents (GAE)(mg GAEg of sample) through a calibration curve [65] ecolor of the wort was determined by the spectrophotometricmethod at a wavelength of 430 nm [66]

24 Determination of Sugars and Organic Acids e sugarsand organic acids were determined by high-performanceliquid chromatography (HPLC) e samples were diluted10 times in ultrapure water and filtered through difluoridepolyvinylidene membranes (045 μm pores 13mm diame-ter) (Millex HV Millipore) Standard curves were built for

2 e Scientific World Journal

the metabolites (glucose fructose maltose maltotriosecitric acid malic acid acetic acid succinic acid and formicacid) (Sigma-Aldrich) which were diluted in ultrapure water(Direct Q3UV Millipore Massachusetts USA) During theanalysis 5 μL of the sample was used An UltiMate 3000(Dionex Germany) system that was equipped with a UV-Visdetector (at a 210 nm wavelength) for the detection of or-ganic acids and with a refraction index detector for thedetection of sugars (Shodex RI-101 Showa Denko Japan)was used for this analysis A Rezex ROAOrganic Acids ionicexchange column (300times 78mm) (Phenomenex TorranceCA USA) was used to separate the compounds at a steadytemperature of 60degC and 0005M sulfuric acid was used as amobile phase at a flow rate of 06mLmin e chromato-grams were integrated using Chromeleon Server monitoringsoftware (Dionex) Identification was performed by com-paring the retention times and the quantification wasestablished according to the standard curve for each analyte

25 Determination of Minerals

251 Sample Digestion For digestion 05 g of the sampleswas weighed accurately in a glass digestion tube and 2mL ofconcentrated nitric acid was added e digestion occurredat 120degC for a period of 4 h and then 15mL of hydrogenperoxide was added e resulting solution was quantita-tively transferred to a plastic flask and filled to 10mL withultrapure water All samples were analyzed in triplicate toeliminate any specific error verify the homogeneity ofsamples and evaluate the repeatability of the procedure eblanks were prepared in the same way however the samplewas omitted for each batch of samples and the certifiedreference material was digested using the same method [67]

252 Inductively Coupled Plasma Optical Emission Spec-troscopy (ICP-OES) for Mineral Determination e ICP-OES operating conditions (Varian Mulgrave Australia)were as follows 15 Lmin argon plasma gas flow rate 15 Lmin auxiliary gas flow rate 07 Lmin nebulizer gas flowrate 13 kW power 40MHz radio frequency (RF) power07mLmin sample flow rate and charge-coupled device(CCD) the detector

All elements were detected in axial mode and the time ofsignal integration was 2 seconds All analytes were measuredat two different emission lines To check for matrix effects onthe sensitivity and selectivity a scan of the emission lines wasobtained for both a standard and a digested sample eemission line was selected taking into account nonspectralinterferences and the best signal to background ratio for allelements and the background was corrected manually for allemission lines selected e calibration standards wereprepared in a 15molL nitric acid solution e calibrationrange for all elements was evaluated from 002 to 10mLexcept for calcium magnesium potassium sodium andphosphorus for which calibration curves were preparedfrom 1 to 100mgLe average value for blank samples wassubtracted from the analytical signals of digested samplesafter interpolation on the corresponding calibration graphs

26 Viscosity of the Cocoa Pulp Enzymatic Hydrolysis of thePectin In addition to the abovementioned analyses it isimportant to highlight that the high viscosity of the cocoapulp necessitated a study to reduce the viscosity to make itsuse in the production of beer possible

e viscosity reduction of the cocoa pulp was performedusing polygalacturonase (7900 PGNUmL) obtained fromAspergillus niger and Aspergillus aculeatus (NovozymesPectinex Ultra Clear) A rotational central compound ex-periment was designed (DCCR) using three variables (en-zyme concentration time and temperature) and evaluatedat two levels using the Statistica 70 program

e maximum temperature was fixed at 80degC as highertemperatures for a longer period would cause significantwater loss thereby concentrating the pulp and leading to anapparent increase in viscosity compared to the initial pulpe response variable studied was the percentage reductionof pulp viscosity after treatment with the enzyme

e experiments consisted of adding the enzyme to 100 gof pure pulp and heating in a static water bath for a pre-established time at a preestablished temperature After thereaction time the samples were cooled to 30degC and exam-ined for viscosity in a viscometer (Rheometer BrookfieldDVII + Pro) ese values were used to calculate the re-duction of viscosity using the following equation

reduction of viscosity (initial viscosity) minus (final viscosity)

(initial viscosity)times 100

(1)

e results of the viscosity reduction were processed bythe Statistica 70 program to obtain a variance analysis tableregression coefficients and response surface graphs as wellas contour curves

27 Yeast Strains e commercial yeast strains used in thiswork were lager (Safale S-23 Belgium) and ale (Safale S-04Belgium)

28 Conditions of Fermentation To evaluate the effect of thecocoa pulp as an adjunct for beer production a follow-up ofthe fermentation was carried out analyzing the followingparameters substrate consumption ethanol production andcellular concentration

e propagation of yeasts was realized using 12degP all-malt wort For initial inoculation the yeasts were weighedaccording to the manufacturerrsquos instructions propagated inan Erlenmeyer flask and incubated in a rotatory shaker(100mL 30degC 150 rpm 24 h) with enough agitation toprovide a cellular concentration of 10times108 cellmL isaimed to ensure the beginning of fermentation with a10times107 cellmL laboratory scale in a 500mL Erlenmeyerflask at a useful volume of 250mL

29 Beer Production Using Cocoa Pulp as an Adjunct eassays for beer production at laboratory scale were per-formed with a combination of three concentrations of cocoapulp (10 30 and 49) as the adjunct and all-malt wort (0)

e Scientific World Journal 3

two commercial yeasts at 15 (Safale S-23 Belgium) and 22degC(Safale S-04 Belgium) and the concentration of the 12degPoriginal wort e cocoa pulp went through an enzymatictreatment for viscosity reduction before being added to thebrewing wort en the cocoa pulp was added to the wort asan adjunct after the boiling phase at the beginning of fer-mentation e fermentations were performed at 15 and22degC in a 500mL Erlenmeyer flask using an airlock valve toseal the system ese fermentations were measured atregular 12 h intervals until the apparent attenuation reachedapproximately 70ndash75 (10degP above the final value of fer-mentable sugars) Statistical analysis (ANOVA) was per-formed by the F-test and the averages were compared byTukeyrsquos test with 5 probability using the statistical softwareSISVAR 50

210 Wort Analyses roughout the fermentation sampleswere collected in triplicate e cells were removed bycentrifugation at 4000 rpm for 10min and the supernatantwas used at 20degC to quantify the apparent extract and theethanol concentration by using beer analyzer equipment(bench hydrometer Rudolph Research Analytical Tecnal)

211 Yeast Analyses e cell concentration in suspensionwas determined using a Neubauer counting chamber andexpressed in cellmL e cellular viability was determinedby a methylene blue staining method [68] Each determi-nation was performed in triplicate

3 Results and Discussion

31 Characterization of the Cocoa Pulp e cocoa pulp wasobtained by mechanical processes and chemically charac-terized to evaluate its contribution to the process of beerfermentation e pulp presented high humidity and anaverage pH value of 35 e cocoa pulp due to its low pH isa product classified as highly acidic (pHlt 45) as are mosttropical fruits [19] e concentrations of total and reducingsugars were 18 and 104 respectively (Table 1) Based onthese results the cocoa pulp can be compared to other fruitswith potential for use in the beverage industry e con-centration of total solids determined through the degBrixvalue was 17 (Table 1) higher than the soluble solids valuefor traditional beers (12degBrix) [24] Studies of cocoa pulpperformed by Dias et al [19] Penha and Matta [69] andPuerari et al [48] found similar values In this case it wasnecessary to dilute the pulp to standardize the process ofbeer production so a sugaring process was not neededwhich according to Dias et al [47] consists of adjusting thesoluble solid content by using a sucrose solution is ad-justment is needed for pulps that have a lower content ofsoluble solids than the wort for the production of beer esoluble solid content is a parameter used as an indirectmeasure of the sugar content [63] Sugars and organic acids(Table 1) are important compounds of the cocoa pulp thatare present during fermentation e glucose and fructosecontents were 5211 and 5235 gL respectively e fer-mentable sugars are those that can contribute to the

nutritional composition of the wort e sugars glucose andfructose which are found in the cocoa pulp will contributeto the fermentative process once they are assimilatedthrough the yeasts by facilitated diffusion stopping thetransport process when the concentrations are equal insideand outside the cell [24 25]

S cerevisiae excretes an extracellular enzyme that hy-drolyses sucrose into glucose and fructose which are thenconducted inside the cell ese compounds found in thecocoa pulp are important sources of carbon for the for-mation of products and subproducts that influence thearoma taste and final characteristics of the beere organicacids citric and malic are commonly found in fermentedbeverages that contain fruits as preservatives and theypossess antimicrobial properties [19ndash25] us the presenceof cocoa pulp can contribute positively in terms of nutrientsto the microorganisms and sensory characteristics in ad-dition to having an interesting commercial appeal In thiswork citric and malic acids (organic acids) were found inhigher quantities at 833 and 610 gL respectively

Acetic acid a product of the yeastrsquos secondary meta-bolism is associated with an ldquooff flavorrdquo in beer and wasfound in lower quantity is substance is metabolizedexclusively by the yeasts which reabsorb it at the end offermentation altering its concentrations and making themvariable at the end of fermentation [24 70] Other importantnutrients in fermentation are the nitrogen compounds[24 57] e value of protein found in the cocoa pulp was062 which can contribute to the amino acids that areessential for yeast nutrition during fermentation as well asflavor compound formation in the beer Carvalho et al [37]who studied the performance of banana in beer production

Table 1 Chemical characterization of cocoa pulp mean values andstandard deviation

Characteristics Meanplusmn standarddeviation

Humidity (g100 g) 8638plusmn 009Ashes (g100 g) 036plusmn 005Raw protein (g100 g) 062plusmn 017Total lipids (g100 g) 145plusmn 020Total sugars (g100 g) 1800plusmn 005Reducing sugars(g100 g) 1041plusmn 005

Soluble solids (degBrix) 1700plusmn 001Glucose (gL) 5211plusmn 030Fructose (gL) 5235plusmn 045pH 350plusmn 001Total titratable acidity(mEqminus 1 in malic acid) 100plusmn 001

Citric acid (gL) 833plusmn 060Malic acid (gL) 610plusmn 050Succinic acid (gL) 045plusmn 034Lactic acid (gL) 043plusmn 050Acetic acid (gL) 040plusmn 044Pectin (g100 g)of calcium pectate 051plusmn 001

Starch (g100 g) 107plusmn 021Polyphenol (g100 g) 021plusmn 002e values are means of three replicates


obtained a pH of 44 to 46 an acidity of 022 to 057 formalic acid a starch content of 09 to 7 and total solublesolids of 28 [71] Comparing those values with those ofcocoa pulp this fruit shows compatible values that make itviable for beer production

32 Mineral Content of the Cocoa Pulp e results obtainedfrom the analysis of the minerals in cocoa pulp are found inTable 2 e elements Ca Cr Cu Fe K Mg Mn Mo Na PSe and Zn were found in higher quantities than the de-tection and quantification limits (Table 2) except for Al andCd which were found in lesser quantities of 174 and014mgkg respectively Co Li Pb and V were not found inthe analyzed sample e elements found in higher quan-tities were K Mg Na and PWith respect to the elements CaandMg themagnesium content found in cocoa pulp was thesame as in studies performed with kaki [67] however thecalcium level was lower ese mineral elements are im-portant during fermentation and act as essential cofactorsfor the activity of several enzymes

Most of the mineral compounds considered importantfor yeast performance during fermentation were found inthe cocoa pulp According to Briggs et al [24] the essentialminerals include B Ca Co Cu Fe K Mo Mn Mg Ni andZn and the concentrations required for the growth of yeastsare normally lower than 10 μM Among these minerals onlyCo was not found in the pulp e performance of the yeastduring fermentation depends on the complex interactionsamong the elements potassium magnesium and calciumDuring beer fermentation Mg has an important role in themaintenance of yeast membrane integrity when the yeastsare exposed to ethanol e increase in the bioavailability ofMg before or during fermentation increases cell growth andviability Supplementing the fermentation medium with Mghas caused an increase in the rate of fermentation andproductivity of ethanol Calcium also contributes to cellgrowth and metabolic responses to external stimulation andis related to flocculation [72] Copper and iron act as co-factors of several enzymes including the redox pigments ofthe respiratory chain In low concentrations copper is anecessary trace element that performs an important andpositive role for almost all organisms [73 74] However inhigh concentrations copper can exert an inhibitory effectand cause toxicity [75] Studies have demonstrated thatwhen the copper concentration is greater than 20mgL thegrowth of S cerevisiae is inhibited which delays the fer-mentative process and reduces alcohol production

According to Sun et al [76] at a concentration of copperbetween 96 and 192mgL there was no impact on thegrowth of S cerevisiae cells in the production of wine whichdiffers from the results of studies performed with highcopper content (32ndash96mgL) [77 78] e concentration ofcopper can reduce the capacity for sugar absorption ethanolproduction and growth of S cerevisiae strains [78] ecomposition of the wort influences the performance ofyeasts during fermentation It is important to highlight thatother components found in this composition can influencethe fermentation performance e cocoa pulp can

contribute to the wort composition by providing funda-mental minerals for the metabolism of yeast in the beerfermentation medium consequently contributing to thefermentative performance

33 Viscosity of the Cocoa Pulp is assay aimed at findingfavorable conditions for the hydrolysis of pectin consequentlyreducing the viscosity of the cocoa pulp in order to facilitate itsuse in beer production wort Examining the rheologicalcharacteristics it was noted that cocoa pulp has a pseudoplasticbehavior Many factors affect the rheological behavior of fruitpulps notably temperature soluble solids and particle sizeStudies show that fruit pulps behave as a pseudoplastic fluid asa result of complex interactions among the soluble sugarspectic substances and suspended solids [79]

e pulp showed an apparent high viscosity of 95 cP at30degC is characteristic indicates the need for an adequatedimensioning of pumps pipes and equipment in order toavoid losses incrustations and contamination duringhandling Carvalho et al [37] used an exogenous enzymaticcomplex to reduce the viscosity of banana pulp in beerproduction and favor its use in the technological process

e rheological behavior and flux properties of fruit pulphave a significant role in the food industry regulating thedevelopment of the product as well as the design and evaluationof equipment In addition knowing the rheological propertiesfundamental to any type of food can be an indication of howthe item behaves under several conditions of a process Beloware the data showing the percentage of viscosity reductionbased on experimental planning using the enzyme poly-galacturonase from Aspergillus niger (Table 3)

Tables 1S and 2S (Supplementary Materials) show theresults of the variance analysis for the assay and the re-gression coefficients with all the factors and their interac-tions respectively e percentage of viscosity reductionvaried from 0 (assay 12) to 6076 (assay 14) with highvariability among the 18 assays e value of R2 (922)considering only the significant factors as shown in Table 3S(Supplementary Materials) and the F-test(Table 4SmdashSupplementary Materials) indicate that theexperiment is significant at the 1 significance level and thatthe model obtained for predicting the viscosity reduction inrelation to the significant variables (equation (2)) can beused to represent the behavior of the viscosity reduction ofthe cocoa pulp when treated with the enzyme poly-galacturonase within the range studied In equation (2) Y isthe response of the percentage of reduction in viscosity of thecocoa pulp and X2 and X3 are the codified factors tem-perature and time of action of the enzyme on the cocoa pulprespectively

1113954Y minus 1292X2 minus 1028X22 minus 464X2X3 + 5357 (2)

34 Model Validation e adequacy of the proposed model(equation (2)) for enzymatic depectinization and conse-quently viscosity reduction was evaluated using threerandom conditions located within the optimal region


(Figure 1) Hydrolyses were performed in triplicate with theaddition of 500 μL of enzymatic solution for 100 g of pulpe experimental conditions (Figure 2) the predicted valuesand the results of the percentage of reduction in viscosityfound are shown in Table 4 Viscosity reductions of 64926475 and 6571 were experimentally achieved for assays1 2 and 3 respectively which is in good agreement with thereduction predicted by the model ere was no significantdifference among the three assays which is in accordancewith expectations as the three conditions point to the samepercentage of reduction in viscosity after analysis ofFigure 1

Finally any condition located within the optimal regioncan be used to treat cocoa pulp to be incorporated in beer

production as the percentage of reduction in viscosity ofapproximately 65 was satisfactory it increased the fluidityof the wort compared to the wort using untreated pulp Inthis experiment it was noted that the increase in temper-ature provided an increase in the fluidity of the cocoa pulpwith the addition of the enzymatic complex reflecting areduction in the apparent viscosity e enzyme acted in thepectic substances which can form gels in an acidic mediumand in the presence of sugar Due to the presence of pecticsubstances the viscosity increases leading to difficulties inclarification and concentration of juices [80]

In the cocoa pulp used in the present study 051 pectinwas found is finding is in accordance with the valueobserved by Dias et al [19] who added an enzymatic

Table 2 Determination of the mineral composition of pulp cocoa sample (mgkg)

Elements Wavelength λ (nm) LODm (mgkg) LOQm (mgkg) Meanplusmn standard deviationAl 167019 0980 3280 1742plusmn 001Ca 422673 1348000 4490 13343plusmn 001Cd 228802 0060 0196 0143plusmn 001Co 228615 0013 0043 mdashCr 206158 0030 0100 0970plusmn 001Cu 213598 0050 0170 0419plusmn 001Fe 259940 0470 1570 3388plusmn 001K 769897 0700 2338 1459842plusmn 001Li 610365 0005 0017 mdashMg 280270 0500 1670 237230plusmn 001Mn 259372 0005 0015 0378plusmn 001Mo 204598 0103 0340 0106plusmn 001Na 330298 3410 11380 98966plusmn 001P 213618 0056 0185 365797plusmn 001Pb 220353 0050 0168 mdashSe 203985 0135 0450 3251plusmn 001V 311837 0007 0022 mdashZn 213857 0017 0058 1535plusmn 001e values are means of three replicates LOD limits of detection LOQ limits of quantification

Table 3 Coded and uncoded values of the variables studied and percentage of reduction in viscosity in cocoa pulp treated with poly-galacturonase from Aspergillus niger

Assay Enzyme(μL100 g) Temperature (degC) Time (min) Enzyme

(μL100 g) Temperature (degC) Time (min) Reductionof viscosity ()

1 minus 1000 minus 1000 minus 1000 2820 400 200 50542 minus 1000 minus 1000 1000 2820 400 500 54283 minus 1000 1000 minus 1000 2820 700 200 30864 minus 1000 1000 1000 2820 700 500 23505 1000 minus 1000 minus 1000 8180 400 200 47186 1000 minus 1000 1000 8180 400 500 58027 1000 1000 minus 1000 8180 700 200 42198 1000 1000 1000 8180 700 500 26999 minus 1682 0000 0000 1000 550 350 571410 1682 0000 0000 10000 550 350 535311 0000 minus 1682 0000 5500 300 350 535312 0000 1682 0000 5500 800 350 00013 0000 0000 minus 1682 5500 550 100 486714 0000 0000 1682 5500 550 600 607615 0000 0000 0000 5500 550 350 546516 0000 0000 0000 5500 550 350 510417 0000 0000 0000 5500 550 350 584018 0000 0000 0000 5500 550 350 5266e values are means of three replicates


complex to reduce viscosity in a study of wine making usingcocoa pulp Manohar et al [81] studied the properties ofmango pulp flow and reported that the pectin content had amarked effect on viscosity a 57 reduction in the content ofpectin reduced viscosity by approximately 50 It is

important to note that the quantification of pectin in al-coholic beverages from fruits is important because of theaction of pectinesterase enzymes which release methanolMethanol inhibits the growth of S cerevisiae even at lowconcentrations [4]

70

60

50

40

30

20

10Redu

ctio

n of

visc

osity

()

100

225

350

475

600

Time (m

in)

800675

550425

300

Temperature (degC)

605040

302010

Figure 1 Response surface

60

50

40

30

20

10

0

Redu

ctio

n of

visc

osity

obs

erve

d (

)

Reduction of viscosity predicted ()0 10 20 30 40 50 60

Figure 2 Observed valuestimes predicted values of percentage of reduction in cocoa pulp

Table 4 Experimental conditions and results of the percentage of reduction in the viscosity of the cocoa pulp using pectinolytic enzymes forvalidation of the model

Assay Temperature (degC) Time (min) Predicted Observed Standard error ()1 550 475 5472 6492a 182 425 600 6437 6475a 0593 425 506 7704 6571a 147


35 Chemical Analysis of the Beer Wort Using Cocoa Pulpe results of the chemical analysis of the wort and thestatistical significances obtained through the F-test areshown in Tables 5 and 6 respectively e mixtures of cocoapulp and wort (10 30 and 49) showed significant dif-ferences compared to the wort without the pulp (0) withrespect to the pH total titratable acid humidity solublesolids protein and color e pH of the wort demonstratedthe interference of the cocoa pulp with the increase inconcentration therefore the total acidity was also influ-enced It was also noted that the formulation with the higherproportion of pulp (49) presented a lower pH and highertotal acidity Since the pH values must have an inverserelationship with the total acidity these results are expectedAn explanation for this finding is that the pulp has a highacidity with the predominance of citric and malic acids(Table 5) that decrease the pH of the mixtures

pte ideal pH to start alcoholic fermentation of beer is from50to 55 therefore it is necessary to correct the pH of the pulp byadding calcium carbonate to standardize the valuee acidity of themedium influences the activity of the yeasts [24] Fruit pulps nor-mally show high humidity and its influence can be observed in themixture with higher pulp content e essential nutrients for thefermentation of yeasts are amino acids and fermentable sugars [82]e protein added to cocoa pulp did not significantly influence themixtures compared to the 100 all-malt wort With regard to theconcentration of total and reducing sugars a significant increase inthe 30 and 49 cocoa pulp mixtures was observed

When analyzing the concentration of the fermentablesugars glucose fructose maltose and maltotriose the in-fluence of the cocoa pulp in relation to glucose and fructosewas noted in higher concentrations e sugars maltose andmaltotriose are only derived from the wort and are thereforereduced in the medium of fermentation with the increase inpulp addition which provides a dilution During fermen-tation the yeasts first consume the hexoses glucose andfructose followed by the more complex sugars maltose andmaltotriose High concentrations of glucose during fer-mentation can interfere with the metabolism of maltoseproducing a beer with a high concentration of residualsugars in addition to interfering negatively with the fer-mentative performance [83 84] e organic acids are otherimportant elements during fermentation e contributionof acetic succinic and formic acids was not observed in theuse of cocoa pulp (Table 6)

An important parameter of beer characterization is thecolor According to Briggs et al [24] the color of beer isrelated to the melanin and caramel present in the malterefore the mixtures with a higher concentration of pulp(less malt in the formulation) showed a lower concentrationof these pigments and were consequently clearer It is im-portant to highlight that the cocoa pulp has a clear colorwhich can also influence the clarification of the mixture

36Analysis of Fermentation of theBeerWortwithCocoaPulpas an Adjunct at Concentrations of 10 30 and 49 eaddition of cocoa pulp as a malt adjunct was used as analternative method to increase the concentration of soluble

solids in the wort favoring fermentation and beer pro-duction with differentiated organoleptic characteristics efermentations were performed at concentrations of 10 30and 49 cocoa pulp using the commercial yeasts to definethe best conditions for the process

In the fermentation at laboratory scale using all-maltwort and cocoa pulp as an adjunct changes in the scale of thewort production of ethanol and cells in suspension wereevaluated during fermentation at 15 and 22degC as shown inFigures 3 and 4

e content of the extract for all fermentations testedwas adjusted to 12degP and the process was ended after thefinal constant concentration Figure 3 reveals the productionof ethanol in the first hours of fermentation with a con-siderable increase from 48 to 36 h at 15 and 22degC respec-tively e use of the adjunct favored this increase inproduction e same behavior was observed in all assayswhere the values of ethanol reached 45 in the final productwhen the concentration of the initial apparent extract was12degP in an all-malt wort [24] During the first 48 h at 15degCthe consumption of extract (measured in terms of apparentextract) was associated with the increase in the number ofcells in suspension (Figures 3 and 4) In this interval (0 to48 h) the apparent extract decreased approximately 50whereas the concentration of cells in suspension increased20 times Consequently the production of ethanol reached4 (vv) At 22degC the consumption of 50 of the substratewas faster (0 to 36 h) and the cells in suspension increasedtheir growth 22 times reaching the same volume of ethanolFrom 60 h when the quantity of saturation in oxygen in themedium probably fell an increase in the production ofethanol was observed for the referred wort e concen-tration of ethanol kept increasing in the medium since it ismore easily formed under anaerobic conditions e end offermentation at 15degC occurred at 120 h whereas at 22degC itoccurred at 96 h with a reduction in the apparent extract andconcentration of cells and with a subsequent increase in thevolume of ethanol

e performance of beer fermentation during produc-tion depends on the capacity of the yeasts to adapt to thechanges and conditions imposed by the medium (oxygenavailability osmotic stress CO2 build-up nutrient limita-tions and ethanol toxicity) [82] An improvement in theperformance of the yeasts during fermentation and a re-duction in the time of the process are desirable for im-proving not only productivity but also the efficiency andsustainability of brewing [85] e analysis of the viable cellsas well as of their performance during fermentation isimportant to ensure the final quality of the product [86]isstudy monitored the percentage of the viable cells in sus-pension and the performance of S cerevisiae using 12degP all-malt wort at 15 and 22degC and compared it to fermentationsusing different concentrations of adjuncts At 22degC theconcentration of cells in suspension reached a maximumvalue of 10times108 cellmL at 48 h and at 15degC the highestnumber of cells was observed at 60 h (18times108 cellmL)After 72 h at 22degC there was a reduction in the number ofviable cells due to the limitation of the substrate and anincrease in the concentration of ethanol At 15degC this


Table 5 Chemical analysis of the wort

ParametersPulp percentage ()

0 10 30 49pH 530aplusmn 001 480bplusmn 0015 450cplusmn 0012 400dplusmn 002Humidity (g100 g) 9086aplusmn 008 9102bplusmn 002 911bplusmn 0021 9180cplusmn 003Total titratable acid (meq NaOH100 g) 176aplusmn 005 181bplusmn 008 185cplusmn 001 195dplusmn 004Reducing sugars (g100 g) 1151aplusmn 002 1164aplusmn 001 1746bplusmn 001 1755bplusmn 003Total sugars (g100 g) 1243aplusmn 003 1222aplusmn 004 1792bplusmn 006 1818bplusmn 009Raw protein (g100 g) 164aplusmn 017 160bplusmn 023 156cplusmn 025 153dplusmn 035Soluble solids (degBrix) 124aplusmn 001 125aplusmn 001 148bplusmn 001 1610cplusmn 001Color (EBC) 151aplusmn 002 1511aplusmn 003 138bplusmn 021 1201cplusmn 015Values marked with the same identification on the same line between the partial means do not differ significantly (pgt 005) according to the Tukey test

Table 6 Analysis of sugars and organic acids in the wort (gL)


0 10 30 49Glucose 987aplusmn 0016 1667bplusmn 002 2518cplusmn 002 3873dplusmn 001Fructose 163aplusmn 005 945bplusmn 005 1185cplusmn 003 1643dplusmn 003Maltose 5863aplusmn 002 5170bplusmn 001 4385cplusmn 001 269dplusmn 001Maltotriose 1462aplusmn 003 1233bplusmn 003 1077cplusmn 002 933dplusmn 001Citric acid 072aplusmn 0015 086aplusmn 002 290bplusmn 004 395cplusmn 006Acetic acid 065aplusmn 012 064aplusmn 033 056bplusmn 035 037cplusmn 032Malic acid 02aplusmn 002 071bplusmn 0015 18cplusmn 0011 25dplusmn 0012Succinic acid 444aplusmn 002 337bplusmn 002 304cplusmn 003 213dplusmn 003Formic acid 091aplusmn 002 091aplusmn 0023 093bplusmn 021 093bplusmn 015Values marked with the same identification on the same line between the partial means do not differ significantly (pgt 005) according to the Tukey test

12

10

8

6

4

2

0

Extr

act a

ppar

ent (

degP)

50

40

30

20

10

0

Etha

nol (

gL)

0 20 40 60 80 100 120 140 160 180Time (h)

0

10

30

49

Extract apparentEthanol




(a)

12

10

8

6

4

2

0

Extr

act a

ppar

ent (

degP)

50

40

30

20

10

0

Etha

nol (

gL)

0 20 40 60 80 100 120 140 160 180Time (h)

0

10

30

49





(b)

Figure 3 Consumption of apparent extract in all-malt wort 0 and wort with cocoa pulp as an adjunct at concentrations of 10 30 and49 and ethanol production at concentrations of 0 10 30 and 49 of cocoa pulp with the following conditions initial concentrationof wort at 12degP and temperature at 15degC (a) and 22degC (b)


reduction was slower and occurred after 84 h Many factorscan affect the capacity of fermentation by the yeasts how-ever the composition of the wort is one of the most im-portant and can vary due to material availability andproduction techniques [24 83] As seen in Figure 4 for allfermentations there was no adaptative long phase once thesubstrates were inoculated with new cells and propagated Inthe all-malt wort fermentation (0) the number of viablecells increased and then fell due to flocculation However inthe fermentation with cocoa pulp as an adjunct under thesame conditions at 15degC and 22degC an increase in the numberof cells was observed characterizing the phase of exponentialgrowth reaching a maximum point of development anddecreasing close to the end of the fermentations With theuse of the adjunct the growth of cells was faster than in theall-malt wort Analyzing the results despite the differenti-ated performances the strains of lager and ale S cerevisiaecan develop in media of different compositions for beerproduction and meet the needs of the industry

e use of an adjunct in this case the cocoa pulp as analternative method to increase the concentration of solublesolids in the wort favors fermentation and aims at producinga beer with differentiated organoleptic characteristics [24]e use of certain types of adjuncts as a substitute for part ofthe malt offers only carbohydrates therefore their use inhigh concentrations can result in the reduction in thecontent of amino acids available in the wort compared tofermentations without adjuncts [87] e two nutrients thatimpact the development of yeasts during fermentation arecarbohydrates and nitrogen e individual assimilation ofnutrients depends on the response of the yeasts to thevarious components e yeasts S cerevisiae can use several

carbohydrates (glucose sucrose fructose maltose galactoseraffinose and maltotriose) with differences between thelager and ale strains as the lager yeasts can ferment meli-biose [83]

At 22degC using the adjunct at the concentrations of 10 30and 49 a higher production of ethanol and a higherconcentration of viable cells were observed As listed inTables 2 and 3 when analyzing the parameters of fermen-tation performed at 15 and 22degC the pulp concentration of30 shows significant differences (plt 005) in relation to theother concentrations with a higher volumetric productivityof ethanol of 048 and 056 gL h (Table 7)

A shorter fermentation time occurred when the cocoapulp was added at the concentration of 30 (Table 8) It isimportant to note that the experiments performed at 22degCoccurred in a shorter time than at 15degC Briggs et al [24]found that an increase in the temperature of fermentationcauses a reduction in the time of attenuation of the wortbecause up to a point the increase in temperature favors themetabolism of the yeasts studiede experiments show thatthe use of 30 cocoa pulp as an adjunct to the malt has thebest results with respect to the time of fermentation ap-parent degree of fermentation consumption of substrateand ethanol production

According to the results described the use of an adjunctin a concentration of 30 favored the ethanol productionconsumption of substrate and concentration of cells insuspension in comparison with the all-malt wort at 15degC and22degC It was therefore noted that the increase in the con-centration of adjunct (up to the limit of 30) contributed tohigher production indicating a positive effect of the nu-trients and sugar profile of the cocoa pulp on the viability

24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)

Concentration of pulp cocoa010

3049

(a)

24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(b)

Figure 4 Growth yeasts S-23 and S-04 in all-malt wort 0 and wort with cocoa pulp as an adjunct at the concentrations of 10 30 and49 in the following conditions initial concentration of 12degP and temperature at 15degC (a) and 22degC (b)


and performance of the yeasts during beer fermentation Inthis case the high concentration of fermentable sugars(glucose and fructose) in the cocoa pulp as an adjunct canexplain the higher ethanol production It is important tonote that at 49 concentration the ethanol production waslower than at 30 which can be related to the reduction ofnutrients in the medium due to the high concentration of theadjunct [83ndash87] Different concentrations of carbohydratescan negatively affect the fermentation particularly in wortsin which glucose is the predominant carbohydrate [84ndash88]In this case the cocoa pulp shows high concentrations ofglucose and fructose at 5211 and 5235 gL respectivelyerefore the concentration of 30 cocoa pulp as an ad-junct was selected to continue pilot studies at the bestperformance temperature at 22degC

4 Conclusions

We have studied the application of cocoa pulp as an adjuvant ofmalt to improve brewing Based on the results of the charac-terization of the cocoa pulp such as pH value of 35 and glucoseand fructose contents of 5211 and 5235 gL respectively thecocoa pulp can be considered to other fruits with potential foruse in the beverage industry e values found for the mineralsare also favorable for fermentation mainly the high contents ofpotassium magnesium and calcium 1459842 237230 and13343mgkg respectively ese minerals can contributepositively to the fermentative performancee pulp showed anapparent high viscosity of 95 cP at 30degC which is not appro-priate to the fermentation process ree conditions used todecrease the viscosity of the pulp through variations between theenzyme concentration (polygalacturonase) time and temper-ature were able to reduce this characteristic by 65 whichmakes the use of any of the conditions for this purpose epresence of the pulp in the wort in different concentrations(10 30 and 49) was able to influence its characteristics

such as pH (530ndash400) glucose (987ndash3873 gL) and fructose(163ndash1643gL) for example Furthermore the concentrationof 30 of the cocoa pulp in the wort was favorable for ethanolproduction consumption of substrate and concentration ofcells in suspension in comparison with the all-malt wort at 15degCand 22degC us the study showed the importance of using thecocoa pulp in the process of obtaining beer

Data Availability

e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

e authors would like to thank the State University of SantaCruz (Bahia) the State University of Feira de Santana(Bahia) the Bahian Federal Institute Catu Campus (Bahia)the National Industrial Learning Service (SENAI-Bahia) theFederal University of the State of Rio de Janeiro and theMars Center for Cocoa Science

Supplementary Materials

Table 1S analysis of variance (ANOVA) for the assay ofviscosity reduction in the cocoa pulp Table 2S coefficients ofregression with all the factors and their interactions for theviscosity reduction analysis assay in the cocoa pulp Table 3Scoefficients of regression with the factors and significantinteractions (plt 005) for the viscosity reduction analysisassay in the cocoa pulp Table 4S F-test for the viscosityreduction analysis assay in the cocoa pulp (SupplementaryMaterials)

Table 7 Average values with standard deviation of volumetric productivity of ethanol by the commercial yeast for fermentation at 15degC and22degC with the addition of cocoa pulp at the concentrations of 10 30 and 49 to the malt wort after 96 h of fermentation

Pulp concentration ()Qp (gLh)

Yeast S-23 Yeast S-040 043aplusmn 000 053aplusmn 00010 047bplusmn 000 055bplusmn 00030 048cplusmn 000 056cplusmn 00049 047bplusmn 000 055bplusmn 000Values marked with the same identification in the same column between the partial means do not differ significantly (pgt 005) according to the Tukey test

Table 8 Comparison of the time and apparent degree of fermentation () for the commercial yeasts S-23 and S-04 in fermentation at 15degCand 22degC respectively in all-malt wort using adjunct

ParametersConcentration of adjunct

0 10 30 49Temperature (degC) 15 22 15 22 15 22 15 22Time (h) 132 84 84 72 72 60 84 72Apparent degree offermentation () 7472aplusmn 01 7472eplusmn 015 7492bplusmn 02 7494fplusmn 018 7496cplusmn 01 7501gplusmn 01 7498dplusmn 02 7507hplusmn 01

Values marked with the same identification in the same column between the partial means do not differ significantly (pgt 005) according to the Tukey test


References

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[2] C L Hii A S Menon C L Chiang and S Sharif ldquoKinetics ofhot air roasting of cocoa nibs and product qualityrdquo Journal ofFood Process Engineering vol 40 no 3 Article ID e124672017

[3] E Teye X Huang J Takrama and G Haiyang ldquoIntegratingNIR spectroscopy and electronic tongue together with che-mometric analysis for accurate classification of cocoa beanvarietiesrdquo Journal of Food Process Engineering vol 37 no 6pp 560ndash566 2014

[4] P B Leite W M Machado A G GuimaratildeesG B M d Carvalho K Teixeira Magalhatildees-Guedes andJ Izabel Druzian ldquoCocoarsquos residual honey physicochemicalcharacterization and potential as a fermentative substrate bySaccharomyces cerevisiae AWRI726rdquo 5e Scientific WorldJournal vol 2019 Article ID 5698089 7 pages 2019

[5] I Abdulai L Jassogne S Graefe et al ldquoCharacterization ofcocoa production income diversification and shade treemanagement along a climate gradient in Ghanardquo PLoS Onevol 13 no 4 Article ID e0195777 2018

[6] H B Daghela Bisseleua D Fotio Yede A D Missoup andS Vidal ldquoShade tree diversity cocoa pest damage yieldcompensating inputs and farmersrsquo net returns in west africardquoPLoS One vol 8 no 3 Article ID e56115 2013

[7] B Trognitz E Cros S Assemat et al ldquoDiversity of cacao treesin waslala nicaragua associations between genotype spectraproduct quality and yield potentialrdquo PLoS One vol 8 no 1Article ID e54079 2013

[8] V Popescu A Soceanu S Dobrinas et al ldquoA study of beerbitterness loss during the various stages of the Romanian beerproduction processrdquo Journal of the Institute of Brewingvol 119 pp 111ndash115 2013

[9] ICCOmdashInternational Cocoa Organization World CocoaProduction 2016 httpwwwiccoorg

[10] E Freire R Schwan and R Mororo ldquoe cocoa pulpagroindustry and the use of its residues in bahia progressachieved in the last ten yearsrdquo in Proceedings of the 12thInternational Cocoa Research Conference Cocoa ProducersAlliance Kuala Lumpur Malaysia pp 1013ndash1020 1999

[11] U V Lopes W R Monteiro J L Pires D ClementM M Yamada and K P Gramacho ldquoCacao breeding inBahia Brazil strategies and resultsrdquo Crop Breeding andApplied Biotechnology vol 11 pp 73ndash81 2011

[12] E S L Santos C B M Cerqueira-Silva G M Mori et alldquoGenetic structure and molecular diversity of cacao plantsestablished as local varieties for more than two centuries thegenetic history of cacao plantations in Bahia Brazilrdquo PLoSOne vol 10 no 12 Article ID e0145276 2015

[13] I M da Veiga Moreira L de Figueiredo Vilela C SantosN Lima and R F Schwan ldquoVolatile compounds and proteinprofiles analyses of fermented cocoa beans and chocolatesfrom different hybrids cultivated in Brazilrdquo Food ResearchInternational vol 109 pp 196ndash203 2018

[14] R F Schwan and A E Wheals ldquoe microbiology of cocoafermentation and its role in chocolate qualityrdquo Critical Re-views in Food Science and Nutrition vol 44 no 4 pp 205ndash221 2004

[15] C da Silva Oliveira Nunes G B M de Carvalho M L C daSilva et al ldquoCocoa pulp in beer production applicability and

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[17] E Meersman J Steensels M Mathawan et al ldquoDetailedanalysis of the microbial population in Malaysian sponta-neous cocoa pulp fermentations reveals a core and variablemicrobiotardquo PLoS One vol 8 no 12 Article ID e81559 2013

[18] C Collar C M Rosell B Muguerza and L MoulayldquoBreadmaking performance and keeping behavior of cocoa-soluble fiber-enriched wheat breadsrdquo Food Science andTechnology International vol 15 no 1 pp 79ndash87 2009

[19] D R Dias R F Schwan E S Freire and R d S SerodioldquoElaboration of a fruit wine from cocoa (5eobroma cacao L)pulprdquo International Journal of Food Science amp Technologyvol 42 no 3 pp 319ndash329 2007

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[23] S Ostergaard L Olsson and J Nielsen ldquoMetabolic engi-neering of Saccharomyces cerevisiaerdquo Microbiology and Mo-lecular Biology Reviews vol 64 no 1 pp 34ndash50 2000

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[26] R Gonzalez A Vian and A V Carrascosa ldquoNote Mor-phological changes in Saccharomyces cerevisiae during thesecond fermentation of sparkling winesrdquo Food Science andTechnology International vol 14 no 4 pp 393ndash398 2008

[27] J Ubeda N Barrajon and A Briones ldquoOptimizing small-scale production of fresh wine yeast biomassrdquo Journal of FoodProcess Engineering vol 36 pp 686ndash693 2013

[28] G M Heard and G H Fleet ldquoe effects of temperature andpH on the growth of yeast species during the fermentation ofgrape juicerdquo Journal of Applied Bacteriology vol 65 no 1pp 23ndash28 1988

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[30] G Donadini M D Fumi E Kordialik-Bogacka L MaggiM Lambri and P Sckokai ldquoConsumer interest in specialtybeers in three European marketsrdquo Food Research Interna-tional vol 85 pp 301ndash314 2016

[31] K Osburn J Amaral S R Metcalf et al ldquoPrimary souring anovel bacteria-free method for sour beer productionrdquo FoodMicrobiology vol 70 pp 76ndash84 2018

[32] H Zabed G Faruq J N Sahu et al ldquoBioethanol productionfrom fermentable sugar juicerdquo 5e Scientific World Journalvol 2014 Article ID 957102 11 pages 2014

[33] L V A Reddy and O V S Reddy ldquoProduction and char-acterization of wine from mango fruit (Mangifera indica L)rdquoWorld Journal of Microbiology and Biotechnology vol 21no 8-9 pp 1345ndash1350 2005

[34] T E Ayogu ldquoEvaluation of the performance of a yeast isolatefrom Nigerian palm wine in wine production from pineapplefruitsrdquo Bioresource Technology vol 69 no 2 pp 189-1901999


[35] V Bridgebassie and N Badrie ldquoEffects of different pectolaseconcentration and yeast strains on carambola wine quality inTrinidad West Indiesrdquo Fruits vol 59 no 2 pp 131ndash1402004

[36] A Arruda A Casimiro D Garruti et al ldquoFermented bananabeverage processingrdquo Revista Ciencia Agronomica vol 34pp 161ndash167 2003

[37] G B M Carvalho D P Silva C V Bento et al ldquoBanana asadjunct in beer production applicability and performance offermentative parametersrdquo Applied Biochemistry and Bio-technology vol 155 pp 356ndash365 2009

[38] G B M Carvalho ldquoBeer obtaining using bana as na adjunctand a flavouring agentrdquo Dissertation for the Degree of Doctorof Industrial Biotechnology Univesity of Satildeo Paulo SatildeoPaulo Brazil 2009

[39] P F Cancalon and M E Parish ldquoChanges in the chemicalcomposition of orange juice during growth of Saccharomycescerevisiae and Gluconobacter oxydansrdquo Food Microbiologyvol 12 pp 117ndash124 1995

[40] M L Corazza D G Rodrigues and J Nozaki ldquoPreparaccedilatildeo ecaracterizaccedilatildeo do vinho de laranjardquo Quim Nova vol 24pp 449ndash452 2001

[41] S Selli A Canbas V Varlet H Kelebek C Prost andT Serot ldquoCharacterization of the most odor-active volatiles oforange wine made from a Turkish cv Kozan (Citrus sinensisL Osbeck)rdquo Journal of Agricultural and Food Chemistryvol 56 no 1 pp 227ndash234 2008

[42] E Polychroniadou M Kanellaki M Iconomopoulou et alldquoGrape and apple wines volatile fermentation products andpossible relation to spoilagerdquo Bioresource Technology vol 87no 3 pp 337ndash339 2003

[43] A A Rossi and C R Barbosa ldquoEstudo da suplementaccedilatildeo comnutrientes na fermentaccedilatildeo do suco de maccedilatilde por Saccharo-myces cerevisiaerdquo Universidade do Vale do Paraıba vol 31pp 52ndash63 2007

[44] J-M Le Quere F Husson C M G C Renard andJ Primault ldquoFrench cider characterization by sensorytechnological and chemical evaluationsrdquo LWT-Food Scienceand Technology vol 39 no 9 pp 1033ndash1044 2006

[45] W F Duarte D R Dias G V de Melo PereiraI M Gervasio and R F Schwan ldquoIndigenous and inoculatedyeast fermentation of gabiroba (Campomanesia pubescens)pulp for fruit wine productionrdquo Journal of Industrial Mi-crobiology amp Biotechnology vol 36 no 4 pp 557ndash569 2009

[46] E H Soufleros I Pissa D Petridis et al ldquoInstrumentalanalysis of volatile and other compounds of Greek kiwi winesensory evaluation and optimisation of its compositionrdquo FoodChemistry vol 75 no 4 pp 487ndash500 2001

[47] D R Dias R F Schwan and L C O Lima ldquoMetodologiapara elaboraccedilatildeo de fermentado de caja (Spondias mombin L)rdquoCiencia e Tecnologia de Alimentos vol 23 no 3 pp 342ndash3502003

[48] C Puerari K T Magalhatildees and R F Schwan ldquoNew cocoapulp-based kefir beverages microbiological chemical com-position and sensory analysisrdquo Food Research Internationalvol 48 no 2 pp 634ndash640 2012

[49] W F Duarte D R Dias J M Oliveira J A TeixeiraJ B de Almeida e Silva and R F Schwan ldquoCharacterizationof different fruit wines made from cacao cupuassu gabirobajaboticaba and umburdquo LWT-Food Science and Technologyvol 43 no 10 pp 1564ndash1572 2010

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productsrdquo Journal of Food Process Engineering vol 30 no 3pp 338ndash356 2007

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[52] G Stewart ldquoe influence of high gravity wort on the stresscharacteristics of brewerrsquos yeast and related strainsrdquo Cerevisiavol 32 pp 37ndash48 2007

[53] A Huuskonen TMarkkula V Vidgren et al ldquoSelection fromindustrial lager yeast strains of variants with improved fer-mentation performance in very-high-gravity wortsrdquo Appliedand Environmental Microbiology vol 76 no 5 pp 1563ndash1573 2010

[54] A I Paz C A Blanco C Andres-Iglesias L PalacioP Pradanos and A Hernandez ldquoAroma recovery of beerflavors by pervaporation through polydimethylsiloxanemembranesrdquo Journal of Food Process Engineering vol 40no 6 Article ID e12556 2017

[55] J K M Mensah and P Twumasi ldquoUse of pineapple waste forsingle cell protein (SCP) production and the effect of substrateconcentration on the yieldrdquo Journal of Food Process Engi-neering vol 40 no 3 Article ID e12478 2017

[56] G Dragone S I Mussatto and J B Almeida e Silva ldquoHighgravity brewing by continuous process using immobilisedyeast effect of wort original gravity on fermentation per-formancerdquo Journal of the Institute of Brewing vol 113 no 4pp 391ndash398 2007

[57] C M Boulton and D Quain Brewing Yeast and Fermenta-tion Blackwell Science Oxford UK 2006

[58] L M Santos F A Oliveira E H Ferreira and A RosenthalldquoApplication and possible benefits of high hydrostatic pres-sure or high-pressure homogenization on beer processing areviewrdquo Food Science and Technology International vol 23no 7 pp 561ndash581 2017

[59] D S Carvalho and R C Zambiazi ldquoAvaliaccedilatildeo do processofermentativo de cerveja pilsen pelo uso de diferentes con-centraccedilotildees de Saccharomyces Cerevisiaerdquo Brazilian Journal ofFood and Nutrition vol 22 pp 351ndash357 2011

[60] IAL - ldquoInstituto Adolfo Lutzrdquo Metodos Fısico-Quımicos paraAnalise de Alimentos -4a Ediccedilatildeo 1a Ediccedilatildeo Digital InstitutoAdolfo Lutz Satildeo Paulo Brazil 2008

[61] G L Miller ldquoUse of dinitrosalicylic acid reagent for deter-mination of reducing sugarrdquo Analytical Chemistry vol 31no 3 pp 426ndash428 1959

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[64] V Singleton and J Rossi ldquoColorimetry of total phenolics withphosphomolybdic-phosphotungstic acid reagentsrdquo AmericanJournal of Enology and Viticulture vol 16 pp 144ndash158 1965

[65] W Peschel F Sanchez-Rabaneda W Diekmann et al ldquoAnindustrial approach in the search of natural antioxidants fromvegetable and fruit wastesrdquo Food Chemistry vol 97 no 1pp 137ndash150 2006

[66] M Biendl and P Anderegg ldquoModification of the methodanalytica-EBC 77 (alpha-and beta-acids in hops and hopproducts by HPLC)rdquo Journal of the Institute of Brewingvol 111 pp 85-86 2005


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[70] L C Basso A J Oliveira V F M Orelli et al ldquoDominanciadas leveduras contaminantes sobre as linhagens industriaisavaliada pela tecnica da cariotipagemrdquo Anais CongressoNacional da STAB vol 5 1994

[71] S C d Jesus M I d S Folegatti F C A U Matsuura andR L Cardoso ldquoCaracterizaccedilatildeo fısica e quımica de frutos dediferentes genotipos de bananeirardquo Bragantia vol 63 no 3pp 315ndash323 2004

[72] G Chandrasena G M Walker and H J Staines ldquoUse ofresponse surfaces to investigate metal ion interactions in yeastfermentationsrdquo Journal of the American Society of BrewingChemists vol 55 no 1 pp 24ndash29 1997

[73] M Azenha M T Vasconcelos and P Moradas-Ferreira ldquoeinfluence of Cu concentration on ethanolic fermentation bySaccharomyces cerevisiaerdquo Journal of Bioscience and Bioen-gineering vol 90 no 2 pp 163ndash167 2000

[74] J Ferreira T M D Du and W J D Toit ldquoe effects ofcopper and high sugar concentrations on growth fermen-tation efficiency and volatile acidity production of differentcommercial wine yeast strainsrdquo Australian Journal of Grapeand Wine Research vol 12 no 1 pp 50ndash56 2006

[75] N J Robinson and D R Winge ldquoCopper metal-lochaperonesrdquo Annual Review of Biochemistry vol 79 no 1pp 537ndash562 2010

[76] X Sun L Liu Y Zhao et al ldquoEffect of copper stress on growthcharacteristics and fermentation properties of Saccharomycescerevisiae and the pathway of copper adsorption during winefermentationrdquo Food Chemistry vol 192 pp 43ndash52 2016

[77] J Du H-L Li H Li et al ldquoInfluence of copper stress onSaccharomyces cerevisiae grape mustrdquo Scientia Agricolavol 43 pp 3259ndash3265 2010

[78] X Sun Y Zhao L Liu et al ldquoCopper tolerance and bio-sorption of Saccharomyces cerevisiae during alcoholic fer-mentationrdquo PLoS One vol 10 no 6 Article ID e01286112015

[79] J Ahmed H S Ramaswamy and N Hiremath ldquoe effect ofhigh pressure treatment on rheological characteristics andcolour of mango pulprdquo International Journal of Food Scienceand Technology vol 40 no 8 pp 885ndash895 2005

[80] M G B Koblitz ldquoBioquımica de alimentos-teoria e aplicaccedilotildeespraticasrdquo Guanabara Koogan Rio de Janeiro Brazil 2008

[81] B Manohar P Ramakrishna and R S Ramteke ldquoEffect ofpectin content on flow properties of mango pulp concen-tratesrdquo Journal of Texture Studies vol 21 no 2 pp 179ndash1901990

[82] B R Gibson S J Lawrence J P R Leclaire C D Powell andK A Smart ldquoYeast responses to stresses associated withindustrial brewery handling figure 1rdquo FEMS MicrobiologyReviews vol 31 no 5 pp 535ndash569 2007

[83] E J Lodolo J L F Kock B C Axcell and M Brooks ldquoeyeast Saccharomyces cerevisiae-the main character in beerbrewingrdquo FEMS Yeast Research vol 8 no 7 pp 1018ndash10362008

[84] G G Stewart ldquoStudies on the uptake and metabolism of wortsugars during brewing fermentationsrdquo Tech Quarterly vol 43pp 265ndash269 2006

[85] J Ekberg J Rautio L Mattinen V VidgrenJ Londesborough and B R Gibson ldquoAdaptive evolution ofthe lager brewing yeast Saccharomyces pastorianus for im-proved growth under hyperosmotic conditions and its in-fluence on fermentation performancerdquo FEMS Yeast Researchvol 13 no 3 pp 335ndash349 2013

[86] L Guido P G Rodrigues J A Rodrigues et al ldquoe impactof the physiological condition of the pitching yeast on beerflavour stability an industrial approachrdquo Food Chemistryvol 87 no 2 pp 187ndash193 2004

[87] M P Piddocke S Kreisz H P Heldt-Hansen K F Nielsenand L Olsson ldquoPhysiological characterization of brewerrsquosyeast in high-gravity beer fermentations with glucose ormaltose syrups as adjunctsrdquo Applied Microbiology and Bio-technology vol 84 no 3 pp 453ndash464 2009

[88] E Cabiscol E Piulats P Echave E Herrero and J RosldquoOxidative stress promotes specific protein damage in Sac-charomyces cerevisiaerdquo 5e Journal of Biological Chemistryvol 275 no 35 pp 27393ndash27398 2000


tropical fruits as it is an abundant product derived from anestablished culture Research aims at obtaining pulp toproduce juice jam compotes fermented beverages andother processed products thereby offering special and at-tractive flavors [19]

Cocoa pulp can be easily fermented by yeasts such asS cerevisiae to produce an alcoholic beverage is yeast hasbeen used in fermentation processes for a long timeaccording to the first historical reports of beer and wineproduction [20ndash27]e wort composition is very importantfor beer production because it ensures the quality of the finalproduct and is a growth medium for the yeasts Severalenvironmental factors influence the production of metab-olites and the survival of the yeasts during industrial fer-mentation e main factors are temperature pH and sugarconcentration as well as pulp characteristics in the case ofbeer production with the use of the adjunct [28ndash31] Similarto cocoa pulp many other fruits can be used in the pro-duction of alcoholic beverages [32] Various experimentshave been carried out with fruits such as mango [33]pineapple [34] star fruit [35] banana [36ndash38] orange[39ndash41] grape [42] apple [43 44] cocoa [19] gabiroba [45]kiwi [46] and caja [47] for the production of fermentedbeverages However due to the different chemical compo-sitions of the fruits further studies are necessary includingthe type of fermented beverage to be produced the idealtemperature of fermentation and the type of treatment thatmust be applied to the fruit pulp or the wort in the pre-fermentative phase andor during fermentation Cocoa pulpconstitutes a very favorable raw material for the productionof beverages by alcoholic fermentation because it is rich incarbohydrates has low acidity and contains importantorganic acids [19 48ndash51]

A relevant aspect in the current beer market is the use ofadjuncts with a high concentration of fermentable sugarsthat contribute to higher production of ethanol from eachdegPlato of extract [52ndash55] e use of certain types of adjunctsonly contributes to the supply of carbohydrates and no othernutrients consequently its addition to the wort at the be-ginning of beer fermentation results in a similar balance ofnutrients to the fermentations without the addition of ad-junct [56ndash58] e main objective of this work was toevaluate the performance of fermentation in the productionof beer using cocoa pulp from the south region of Bahia(Brazil) as an adjunct for malt after the determination of itschemical composition

2 Materials and Methods

21 Obtaining and Storing the Cocoa Pulp Cocoa fruits wereobtained from clone VP 1151 which was supplied by theMars Center for Cocoa Science located in the south region ofthe state of Bahia (Ilheus Brazil) the cocoa fruits wereharvested at their optimum ripeness for consumption eselection step consisted of the rejection of defective fruits toavoid compromising the quality of the final pulp and size-and color-based selection from the commercially availablefruitse selected fruits were washed with clean tap water toremove impurities sanitized for 30min in a 100mgmiddotmLminus 1

free chlorine solution and rinsed with clean tap water toremove excess free chlorine en the cocoa fruits werebroken pulped using a bench depulper (model DM-Ji-05Macanuda) fractionated and stored in flexible polyethylenebags e pulp fractions were then frozen and stored atminus 18degC until chemical analysis

22 Wort Production e wort was produced according tothe conventional techniques of beer production at theLaboratory of Fermentation of the State University of Feirade Santana (UEFS) Bahia Brazil During this process 88 kgof malt (Chateau Pilsen 325 EBC from Belgium) wasground in a bench grinder to decrease the grain size andfacilitate hydrolysis catalyzed by enzymes during wortproduction e wort production ramp used was adaptedfrom Carvalho and Zambiazi [59]e initial pH value of thewort was adjusted to 53 by adding food-grade lactic acide wort was hot-packed (gt90degC) and stored at 0degC until use

23 Chemical Analysis of the Pulp and Wort e chemicalanalysis of the cocoa pulp and wort was performed at theLaboratory of Physical-Chemical Analysis of the Depart-ment of Technology State University of Feira de SantanaBahia e humidity was determined by direct drying in anoven at 105degC and ashes were obtained by incinerating thesamples in a muffle oven at 550degC Protein was determinedby the Kjeldahl method and lipids by gravimetry after ex-traction with petroleum ether using a Soxhlet extractor andstarch Pectin and titratable acidity were determinedaccording to the methodology recommended by Adolfo LutzInstitute [60] e sugars were determined by spectropho-tometry with the reducing sugars established by the DNSmethod (35-dinitrosalicylic acid) [61] and the total sugarsby the phenol-sulfuric method [62] e pH was determinedby the potentiometric method using a digital InstruthermpH meter (model PH-1700) and titratable acidity by neu-tralization with the results of the titration expressed in g ofmalic acid per 100 g of pulp according to the methodologydescribed by Carvalho et al [63]

e total phenolic content of the cocoa pulp was de-termined according to Singleton and Rossi [64] by the re-action between the pulp extract and FolinndashCiocalteu reagentin the presence of sodic carbonate based on the appearanceof blue due to the oxidation of phenols in the basic mediume absorbance was measured in a spectrophotometer(Shimadzu model UV mini 1240) at 765 nme quantity oftotal phenols was expressed in gallic acid equivalents (GAE)(mg GAEg of sample) through a calibration curve [65] ecolor of the wort was determined by the spectrophotometricmethod at a wavelength of 430 nm [66]

24 Determination of Sugars and Organic Acids e sugarsand organic acids were determined by high-performanceliquid chromatography (HPLC) e samples were diluted10 times in ultrapure water and filtered through difluoridepolyvinylidene membranes (045 μm pores 13mm diame-ter) (Millex HV Millipore) Standard curves were built for













(1)















































70

60

50

40

30

20

10Redu

ctio

n of

visc

osity

()

100

225

350

475

600

Time (m

in)

800675

550425

300

Temperature (degC)

605040

302010


60

50

40

30

20

10

0

Redu

ctio

n of

visc

osity

obs

erve

d (

)






















12

10

8

6

4

2

0

Extr

act a

ppar

ent (

degP)

50

40

30

20

10

0

Etha

nol (

gL)

0 20 40 60 80 100 120 140 160 180Time (h)

0

10

30

49





(a)

12

10

8

6

4

2

0

Extr

act a

ppar

ent (

degP)

50

40

30

20

10

0

Etha

nol (

gL)

0 20 40 60 80 100 120 140 160 180Time (h)

0

10

30

49





(b)









24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(a)

24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(b)




4 Conclusions



Data Availability




Acknowledgments












References









































































































(1)















































70

60

50

40

30

20

10Redu

ctio

n of

visc

osity

()

100

225

350

475

600

Time (m

in)

800675

550425

300

Temperature (degC)

605040

302010


60

50

40

30

20

10

0

Redu

ctio

n of

visc

osity

obs

erve

d (

)






















12

10

8

6

4

2

0

Extr

act a

ppar

ent (

degP)

50

40

30

20

10

0

Etha

nol (

gL)

0 20 40 60 80 100 120 140 160 180Time (h)

0

10

30

49





(a)

12

10

8

6

4

2

0

Extr

act a

ppar

ent (

degP)

50

40

30

20

10

0

Etha

nol (

gL)

0 20 40 60 80 100 120 140 160 180Time (h)

0

10

30

49





(b)









24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(a)

24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(b)




4 Conclusions



Data Availability




Acknowledgments












References






































































































































70

60

50

40

30

20

10Redu

ctio

n of

visc

osity

()

100

225

350

475

600

Time (m

in)

800675

550425

300

Temperature (degC)

605040

302010


60

50

40

30

20

10

0

Redu

ctio

n of

visc

osity

obs

erve

d (

)






















12

10

8

6

4

2

0

Extr

act a

ppar

ent (

degP)

50

40

30

20

10

0

Etha

nol (

gL)

0 20 40 60 80 100 120 140 160 180Time (h)

0

10

30

49





(a)

12

10

8

6

4

2

0

Extr

act a

ppar

ent (

degP)

50

40

30

20

10

0

Etha

nol (

gL)

0 20 40 60 80 100 120 140 160 180Time (h)

0

10

30

49





(b)









24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(a)

24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(b)




4 Conclusions



Data Availability




Acknowledgments












References














































































































12

10

8

6

4

2

0

Extr

act a

ppar

ent (

degP)

50

40

30

20

10

0

Etha

nol (

gL)

0 20 40 60 80 100 120 140 160 180Time (h)

0

10

30

49





(a)

12

10

8

6

4

2

0

Extr

act a

ppar

ent (

degP)

50

40

30

20

10

0

Etha

nol (

gL)

0 20 40 60 80 100 120 140 160 180Time (h)

0

10

30

49





(b)









24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(a)

24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(b)




4 Conclusions



Data Availability




Acknowledgments












References




































































































24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(a)

24

22

20

18

16

14

12

10

8

6

4

2

0

Cell

mL

times 10

7

0 20 40 60 80 100 120 140 160 180Time (h)


3049

(b)




4 Conclusions



Data Availability




Acknowledgments












References































































































4 Conclusions



Data Availability




Acknowledgments












References






























































































References






























































































potentialapplicabilityofcocoapulp(theobromacacao l)asan...

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