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Physicochemical characterization of special persimmon fruit beers using bohemian pilsner malt as a base

2017; Wiley; Volume: 123; Issue: 3 Linguagem: Inglês

10.1002/jib.434

2050-0416

Alejandro Martínez‐Moreno, Salud Vegara, Nuria Martí, Manuel Valero, Domingo Saura,

Horticultural and Viticultural Research

Journal of the Institute of BrewingVolume 123, Issue 3 p. 319-327 Research articleFree Access Physicochemical characterization of special persimmon fruit beers using bohemian pilsner malt as a base Alejandro Martínez, Alejandro Martínez IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, SpainSearch for more papers by this authorSalud Vegara, Salud Vegara IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, SpainSearch for more papers by this authorNuria Martí, Nuria Martí IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, SpainSearch for more papers by this authorManuel Valero, Corresponding Author Manuel Valero m.valero@umh.es IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, SpainCorrespondence to: M. Valero, IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, Spain. E-mail: m.valero@umh.esSearch for more papers by this authorDomingo Saura, Domingo Saura IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, SpainSearch for more papers by this author Alejandro Martínez, Alejandro Martínez IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, SpainSearch for more papers by this authorSalud Vegara, Salud Vegara IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, SpainSearch for more papers by this authorNuria Martí, Nuria Martí IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, SpainSearch for more papers by this authorManuel Valero, Corresponding Author Manuel Valero m.valero@umh.es IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, SpainCorrespondence to: M. Valero, IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, Spain. E-mail: m.valero@umh.esSearch for more papers by this authorDomingo Saura, Domingo Saura IBMC.-JBT Corp., FoodTech R&D Alliance, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández (UMH)-Campus de Orihuela, Carretera de Beniel km 3.2, 03312 Orihuela, Alicante, SpainSearch for more papers by this author First published: 05 June 2017 https://doi.org/10.1002/jib.434Citations: 25AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract This study involved the production of special fruit ale beers with different concentrations (100:0%, 75:25%, 50:50% and 25:75% v/v) of barley malt and persimmon juice from the ‘Rojo Brillante’ variety. Fermentation took place under beer quality control parameters and the influence of persimmon juice on beer quality was investigated. Colour, turbidity, pH, titratable acidity, total soluble solids, sugars, organic acids, total phenolic compounds, antioxidant capacity and ethanol formation were determined during the fermentation process. These fruit beers, whose alcoholic contents were within the standards (3.6–5.63% v/v ethanol), were characterized by a normal acid pH (3.97–4.13) with citric and lactic acids the most abundant organic acids, a clear golden colour without turbidity [2.05–2.83 European Brewery Convention units], intermediate total phenolic compound values (283.0–327.1 mg GAE/L) and antioxidant activities between 1.65 and 5.78 mm TE/L. The persimmon beverage which contained 75% fruit juice was the most valued and preferred by the panelists followed by the 50:50% wort–persimmon beer. Copyright © 2017 The Institute of Brewing & Distilling Introduction Beer is a popular drink and represents the most widely consumed alcoholic beverage in the world 1. It is a nutritive, digestive, soothing and sedative tonic or beverage 2. It is consumed as a refreshing drink. In the summer months, beer drinkers occasionally dilute their beers with lemonade or fruit soda to obtain refreshing radlers and shandys. However, introducing actual fruit into the brewing process is relatively novel. It was made possible for the first time when the German Purity Law (Reinheitsgebot) — which requires that beer contains only water, yeast, malt and hops — was circumvented by the addition of cherry or raspberry to Belgian lambic sour beers. Lambic sour beers are among the oldest types of beers still brewed and are the product of a spontaneous fermentation process that lasts for 1 to 3 years before bottling 3, 4. The end product is a non-carbonated sour beer that mainly serves as a base for gueuze or fruit lambic beers 5. Fruits have been used as beer adjuncts for centuries, especially with Belgian lambic sour beers 6. The addition of whole fruit to this style of beer is traditionally practised in Belgium for the production of cherry lambic (‘Kriek’) or raspberry lambic (‘Framboise’) by adding, respectively, sour cherries (Prunus cerasus L.) or raspberries (Rubus idaeus L.) to fermenting lambic in casks 3, 7. The sugar in the fruits triggers a secondary fermentation 8 and the fruit flavours are slowly extracted from the fruit solids. The benefit of adding the fruit after primary fermentation, as opposed to the boil, or even in the mash, is that volatile aroma compounds are not boiled off and fruit flavours do not take on a cooked flavour. Nowadays, we can find fruit beers with banana, blueberry, strawberry, apricot, peach, tangerine, blackcurrant, apple or loquat among others. Few weeks of contact time with the fruit is sufficient and enough for the beer and the fruit solids to be separated. In some cases, however, pure fruit juice is added. For example, Früli is a high-quality fruit beer produced at a craft brewery by blending Belgian wheat beer (70%) and strawberry juice (30%). Together with being small scale and independent, the main characteristic of craft breweries is to put emphasis on flavour and the brewing techniques 9, 10. They compete with the mass-market breweries on the basis of quality and diversity, instead of low prices 11. In the Mediterranean regions of Spain, persimmon fruit (Diospyros kaki Thunb.) is an emergent crop that is increasing in production. The total production area of persimmon in Spain is 7243 ha and it is the autonomous Region of Valencia which has the largest production, with 6531 ha in 2011 12. The high quantities of damaged and surplus fruit for fresh consumption requires the development of new products from ‘Rojo Brillante’ persimmon fruits, the main cultivar grown in the Valencia region. Considering the adequate nutritional and functional profile of persimmon juice 13, 14, the aim of this study was the production of special fruit ale beers with different juice concentrations. Wort fermentation took place under beer quality control parameters. Colour, turbidity, pH, titratable acidity (TA), total soluble solids (TSS), sugars, organic acids, total phenolic compounds (TPC), antioxidant capacity and ethanol formation were determined during the fermentation process. Sensory analysis was also performed on the developed product. Materials and methods Materials The base malt used for brewing was Weyermann® Bohemian Pilsner Malt (Weyermann Malting Company, Brennerstraβe, Bamberg, Germany) produced from barley variety ‘Hanka’. It was ground using a corona-style mill. Persimmon juice from ‘Rojo Brillante’ fruits was obtained by the pressed extraction method after grinding and enzymatic treatment. An enzyme preparation of Amyloglucosidase (Fluka-A9913, Sigma-Aldrich Corp., St Louis, MO, USA) and Diazyme® (from Danisco AS, Copenhagen, Denmark) was used for saccharification of the mash. Commercial pelleted hops [Lúpulo P. (CZ) SAAZ (AA 3%)] were added to the sweet wort. Saccharomyces cerevisiae Safale S-04 (Fermentis, Marcq-en-Baroeul, France), a well-known, commercial dry ale yeast, selected for its fast fermentation character and its ability to form a very compact sediment, was used for fermentation of the wort. Juice extraction Persimmon fruits of the variety ‘Rojo Brillante’ were harvested at ripening, yellow-orange state (in autumn 2014) in Carlet, Valencia (Spain). To remove their astringency, the fruits were treated in a chamber with 95% carbon dioxide for 24 h at 20°C and 90% relative humidity (RH). Fruits were washed in cold tap water, drained, cut in half and woody peduncles discarded. Juice extraction was performed according to the method reported by González et al. 13. Briefly, fruit pieces were ground into slurry in a Nutrifaster N350 blender (Nutrifaster Inc., Seattle, WA, USA). Then the slurry was reacted with 200 mg/kg macerating enzyme pectinase (Novozymes, Bagsvaerd, Denmark) for 24 h at room temperature. Subsequently the mixture was heated to 95°C for 5 min, pressed with a laboratory pilot press (Zumonat C-40; Somatic AMD, Valencia, Spain), and after cooling, the liquid fraction was centrifuged at 8,000 g for 15 min. The supernatant was collected as persimmon juice, bottled and stored at 4°C for further use. Brewing Brewing is a critical operation and takes the following steps: grinding the malt, mashing, extracting the wort, boiling and cooling the wort. During mashing the milled malt was mixed with water (1:3 w/v) in a controlled heating process 15. Mashing was performed using a ramped temperature profile from approximately 45 to 75°C within 3 h. In connection with the different levels of mashing temperature, biochemical and physicochemical transformations of starchy biomass occur as a function of operating conditions 16, 17. This stage of saccharification ended by heating at 80°C to inactivate enzymatic activities and filtering to eliminate the residual solids in a clarifier tank. The resulting sweet wort was then hopped with 1 g/L commercial pelleted hops (Humulus lupulus L.) by heating to 96 ± 1°C with agitation for 1 h and later cooled at 20°C. After cooling, the hopped wort was stored at 4°C. Fermentation Four different formulations for wort and persimmon juice were made. The mixtures were as follows: 100:0%, 75:25%, 50:50%, and 25:75% wort and persimmon juice, respectively. The volume of each of these formulations was 10 L. A commercial dry yeast strain of S. cerevisiae Safale S-04 (Fermentis) was inoculated at ratio of 80 g/hL after its activation in sterile water (1:10 w/v) at 27 ± 3°C for 30 min rest and 30 min in soft agitation for cooling the resultant cream. The mixtures were placed into fermentation bioreactors with pressure reducing valves, refrigeration systems and sampling ports. Fermentation process was performed at 20°C for 6 days and then yeasts were separated from beer by cold precipitation for 24 h at 4°C. Samples were taken daily to observe changes in the various parameters analysed, from day 0 to day 6 of the fermentation period. Determination of TSS, pH and TA TSS, expressed as °Brix, and pH were measured by using a WAY-S digital Abbe refractometer (Optic Ivymen® System, Biotech SL, Barcelona, Spain) and a pH meter GLP 21 (Crison Instruments S.A., Alella, Barcelona, Spain), respectively. TA was measured by titrating 10 g of beer with 0.5 mol/L NaOH. Results are expressed as grams of malic acid per kilogram. Colour and turbidity measurements Beer colour was determined by using two colour measurement methods: the CIE (Committee International d'Eclairage) Lab colour system and the Standard Reference Method (SRM). A ColorFlex EZ-45/0 Spectrophotometer (Hunter Associates Laboratory Inc., Reston, VA, USA) was used for colour measurements using the CIELab colour notation system 18. Hue angle (h) was calculated from tan−1 (b/a) and chroma, colour intensity or saturation (C) was calculated as (a2 + b2)1/2. Determination of SRM value involves measuring the attenuation of light of a particular wavelength (430 nm) in passing through 1 cm of beer, expressing the attenuation as absorption and scaling the absorption by a constant: 12.7 for the American Society of Brewing Chemists and 25 for the European Brewery Convention (EBC). The turbidity value of the beer samples was determined by reading the absorbance at a wavelength of 700 nm in a SPECTROstar Omega UV/Vis microplate reader (BMG Labtech GmbH, Offenburg, Germany). Analysis of ethanol, organic acids and sugars Samples were centrifuged at 15,000 g for 5 min and then the supernatant was filtered through a 0.45 μm PVDF syringe filter. After that, it was passed through a solid-phase separation C18 Sep-Pak cartridge (Waters Associates, Milford, MA, USA) pre-activated with equal volumes of methanol, air and water (10:10:10, v/v). Sugars, organic acids and ethanol were determined by HPLC 19, 20 using a HP 1100 series system provided with an automatic injector and an UV detector, set at 210 nm wavelength, coupled with a refractive index detector (Hewlett-Packard, Palo Alto, CA, USA). A 10 μL sample was injected into a C610H Supelcogel (30 cm × 7.8 mm) column preheated at 30°C and protected with a C610H Supelcogel (5 cm × 4.6 mm) guard column (Supelco, Bellefonte, PA, USA). The mobile phase consisted of 0.1 g/100 mL phosphoric acid at a flow rate of 0.5 mL/min. Glucose, fructose and maltose, citric, malic and lactic acids, as well as ethanol were characterized by chromatographic comparison with analytical standards and quantified by the absorbance of their corresponding peaks. Calibration curves were obtained using different standard solutions. Results were expressed as g/L for sugars and organic acids, and g/100 mL for ethanol. Determination of TPC TPC was determined by the Singleton and Rossi 21 method, adapted to a micro-scale, in a SPECTROstar Omega UV/vis microplate reader (BMG LABTECH GmbH). For TPC determinations, beer dilutions in water (1:2 v/v) were used. TPC were determined by mixing 50 μL Folin–Ciocalteu reagent (Sigma–Aldrich Corp.), 10 μL diluted sample, 100 μL aqueous 20% Na2CO3 (Panreac Química S.A., Barcelona, Spain) and 100 μL distilled water. The mixture was allowed to stand for 30 min at room temperature before the absorbance was measured at 750 nm. Gallic acid (Sigma–Aldrich Corp.) was used as standard. Results were expressed as gallic acid equivalents (mg GAE/L). Antioxidant capacity Antioxidant capacity of beers was evaluated by the ABTS [2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid); Sigma-Aldrich Corp.] radical scavenging assay 22, 23. The radical cation was prepared by the reaction between a 7 mmol/L solution of ABTS in water mixed with a 2.45 mmol/L solution of potassium persulfate. The mixture was incubated for 24 h in the dark at room temperature. Then the solution obtained was diluted with water to reach an absorbance of 0.7 ± 0.02 at 734 nm, measured in a SPECTROstar Omega UV/vis microplate reader (BMG LABTECH GmbH). To determine the antioxidant capacity of beer, 200 μL of the ABTS+ dissolution was mixed with 20 μL of beer and after 6 min the absorbance was measured at 734 nm, obtaining the value of the decrease in absorbance. This determination was carried out with a 1:10 dilution of beer. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Fluka Chemika, Neu-Ulm, Germany) was used as standard and results were expressed as Trolox equivalents (mm TE/L). Sensory analysis A panel of 20 untrained participants, showing a predetermined quota with respect to gender (equal numbers of females and males) and aged between 25 and 40 years, evaluated the samples for their liking (acceptance) using a nine-point hedonic scale where 9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like nor dislike, 4 = dislike slightly, 3 = dislike moderately, 2 = dislike very much, and 1 = dislike extremely 24. The beer samples were refrigerated to 10 ± 2°C prior to serving 25. Each sample was coded using a random number, poured into a tulip-shaped glass cup (AFNOR cup, French Standards Association) and presented with approximately the same level of foam. The panelists evaluated all sample under standardized situations 26. Statistical analysis Fermentation was performed in duplicate and all analyses were in triplicate. Results are presented as means ± standard deviation (SD). Statgraphics® Plus for Windows 3.0 (Statistical Graphic Corp. and Graphic Software Systems Inc., Rockville, MD, USA) was used for Statistical Analysis of data, including Analysis of Variance (ANOVA), Fisher's least significant difference (LSD) procedure to discriminate among the means, and Regression Analysis to describe the relationship between variables. Results and discussion pH, TA and TSS According to Kunze 27, the initial pH values of the wort vary from 5.3 to 5.6 depending on the feedstock used. In this study, it was observed that at the beginning the samples exhibited pH values between 4.58 ± 0.08 and 5.73 ± 0.14, with the lower (p = 0.0028) values ​​in the cases of the 50:50% and 25:75% wort–persimmon beers. Thus samples containing lower percentages of persimmon juice had higher pH values. These differences may therefore be due to the pH range (4.43–4.79) of persimmon juice 13. After the first 24 h the pH value decreased and remained constant throughout the fermentation time (Fig. 1A). This pH decrease may be due to the formation of volatile acids (acetic and formic acids) and non-volatile acids (pyruvic, malic, citric and succinic acids) by deamination, consumption of primary phosphates, absorption of potassium and ammonium ions, and delivery of hydrogen ions by the yeasts to the wort at the beginning of the logarithmic growth phase 27. The final pH values obtained in the beers were between 3.97 ± 0.11 and 4.13 ± 0.11, which are in agreement with similar studies by Diakabana et al. 2 and Swistowiez 28, but are slightly lower than those reported by Barredo Moguel et al. 29 and Kunze 27. Beers obtained in this study are, therefore, slightly acidic although others like lambic ones have a pH much lower, around 3.0. Such pH values may promote an acid taste, but could inhibit certain spoilage microorganisms and increase the biological stability of beer. Figure 1Open in figure viewerPowerPoint Evolution of (A) pH, (B) titratable acidity and (C) total soluble solids in fermenting beer samples: (♦) 100:0%, (∎) 75:25%, (▲) 50:50% and (×) 25:75% wort–persimmon juice. Initial TA values of beers varied as result of the percentage of persimmon juice present in the formulations (p = 0.0149); as juice content increased the TA did also (from 0.60 ± 0.11 g malic acid/L in 100% malt beer to 1.49 ± 0.06 g malic acid/L in 25:75% wort–persimmon beer). During the three early days of the fermentation process, TA increased significantly and stabilized thereafter (Fig. 1B). This TA increase may be due to the formation of organic acids like acetic, shikimic, ketoglutaric and succinic acids generated by yeasts during the first stages of alcoholic fermentation 30, 31. In rice beer, TA grew steadily throughout the fermentation from minimum values of 0.4 g tartaric acid/L to maximum values ​​up to 0.8 g tartaric acid/L 32, in a very similar way to what happened in our formulation free of fruit juice, which showed initial and final TA values of 0.60 ± 0.11 and 1.47 ± 0.08 g malic acid/L, respectively. Regarding the final TA values (1.73 ± 0.05, 2.01 ± 0.08 and 2.51 ± 0.07 g malic acid/L), differences were also observed depending on the increased percentage of persimmon juice present in fruit beers. In the same way as pH, the TA has a decisive influence on sourness of the beer and its level may be used as an index of shelf-life. The evaluation of TSS content in fermenting beer samples is shown in Fig. 1(C). Differences between TSS values in the sweet formulations were observed (p = 0.0077), ranging from 16.9 ± 0.3 to 18.0 ± 0.5°Brix. Within the first 24 h of fermentation, TSS content of 100% malt wort displayed a rapid reduction and remained constant afterwards. The final TSS value was 12.9 ± 0.22°Brix. The decrease of TSS in the 75:25 and 50:50% wort–persimmon formulations stabilized after 48 and 72 h of fermentation and remained constant up to the end, with values of 10.4 ± 0.46 and 8.8 ± 0.33°Brix, respectively. The greater decrease in TSS was observed in the 25:75% wort–persimmon formulation, reaching a final value of 6.8 ± 0.19°Brix. Since TSS takes into account both fermentable sugars and non-fermentable compounds, decline in its value was due to the assimilation of sugars such as maltose, glucose and fructose by the yeasts. Initial glucose and fructose concentrations increased in accordance with the percentage of persimmon juice present in the formulations. This may be because fructose and glucose are the most abundant sugars in raw persimmon juice with a G/F ratio ranging from 0.90 to 0.94 13. Colour and turbidity Colour during fermentation of sweet wort was assessed using the CIELab system of chromatic coordinates, an objective tool for colour perception. Results are presented in Fig. 2(A–E). Statistical differences were found for CIELab parameters between the three formulations containing persimmon juice and that free of fruit juice. The 25:75% wort–persimmon juice formulation was observed to have the highest lightness (L = 33.6 ± 0.0), redness (a = 2.7 ± 0.1), yellowness (b = 12.5 ± 0.1) and colour intensity (C = 12.4 ± 0.0), which reflected that the colour saturation might be caused by the abundance of carotenoids in persimmon juice 33. Higher value of the positive a/b ratio indicated that the colour was redder. Increases in all CIELab values were the characteristic changes in the beer colour during fermentation with the exception of the 100% malt beer that it became more green (a = −1.2 ± 0.1); also, brightness was kept constant during fermentation in the 25:75% wort–persimmon beer. Alternatively, the colour could be well described by h as follows: 0° for red-purple, 90° for yellow, 180° for bluish-green and 270° for blue. Since the average h values of the 25:75% wort–persimmon juice and 100% malt beers were 77.9 and 97.1°, the colours of these tended to be light orange and yellow, respectively. Figure 2Open in figure viewerPowerPoint Changes in (A–E) CIELab colour parameters, (F,G) SRM and EBC colour values and (H) turbidity in fermenting beer samples: (♦) 100:0%, (∎) 75:25%, (▲) 50:50% and (×) 25:75% wort–persimmon juice. The SRM and EBC colour values for the fermenting beer samples are shown in Fig. 2(F and G). Higher SRM and EBC values and, therefore, darker colours were obtained for the 100% malt beer. The final SRM and EBC values for this beer after fermentation were 2.16 ± 0.15 and 4.25 ± 0.30. Furthermore, when increasing the persimmon juice percentage in the formulation of the beers, the SRM and EBC values were decreased. The EBC colour value (5.49 ± 0.20) obtained by Goode and Arendt 34 for sorghum beer was similar to that obtained in this study for the 100% malt beer. In any case, the results expressed as SRM or EBC units depend on the raw material and the type of brewing. A brown colour (25 EBC units) of a traditional beer from maize was related to the colour of corn malt used 2. According to the Beer Judge Certification Program (BJCP) 2015 Style Guidelines (www.bjcp.org), a beer defined as golden is one whose colour is about 5–6 SRM units, but Randy Mosher 35 specifies the 4 and 6 SRM values as pale and deep golden, respectively. In our case, the mean SRM colour values obtained are below these values and, therefore, only the 100% malt beer (3.96 ± 0.13 SRM) could be classified as pale golden ale. Persimmon beers had pale straw colour. Beer stability is defined as time units elapsed to reach a certain level of turbidity. The loss of brightness, the decline of transparency, degree of clouding, even the flocculation, precipitation and sedimentation are successive visual manifestations of the lack of stability or instability of the beer. Turbidity values of beers, expressed as the absorbance measurement at 700 nm wavelength, are shown in Fig. 2(H). The formulation with the highest values ​​of suspended solids was the 100% wort from barley malt. Turbidity decreased along fermentation, probably owing to precipitation on the bottom of the fermentation tank. The 25:75% and 50:50% wort–persimmon fruit beers had lower turbidity values (0.03 ± 0.0) and this could be because persimmon juice contains more molecules which flocculate to the bottom, dragging other smaller molecules with them. Evaluation of sugars and ethanol production Monitoring the reducing sugar concentration during fermentation is a very important parameter for beer production. In this study, the initial concentrations of maltose, glucose and fructose as well as during fermentation were determined (Fig. 3A–C). The initial glucose concentration in formulations ranged from 28.08 ± 1.72 to 67.06 ± 2.97 g/L, increasing in accordance with the percentage of persimmon juice present. Because glucose is the sugar that is easily and quickly assimilated by yeasts, at day 1, glucose consumption was complete in the 100% malt beer, and it extended until day 2 in those containing 25 and 50% persimmon juice (Fig. 3A). The glucose concentration diminished significantly almost disappeared (2.26 ± 0.75 g/L) after 3 days of fermentation in the formulation containing larger amount of fruit juice (25:75% wort–persimmon juice). Figure 3Open in figure viewerPowerPoint Kinetic changes of reducing sugar consumption: (A) glucose, (B) maltose and (C) fructose, and (D) ethanol production in fermenting beer samples: (♦) 100:0%, (∎) 75:25%, (▲) 50:50% and (×) 25:75% wort–persimmon juice. Maltose, another important sugar in fermenting beer, is derived from the hydrolysis of barley starch during mashing. The initial maltose content in formulations ranged from 38.41 ± 2.75 to 10.69 ± 1.09 g/L, decreasing in accordance with the percentage of persimmon juice present. In general, during the first 2 days of fermentation, great quantities of maltose were used (Fig. 3B), but there were still maltose residues in the three fruit beers. Fructose is the most abundant fermentable sugar in raw persimmon juice 13. This explains why the higher fructose content before fermentation (45.16 ± 2.40 g/L) was detected in the formulation containing more fruit juice. In comparison with the other sugars, fructose was slowly consumed by the yeasts (Fig. 3C) and residual quantities (4.20 ± 0.62 and 8.06 ± 1.03 g/L) were determined after 3 days of fermentation in the 50:50% and 25:75% wort–persimmon juice formulations. Kinetic changes in reducing sugar consumption and ethanol production for fermenting formulations were similar. The slope of the ethanol production (Fig. 3D) changed at the initial stages of fermentation just after yeast inoculation and was maintained until reducing sugars were depleted, being at day 1 for the 100% malt beer, at day 2 for the 75:25% and 50:50% wort–persimmon beers, and at day 4 for the 25:75% wort–persimmon beer. The final ethanol content of the beers depended on the initial reducing sugar concentration in the formulations and, therefore, those containing more persimmon juice contained more alcohol. At the end of the fermentation process, 100:0, 75:25, 50:50 and 25:75% wort–persimmon beers contained 2.91 ± 0.13, 3.64 ± 0.11, 4.72 ± 0.15 and 5.63 ± 0.16 ethanol (% v/v), respectively. In comparison with some commercial beers 36, the low alcohol content of 100% malt beer could be due to insufficient saccharification of starchy biomass of barley. In turn, this could be due to inadequate choice of enzymes or rapid thermal inactivation thereof. Evaluation of organic acids during fermentation The organic acids profile is a very important factor to determine the quality of beer since it may alter the organoleptic perception of the product. These acids, in concert with carbonic acid, are responsible for the pleasurable sensation of tartness 37. Therefore, we investigated the kinetic changes of organic acids (acetic, citric, malic, lactic and succinic acids) in fermenting beers and results are shown in Fig. 4(A–C). Malic acid was found to be the major organic acid in 100% malt wort (1.29 ± 0.16 g/L) followed by citric acid (0.46 ± 0.02 g/L). The major organic acids detected in raw persimmon juice were also malic and citric acids, but they were found 13 in higher concentrations (1.5–1.7 and 0.6–07 g/L, respectively). These differences may explain the