FEDERAL UNIVERSITY OF RIO DE JANEIRO

ENCAPSULATION OF WINE INDUSTRY BY-PRODUCT TO BE USED AS ADDITIVE IN BEER AND DEVELOPMENT OF GLUTEN-FREE BEER

ANNA CAROLYNA GOULART VIEIRA

2019

FEDERAL UNIVERSITY OF RIO DE JANEIRO

ENCAPSULATION OF WINE INDUSTRY BY-PRODUCT TO BE USED AS ADDITIVE IN BEER AND DEVELOPMENT OF GLUTEN-FREE BEER

ANNA CAROLYNA GOULART VIEIRA

PhD Thesis presented to the Graduate Program in Food Science of the Federal University of Rio de Janeiro, as part of the necessary requirements to obtain the title of Doctor in Food Science.

Advisor: DSc. Priscilla Filomena Fonseca Amaral Co-advisors: DSc. Gizele Cardoso Fontes Sant’Ana DSc. Maria Helena Miguez da Rocha Leão

Rio de Janeiro August/2019

CATALOG CARD

ENCAPSULATION OF WINE INDUSTRY BY-PRODUCT TO BE USED AS ADDITIVE IN BEER AND DEVELOPMENT OF GLUTEN-FREE BEER

ANNA CAROLYNA GOULART VIEIRA

Advisors: DSc. Priscilla Filomena Fonseca Amaral, DSc. Gizele Cardoso Fontes Sant’Ana e DSc. Maria Helena Miguez da Rocha Leão

PhD Thesis presented to the Graduate Program in Food Science of the Federal University of Rio de Janeiro, as part of the necessary requirements to obtain the title of Doctor in Food Science.

Approved by:

President, Professor DSc. Priscilla Filomena Fonseca Amaral

Professor DSc. Maria Alice Zarur Coelho

Professor DSc. Renata Valeriano Tonon

Professor DSc. Ellen Cristina Quirino Lacerda

Professor DSc. Thiago Rocha dos Santos Mathias

Rio de Janeiro August/2019

I dedicate this thesis to my family that is my base and to God, because without them I would be nothing.

ACKNOWLEDGMENT

I thank God for allowing me to get here and for being so generous with me and putting angels in my path, because when everything was difficult I always had support.

To my husband, best friend and great love, David Sodré da Silva Ferreira, who was my partner in this Thesis, helping me with calculations and going to the lab with me on weekends and holidays. You were crucial in the journey of mine!

To my parents, Rogério Vieira da Silva and Ana Maria Goulart Vieira, who never measured their efforts to provide me with a good education and for always supporting me in everything to achieve my goals. Mom, thanks for not giving up on me! When I could not believe I could do it myself, you spent the afternoon and sleepless studying with me, and now I'm finishing the Doctorate, who would say? Dad, thank you for being an example of hard worker and determination, you are my hero. Thank you both for the biggest and best gift, my sister!

To my sister and brother-in-law, Anna Clara Goulart Vieira and Breno Correa, for all their support, encouragement, friendship and cheering. Surely, this walk would be lonelier without you!

To my brothers-in-law Marcelle and Daniel, for their friendship and encouragement, in addition to giving me the best gift of being the aunt of the beautiful Alice and Sofia, where I find the strength to write my articles.

To my parents-in-laws who gave me, the best husband that, I could dream of. Thank you so much for all the support and encouragement when I thought of giving up!

To the other members of my family who always cheered for me a lot, thank you very much!

To my coach Carmen Lima who showed me how to rearrange my time by prioritizing what really mattered, and helping me identify my weaknesses that bothered me so much and I didn't realize it. In this process of self-awareness, I went to seek treatment for depression that I didn't even know I had! Thank you!

To my dear spiritual directors Father Gustavo and Glauberto who strengthened me with their prayers.

To my circle and members of the Meeting of Couples with Christ of the Parish of Our Lady of Conception and St. Joseph, as well as the Pastoral of Acolhida and Team 18

of the Teams of Our Lady movement who always understood my absences and still prayed for my doctorate.

To my advisor, Prof. Priscilla Amaral, for her patience and opportunity to guide someone from such a distant area. Thank you not only for the guidance, but also for the daily example of professional, healthy living and for all the tips that have made me grow professionally over the years.

To my co-adviser, Prof. Gizele Fontes, for all her strength the moment I thought of giving up everything, when I felt inferior and she said I was just like everyone else. Thank you also for making UERJ laboratories available for characterization analysis.

To my co-adviser, Prof. Maria Helena Miguez, because six years ago she believed in me and opened the doors of the Graduate Program in Food Science.

To Professor Maria Alice and all the students in Lab 123 who have always been willing to help with my research.

To Professors Tatiana Felix and Elcio Ribeiro for their friendship and for always helping me when I needed it, your positive energy is contagious.

To Professor Marcia Feijó who not only opened the laboratory doors at UFF, but also went to the bench with me to continue the analysis.

To Professors Monica Valle, Rosa Maria de Sá Alves, Rinaldini Tancredi and Chef Renato Freire who encouraged me at the beginning of the walk, the first step was the hardest and you were holding it by my hand. Now I'm running this marathon thanks to you! You live in my heart!

To Professor Thiago Mathias, who since qualification has always been available for questions and to use the equipment in his laboratory at IFRJ.

To Professor Daniel Perrone for making his laboratory available for analysis of my sample and for active participation in the article.

The researchers Edna Maria Morais Oliveira and Anna Beatriz Robotton Ferreira were amazing looking for solutions for gluten analysis, even if it didn't work out, the partnership was wonderful and I hope one day to repay.

To my friend Genilton, who taught me how to handle the HPLC device and integrate graphics, you are an amazing person who deserves all the best in the world.

To my villain friends, Cecilia, Renata and Gisele Lulu's mothers, Rafa and Pedro who are wonderful and are always by my side when I need it.

My friends from high school, Marianna, Luisa and Monique, who accompany me since the time of the college entrance exams, supporting me and giving strength in all the difficult moments.

To my college friends Gabi and Dani who went out with me making my weeks more fun, thank you so much for all the support.

To my friend Ellen Lacerda, who I met in the Masters and helped me with antioxidant analysis when I was desperate and she calmly always putting me up. You are wonderful!

To my great friend, Adejanildo (Deja), whom I met during the Doctorate and to whom I will be eternally grateful for teaching me various analyzes and statistics, as well as daily support. Thanks for everything you are my best friend!

To Luana, Jully and Fabiane who were always by my side giving strength, because together we are stronger. Thanks for everything girls! Thank you for living together all this time!

To Gabriel, my undergraduate student, who arrived in the second year of my doctorate and was essential in the most crucial stages. Thank you for your efforts and for your efforts to help me when I needed it, including beer brewing on Saturday.

To the students of IC, Adrian, Karine and Mariane, for their help and for making the days lighter in the lab with play and relaxation. Thanks for the friendship!

To the friends of Lab123, Jonas, Alanna, Fabi, Vanessa, Carol, Naty, Roberta, Marcos and Nanci, for never measuring efforts to help me when I needed and of course for being part of this family that welcomed me with great affection.

To Douglas from LADEBIO who helped me in the 48 minutes of the second half, fitting my samples to his laboratory HPLC so that I wouldn't be late any longer. Thank you so much, you are a cute!

To UFRJ, in particular to the Graduate Program in Food Sciences, for offering the necessary structure for the development of my work.

To CAPES for granting the scholarship throughout the doctorate period.

Finally, I thank everyone who contributed in one way or another with their friendship and with effective suggestions for this work. Gratefulness is the word!

ABSTRACT

ENCAPSULATION OF WINE INDUSTRY BY-PRODUCT TO BE USED AS ADDITIVE IN BEER AND DEVELOPMENT OF GLUTEN-FREE BEER

ANNA CAROLYNA GOULART VIEIRA

Advisors: Professor DSc. Priscilla Filomena Fonseca Amaral, Professor DSc. Maria Helena Miguez da Rocha Leão and Professor DSc. Gizele Cardoso Fontes Sant’Ana

Summary of Doctoral Thesis submitted to the Graduate Program in Food Science of the Federal University of Rio de Janeiro, as part of the necessary requirements to obtain the title of Doctor in Food Science.

The present work aimed to add phenolic compounds in the gluten-free beer produced from buckwheat and quinoa with wine industry by-product (WIBP) encapsulated in alginate beads. Best conditions to obtain beads with simultaneously lower swelling (606.7 %) and erosion degree (7.6 %) are: 1.5% sodium alginate, 4% WIBP, 0.26 M calcium chloride and 26 min complexion time (CT). The antioxidant activity of WIBP was demonstrated by ferric reducing ability of plasma (0.13 mmol Fe2+/g) and by (ABTS•+) scavenging capacity (0.078 mmol Trolox/g). The antioxidant activity of the craft wheat beer was in the range reported for Brazilian beers, which was increased by WIBP. The phenolic profile of craft beer was not altered by incorporation of beads, maintaining its six phenolic compounds (gallic, 3,4-dihydroxyphenylacetic, 4- hydroxybenzoic, 2,4-dihydroxybenzoic, ferulic and salicylic acids). Phenolic compounds of WIBP and absent in the craft beer were only detected after simulated digestion, showing that WIBP encapsulation protects the bioactive compounds until its consumption. Total phenolic content increased continuously from 1.53 to 14.43 mg/L during gastrointestinal tract. Mean values for commercial beers without gluten were less than 3.75 for alcohol (% w / w), 11.52 for real extract (% w / w), 4.24 for original extract (w / w) and 64.66 for the actual degree of fermentation (RDF %). Beers produced from quinoa (Q beer), buckwheat (B beer) or quinoa and buckwheat (QB beer) had alcohol content similar to commercial gluten free beers, as real degree of fermentation. Only QB beer had statistically similar pH to that of a commercial sample. FAN fraction of the wort decreased during beer production as expected due to fermentation. In overall, quinoa had a great significance in the final composition parameters, since considerable differences were observed between compositions comprising quinoa to others without it. All three types of beer had alcohol content similar to barley beer (alcohol = 3.92 % wt). The beer produced with buckwheat was the one real degree of fermentation (RDF = 77.96%) and closer to a barley beer (RDF = 80.05%). All three beverage compositions had shown mean values near the expected, considering the barley malt parameters, which indicates the potential of using this raw material to produce a gluten-free beer.

Key-words: wine industry by-product, encapsulation, gluten-free beer, quinoa, buckwheat, inovation. Rio de Janeiro August/2019

LIST OF FIGURES CHAPTER 1 Figure 1. Scanning electron microscopy of saccharomyces cerevisiae. Source: Wheals, 2007 ...... 29 Figure 2. Structural difference between microcapsules (a), multinuclear microcapsules (b) and microspheres (c). Source: Adapted from Tyagi et al. (2011)...... 33 Figure 3. Sodium alginate chemical structure. Source: Alginate Industry Co., Ltd...... 34 Figure 4. Representative scheme of the encapsulation method based on the gelation property of alginate in the presence of di and trivalent cations. Source: Adaptaded from Finotelli et al. (2006)...... 35 Figure 5. Egg-box model of the Alginate gelation process. Source: Adapted from Calvo et al. (2011)...... 36 CHAPTER 2

Figure 1. Response surface for the degree of erosion (E %) after 15 immersion days in wheat beer (a), (b): as a function of wine industry by-product (WIBP) concentration, CaCl2 concentration and complexation time (CT)...... 46

Figure 2. Response surface for the swelling behavior (SW %) as a function of wine industry by-product (WIBP) concentration, CaCl2 concentration and sodium alginate concentration (SA)...... 47

CHAPTER 3

Figure 1. Picture of the beads containing WIBP visually spherical and with dark purple color...... 58 Figure 2. Scanning electron micrographs of calcium alginate beads with wine industry by-product (WIBP) at 43x (a) and 2000x (b) magnification...... 58 Figure 3. X-ray diffraction patterns for sodium alginate (a), wine industry by-product (WIBP) (b) and beads containing WIBP (c)...... 59

CHAPTER 4

Figure 1. Moisture evolution during malting process for quinoa seeds and buckwheat grains...... 80 Figure 2. Germinated quinoa (a) and germinated buckwheat (b)...... 80 Figure 3. FAN for beers and wort: B beer: beer made from malted buckwheat grains; Q beer: beer made from malted quinoa seeds; QB beer: beer made from malted quinoa seeds and buckwheat grains; Commercial beer A is a Pilsen type beer with moderate bitterness made from barley, with an enzymatic gluten degradation process during fermentation; Commercial beer B is a Premium American Lager type beer made from barley, with an enzymatic gluten degradation process during fermentation. Values are expressed as mean ± standard deviation of six independent experiments (n=6)...... 84 Figure 4. Enzyme immunoassay for the quantitative determination of gliadins in

xi triplicates of B beer, Q beer, QB beer and commercial gluten beer...... 89

xii LIST OF TABLES CHAPTER 2

Table 1. Factors and levels of experimental runs for Central composite rotatable design (CCRD) for degree of swelling and erosion...... 40 Table 2. Matrix of experimental runs for central composite rotatable design (CCRD) for swelling behavior of the beads and erosion degree after 15 or 90 immersion days in a wheat beer...... 42 Table 3. Analysis of variance (ANOVA) for composite rotatable design (CCRD) to evaluate degree of erosion in 15 and 90 days...... 43 Table 4. Analysis of variance (ANOVA) for composite rotatable design (CCRD) for swelling behavior of the beads ...... 44

CHAPTER 3

Table 1. Soluble and insoluble phenolic compounds (mg/100g) from wine industry by- product (WIBP)...... 62 Table 2. Phenolic compounds (mg/L) in beer without wine industry by-product (WIBP) beads and with WIBP beads immersed for 15, 30 and 60 days1...... 63 Table 3. Bioacessibility of phenolic compounds (mg/L) during simulated digestion: gastric digestion (GD), small intestine digestion (SID) and exvivo colonic fermentation (CF)1...... 66

CHAPTER 4 Table 1. Centesimal composition of quinoa seed and buckwheat grains compared to barley ...... 79 Table 2. Mean values of alcohol (% w/w), real extract (% w/w),original extract (% w/w), real degree of fermentation (RDF) (%), pH, Bitterness units (BU) and color for beers produced from buckwheat (B beer), quinoa (Q beer) or buckwheat and quinoa (QB beer) malts comparing with two commercial gluten-free beers...... 82 Table 3. Phenolic compounds (mg/L) of beer produced with buckwheat (B beer), quinoa (Q beer) or buckwheat and quinoa (QB beer) comparing with two commercial gluten-free beers ...... 86

xiii LIST OF ACRYNOMS AA ANTIOXIDANT ACTIVITY 2,2’- AZINOBIS (3-ETHYLBENZOTHIAZOLINE-6-SULFONIC) ABTS ACID RADICAL CATION ASBC AMERICAN SOCIETY OF BREWING CHEMISTS ALG SODIUM ALGINATE ANOVA ANALYSIS OF VARIANCE AOAC OFFICIAL METHODS OF ANALYSIS B BUCKWHEAT BEER BU BITTERNESS UNIT

CaCl2 CALCIUM CHLORIDE CCRD CENTRAL COMPOSITE ROTABLE DESIGN CD CELIAC DISEASE CT COMPLEXION TIME CF COEFFICIENT OF VARIATION CF EXVIVO COLONIC FERMENTATION DAD DIODE ARRAY DETECTOR DF DEGREE OF FREEDOM E PERCENTAGE EROSION EROSION DEGREE AFTER 15 IMMERSION DAYS IN A E15 days WHEAT BEER EROSION DEGREE AFTER 90 IMMERSION DAYS IN A E 90 days WHEAT BEER EBC EUROPEAN BREWERY CONVENTION FOOD AND AGRICULTURE ORGANIZATION OF THE FAO UNITED NATIONS FAN FREE AMINO NITROGEN FRAP FERRIC REDUCING ANTIOXIDANT POWER F-test FISHER'S STATISTICAL TEST G GULURONIC ACID GD GASTRIC DIGESTION GRAS GENERALLY REGARDED AS SAFE H+ HYDROGEN ION CONCENTRATION

xiv HDL HIGHD DENSITY LIPOPROTEIN CHOLESTEROL HPLC HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY IAL ADOLFO LUTZ INSTITUTE L LINEAR TERM LC LIQUID CHROMATOGRAPHY M MANNURONIC ACID MG BLOCS WITH MANNURONIC ACID AND GULURONIC ACID ND NOT DETECTED NIFEXT NITROGEN FREE EXTRACT OD ORAL DIGESTION pH HYDROGEN POTENTIAL Q QUADRATIC TERM Q QUINOA BEER QT QUINOA AND BUCKWHEAT BEER RDC RESOLUTION OF THE COLLEGED BOARD RDF REAL DEGREE OF FERMENTATION SA SODIUM ALGINATE SD STANDARD DEVIATION SEM SCANNING ELECTRON MICROSCOPE

SW SWELLING DEGREE SID SMALL INTESTINE DIGESTION T TEMPERATURE TPTZ 2,4,6-TRI-2-PYRIDYL-1,3,5-TRIAZINE USDA U.S. DEPARTMENT OF AGRICULTURE UV ULTRA VIOLET

Wi INITIAL WEIGHT OF THE BEADS

Wf FINAL WEIGHT OF THE BEADS

WiDB WEIGHT OF DRIED BEADS WIBP WINE INDUSTRY BY-PRODUCT XRD X-RAY DIFFRACTION

xv SUMMARY

LIST OF FIGURES ...... xix LIST OF TABLES ...... xix LIST OF ACRYNOMS ...... xix SCIENTIFIC PRODUCTION ...... xix

CHAPTER 1 ...... 21 1 INTRODUCTION ...... 22 2 OBJECTIVE ...... 24 2.1 General Objective ...... 24 2.2 Specific Objective ...... 24 3 THEORETICAL FRAMEWORK ...... 25 3.1 Celiac disease ...... 25 3.2 Gluten-free beer ...... 27 3.2.1 Saccharomyces cerevisiae ...... 28 3.2.2 Buckwheat ...... 29 3.2.3 Quinoa ...... 30 3.3 Agro-industrial waste from the wine industry ...... 31 3.3.1 Encapsulation ...... 32 3.3.1.1 Alginate as encapsulating material ...... 33 3.3.1.2 Encapsulation by ionotropic gelation ...... 34

CHAPTER 2 ...... 37 ABSTRACT ...... 38 1 INTRODUCTION ...... 38 2 METHODOLOGY ...... 39 2.1 Materials ...... 39 2.2 Encapsulation of wine industry by-product ...... 39 2.3 Analytical Methods ...... 40 2.3.1 Swelling Behavior ...... 40 2.3.2 Degree of erosion...... 41 3 RESULTS ...... 41 4 CONCLUSIONS ...... 47

CHAPTER 3 ...... 49 ABSTRACT ...... 50 1 INTRODUCTION ...... 50

xvi 2 MATERIALS AND METHODS ...... 51 2.1 Standards and chemicals ...... 51 2.2 Wine industry by-product and craft beer ...... 52 2.3 Encapsulation of WIBP by ionotropic gelation ...... 52 2.4 Morphological and physical characterization of WIBP beads ...... 52 2.4.1 Scanning electron microscopy ...... 52 2.4.2 X-ray Diffraction ...... 53 2.5 Antioxidant activity ...... 53 2.6 Stability of WIBP beads in beer...... 54 2.7 Phenolic profile ...... 54 2.7.1 Samples preparation ...... 54 2.7.2 Phenolic compounds analysis ...... 55 2.8 Simulated digestion of WIBP beads ...... 55 2.8.1 In vitro simulated gastrointestinal digestion ...... 56 2.8.2 Ex vivo colonic fermentation ...... 56 2.9 Statistical analysis ...... 57 3 RESULTS AND DISCUSSION ...... 57 3.1 Physical and Morphological analysis of WIBP beads ...... 57 3.2 Antioxidant activity ...... 60 3.3 Phenolic profiles of WIBP and wheat beer ...... 61 3.4 Release of phenolics to beer ...... 64 3.5 Bioaccessibility of phenolic compounds ...... 65 4 CONCLUSIONS ...... 67 CHAPTER 4 ...... 69 1 INTRODUCTION ...... 70 2 MATERIALS AND METHODS ...... 72 2.1 Materials ...... 72 2.2 Beer Production ...... 73 2.2.1 Malting procedure ...... 73 2.2.2 Milling, mashing and boiling procedures ...... 73 2.2.3 Fermentation, maturation and packaging ...... 74 2.3 Germination ...... 74 2.4 Physical-chemical analysis ...... 75 2.4.1 Real extract, ethanol extract and real degree of fermentation ...... 75 2.4.2 Color ...... 75 2.4.3 Bitterness ...... 75 2.4.4 pH ...... 76 2.4.5 Free amino nitrogen (FAN) ...... 76 2.5 Phenolic profile ...... 76

xvii 2.6 Centesimal composition of buckwheat and quinoa ...... 77 2.7 Enzyme immunoassay for the quantitative determination of gliadins ...... 77 2.8 Statistics ...... 78 3 RESULTS AND DISCUSSION ...... 78 3.1 Quinoa and buckwheat characterization ...... 78 3.2 Buckwheat and quinoa malting process ...... 79 3.3 Physical-chemical parameters of gluten free beer ...... 81 3.4 Phenolics of gluten free beer...... 85 3.5 Enzyme immunoassay for the quantitative determination of gliadins ...... 88 4 CONCLUSIONS ...... 89 FINAL CONSIDERATIONS ...... 91 SUGGESTIONS FOR FUTURE WORK ...... 92 REFERENCE ...... 93

xviii

SCIENTIFIC PRODUCTION

FULL ARTICLES PUBLISHED

Vieira, A.C.G., Jong, G.A., Pereira, A.D.S., Fontes-Sant’Ana, G.C., Tonon, R.V., Amaral P.F., Rocha-Leao M.H.M., 2019, Encapsulation of Wine Industry By-product by Ionotropic Gelling for a Wheat Beer, Chemical Engineering Transactions, 75, 601-606.

Jong, G. A., Vieira, A.C.G., Fontes-sant’ana, G.C., Rocha-Leão, M.H.M., Amaral, P.F.F., 2019, Análise físico-química de cervejas comerciais sem glúten, Higiene Alimentar, V.33 – NS. 288/289, 1210-1213.

Jong, G.A., Vieira, A.C.G., Fontes-sant’ana, G.C., Rocha-Leão, M.H.M., Amaral, P.F.F., 2019, Preparo de curva padrão para inativação térmica da cepa de levedura comercial saccharomyces cerevisiae WB-06, Higiene Alimentar, V.33–NS. 288/289, 2221-2225.

FULL WORKS PUBLISHED IN CONGRESS ANNUALS

Vieira, A. C.G., Jong, G. A., Santana, G. C. F., Leão, M.H.R., Amaral, P. F. F. Viabilidade da maltagem da quinoa (Chenopodium quinoa) e do Trigo Sarraceno (Fagopyrum esculentum) para a produção de bebida fermentada tipo cerveja sem glúten. In: Congresso Brasileiro de Ciência e Tecnologia de Alimentos, 2018, Belém-PA.

Vieira, A. C.G., Lacerda, E.C.Q., Jong, G. A., Santana, G. C. F., Leão, M.H.R., Amaral, P. F. F. Atividade Antioxidante de Cerveja Comercial de Trigo. In: Congresso Brasileiro de Ciência e Tecnologia de Alimentos, 2018, Belém-PA.

Vieira, A. C.G., Santana, G. C. F., Leão, M.H.R., Amaral, P. F. F. Comparação do crescimento celular e da capacidade fermentativa de duas cepas de leveduras: Saccharomyces cerevisiae S-23 E WB-06 em meio sintético de trigo sarraceno e meio rico. In: Congresso Brasileiro de Ciência e Tecnologia de Alimentos, 2016, Gramado-RS.

ABSTRACTS PUBLISHED IN CONGRESS ANNUALS

Vieira, A. C.G., Santana, G. C. F., Leão, M.H.R., Amaral, P. F. F. Centesimal composition of buckwheat and quinoa for the production of gluten-free brewed beer.In: Simpósio Latino Americano de Ciênciasde Alimentos, 2017, Campinas-

xix SP.

BOOK CHAPTER

Vieira, A. C.G., Santana, G. C. F., Leão, M.H.R., Amaral, P. F. F. Comparação do crescimento celular e da capacidade fermentativa de duas cepas de leveduras: Saccharomyces cerevisiae S-23 E WB-06 em meio sintético de trigo sarraceno e meio rico. Ed. Poisson (october publication forecast).

Jong, G.A., Vieira, A.C.G., Fontes-sant’ana, G.C., Rocha-Leão, M.H.M., Amaral, P.F.F., 2019, Preparo de curva padrão para inativação térmica da cepa de levedura comercial saccharomyces cerevisiae WB-06. Ed. Athenas (november publication forecast).

Jong, G.A., Vieira, A.C.G., Fontes-sant’ana, G.C., Rocha-Leão, M.H.M., Amaral, P.F.F., 2019, Análise físico-química de cervejas comerciais sem glúten. Ed. Athenas (november publication forecast).

xx CHAPTER 1

INTRODUCTION

OBJECTIVE

THEORETICAL REFERENCE

21 1 INTRODUCTION

The global beer market is approximately $ 250 billion, larger than that of wine. As beer is cheaper and less alcoholic, the differences in volume consumption are much larger (Swinnen and Briski, 2017) and among alcoholic beverages, it is the most consumed in the world (Colen & Swinnen, 2016). Despite this beverage competition, the wine and beer industries can help each other. While winemakers face the problem of discarding solid by-products, the beer industry is looking for innovation as many craft beers with different flavors and additives are invading the market. Waste from the wine industry, which consists mainly of solid by-products, includes skin, seeds and stems, and can represent on average nearly 30% (w / w) of grapes used for wine production (Makris et al., 2007; Colussi et al., 2012). Although biodegradable, this solid waste requires minimal time to be mineralized constituting a source of environmental pollutants (Christ and Burrit, 2013, Cuccia, 2015).The importance of reusing the solid by-product of the wine industry is related to its phenolic compounds, antioxidants, anthocyanins and dyes, which are of great importance to the pharmaceutical, food and cosmetic industries (Rockenbach et al., 2011). Encapsulating antioxidants is an appropriate way to prevent their degradation by light and oxygen (Ferreira et al., 2007). Encapsulating the by-product of the wine industry into alginate pearls would fit a nobler destination and beer could be one of its applications, enhancing the antioxidant properties of this beverage. It should also be noted that the application of WIBP without being encapsulated could have a sandy effect on the texture of the beer, in addition to its color and taste alteration, as the anthocyanins present would give a brown color in the drink. Alginates form a family of gel salts made from naturally occurring alginic acids. These acids are copolymers consisting of unbranched straight chains of 1,4-linked p-D- manuronic acid and α-L-guluronic acid residues (Martinsen et al., 1992). An alginate sphere is prepared by gelation, which occurs by cross-linking uronic acids with divalent cations, such as calcium. Calcium alginate beads are formed by ionotropic gelation when a sodium alginate salt is added dropwise to a calcium chloride solution (Narra, 2012). An "egg box" model has been proposed to explain the nature of this interaction (Narra, 2012). The great advantage of encapsulation is that the inert environment within the alginate polymer network allows the entrapment of a wide range of substances with minor interactions between the biopolymer and the substances (Griffith, 2000).

22 Thus, the objective of this work was to develop a gluten-free fermented beer drink using malted cereals such as buckwheat and quinoa with the introduction of spheres containing the processing residue of the wine industry, to increase phenolic compounds content in the beer. This reduces the problem of wine industry and is an alternative for the use of this antioxidant-rich to make an innovative product with high added value and nutritionally for the celiac public and adherents of gluten-free food. In addition to continuing that developed by Vieira (2014), in which the potential application of spheres containing grape residue in commercial rosé sparkling wine was glimpsed.

23 2 OBJECTIVE

2.1 General Objective

Contribute to the development of a new product for producing gluten-free beer- added with alginate spheres containing the wine industry by-product.

2.2 Specific Objective

• Determine the nutritional quality of wine industry residue, buckwheat and quinoa;

• A comparision with buckwheat and quinoa by proximate composition to barley;

• Analyze the fermentation processes of different formulations of brewer wort, produced under alternative mashing condition, on bench scale, using quinoa, buckwheat and wheat comparing with comercial gluten-free beers;

• Produce a gluten-free beer with no barley;

• Add the spheres containing the residue of the wine industry to beer, so that the beverage has a higher concentration of phenolic compounds, equivalent to red wine;

• Characterize by means of physicochemical analysis the drinks and spheres obtained;

24 3 THEORETICAL FRAMEWORK

3.1 Celiac disease

Celiac disease (CD) is an immune-mediated inflammatory enteropathy triggered by gluten exposure in genetically susceptible individuals, affecting around 1% of the world population (Al-Bawardy et al., 2017). This results in the atrophy of the mucosal villi, which are the intestinal extensions accountable for absorption of nutrients, conducting to malabsorption and malnutrition (Freeman et al., 2011). The celiac patients have to consumption gluten-free food, it is recommended for these patients lack of an intestinal peptidase responsible for removal of gliadin. Accumulation of gliadin in intestine trigger immune reactions and inflammation of tissue and its elimination from the diet generally leads to rapid attenuation of gastrointestinal symptoms, as well as a slower recovery of intestinal mucosa. Main symptoms are bulky stools, breathlessness, fatigue, flatulence and diarrhea, more specifically, steatorrhea (Laurikka; Lindfors; Oittinen, 2018). Determinate kind of foods must to be avoided such as wheat, rye, triticale, barley, and oats. In the case of barley and wheat, which are largely applied in beer production, there are many studies proposing avoidance of these cereals or food derived from them. There is a necessity to guard against unintended gluten ingestion, specially since persevering uncontrolled gluten exposition is known to lead to perpetual health issues and comorbidities such as anemia, malnutrition, and lymphoma (Bethune; Khosla, 2008). In the past, Campbell (1982) suggested barley consumption allowance for celiac disease patients, namely 7 centers in Great Britain of a total of 14. Certain groups still against it consumption specially due to harmful effects even without symptoms. It is now known that this conduct was harmful, which hindered the treatment and relief of symptoms, being necessary the restriction of cereals. As a solution to cereal restrictions, possible alternatives are always being studied and tested to get as close as possible to the original recipes. Briefly, there are no evidence for gluten in flavorings and food additives (e.g., monosodium glutamate). A few cereals without significant gluten content are described; among them are corn, rice, buckwheat, millet, and sorghum. These cereals are not toxic for celiac disease patients and can be used for gluten-free diets (Syage et al., 2018). Latter quinoa was included and is commonly accepted as gluten-free pseudo cereal. Overdue to its excellent nutritional value and a potential for production in various climates, quinoa has been classified as one

25 of the humanity’s most promising crops (Jacobsen; Mujica; Ortiz, 2006; FAO, 2011). The average Western diet contains about 5–15 g gluten/d (Hopp et al., 2015). Gluten ingestion as low as 50 mg/d can be adverse to some celiac patients (Catassi et al., 2007). The removal of 99% of gluten from a diet can even be inefficient to avoid symptoms and histologic damage (Syage et al., 2018). Normally, the types of beer available on the market are made from barley malt, being a very limited, with poor quality and expensive gluten-free beer product. Thus, the public with intolerance to this protein or its dietary presents difficulties in the consumption of beer-type fermented beverages, mainly with sensing similar to the conventional product (Zarnkow; Becker; Jo, 2014). Regardless of a gluten-free diet is a well settled solution for coeliac disease, actually it has also been suggested for the prevention and treatment of other diseases, such as rheumatoid arthritis, type 1 diabetes mellitus, obesity and insulin resistance in non coeliac individuals (Ringertz et al., 2001; Mojibian et al., 2009; Jonsson et al., 2005; Lacerda et al., 2012). People with this condition that eliminated gluten obtained an improvement in the quality of life, they ended up throwing a tendency to eliminate it also from the diet of healthy people (Witczak et al., 2015). Other main focus of publications are in alternative raw material for malting process as pointed out by buckwheat and quinoa in the manufacture of gluten-free brewed beer have been published in recent years, studies in this area are still limited. In particular, the relation between the malting process (Phiarais et al., 2005) and optimization of conditions. Such as, increased activity of amylases and decreased viscosity of the fermented beverage, compared to barley malt and that the addition of increasing levels of alpha and beta-amylases improved the quality of beer produced (Phiarais et al., 2018), indicating that more studies are needed to optimize the process and commercially produce gluten-free beer (Giménez-Bastida et al., 2015). However, there are difficulties with actually approved analytical methods for the detection and quantification of gluten in determinate foods (fermented and hydrolyzed foods) (Diaz-amigo; Popping, 2012). Some processed foods may not include gluten, as during the process proteins are removed, such as spirits and other distilled alcoholic beverages (Campbell, 1982). In counterpart, fermentative processes do not retain all gluten. Specially in beer, high levels of these protein are detected in the final product, and research aiming to reduce final content to values under 20 ppm are widely published. In this way, with the strong growth of the beer market in recent years, the profile

26 of consumers has changed. Currently, the pub is looking for craft or differentiated beers, including in this group an expressive increase of people intolerant to gluten or adherents of the gluten-free diet. It is necessary to develop quality products to meet the demands of this group, which makes research relevant in this topic.

3.2 Gluten-free beer

Beer is a millenary drink originating from classical Mediterranean culture, linked from ancient times to therapeutic purposes (Mataix, 2004). The earliest historical references have been in existence for over 6,000 years, and show that beer, being a fermented drink, was consumed by civilization in order to prevent infectious diseases acquired by drinking unhygienic water (Saura, 2003). With the average consumption of 511 million liters a day, beer is the most consumed alcoholic beverage in the world. Today, consumers are looking for artisanal or differentiated beers (Colen & Swinnen, 2016). Beer is defined as the alcoholic fermentation product of wort from barley malt (Brasil, 2009). Barley beer contains gluten at almost 50 ppm, which discards this beverage as an option for people with celiac disease (permanent intolerance to gluten), specially when used in combination with wheat malt. International food standards establish a maximum limit of 20 ppm in gluten-free foods, and of 100 ppm in foods with low gluten content (FAO, 2015). The introduction of the German Purity Law, established in 1516, defined that beer could not be produced with a different grain than barley (Chen et al., 2013). In recent decades there has been a growth in the world beer market, which has leveraged the search for different means of production in order to please the increasingly demanding public, that includes, celiacs and people who adopt a gluten-free diet (Witczak et al., 2015). In this context, specially aiming at serving a target market niche different from the big companies, alternative raw materials, such as buckwheat and quinoa, have been studied for beer-like production due to the increasing competitiveness of the market, both to reduce costs and to introduce new products to attempt a new demand (Rubio-Flores & Serna-Saldivar, 2016). There are several alternative raw materials that can be used to make a functional gluten-free beer could be an option for persons with celiac disease (Witczak et al., 2015). However, when these alternative raw materials are used, it should be noted that in addition to the fermentable sugars from the starch, other components are important for healthy growth such as FAN (Free Amino Nitrogen).Which are compounds assimilable nitrogen

27 by yeast (free amino acids, ions of ammonium, dipeptides and tripeptides), oxygen, vitamins, phosphorus ions, sulfur, calcium and magnesium, and trace elements such as copper ions and zinc (Lewis & Young, 1995). Moderate consumption of alcohol, particularly wine and beer, is associated with decreased mortality from cardiovascular disease (Gronbaek, 1995). Bobak et al. (2000) analyzed the possible protective effects of moderate beer consumption, eliminating factors related to alcohol consumption habits. They concluded that the protective effect of beer is similar to that of wine due to reduced blood coagulation and increased high- density lipoprotein cholesterol - HDL - and confirmed previous studies in which the cardioprotective effect limiting people is indicated drink moderately. Thus, the demand for inputs to make a beer-type beer without gluten becomes a good field of study (Phirais & Arendt, 2005).

3.2.1 Saccharomyces cerevisiae

Saccharomyces cerevisiae (Figure 1) is a facultative anaerobic yeast that can use both airway aerobic and fermentative, unicellular eukaryotes, devoid of chlorophyll and belong to the Fungi Kingdom (Shwan; Castro, 2001). This yeast is the most common used in the bakery and alcoholic beverage industry. Specially for fermented alcoholic beverages, strains are selected for each style of product and for which process is intended. Each variation has unique genetic and phenotypic identity by cultivation characteristics and their metabolism (Carrau; Gaggero; Aguilar, 2015). Saccharomyces cerevisiae is a yeast that reproduces through budding where carbon dioxide produced it is also used in the gasification of beers, sparkling wines and other alcoholic beverages (Kurzman e Fell, 1998).

28

Figure 1. Scanning electron microscopy of saccharomyces cerevisiae. Source: Wheals, 2007. In the 1980s, Emil C. Hansen's work from the Carlsberg brewery laboratory, Denmark, was the first to classify S. cerevisiae as a high fermentation yeast (ale). A traditional difference between yeast strains ale and lager is that ale strains are not able to metabolize sugar melibiosis. This ability depends on the presence of the α-galactosidase enzyme that hydrolyses melibiosis in galactose and glucose (Carvalho et al., 2006; Russel, 2006). S. cerevisiae strains have the ability to ferment a vast diversity of sugars, like sucrose, glucose, fructose, maltose and maltotriose in this priority sequence, while some degree of overlap occurs and produce ethanol as a metabolite (Silva, 2005). Concerning the metabolic pathway S. cerevisiae even has a peculiar behavior called diauxism, which in the presence of over critical concentrations, glucose represses the expression of genes that encode Krebs cycle enzymes, respiratory chain enzymes and mitochondrial structures (Barnett, 1976 & Porro et al., 1994). Therefore, since mitochondrial activity is reduced in S. cerevisiae, the route to be followed by pyruvate is anaerobic with ethanol formation. On the other hand, at below critical glucose concentrations and in the presence of O2, pyruvate will follow the aerobic route because, if the genes in question are not repressed, S. cerevisiae will have high mitochondrial activity (Fiechter; Fuhrmann; Käppeli, 1981).

3.2.2 Buckwheat

Fagopyrum esculentum Moench (common buckwheat), belonging to the Polygonaceae family. It is an important pseudo cereal, showing both differences and similarities with cereals, found almost everywhere but grows mainly in the northern hemisphere (Sanchez et al., 2011). Russia and China are the main producers of buckwheat in the world (Li & Zhang, 2001) and it`s consumption has become increasingly popular

29 in the United States, Canada, and Europe (Stember, 2006). The main structural difference is that buckwheat is a dicotyledonic plant, while the cereals are monocotyledonic. In a buckwheat, the embryo seed is located in the center of the endosperm and possesses two cotyledons (Pomeranz 1983; Steadman et al 2001a). Owing to the macromolecular components inclusive starch and proteins, the potential use of buckwheat as a gluten-free diet in human consumption and as animal forage have been protected (Guo et al., 2007; Benvenuti et al., 2012). Buckwheat is a high source of dietary fibre, with 10g per 100g (USDA, 2008). The grains compose of several polyphenolic antioxidant compounds such tannins and catechin dominated by rutin (Kreft & Jane, 2013). The high agricultural and medicinal values make buckwheat potentially an attractive brewing cereal (Park et al., 2017; Kordialik-Bogacka et al., 2018).

3.2.3 Quinoa

Chenpodium quinoa Willd as an annual herbaceous flowering plant belonging to Chenopodiaceae family is considered as a pseudo cereal and dicotyledonous as buckwheat (Abugoch, 2009; Yildiz et al., 2014; Navruz-Varli & Sanlier, 2016). Pseudo cereal because, although not belonging to the Gramineae family, it produces seeds that can be milled into flour and used as a cereal crop (Encina-Zelada et al., 2017; Vilcacundo & Hernández-Ledesma, 2017). Quinoa are a native food plant of the Andean region (Argentina, Colombia, Bolivia, Peru, Ecuador, Chile, Venezuela), there are records of its consumption and agricultural history dating back to approximately 5,000-7,000 AD (Abugoch, 2009; Vega- Galvez et al., 2010; Geren et al., 2014). It remains as the principal protein source in many areas, while the consumption of quinoa in these places has been substituted by meat (Tapia, 2000). Nowadays, their farming has disseminate for too many countries as Australia, Canada, China, England and others (Hu et al., 2017; Aziz, Akram, & Ashraf, 2018), because quinoa has an elevated resistance to abiotic stresses resembling as meteorological conditions too hot during the day and below 0°C at nights, the lands with different pH degrees and that are arid, salty, or poor, and the exposure to the radiations emitted from the sun lights (Gonzalez et al., 2009; Repo-CarrascoValencia & Serna, 2011; Keskin & Kaplan Evlice, 2015). With higher nutritional properties owing to its larger amount of essential amino acids (lysine, tryptophan and cysteine) (Ruales & Nair, 1994), vitamins (vitamin E, B, C)

30 and minerals (calcium, iron, manganese, magnesium, copper and potassium) (Konishi, Hirano, Tsuboi, & Wada, 2004), it is rich in antioxidant compounds such as polyphenols and includes a considerable amount of fiber (Hirose et al., 2010; Repo-Carrasco-Valencia & Serna, 2011; RepoCarrasco-Valencia, Hellström, Pihlava, & Mattila, 2010). The United Nations declared the year 2013 as "International Quinoa Year" in order to increase the interest in quinoa, because it was as one of the seeds promising for humanity by notifying the high nutrient value and genetic diversity that may contribute to food safety in the 21st century (FAO, 2014). Conservative brewers should become interested in pseudo cereals as quinoa because it is an alternative for health considerations as absence of gluten, high levels of fatty acids, vitamins, minerals, dietary fibers, and proteins with more amino acids (Watanabe et al., 2014;Vilcacundo & Hernández-Ledesma, 2017; Kordialik-Bogacka et al., 2018).

3.3 Wine industry by-product

Brazil is a country of great agriculture activity, one of those that produce the waste agribusiness and the search for alternatives to use of the organic matter generated has been growing within several research centers. The wine industry face the problem of disposal of the residual biomass that, although biodegradable, it requires a minimum of time to be mineralized, constituting a source of environmental pollutants (Boeira, 2008). It`s estimated that, after processing of the wine industry, about 13% of the total weight of the grapes are discarded (Renaud; De Lorgeril, 1992; Torres et al., 2002) The linkage of the wine industry are used as animal feed or as a vineyard fertilizer, but its greater amount is still wasted. The importance of reusing the bagasse is because its content is rich in phenolic compounds among others with phytotherapeutic activities, which are of great importance for the pharmaceutical, food and cosmetic industries (Rockenbach et al., 2011). Polyphenols are substances that make wine a drink different from all others. More than 8,000 types of these chemical compounds are known to exist in plants (Araujo et al., 2005; Mamede; Pastore, 2004). They protect plants from physical attacks such as ultraviolet radiation from the sun and biological attacks by fungi, viruses and bacteria. In the case of wines, 200 polyphenols with important antioxidant effects have been identified (Anjo, 2004; Araujo et al., 2005; Mamede; Pastore, 2004) and are distributed on the leaves of the vine, in the seeds and mainly in the bark of the grapes. All these substances are antioxidants generally derived from seeds and grape skin.

31 The human organism is subject to several reactions of imbalance that lead to the formation of free radicals, which in turn can cause various cellular damages such as the degeneration of lipid membranes (Nepomuceno et al., 1999). In order to balance, prevent this type of cellular damage, the body uses endogenous enzymes (such as superoxide dismutase, glutathione peroxidase and catalase, for example) capable of catalyzing reactions to inactivation of free radicals. Valduga et al. (2008) determined the maximum purity of anthocyanins and the maximum amount of 300 mg of 3-glycoside cyanidin / 100 g of 'Isabel' grape marc. Therefore, due to the presence of anthocyanin compounds in grape marc, this residue can have a nobler destination, representing an alternative for the elaboration of natural dyes. The encapsulation of the wine industry by-product would fit into a nobler destination and the alginate is widely used encapsulating matrix.

3.3.1 Encapsulation

Encapsulation is a technique capable of coating or packaging solid, liquid or gaseous substances through a polymeric covering (Suave et al., 2006). This technique has been studied to update delivery systems in pharmaceutical (Fontes et al., 2013), food- science (Costa Neto et al., 2017), medical (Bhujbal, de Vos & Niclou, 2014), and technological (Sarkar, Sahoo, Das, Prusty & Swain, 2017) applications by conserving bioactive compounds from environmental conditions (Lacerda et. al, 2016). This packaging process can form capsules or spheres, which will be classified according to their structure, as we can better see in figure 2. The capsules are comprised of a nuclear region surrounded by a continuous polymeric layer. The beads have an irregular internal geometry, where small particles of active ingredient are dispersed in an encapsulating agent matrix. However, the sense of encapsulation encompasses both the concept of spheres and capsules (Araújo, 2011). According to their size, the capsules are classified in three ways: macrocapsules (> 5000 µm), microcapsules (0.2-5000 µm) and nanocapsules (<0.2 µm) (Azeredo, 2005).

32 Figure 2. Structural difference between microcapsules (a), multinuclear microcapsules (b) and microspheres (c). Source: Adapted from Tyagi et al. (2011).

The use of encapsulation to immobilize biologically active substances, such as bioactive compounds, has become increasingly popular for providing a number of benefits such as increased storage, temperature and pH stability. These benefits have attracted more interest from the scientific community, and in the last decades several works on the encapsulation of these compounds for different applications have been published (Moschona, A., & Liakopoulou-Kyriakides, M., 2018). The encapsulations of a bioactive ingredient purpose not only protect it against any unwelcome interactions with the environment, but also enhance their stability and to assure that the taste and odor of the food will keep unaltered. Through encapsulation the bioavailability of the entrapped component is ensure and it is possible to increase the effectiveness of functional foods, thus finding fertile ground in both the food and pharmaceutical industries (Nedović et al., 2013).

3.3.1.1 Alginate as encapsulating material

Alginate is used in encapsulation due to its ease handling, bio degradability, low- cost, and non-toxicity, and is deemed as ‘generally recognized as safe’ (GRAS) (Zazzali et al., 2018). It is a naturally occurring anionic and hydrophilic linear polymer, derived primarily from brown seaweed and bacteria. Alginate contains blocks of (1–4)-linked β- D-mannuronic acid (M) and α-L-guluronic acid (G) forming regions of M and G blocks as well as alternating MG blocks (Figure 3) (Lee & Mooney, 2012).

33

Figure 3. Sodium alginate chemical structure. Source: Alginate Industry Co., Ltd.

The shapes of the monomers and their mode of binding in the polymer are different, as are the geometry of the regions (G) and (M) and their alternation, with the composition and extension of these sequences and the molecular mass determining factors physicochemical properties of alginates (George & Abraham, 2006; Dentini et al., 2007). According to Amici et al. (2008), the physical properties of alginate depend on the composition of uronic acid and the relative amount of the three sequences, M, G and MG. The biocompatibility and / or immunogenicity of alginates varies with the proportion of M / G residues, and G-rich alginate generally has a higher biocompatibility than M-rich polymers (Tonnesen & Karlsen, 2002). Studies demonstrated that the strength of alginate gel is determined by the degree of interaction between cations and G sequences and is directly proportional to alginate concentration, cation concentration and duration of interaction between alginate and cation (gelation time / hardening time) (Mammarella, Vicin, & Rubiolo, 2002; Kaklamani,Cheneler, Grover, Adam, & Bowen, 2014).

3.3.1.2 Encapsulation by ionotropic gelation

Encapsulation methods can be classified into physical, chemical and physical- chemical. Among physical methods, we have spray drying, freeze-drying and precipitation with supercritical fluid; chemical methods such as interfacial polymerization and molecular inclusion complexation; and physicochemical methods, including complex coacervation, liposomes and ionic gelation (Ozkan et al., 2018). Ionic gelation is an encapsulation method that have more advantages and with an easy execution and practicality, avoiding high temperatures and organic solvents besides a lower cost. Particulate forms of gel present some profitable applications: structuring, strengthening and texturizing agents in food matrices and the capacity of enhancing the visual acceptability of products. In addition, they are able to adapt in shape and size,

34 enabling controlled release of the active in agricultural, pharmaceuticals or food product (Da Silva Carvalho, 2019). The gelation process occurs by diffusion of these ions into the hydrocolloid solution, in which the droplets in contact with the ionic solution provide instantaneous formation of spherical gel structures containing the active material (Smrdel, 2008). Ion gelation is an encapsulation technique based on the ability of crosslinking with divalent cations, such as Zn2+, Ca2+, and Ba2+, is regularly employed as a method to prepare alginate hydrogels from an aqueous solution under gentle conditions. Divalent and trivalent cations bind into the cavities of contiguous G blocks of alginate (Stokke et al., 2000) in a highly cooperative manner, thus forming a gel, with a structure commonly known as “egg-box” (He, Liu, Li & Li, 2016; Narayanan, Melman, Leorneau, Mendelson & Melman, 2012; Sonego, Santagapita, Perullini & Jobbágy, 2016). This process can be performed by extrusion or emulsification / gelation (Lupo et al., 2015), with extrusion being the most common method (Figure 4) (Paques et al., 2014).

Figure 4. Representative scheme of the encapsulation method based on the gelation property of alginate in the presence of di and trivalent cations. Source: Adaptaded from Finotelli et al. (2006).

Alginate forms a gel matrix in the presence of divalent cations such as Ca2+, and the cations bind to G sequences of alginate creating a heat-stable three dimensional gel network (Smidsrod & Skjak-Braek, 1990; Agulhon, Robitzer, David, & Quignard, 2012). One calcium ion binds to four G units and forms an “egg-box” gel structure (Figure 5).

35

Figure 5. Egg-box model of the Alginate gelation process. Source: Adapted from Calvo et al. (2011).

Beads produced by ionic gelation have a porous gel matrix, which allows a fast and easy diffusion of water or other fluids into or out of the particle structure (Gouin, 2004; Burey et al., 2008).This particle porosity decreases encapsulation efficiency and represents a disadvantage for encapsulating hydrophilic materials. In order to improve these and other properties of alginate beads, a study was done to find the best encapsulation condition and reducing any loss of WIBP in beer (Vieira, 2019), as well as simulating the release of bioactive compounds in vitro. (Vieira, in press). The encapsulation of wine industry by-product using the ionotropic gelation technique has already been used with the Vitis labrusca variety by Vieira (2014) and the Malagouzia and Syrah varieties in the study by Moschona et al. (2018). However, it had not yet been tested for beer application, which is proposed in this paper (Vieira, in press).

36 CHAPTER 2

ARTICLE

Encapsulation of Wine Industry By-product by Ionotropic Gelling for a Wheat Beer

Chemical Engineering Transactions

Anna Carolyna Goulart Vieira, Gabriel Alves Jong, Adejanildo da Silva Pereira, Gizele Cardoso Fontes Sant’Ana, Renata Valeriano Tonon, Maria H. M. Rocha-Leão, Priscilla Filomena Fonseca Amaral

Published: 06/15/2019

37 ABSTRACT

Best encapsulation conditions of wine industry by-products (WIBP) with sodium alginate by ionotropic gelling with Ca2+ ions were selected by a 24 central composite rotational design. The erosion degree of the beads immersed for 15 and 90 days in a wheat beer was reduced with lower CaCl2 concentrations, higher WIBP amounts and reduced complexation times (CT). The swelling behavior of the beads was influenced by CaCl2, sodium alginate, CT and WIBP concentrations. Best conditions to obtain beads with simultaneously lower swelling (606.7 %) and erosion degree (7.6 %) are: 1.5% sodium alginate, 4% WIBP, 0.26 M calcium chloride and 26 min CT. Beads containing the residue of the wine industry can be considered potential additives for a wheat beer to increase the antioxidant activity of this beverage benefiting the consumer.

KEYWORDS: Craft beer, Saccharomyces cerevisiae, wine industry by-product, antioxidant, sodium alginate.

1 INTRODUCTION

The global beer market is larger than the global wine market. Since beer is cheaper and with lower alcohol content, the differences in volume are much larger (Swinnen and Briski, 2017) and, among the alcoholic beverages, it is the world's most consumed one (Colen & Swinnen, 2016). Despite this beverage competition, wine and beer industries can help each other. While wine producers face the problem of disposal of the solid by-products, beer industry is in search of innovation since many artisanal beers with different tastes and additives are invading the market. Wine industry wastes, which consist mainly of solid by-products, include grape pomace, and may account on average for almost 30% (w/w) of the grapes used for wine production (Colussi et al., 2012). Although biodegradable, this solid waste requires a minimum time to be mineralized, constituting a source of environmental pollutants (Christ and Burrit, 2013). The importance of reusing the solid by-product of wine industry is related to its phenolic compounds which are of great importance for the pharmaceutical, food and cosmetic industries (Rockenbach et al., 2011). Encapsulation of antioxidants is a proper way of avoiding its degradation by light and oxygen (Ferreira et al., 2007). The encapsulation of the wine industry by-product in alginate beads would fit into a nobler destination and beer could be one of its applications, increasing antioxidant properties to this beverage, benefiting the consumer. This approach to increase beer nutritional properties has never been reported in

38 literature. Alginates are produced from naturally occurring alginic acids, which are copolymers comprised of linear, unbranched chains of 1,4-linked β-D-mannuronic and α- L-guluronic acid residues (Martinsen et al., 1992). An alginate bead is prepared by gelation, which occurs by cross-linking the uronic acids with divalent cations, such as calcium. Calcium alginate beads are formed by ionotropic gelation when a sodium alginate salt is added dropwise to a solution of calcium chloride (Narra, 2012). The great advantage of encapsulation is that the inert environment within the alginate polymer network allows the entrapment of a wide range of substances with minor interactions between the biopolymer and the substances (Griffith, 2000). Therefore, this work evaluated the use of WIBP encapsulated in alginate beads as an ingredient in a wheat beer. The best conditions of the encapsulation conditions was performed using a central composite rotatable design (CCRD) in order to reduce the degree of erosion and swelling behavior of the capsules when applied in the beer.

2 METHODOLOGY

2.1 Materials

Sodium alginate was purchased from Sigma®, with a mannuronic acid to guluronic acid residues ratio of 0.4 to 1.9 (a sodium alginate solution (2%, w/v) in water has a viscosity at 25 °C and 60 rpm (spindle no. 2) of 100- 300 Pa.s). The calcium chloride was obtained from Vetec®. The artisanal wheat beer (Rio de Janeiro, Brasil, Alcohol by Volume 4.3%, international bitterness units scale 12, Initial specific gravity (SG) 1048, Final SG 1014) was purchased at a local fair for small producers (Rio de Janeiro, Rio de Janeiro, Brasil). The wine industry by-product (WIBP) consisted of Alicante Bouschet grape pomace from red wine production and was provided by Rio Sol Winery (Lagoa Grande, Pernambuco, Brazil). The WIBP was dried at 60°C for 24 h and the solid material obtained was ground and sieved with particle size less than 0.80 mm.

2.2 Encapsulation of wine industry by-product

Encapsulation conditions to obtain WIBP beads were best conditions by central composite rotatable design (CCRD) aiming to obtain better swelling behavior and erosion degree in wheat beer. The general encapsulation process by inotropic gelation was performed as described by Fontes et al. (2013). Sodium alginate was dissolved in distilled water with WIBP (concentrations determined by CCRD). The polymer solution with

WIBP was then added drop-wise into gelation media consisting of 250 mL of CaCl2

39 solution of different concentrations (% w/v) using a 25 mL hypodermic syringe (without needle), under constant stirring at room temperature with a 10 cm of distance from calcium chloride solution. The beads, thus formed, were left in the gelation medium for a certain period (complexation time), then collected by filtration, followed by a washing procedure with distilled water. Just after preparation, with the aid of a pachymeter the size of the beads was measured (mean size of 0.32 cm). Thus, five beads were immersed in 15 mL amber bottles with 10 mL wheat beer at room temperature, and samples were taken every 15 days for 90 days to measure the degree of erosion. A 24 central composite rotatable design (CCRD) was carried out to verify the effects and interactions of sodium alginate, WIBP and calcium chloride concentrations, as well as reticulation time in the swelling behavior and erosion degree of the beads immersed in wheat beer. Table 1 shows the limits for each parameter studied. The experiments were performed at random conditions. “STATISTICA” (version 7.0) software was used for regression and graphical analyses of the obtained data.

Table 1.2. Factors and levels of experimental runs for Central composite rotatable design (CCRD) for degree of swelling and erosion.

Levels Independent variables -2 -1 0 +1 +2 Sodium alginate (% w/v) 1.5 2.5 3.5 4.5 5.5 WIBP (% w/v) 0 1 2 3 4 Calcium chloride (M) 0.15 0.25 0.35 0.45 0.55 Complexation time (min) 5 15 25 35 45

2.3 Analytical Methods

2.3.1 Swelling Behavior

The increase in weight due to absorbed liquid (swelling behavior) was determined for wet beads, after 90 days of immersion in 10 mL of a wheat beer. Initial weight was determined after washing the beads with Milli-Q water just after being produced and removing the excess of water with filter paper (Ww). Then, beads were weighted after drying them at 40 °C until constant weight was achieved (WD). The swelling degree (Sw) was determined by Eq (1) (Fontes, 2013).

40 푊푊 − 푊퐷 푆푤 = ∗ 100 (1) 푊퐷 where WW is the weight of the wheat beads in the swollen state (after 90 days immersed in beer) and WD is the weight of the dry beads. The analysis was performed with four replicates.

2.3.2 Degree of erosion

The degree of erosion was determined after the immersion of beads in 10 mL of a wheat beer. After a selected time interval (15 and 90 days), the beads were withdrawn, filtered and the excess of water removed with filter paper. Three different samples were weighted for each time, and fresh samples were used for each individual time. The percentage erosion (E) was estimated as Eq (2) (Efentakis et al, 2000).

푊 − 푊 (2) 퐸(%) = 푖 푓 ∗ 100 푊푖 where, Wi is the initial weight of the beads (just after preparation) and Wf is the final weight of the same partially eroded sample. The analysis was performed with three replicates.

3 RESULTS

The characteristics of hydrophilic polymers and their ability to hydrate and form a gel layer are well known and essential to sustain and control the release of internal material from matrices. The rate and extent of this release are also dependent on the swelling (Sw) and erosion (E) of the hydrated polymer mass (Efentakis et al, 2000). Central composite rotatable design was performed to evaluate the influence of sodium alginate (SA), wine industry by-product (WIBP) and calcium chloride (CaCl2) concentrations, as well as complexation time in Sw of the prepared beads and in E of the beads immersed in a wheat beer for 15 and 90 days, as shown in Table 2.

41 Table 2.4. Matrix of experimental runs for central composite rotatable design (CCRD) for swelling behavior of the beads and erosion degree after 15 or 90 immersion days in a wheat beer.

Real values (corresponding coded values) E90days Sw E15days Run WIBP CaCl2 CT SA(%w/v) (%) (%) (%) (%) (M) (min)

1 2.50(-1) 1.0(-1) 0.25(-1) 15.00(-1) 3.78 13.34 1331.82

2 2.50(-1) 1.0(-1) 0.25(-1) 35.0(+1) 16.23 20.40 1050.38

3 2.50(-1) 1.0(-1) 0.45(+1) 15.0(-1) 9.14 17.81 931.62

4 2.50(-1) 1.0(-1) 0.45(+1) 35.0(+1) 1.64 7.16 793.79

5 2.50(-1) 3.0(+1) 0.25(-1) 15.0(-1) 1.18 7.36 720.40

6 2.50(-1) 3.0(+1) 0.25(-1) 35.0(+1) 2.57 9.33 791.24

7 2.50(-1) 3.0(+1) 0.45(+1) 15.0(-1) 10.23 17.84 747.96

8 2.50(-1) 3.0(+1) 0.45(+1) 35.0(+1) 8.42 15.20 787.18

9 4.5(+1) 1.0(-1) 0.25(-1) 15.0(-1) 4.84 12.21 812.41

10 4.5(+1) 1.0(-1) 0.25(-1) 35.0(+1) 5.69 13.48 790.79

11 4.5(+1) 1.0(-1) 0.45(+1) 15.0(-1) 7.09 13.06 803.10

12 4.5(+1) 1.0(-1) 0.45(+1) 35.0(+1) 8.30 13.9 801.38

13 4.5(+1) 3.0(+1) 0.25(-1) 15.0(-1) 3.95 9.65 863.40

14 4.5(+1) 3.0(+1) 0.25(-1) 35.0(+1) 14.7 20.6 823.26

15 4.5(+1) 3.0(+1) 0.45(+1) 15.0(-1) 12.15 16.90 779.59

16 4.5(+1) 3.0(+1) 0.45(+1) 35.0(+1) 7.61 11.96 654.11

17 1.5 (-2) 2.0(0) 0.35(0) 25.0(0) 9.26 19.98 864.63

18 5.5(+2) 2.0(0) 0.35(0) 25.0(0) 0.36 9.57 597.21

19 3.5(0) 0.0(-2) 0.35(0) 25.0(0) 0.69 10.50 1000.45

20 3.5(0) 4.0(+2) 0.35(0) 25.0(0) 7.68 15.30 598.49

21 3.5(0) 2.0(0) 0.15(-2) 25.0(0) 1.90 30.32 578.00

22 3.5(0) 2.0(0) 0.55(+2) 25.0(0) 3.46 15.30 653.49

23 3.5(0) 2.0(0) 0.35(0) 5.0(-2) 5.87 13.68 781.08

24 3.5(0) 2.0(0) 0.35(0) 45.0(+2) 11.37 17.62 696.04

25(C) 3.5(0) 2.0(0) 0.35(0) 25.0(0) 8.80 16.51 790.06

26(C) 3.5(0) 2.0(0) 0.35(0) 25.0(0) 7.55 12.25 797.92

27(C) 3.5(0)) 2.0(0) 0.35(0) 25.0(0) 6.99 11.94 797.93

42 SA: sodium alginate concentration; WIBP: wine industry by-product concentration; CaCl2: calcium chloride concentration; CT: complexation time; E15 days: erosion degree after 15 immersion days; E90 days: erosion degree after 90 immersion days; Sw: swelling degree.

From the experimental data presented in Table 3, it is possible to notice that E15 days ranged 16 % and E90 days 13 %. Higher erosion was already expected for more immersion days because there is more time for the polymer to dissolute. Swelling degree ranged almost 754 %, a wider range than the variation at central point (8 %). The analysis of variance was performed (Tables 3 and 4) to verify the variables that influenced the results and to obtain a predictive model. The significance of the model was verified by Fisher's statistical test (F-test), considering the level of significance of 10% (p

Table 3.6. Analysis of variance (ANOVA) for composite rotatable design (CCRD) to evaluate degree of erosion in 15 and 90 days.

DF Sum of square Mean square F-value p-value Factor E15 days E90 days E15 days E90 days E15 days E90 days E15 days E90 days E15 days E90 days (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (2)WIBP(%)(L) 1 13.59 13.59 15.82 0.06

(3)CaCl2(M)(L) 1 1 9.08 21.27 9.08 21.27 10.57 3.26 0.08 0.22

CaCl2(M)(Q) 1 1 11.65 106.13 11.65 106.13 13.56 16.26 0.07 0.06 (4)CT(min)(L) 1 1 23.62 5.74 23.62 5.74 27.49 0.88 0.03 0.45 CT (min)(Q) 1 16.79 16.80 19.55 0.05 2L by 3L 1 25.98 25.98 30.23 0.03 3L by 4L 1 1 90.59 93.39 90.59 93.39 105.41 14.31 0.01 0.06 Lack of Fit 17 20 248.09 380.31 14.59 19.02 16.98 2.92 0.06 0.29 Pure Error 2 2 1.72 12.05 0.86 6.53 Total SS 26 26 448.14 619.88 13.59 21.27

WIBP: wine industry by-product concentration; CaCl2: calcium chloride concentration; CT: complexation time; L: Linear term; Q: Quadratic term

43 Table 4.8. Analysis of variance (ANOVA) for composite rotatable design (CCRD) for swelling behavior of the beads

Factor DF Sum of square Mean square F-value p-value Factor DF Sum of square Mean square F-value p-value

(1) Alg. (%)(L) 1 77199.5 77199.5 3743.69 0.0003 1L by 2L 1 59230.7 59230.7 2872.32 0.0003 Alg. (%)(Q) 1 367.2 367.2 17.81 0.0518 1L by 3L 1 9101.5 9101.5 441.36 0.0023 (2)WIBP 1 158772.4 158772.4 7699.47 0.0001 1L by 4L 1 903.6 903.6 43.82 0.0221 (%)(L) WIBP (%)(Q) 1 9667.2 9667.2 468.80 0.0021 2L by 3L 1 11345.4 11345.4 550.18 0.0018

(3)CaCl2 1 22447.7 22447.7 1088.57 0.0009 2L by 4L 1 9363.0 9363.0 454.05 0.0021 (M)(L)

CaCl2 (M)(Q) 1 12956.3 12956.3 628.30 0.0015 (4)CT (min)(L) 1 18607.9 18607.9 902.37 0.0011 CT (min)(Q) 1 783.4 783.4 37.99 0.0253 Lack of Fit 11 204310.7 18573.7 900.71 0.0010 Pure Error 2 41.2 20.6 3743.69 Total SS 26 606734.5 Alg.: sodium alginate concentration; WIBP: wine industry by-product concentration; CaCl2: calcium chloride concentration; L: Linear term; Q: Quadratic term; DF: degree of freedom.

44 The results presented in Table 3 showed that there was a lack of fit (p <0.10) for the degree of erosion for 15 days. However, the lack of fit is not important for the development of a predictive model when the pure error presents a very low value (Rodrigues & Iemma, 2014), as observed in the present study. Another important point is that SA concentrations did not influence erosion of the beads. The mathematical models with the real variables to predict the variation of the degree of erosion in 15 and 90 immersion days in a wheat beer are represented in Eq(3) and (4).

2 퐸15 푑푎푦푠 = −14.64 – 3.71 ∗ WIBP + 87.38 ∗ CaCl2 − 67.47 ∗ CaCl2 + 0.53 ∗ CT (3) 2 + 0.0081 ∗ CT + 12.74 ∗ WIBP ∗ CaCl2– 2.38 ∗ CaCl2 ∗ CT

2 퐸90 푑푎푦푠 = −18.16 – 88.66 ∗ 퐶푎퐶푙2 + 199.49 ∗ CaCl2 + 0.89 ∗ CT– 2.42 ∗ CaCl2 ∗ CT (4) where E15days and E90days represents erosion degree in 15 and 90 immersion days, respectively, WIBP represents wine industry by-product concentration; CaCl2 is for calcium chloride concentration and CT stands for complexation time.

The data in Table 4 shows that there was lack of fit (p < 0.10) for the swelling behavior of the beads. However, as for the Erosion degree, the lack of fit is not important for the development of a predictive model because the pure error presents a very low value, which indicates that the model obtained is adequate to explain the process. Eq. (5) represents the mathematical model obtained using the real variables.

2 2 푆푤(%) = 22296.27 − 309.71SA + 4.15SA − 533.11WIBP + 21.29WIBP + 51.91CaCl2 2 2 − 2464.40CaCl2 − 13.28CT + 0.06CT + 60.84SA ∗ WIBP + 238.50SA (5)

∗ CaCl2 + 0.75SA ∗ CT + 266.29 WIBP ∗ CaCl2 + 2.42WIBP ∗ CT where Sw represents swelling behavior of the beads, SA is sodium alginate concentration,

WIBP represents wine industry by-product concentration and CaCl2 is for calcium chloride concentration and CT stands for complexation time. From the models of Eq. (3) and (4), response surfaces were obtained (Figure 1).

45

(a) (b)

Figure 1. 6Response surface for the degree of erosion (E %) after 15 immersion days in wheat beer (a), (b): as a function of wine industry by-product (WIBP) concentration,

CaCl2 concentration and complexation time (CT).

Figure 1 (a) depicts that when lower calcium chloride concentrations and higher WIBP concentrations were used, or the opposite, lower degrees of erosion were obtained, which is better for the product. Due to greater complexation of alginate chains by Ca2+ ions, the solubility of the polymer decreases, which also results in reduced erosion. For the complexation time (Figure 1b), 20 min would be better, considering different WIBP concentrations. Therefore, to reduce erosion, lower CaCl2 concentrations, higher WIBP amounts and reduced CT would be good encapsulation conditions. For the swelling behavior, Eq (5) was used to obtain the response surfaces. Figure 2(a, b) indicates that lower swelling was observed when larger concentrations of sodium alginate, medium to lower amounts of WIBP and lower CaCl2 concentration were used to produce the beads. Therefore, for lower Sw, high sodium alginate and lower CaCl2 concentrations should be used as well as medium WIBP and CT.

46

(a) (b)

Figure 2.7Response surface for the swelling behavior (SW %) as a function of wine industry by-product (WIBP) concentration, CaCl2 concentration and sodium alginate concentration (SA).

A desirability function (Statistica 7.0) was used to obtain lower degree of erosion in 90 immersion days simultaneously with lower swelling behavior. A greater degree of importance was given to the lower values of the responses, because it is important that WIBP beads should not disintegrate in wheat beer before consumption. For sodium alginate, a range of 1.5 to 4% was used in the desirability function, aiming at a better formation of the spheres, since low concentrations generates imperfect beads, whereas concentrations above 4% results in difficulties in the extrusion of the polymer solution in the syringe. In order to obtain the lowest values of degree of erosion in 90 immersion days and lower swelling behavior of the beads, 1.5% of sodium alginate, 4% of WIBP, 0.26 M of CaCl2 and 26 min of CT should be used to obtain the beads. In those conditions it will be possible to obtain a degree of erosion at 90 days of 7.6 % and a Sw of 606.7 %.

4 CONCLUSIONS

The production conditions of alginate beads with wine industry by-product

(WIBP) is influenced by WIBP and CaCl2 concentrations as well as complexation time (CT) when erosion degree of the beads immersed by 15 days is considered. When the beads are immersed for longer periods (90 days) only calcium chloride concentration and CT influence erosion. Considering 15 to 90 immersion days, the encapsulation conditions to reduce the degree of erosion in wheat beer are lower CaCl2 concentrations, higher

WIBP amounts and reduced CT. To avoid excessive swelling, CaCl2 should be used in lower concentrations with high sodium alginate and medium amount of WIBP and medium CT. Best conditions to obtain beads with simultaneously lower swelling (606.7

47 %) and erosion degree (7.6 %) are: 1.5% sodium alginate, 4% WIBP, 0.26 M CaCl2 and 26 min CT. Beads containing the residue of the wine industry are potential additives for wheat beer increasing the consumption of antioxidants by the final consumer.

48 CHAPTER 3

ARTICLE

Bioaccessibility of phenolic compounds from wheat beer enriched with alginate- encapsulated wine industry by-product

Anna Carolyna Goulart Vieira, Genilton Alves da Silva, Renata Valeriano Tonon, Gizele Cardoso Fontes Sant’Ana, Maria Helena Miguez da Rocha Leão, Daniel Perrone Moreira, Priscilla Filomena Fonseca Amaral

Food Hydrocolloids Submitted

49 ABSTRACT

Wine industry by-product (WIBP) encapsulated in alginate beads was incorporated to beer in order to increase its antioxidant properties. The antioxidant activity of WIBP was demonstrated by ferric reducing ability of plasma (0.13 mmol Fe2+/g) and by the 2,2′- azinobis (3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS•+) scavenging capacity (0.078 mmol Trolox/g). The antioxidant activity of the craft wheat beer was in the range reported for Brazilian beers, which was increased by WIBP. The phenolic profile of craft beer was not altered by incorporation of beads, maintaining its six phenolic compounds (gallic, 3,4-dihydroxyphenylacetic, 4-hydroxybenzoic, 2,4- dihydroxybenzoic, ferulic and salicylic acids). Phenolic compounds of WIBP and absent in the craft beer were only detected after simulated digestion, showing that WIBP encapsulation protects the bioactive compounds until its consumption. Total phenolic content increased continuously from 1.53 to 14.43 mg/L during gastrointestinal tract. The enrichment of beer with alginate beads containing the WIBP increased the phenolic compounds content.

KEYWORDS: In vitro Digestion; antioxidant activity; bioactive compounds; ionotropic gelation; beads.

1 INTRODUCTION

The wine industry has faced a huge problem for years: the disposal of the solid residue generated by winemaking activities, which includes grape pomace, lees, stalk and sludge (Christ and Burrit, 2013). Grape pomace is the main solid by-product and is made of seeds (38 – 52 %) and seedless pomace (48 – 62 %), consisting of stems, skins and residual pulp (Beres et al., 2017). Although subject to less concern when compared with other industries such as chemicals and mining (Christ and Burrit, 2013), with the increase of environmental worry by governmental entities and population, and to follow the circular economy growth model, this sector has been challenged to bring alternatives to traditional disposal methods. Wine industry by-products (WIBP) are usually used as animal feed or as a vineyard fertilizer, but the majority is still wasted (Ruggieri et al., 2009). Additionally, the waste of this material is a real missed opportunity, since it is rich in bioactive compounds (Cataneo et al., 2008). Grapes are recognized as a health promoting fruit, specially because of their high amounts of polyphenols, mainly anthocyanins and tannins, vitamins and others (Rockenbach et al. 2011). Over the last decade, there is a growing interest on the valorization of the winery by-products, and many works focus on their phenolic compounds (Teixeira et al., 2014). This interest is due to their recognition as important phytochemicals, related to their antioxidant and antimicrobial activities (García-Ruiz et al., 2009). Recently, we have optimized the encapsulation of wine industry by-product

50 (WIBP) by ionotropic gelation with sodium alginate with regards of beer incorporation, reducing erosion degree and swelling behavior of the beads (Vieira et al., 2019). Beer is one of the most consumed alcoholic beverages in the world and can be a major source of phenolic compounds deriving from malt and hops, which contribute to flavor and color (Piazzon et al., 2010; Veljovic, 2015). Associated to a low ethanol content (usually 4–6%) these characteristics contribute to its functional value (Piazzon et al., 2010). Although additional antioxidants have been added to beer to increase stability, it appears that this is not the best strategy because of consumers’ criticism and firmer regulations (Zhao et al., 2010). Veljovic et al. (2015) have shown that adding grapes to beer fermentation increased the phenolic content and antioxidant capacity of beers, indicating that grape beer is a better dietary source of natural antioxidants than regular lager beer. To enhance retention of phenolics in the intestinal tract and control its release in the body, several encapsulation strategies have been proposed (Bonat-Celli et al., 2016; George & Abraham, 2006). Ionic gelation constitutes a simple, efficient and low-cost encapsulation technique that does not require specialized equipment, high temperature, or organic solvents, making it appropriate for both hydrophobic and hydrophilic compounds (Đorđević et al., 2015). As an anionic polymer with carboxyl end groups, alginate is a good mucoadhesive agent. In gastric fluid (pH ± 1.2), the alginate hydrogel becomes a porous insoluble layer that protects the encapsulated compounds, turning into a soluble viscous film at the higher pH of the intestinal tract (pH ± 7.4), resulting in faster release of encapsulated compounds (Zhao et al., 2013). Moschona and Liakopoulou-Kyriakides (2018) evaluated the bioaccessibility of extrusion-encapsulated wine waste phenolic compounds, using different concentrations of biopolymers. The authors observed that the encapsulation efficiency was slightly decreased as the diameter of beads increased, which is smaller particle size beads released higher concentration of phenolics. Therefore, the aim of the present study was to evaluate the bioaccessibility of phenolic compounds from wheat beer enriched with alginate-encapsulated WIBP.

2 MATERIALS AND METHODS

2.1 Standards and chemicals

Sodium alginate was purchased from Sigma® (Saint Louis, USA), with mannuronic to guluronic acid residues ratio of 0.4 to 1.9. An aqueous sodium alginate solution (2%, w/v) at 25 °C and 60 rpm (spindle no. 2) had a viscosity of 100-300 Pa.s).

51 Calcium chloride was obtained from Vetec® (Duque de Caxias, Rio de Janeiro). FRAP and ABTS•+ analysis was performed with TPTZ and HCl purchased from ® Sigma-Aldrich (St. Louis, MO), the FeCl3 and K2S2O8 was obtained from Vetec (Duque de Caxias, Rio de Janeiro). Commercial standards of the following phenolic acids were acquired from Sigma- Aldrich (St. Louis, MO): 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, caffeic acid, gallic acid, quercetin, ferulic acid, rutin, syringic acid, m-coumaric acid, 3,4- dihydroxyphenylacetic acid, p-coumaric acid, salicylic acid, 4-hydroxybenzoic acid. Sodium carbonate was purchased from Spectrum Chemical Manufacturing Corp. (Gardena, CA). All solvents were HPLC grade from Tedia (Fairfield, OH). HPLC grade water (Milli-Q system, Millipore, Bedford, MA) was used throughout the experiments.

2.2 Wine industry by-product and craft beer

The wine industry by-product (WIBP) was Alicante Bouschet grape pomace from red wine production, provided by Rio Sol Winery (Lagoa Grande, Pernambuco, Brazil) a semi-arid region. Pomace was dried at 60 °C in a convective dryer for 24 h and grinded in a Willye-type Micro mill (Model: TE-648) with 20 mesh stainless steel screen. Craft wheat beer was purchased at a local marketplace for small producers (Rio de Janeiro, Brazil).

2.3 Encapsulation of WIBP by ionotropic gelation

The encapsulation process by inotropic gelation was performed as described by Vieira et al. (2019). Sodium alginate aqueous solution (1.5 % w/v) was mixed with wine industry by-product (WIBP) (4 % w/v) by gentle hand movements to prevent lump and bubble formation. Then, polymer solution was added dropwise into gelation media (250 mL of 0.26 % w/v CaCl2 solution) under constant stirring at room temperature using a 25 mL hypodermic syringe (without needle) of 16 mm diameter. The distance between the syringe and the CaCl2 bath was 10 cm. Once formed, WIBP beads were left in the gelation medium for 26 minutes (complexation time), collected by filtration and washed with distilled water.

2.4 Morphological and physical characterization of WIBP beads

2.4.1 Scanning electron microscopy

The morphology of the WIBP beads was observed in a JSM-6510LV scanning

52 electron microscope (SEM) (Jeol, USA). The beads were dried in an oven (50 °C) and fixed to a 10 mm diameter metal cylindrical support using a double-sided carbon adhesive tape. The acceleration voltage was 15 or 20 kV.

2.4.2 X-ray Diffraction

X-ray diffraction (XRD) profiles of the dried WIBP beads, alginate and WIBP were obtained using a bench dust diffractometer (D2 Phaser-BRUKER, Germany) equipped with a linear detector (LYNXEYE), X-ray tube with anode (wavelength of 1.541 Å, voltage of 30 kV, current of 10 mA and maximum power of 300 W) and scanning with angular precision of ± 0.002° in the range of -2° to 150° in 2θ. Phase identification was performed using Diffrac software (EvaTM of BRUKER). Prior to analysis, WIBP beads were dried at 40 °C for 24 hours and ground in a ceramic mortar.

2.5 Antioxidant activity

The antioxidant activity of the wheat beer and the dried and grinded grape pomace (WIBP) were evaluated by the 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acidradical cation (ABTS•+) scavenging capacity and FRAP (Ferric Reducing Antioxidant Power) methods. For WIBP, an extraction procedure was performed as described by Pérez-Jiménez et al (2008). The FRAP assay was performed according to Moreira et al. (2005). FRAP reagent was prepared by mixing 2 mL of 10 mM of 2,4,6-tripyridyl-S-triazine (TPTZ) solution in

6 M HCl with 2 mL of 20 mM of FeCl3 solution and 20 mL of 300 mM acetate buffer (pH 3.6) and warmed at 37 °C prior to analysis. Twenty microliters of sample were mixed with FRAP reagent (180 µL) and mixed. Then it was left to stand for 6 min at 37 °C. The absorbance was then measured at 595 nm. Quantification was performed using a standard +2 curve prepared with FeSO4. Results were expressed as mmol of Fe equivalents per g or liter. Each sample was analyzed in triplicate. The ABTS+ method was performed according to Re et al. (1999) with some modifications. The ABTS (2,2′-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt) radical solution was prepared by reacting K2S2O8 and ABTS for 12– 16 h prior to use. ABTS radical stock solution was diluted in water (1:50) to an absorbance of 0.70 ± 0.02 at 720 nm. Ten microliters of sample were mixed with ABTS radical solution (190 µL). Then, it was left to stand for 6 min at 37 °C. Sample absorbance was then read at 720 nm and subtracted from solvent blank absorbance. Quantification was

53 performed using a standard curve prepared with Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid). Results were expressed as mmol of Trolox equivalents per g or liter. Each sample was analyzed in triplicate.

2.6 Stability of WIBP beads in beer

Immediately after preparation, five WIBP beads (approximately 0.32 g) were immersed in 15 mL amber bottles with 10 mL wheat beer at room temperature and for 60 days. One milliliter sample was withdrawn from beers containing WIBP beads after 15, 30 and 60 days of immersion and stored at -20 °C until analysis. Bottles were closed with screw cap and opened just for sampling.

2.7 Phenolic profile

2.7.1 Samples preparation

To investigate whether phenolic compounds were released from beads to beer, beer samples were clarified by the procedure described by Perrone et al. (2012). Briefly, 1 mL of sample was mixed with 10 µL of each Carrez’s solutions (Carrez’s solution I:

0.3 M K2Fe(CN)6; Carrez’s solution II: 1.0 M zinc acetate). After centrifugation (2500×g, 10 min, 10 °C), the supernatant was stored at -20 °C until analysis. For the analysis of the WIBP, extraction of soluble and insoluble phenolic compounds was performed as described by Dinelli et al. (2011), with modifications. Twenty milliliters of chilled ethanol: water solution (80:20, v/v) were added to 1 g of sample for the extraction of free phenolic compounds. The solution was vortexed for 10 min, centrifuged (2500×g, 10 min, 10 °C) and the supernatant was placed in a round flask. The solid residue was extracted again following the same procedure and the supernatants were combined and evaporated in rotary evaporator (R-215, Büchi®, Flawil, Switzerland). The dried residue was reconstituted in 10 mL of water and stored at -20 °C until analysis. Insoluble compounds were extracted from the solid residue obtained after the extraction of free phenolics. Alkaline hydrolysis was performed by adding 12 mL of water and 5 mL of 10 M NaOH to this solid and keeping this mixture under agitation in an orbital shaker (IKA KS 4000i control, Staufen, Germany) at dark for 16 h. After this period, the pH was adjusted to 2 with concentrated HCl and 15 mL of ethyl acetate were added. After vortexing for 30 s, the mixture was centrifuged (2500×g, 5 min, 10 °C) and the supernatant was placed in a round flask. The extraction procedure was repeated twice,

54 and the supernatants were combined and evaporated in rotary evaporator. The dried residue was reconstituted in 10 mL of methanol: water solution (80:20, v/v) and stored at -20 °C until analysis. Acid hydrolysis was performed in the solid residue remaining after the alkaline hydrolysis by adding 2.5 mL of concentrated HCl and incubating in a water bath at 85 °C for 30 min. The same extraction procedure using ethyl acetate previously described for the alkaline hydrolysis was performed. The whole extraction process (free phenolics, alkaline and acid hydrolysis) was performed in duplicate. The calcium chloride solution remaining after the encapsulation process was also analyzed for phenolic compounds to verify any loss of phenolic compounds during encapsulation. Prior to injection in the High-performance liquid chromatography (HPLC) equipment, all samples were filtered through a 0.45 µm PTFE filter unit (Millipore, Barueri, Brazil).

2.7.2 Phenolic compounds analysis

Phenolic compounds were analyzed by HPLC with diode array detector (DAD). The LC (liquid Chromatography) system (Shimadzu, Kyoto, Japan) comprised a LC- 10ADvp quaternary pump, a CTO-10ASvp column oven, a manual injector (Rheodyne) with a 20 µL loop and an SPD-M10Avp DAD. Chromatographic separations were achieved using a Kromasil® C18 column (5 μm, 250 mm × 4.6 mm) coupled to a Kromasil® C-18 pre-column (5 μm, 10mm × 3 mm) maintained at a constant temperature of 40 °C. The LC mobile system consisted of a gradient of water with 0.3% formic acid (eluent A), methanol (eluent B) and acetonitrile (eluent C, kept at 1% during the whole run), with a constant flow rate of 1.0 mL/min. Prior to injection, the column was equilibrated with 81% A. After injection of sample, this proportion was decreased to 79% in 1 min, 56% in 18 min and 14% in 23 min and kept constant until the end of the 30 min run. Between injections, 10 min intervals were used to re-equilibrate the column with 81% A. Phenolic compounds were monitored by DAD between 190 to 370 nm and identified by comparison of their retention times and UV spectra with those of commercial standards. Quantification was performed by external standardization. Data were acquired by LCMS solution software (Shimadzu Corp., version 2.00, 2000). Results were expressed as mg of compound per 100 g.

2.8 Bioacessibility of WIBP beads

55 The bioaccessibility of phenolic compounds of the wheat beer containing WIBP beads immersed for 60 days was evaluated by simulated digestion.

2.8.1 In vitro simulated gastrointestinal digestion

The in vitro human simulated gastrointestinal digestion, including the oral (OD), gastric (GD) and small intestine digestion (SID) phases, was performed according to Silva et al. (2019) with modifications. For the OD simulation, aliquots of 1 g of the WIBP beads were placed in a glass vial followed by the addition of 3 mL of human saliva, donated spontaneously by two volunteers and incubated at 37°C for 1 min under orbital agitation at 260 rpm to obtain the OD fluid. A 2.5 mL aliquot of artificial gastric fluid (prepared by dissolving 2.75 g of NaCl,

0.27 g of NaH2PO4, 0.82 g of KCl, 0.42 g of CaCl2, 0.31 g of NH4Cl, 0.65 g of glucose, 0.085 g of urea, 3.0 g of mucine, 2.64 g of swine gastric pepsin, 1.0 g of bovine albumin and 8.3 mL of HCl to a final volume of 500 mL, after which the pH was adjusted to 2.0 with 5 M HCl) was added to the OD fluid. The glass vials were then sealed with a rubber septum, the atmosphere was saturated with N2 and incubated at 37 ºC for 2 h under orbital shaking at 260 rpm to obtain the GD fluid.

The pH of the GD fluid was adjusted to 6.0 with NaHCO3 and 2.0 mL of artificial small intestine fluid (prepared by dissolving 6.75 g of NaCl, 0.517 g of KCl, 0.205 g of

CaCl2, 3.99 g of NaHCO3, 0.06 g of KH2PO4, 0.0375 g of MgCl2, 0.1375 g of urea, 25.0 g of swine bile, 4.0 g of swine pancreatin, 1.2 g of albumin bovine and 0.185 mL HCl to a final volume of 500 mL) were added. The glass vials were then resealed, and the atmosphere was saturated with N2 and incubated at 37 ºC for 2 h under orbital shaking at 260 rpm to complete the SID. All steps were performed in triplicate and independently. At the end of the representative of each stage of digestion (OD, GD, SID), aliquots were collected and centrifuged (3000×g, 15 min, 25 °C). The supernatants were analyzed by HPLC-DAD as described in section 2.6.

2.8.2 Ex vivo colonic fermentation

The ex vivo colonic fermentation was performed according to the methodologies described by Hu et al. (2004) and Mosele et al. (2015), with modifications. The feces were homogenized in a nutrient-rich medium (0.5 g in 10 mL) described by McDonald et al. (2013), and 5.0 mL of this mixture was added to the in vitro digested material at the end of the entire simulated digestion process, incubated at 37 ºC and 50 rpm for 48 h. All steps

56 were performed in triplicate. Feces were donated by one volunteer who filled the inclusion criteria: age between 18 and 35 years, eutrophic (body mass index between 18.5 and 24.9 kg/m2), no gastrointestinal disease, regular bowel function and without the use of nutritional supplements, antibiotics, probiotics, prebiotics or symbiotic in the three months prior to collection of feces. The volunteer was instructed to avoid, for at least two days prior to feces collection, the ingestion of foods rich in phenolic compounds as fruits in general, black beans, juices, soy-derived products, alcoholic beverages rich in phenolic compounds like beer and wine. The study protocol was approved by the Ethics and Research Committee (Nº 512847) of the University Hospital Clementino Fraga Filho/Universidade Federal do Rio de Janeiro. At the end of the ex vivo colonic fermentation, aliquots were collected and centrifuged (3000×g, 15 min, 25 °C). The supernatant was analyzed by HPLC-DAD as described in section 2.6.

2.9 Statistical analysis

All of experiments were carried out in triplicate. Data were reported as means ± standard deviation (SD) for triplicate determinations. Analysis of variance and significant difference tests were conducted to identify differences among means by one-way ANOVA using Statistica software (version 7.0, StatSoft Inc., Tulsa, OK). Differences were considered significant when p<0.05. Analyzes of variance (ANOVA) and the Tukey test were performed to identify significant differences among the means, using the Statistica 7.0 program. Differences between means at the 5% level (P <0.05) were considered significant.

3 RESULTS AND DISCUSSION

Physical and morphological analysis of WIPB beads was performed as well as its immersion in a craft wheat beer to evaluate phenolics release and their bioaccessibility by simulated digestion.

3.1 Physical and Morphological analysis of WIBP beads

Beads containing WIBP were visually spherical and assumed the color of the WIBP (dark purple), as shown in Figure 1.

57

Figure 1.8Picture of the beads containing WIBP visually spherical and with dark purple color.

Scanning electron microscopy (SEM) revealed a spherical shape of WIPB beads (Figure 2A) with a relatively rough surface and markedly open and large pores with craters bordered by prominent walls (Figure 2B).This morphology is similar to that of alginate beads reported by Lotfipour et al. (2012). No cracks were observed in the surface of the beads, which could affect preventing the erosion and a consequent release of the solid material (WIBP) from the beads, which is the spreading of phenolic compounds.

(a) (b)

Figure 2.9Scanning electron micrographs of calcium alginate beads with wine industry by-product (WIBP) at 43x (a) and 2000x (b) magnification.

Figure 3 shows the x-ray diffraction (XRD) patterns of sodium alginate (a), WIBP (b) and WIBP beads (c).

58

(a) (b)

(c) Figure 3.10X-ray diffraction patterns for sodium alginate (a), wine industry by-product (WIBP) (b) and beads containing WIBP (c).

The presence of crystalline regions with two broad peaks at 2θ = 13° and 23° (Figure 3a) is observed for sodium alginate, which is consistent with previously reported data (Fontes et al., 2013). These peaks match to the lateral packing of alginate molecules and to the layer spacing along the molecular chain direction, respectively (Vreeker et al., 2008). Regarding WIBP (Figure 3b), XRD revealed the presence of crystalline regions in one broad peak at 2θ of 21.7° representing the cellulose crystallographic plane (Chiriac et al., 2014),besides many evident peaks, which indicates the crystallinity intensity in this material. WIBP beads (Figure 3c) shows XRD patterns depicting the disappearance of one alginate peak (2θ = 13º) and the intensity reduction of the other peak (2θ = 23º) after the formation of the beads (when alginate crosslinks with Ca2+). The WIBP peaks, including the broad one, are also reduced when the material is encapsulated by calcium alginate beads. This could be the result of the strong interaction between alginate and Ca2+, which destroyed the close packing of the alginate molecules for the formation of regular crystallites, as reported by Cho et al. (2014).

59 3.2 Antioxidant activity

Phenolic compounds are considered very important antioxidant sources in beer. They also play critical roles both in flavor stability and in colloidal stability of beer (Vanderhaegen et al., 2006). Therefore, antioxidant activity of the craft beer and the WIBP were evaluated by ferric reducing ability of plasma (FRAP) and by ABTS+ cationic radical scavenging capacity. The antioxidant activity of the craft beer was 2.49 ± 0.06 mmol Trolox/L for ABTS+ and 1.16 ± 0.09 mmol Fe2+/L for FRAP. Although both methods evaluate antioxidant activity, FRAP is generally based on electron transfer reaction mechanisms and ABTS+ is related to the transfer of hydrogen atom and electrons simultaneously, thus resulting in different values (Li et al., 2012). Moura-Nunes et al. (2016) reported that the antioxidant capacity of Brazilian beers measured by FRAP and ABTS+ assays varied between types and styles, ranging from 0.81 mmol Fe+2/L to 6.37 mmol Fe+2/L and from 0.40 mmol Trolox/L to 3.02 mmol Trolox/L, respectively. The results of the present study are in agreement with the values reported by this paper. Pellegrini et al. (2003) evaluated the antioxidant capacity of alcoholic beverages from Italy and found 9 to 12 times higher antioxidant activities by TEAC and FRAP for red wines in relation to a lager beer (1.04 mmol Trolox/L and 2.78 mmol Fe2+/L). The high antioxidant capacity of red wine is related to the prolonged contact between juice and pomace (Pellegrini et al., 2003) and is known for its health protective effect towards consumers (Beres et al., 2017). Grape pomace is a by-product from wine industry with high antioxidant capacity (Beres et al., 2017). WIBP was evaluated for antioxidant activity and values of 0.078 mmol Trolox/g and 0.13 mmol Fe2+/g were obtained for TEAC and FRAP assays, respectively. A higher FRAP value (0.27 mmol Trolox/g) was reported for the red grape pomace produced in the region of Manzanares, Spain (Pérez-Jiménez et al., 2008), which was attributed to the individual redox potential of the diverse phenolic compounds and their structural properties (Pulido et al., 2000). Rockenbach et al. (2011) reported significant differences between the total content of phenolic compounds in extracts of different red grape pomaces. Higher total phenolic contents for the Cabernet Sauvignon extract than the Isabel, the Pinot Noir and the Regente extracts resulted also in higher antioxidant activity for this variety. Martins et al. (2016) determined the FRAP values for

60 red, white and mixed grape pomaces and reported a range from 0.05 to 0.25 mmol Trolox Equivalent/g, with the mixed grape pomace with the lower values. Since the first observations of the “French paradox” (Renaud & Lorgeril, 1992) wine was studied for its health promoting effects, specially for its relation to cardiovascular system. These benefic effects might be related to the presence of phenolic compounds of grape, which are also found in grape pomace, as shown by some authors (Pérez-Jiménez et al., 2008; Rockenbach et al. 2011). Therefore, grape pomace can be considered a valuable source of phenolic compounds, which can be used as functional food to supply the antioxidant activity deficiency in beer in relation to wine.

3.3 Phenolic profiles of WIBP and wheat beer

Ten phenolic compounds were identified in the red grape pomace residue (WIBP) (Table 1): eight compounds (gallic, 2,4-dihydroxybenzoic, p-coumaric, caffeic, 3,4- dihydroxybenzoic and syringic acids, rutin, and quercetin) were observed in the soluble fraction and four compounds (gallic, 3,4-dihydroxybenzoic, 3,4-dihydroxyphenylacetic and m-coumaric acids) were observed in the insoluble fraction. Only gallic and 3,4- dihydroxybenzoic acids were present in both fractions. Gallic acid was the most abundant phenolic compound, present in both soluble and insoluble fractions, but in higher concentration in the insoluble fraction (45.3%), followed by 3,4-Dihydroxyphenylacetic acid (31.45%) present in the insoluble fraction (Table 1). In fact, Rockenbach et al. (2011) concluded that red pomaces from the semi-arid region had highest concentrations of gallic acid. Total phenolic compound (Table 1) was lower (3.2 mg/g) to that found for red grape marc (22 mg/g) (Moschona and Liakopoulou-Kyriakides, 2018), which might be related to Folin–Ciocalteu colorimetric method that was used to determine total phenolic content, which is less precise than HPLC. Trošt et al. (2016) also found values of the same magnitude of the present study for Cabernet Sauvignon grape by-products (1.7 mg/g) using similar extraction procedure and HPLC determination method.

61 Table 1.9Soluble and insoluble phenolic compounds (mg/100g) from wine industry by- product (WIBP).

Phenolic compounds Soluble mg/100g CV1 Gallic acid 3.4 ± 0.1 2.1 3,4-Dihydroxybenzoic acid 0.6 ± 0.1 16.7 p-Coumaric acid 0.8 ± 0.02 2.9 5-Caffeoylquinic acid 3.1 ± 0.3 8.1 2,4-Dihydroxybenzoic acid 5.8 ± 0.6 11.2 Syringic acid 7.9 ± 0.01 0.1 Rutin 10.7 ± 1.0 9.2 Quercetin 14.8 ± 1.7 11.6 Insoluble Gallic acid 153.4 ± 1.4 0.9

3,4-Dihydroxybenzoic acid 30.8 ± 8.7 28.2 3,4-Dihidroxyphenylacetic acid 106.5 ± 4.0 3.7 m-Coumaric 47.9 ± 6.4 13.3 Total (soluble + insoluble) 385.7 ± 7.5 2.3 1 Coefficient of variation of 3 true replicates.

Around twelve percent of the total phenolic compounds of beer correspond to soluble compounds, while 88% are insoluble (Table 2). In the soluble fraction quercetin (31.42%) was the major phenolic compound followed by rutin (22.71%), as also shown by Pérez-Navarro et al. (2019). These authors reported that quercetin-type flavonols generally dominates flavonol profile in Vittis Vinifera red grapes. Different phenolic compounds compositions of winery by-products (skins, seed, pomace, etc.) from several cultivars have been reported, including anthocyanins, flavanols, stilbenes (resveratrol) and phenolic acids (Pinelo et al., 2005; Spigno et al., 2007; Trošt et al., 2016). Trošt et al. (2016) described interesting phenolic profiles from winery by-products extracts, stating that they are influenced by grape cultivars and extraction conditions. When WIBP was used as core material for the encapsulation, it was mixed with sodium alginate and then added to a CaCl2 solution dropwise. After the formation of the beads, the content of phenolic compounds was determined in the remaining solution. No detectable amount of phenolics (by HPLC) was found in this sample, indicating that all phenolic compounds were encapsulated. Phenolic compounds in beer without WIBP beads and with WIBP beads immersed for 15, 30 and 60 days are presents in Table 2.

62 Table 2. 10Phenolic compounds (mg/L) in beer without wine industry by-product (WIBP) beads and with WIBP beads immersed for 15, 30 and 60 days1.

With WIBP beads no WIBP beads Phenolic Compounds 15 days 30 days 60 days mg/L CV3 mg/L CV mg/L CV mg/L CV Gallic acid 4.8 ± 0.1a 2.4 4.8 ± 0.1a 2.5 4.7 ± 0.1ab 2.4 4.4 ± 0.1b 2.7 3,4-hidroxyphenylacetic 6.7 ± 0.2a 2.7 6.6 ± 0.2a 2.3 6.7 ± 0.1a 1.7 6.6 ± 0.1a 2.2

4-Hidroxybenzoic acid 7.3 ± 0.4a 5.7 7.2 ± 0.5a 6.4 7.2 ± 0.4a 5.2 7.0 ± 0.2a 3.0 acid 2,4-Dihydroxybenzoic acid 1.9 ±0.1a 5.5 1.9 ± 0.1a 6.4 1.9 ± 0.1a 5.4 1.7 ± 0.03a 1.9 FerulicSalicylic acid Acid 0.5 ± 0.0a 0.5 0.7 ± 0.0a 0.7 0.7 ± 0.0a 2.3 0.4 ± 0.06a 14 Salicylic acid 20.5 ± 1.5a 7.4 19.6 ±0.9a 4.6 19.3 ± 0.8a 4.0 18.7 ± 0.1a 0.7 Total Phenolic Compounds 41.7 ± 0.71a 1.7 40.8 ± 0.53ab 1.3 40.5 ± 0.42ab 1.04 38.8 ± 0.56b 1.44 1 Results expressed as mean ± standard deviation of two replicates. Different superscript letters in the same line indicate significant difference between different immersion times (ANOVA followed by Tukey’s multiple comparison post hoc test, p < 0.05). 2 Values followed by the same letter in the line do not differ significantly at 5% significance by the Tukey test. 3 Coefficient of variation.

63 For the wheat craft beer, six free phenolic compounds were identified (Table 2): gallic, 3,4-dihydroxyphenylacetic, 4-hydroxybenzoic, 2,4-dihydroxybenzoic, ferulic and salicylic acids, with salicylic acid being the major phenolic compound (50% of the total phenolic compounds). Total phenolic compounds content was higher (41.7 mg/L) than was reported for Brazilian beers (13 mg/L, on average) (Moura-Nunes et al., 2016). Piazzon et al. (2010) showed a different phenolic profile for wheat beer, being ferulic acid the major compound (10.4 mg/L), accounting for more than 50% of the total phenolic compounds. This was the most abundant phenolic acid for the 21 different commercial beers analyzed in that study, from 9 different European countries (Germany, Belgium, Italy, The Netherlands, England, France, Austria, Czech Republic, and Ireland).Gallic acid was the most abundant phenolic compound in commercial Brazilian beers, as reported by Moura-Nunes et al. (2016). According to Zhao et al. (2010), in addition to gallic acid, ferulic acid was also abundant in Chinese commercial beers. On the other hand, gallic acid was not reported in European beers (Piazzon et al., 2010). The amount and quality of raw materials is dependent on the industrial brewing process, which can influence the phenolic compounds contents (Rodrigues and Gil, 2011). The addition of WIBP encapsulated in alginate beads to beer can be an alternative to fill the phenolic gaps of some beers and to increase its content.

3.4 Release of phenolics to beer

WIBP beads were immersed in the craft wheat beer for 60 days. Table 2 shows that there was no change in the phenolic profile of the beer during this period, indicating that there was no phenolic compounds release from the WIBP beads. This result suggests that the encapsulation in alginate beads may prolong the stability of the phenolic compounds from the WIBP until its consumption. Moschona and Liakopoulou- Kyriakides (2018) also encapsulated wine by-products by ionotropic gelling and tested the phenolic release to ethanol and buffer solutions. At room temperature (25 ºC) 15% to 25% of phenolic compounds were released to buffer and ethanol solutions after 24 h, respectively. The WIBP encapsulation process used in the present study, including the concentration of alginate and CaCl2, which were optimized previously (Vieira et al., 2019) in order to reduce swelling and erosion of the beads, might have helped the more efficient retention of the core material. The encapsulation of WIBP also intended to protect the bioactive compound against any undesirable interactions with the environment, enhance

64 their stability and also to ensure that the taste and odor of the beer would remain unchanged.

3.5 Bioaccessibility of phenolic compounds

WIBP beads immersed in the craft wheat beer for 60 days were collected to further evaluate their phenolic compounds bioaccessibility after simulated digestion (in vitro gastrointestinal digestion and ex vivo colonic fermentation). In the saliva (oral digestion – OD), there was no phenolic compounds as capsules prevented it from being released. Table 3 shows that two phenolic compounds were found in the gastric (GD) and small intestine (SID) phases: gallic acid and 3,4-dihydroxyphenylacetic acid. These compounds are present mainly in the insoluble part of the WIBP (Table 1), being released during digestion.

65 Table 3.11Bioacessibility of phenolic compounds (mg/L) during simulated digestion: gastric digestion (GD), small intestine digestion (SID) and exvivo colonic fermentation (CF)1.

CF (mg/L) Phenolic Compounds GD (mg/L) SID (mg/L) 4 h 24 h 48 h Gallic acid 1.32d 6.43b 4.33c 7.16a 4.08c 3,4-Dihidroxyphenylacetic acid 0.21b 1.59b 16.41a 0.50b 0.68b Quercetin nd2 nd 0.60c 0.89b 4.02a Rutin nd nd nd 4.28b 4.57a 3,4-Dihydroxybenzoic acid nd nd nd 0.25a nd Syringic acid nd nd nd 0.33b 1.08a Total 1.53d 8.02c 21.34a 13.41b 14.43b

1 Different superscript letters in the same line indicate significant difference between GD, SID and CF (4 h, 24 h and 48 h) (ANOVA followed by Tukey’s multiple comparison post hoc test, p < 0.05). 2 Not detected.

66 Zhao et al. (2013) reported that in the gastric juice, the pH is around 2 resulting in exchange of calcium ions in the beads cross-linking the alginates with H+, which could enhance crosslinking in the beads. However, due to the increase of pH to above 8.1 in the small intestine, the compounds present in the beads can be released. During colonic fermentation, 3,4-dihydroxyphenylacetic acid was the most released compound after 4 h. Appeldoorn et al. (2009) and Cueva et al. (2013) proposed that the formation of 3,4-dihydroxyphenylacetic acid could be the result of the catabolism of dimeric procyanidins. In the present work, the insoluble part of WIBP could be the source of this compound, since it was detected in large amounts in this by-product (Table 1) and but it could also come from catabolism of procyanidins which have been detected in red grape pomace (Monrad et al., 2010). Other compounds were also detected in this stage (colonic fermentation), including quercetin, rutin and syringic acid. These compounds were not found in the craft wheat beer (Table 2), but were present in the WIPB (Table 1), suggesting their release from the beads during colonic fermentation. Ducruet et al. (2017) showed that the addition of goji berries prior to fermentation during beer production increased substantially the content of phenolic acids in beer. The addition of grape during beer fermentation was also reported in another study and the authors concluded that grape beer was a better source of phenolic compounds and antioxidants than regular lager beer (Veljovic et al., 2015). However, no bioaccessibility test was performed in these studies. As far as we know, this is the first report in the literature describing the addition of encapsulated wine industry by-product to beer to increase of phenolic compounds content of this beverage. Additionally, the encapsulation of the wine industry by-product for the purpose of increasing the health qualities of a beverage helps protect the active substances during the storage period prior to their release in the digestion processes. In this sense, the target the bioactive component is absorbed by the human organism, fulfilling the nutritional purpose of the encapsulation (Fang and Bhandari, 2010).

4 CONCLUSIONS

The incorporation of encapsulated phenolic compounds into beer was observed for the first time in the present study. The red grape pomace (WIBP) used as core material for the beads was rich in antioxidants with high concentration of phenolic compounds. These enriched beads were added to a craft wheat beer and the phenolic compounds were not released during the 60 days of storage, protecting the phenolic content and the

67 antioxidant activity of the core material. The release of phenolic compounds from the beads during simulated digestion, specially of WIBP components that were not present in beer, shows the advantage of using these beads as enrichment material to beverages in general.

68 CHAPTER 4

ARTICLE

Gluten-free beer produced from quinoa (Chenopodium quinoa Willd.) and buckwheat (Fagopyrum esculentum): physical-chemical characterization and phenolic profile

Anna Carolyna Goulart Vieira, Gabriel Alvez de Jong, Genilton Alves da Silva, Gizele Cardoso Fontes Sant’Ana, Maria Helena Miguez da Rocha Leão, Daniel Perrone Moreira, Thiago Rocha dos Santos Mathias, Priscilla Filomena Fonseca Amaral.

69 ABSTRACT

The availability of gluten free food has increased in the market because of the demand for foods that can be ingested by celiac patients, and also dietary restricted consumers. Traditionally produced beer contains gluten and, therefore, is not an option for this growing market. Therefore, three types of gluten-free beers were produced (buckwheat B beer, quinoa Q beer and buckwheat with quinoa QB beer) and were still analyzed two commercial brands that make the hydrolysis of gluten from barley. Anton Paar Beer Analyzer was used to determine important beer physical-chemical parameters. Mean values for commercial beers without gluten were less than 3.75 for alcohol (% w / w), 11.52 for real extract (% w/w), 4.24 for original extract (w / w) and 64.66 for the actual degree of fermentation (RDF %). Alcohol content of all three types of beer produced in the present work was similar to commercial gluten free beers, as well as the real degree of fermentation. Only for QB beer the pH was statistically similar to that of a commercial sample. Free amino nitrogen (FAN) fraction of the wort decreased during beer production, as expected due to fermentation. As far as color the only beer that was statistically similar to commercial beers was beer 50% malt quinoa 50% buckwheat malt. None of the three beers produced was statistically similar to the commercial samples, however, samples of buckwheat and 50% malt of buckwheat 50% quinoa malt were found to be higher than commercial samples. Already the means for the concentration of various phenolic compounds showing the potential of using this raw material to produce gluten-free beer.

KEYWORDS: Phenolic; beer; Beer analyzer; gluten free beer.

1 INTRODUCTION

Beer is the most consumed alcoholic beverage in the world, with an average consumption of 511 million liters a day. Even with a guaranteed market, this beverage sector is innovating every day since consumers are looking for craft beers or beers with different tastes (Colen and Swinnen, 2016). Beer is manly composed of water, carbohydrates and ethanol. These three parameters are usually used for the quality control in the brewing industry under the name of real extract, original extract and alcoholic content. Being the latter, a key aspect to determine affecting the beer classification and its taste (Llario et al., 2006). The main raw material used for beer production, barley, contains hordeins, which is a gluten protein. Proteins are present in beer at a concentration of almost 500mg/L, which can be a problem for people with celiac disease (permanent intolerance to gluten) (Rubio-Flores and Serna- Saldivar, 2016). International food standards establishes a maximum limit of 20 ppm for gluten-free foods, and 100 ppm for foods with low gluten content (FAO, 2015). Gluten has attracted a lot of attention in recent decades due to increasing number of patients diagnosed with intolerance to this protein fraction. This increase can be related to the improvement of detection methods sensitivity as well as the dissemination of data

70 on this disease. Three pathologies are associated with gluten consumption: (i) wheat allergy; (ii) celiac disease, which is an autoimmune pathology caused by ingestion of gluten from different origins, not only wheat and (iii) sensitivity to gluten, which is not allergy or autoimmune dysfunction (Rosell et al., 2014). Not only hordein from barley can trigger celiac disease, but also gliadin, secalin and avenins from wheat, rye and oats, respectively (Wieser and Koehler, 2008), which makes it difficult to find new raw material for beer. The production of gluten free beers without barley malt is also challenging because it is difficult to find replacement malts with adequate diastatic activity, specially in terms of α-amylase (Rubio-Flores and Serna-Saldivar, 2016). Brewing industry have been testing alternative cereals, gluten-free, such as rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor) and pearl and finger millets (Pennisetum americanum and Eleusinecoracana) and pseudo cereals such as buckwheat (Fagopyrum esculentum). Alternatively, these beers can be manufactured from gluten-containing cereals by reducing the level of gluten to below 20 ppm (Hager et al., 2014; Rubio-Flores and Serna- Saldivar, 2016). However, the still scarce gluten-free beer choices available in the market and their poor quality represent major determinants for both industries and academy to intensify research to improve its taste and acceptability (Deželak et al., 2014). The use of buckwheat (Fagopyrum esculentum) malt for beer production is becoming increasingly popular (NicPhiarais et al., 2010). Malting and mashing procedures, along with the addition of commercial enzymes to buckwheat has been optimized (Nic Phiarais et al., 2005; Wijngaard and Arendt, 2006). Nic Phiarais et al. (2010) brewed top fermented beer from 100% buckwheat malt resulting in a beer with pH, FAN (free amino nitrogen), fermentability and total alcohol comparable to wheat beer. Maccagnan et al. (2004) describes a mixture composed of buckwheat (40 to 60%) and syrup obtained by the hydrolysis of gluten free starch (20 to 60%) to obtain gluten free beer with organoleptic properties similar to beer made from barley. Quinoa (Chenopodium quinoa Willd.) is a highly nutritious food product, being cultivated for several thousand years in South America, with an outstanding protein quality and a high content of vitamins and minerals (Jacobsen, 2003). Hager et al. (2014) revealed that only few publications exist on the utilization of this grain for brewing purposes and they reported that quinoa beer was produced by Zweytick et al. (2005).These authors described a slightly opaque yellow product with acceptable foam and taste. Meo et al. (2011) also evaluated quinoa for malting process but didn’t produce the beer from

71 it. Physical-chemical parameters are key economic concern for brewers, and it is the subject of a considerable amount of research. According to American Society of Brewing Chemists (ASBC) Methods of Analysis, real degree of fermentation (RDF) is considered to have a higher reliability to indicate fermentable components depletion in wort, namely fermentability (Huerta-Zurita et al., 2019). Although many studies assessed ethanol, original extract, real extract and RDF in beer using Anton Paar's analyzer (Olšovská et al., 2015; Kryl et al., 2012). Studies characterizing gluten-free beer made of alternative raw materials are still scarce. FAN is believed to be a good index for potential yeast growth and fermentation efficiency (Taylor et al., 1986). In addition, parameters such as color, bitterness unit (BU) and pH are necessary to characterize the beer, not only by determining the quality standard, but also by the search for closeness with the original barley product. Therefore, this study aims to determine the physicochemical parameters of three types of gluten-free beers produced from 100% buckwheat, 100% quinoa or 50% buckwheat and 50% quinoa, to compare with traditional beer made of barley. Phenolic profile of the beers was also accessed to evaluate its antioxidant properties.

2 MATERIALS AND METHODS

2.1 Materials

Buckwheat (Fagopyrum esculentum) and quinoa (Chenopodium quinoa) were obtained from Zona Cerealista® (São Paulo, Brazil). Hops pellets (Hallertau Magnum, 12.10% p/p in α-acids) were obtained from BWS® (Huell, German) and the yeast SaflagerW-06 (German ale) was obtained from Fermentis® (MarcqenBarceul, France). The enzymes liquozyme (α-amylase) and AMG (Glucoamylase) used in the mashing stage were obtained from Novozymes® (Rio Grande do Sul, Brazil).The compounds used for phenolic analysis were: octyl alcohol, isooctane (2,2,4-trimethylpentane), ninhydrin, glycine, Carrez’s solution I (0.3 M K2Fe(CN)6), Carrez’s solution II (1.0 M zinc acetate), formic acid, methanol and acetonitrile, all from Sigma® (Germany). Commercial gluten-free beers were used as comparison. Commercial beer A is a Pilsen type beer with moderate bitterness made from barley, with an enzymatic gluten degradation process during fermentation. Commercial beer B is a Premium American Lager type beer made from barley, with an enzymatic gluten degradation process during fermentation.

72 2.2 Beer Production

Three types of gluten-free beers were produced following the procedure adapted from Dragone and Almeida e Silva (2010), using buckwheat (100%) – B beer, quinoa (100%) – Q beer, or quinoa (50%) and buckwheat (50%) – QB beer.

2.2.1 Malting procedure

Malt production from buckwheat and quinoa was adapted from Meo et al. (2011). Buckwheat grains and quinoa seeds were washed to remove dirty matter. Steeping was performed by soaking 10 g of the grain or the seed in 300 mL of filtered mineral water in a 500 mL beaker flask, maintaining the aeration with an aquarium pump for 24 hours. After that, water drained and a self-irrigating vessel was used to germinate the grains/seeds in an incubator at 20 °C for 5 days. Then, kilning started by drying the germinated grains/seeds in a heating oven in the following times and temperatures: 50 °C for 16 h, 60 °C for 1 h and 74 °C for 5 h. After rootlets removal, quinoa and buckwheat malts were packed in plastic bags and stores at 8ºC. The evaluation of moisture and germination of quinoa seeds and buckwheat grains was carried out during the process.

2.2.2 Milling, mashing and boiling procedures

The preparation of the brewer's wort was carried out on a bench scale (up to 1 liter), following the methodology of Dragone and Almeida e Silva (2010). Malted buckwheat grains (360 g) and malted quinoa seeds (360 g) were milled separately in a mill ( COD 000934, Botini) at a setting of 0.75 mm (particles size) (Mousia et al., 2004). The milled grist (222 g) was infused in water in a volume ratio of 3:1 (water: malt) in 2 L beakers and controlled conditions of temperature were maintained in hot plates by thermometer control for the mashing procedure. This infusion was performed for quinoa, buckwheat or the mixture of both. The enzymes liquozyme® (α-amylase) 0.175 mL and AMG® (Glucoamylase) 0.168 mL were added in this step and a temperature ramp was performed as follows: 62°C for 30 min, 72 °C for 30 min and 78 ºC for 30 min (Kordialik- Bogacka, 2018). Saccharification was monitored during mashing by transferring a drop of the mash sample to a porcelain plate, cooling the sample to 20°C, and adding a drop of iodine solution. The clarified and filtered wort was transferred to the boiling beaker. During transfer, water was added to the filtration beaker at 78 ºC so that the resulting malt is washed, and this liquid was transferred to the boiling beaker, until a final volume of

73 405 mL. At the beginning of the boiling process (after 5 min at 100 ºC), hops pellets (Hallertau Magnum) were added in amount necessary to obtain10 mg/L of iso- α -acids, corresponding to 10 BU (Bitterness Units), considering isomerization of approximately 30% of the α-acid, according to Jaskula-Goiris et al. (2010). After boiling for 60 min, the precipitation of proteins, polyphenols and other insoluble compounds occurred, forming the trub, which was separated from the liquid wort. Subsequently the wort (700 mL) was cooled to the fermentation temperature (23 ºC) and transferred to the 3 L Erlenmeyer (fermenter).

2.2.3 Fermentation, maturation and packaging

The yeast (SaflagerW-06 of the German ale type) was rehydrated in distilled water for 30 min and inoculated (107 cells/mL) by pitching in the wort. The fermentations was carried out in 3 L Erlenmeyer flasks, without stirring, at a temperature of 23 ± 1.0 °C for 7 days. Fermented media was transferred to another 3 L Erlenmeyer flask for maturation, leaving sedimented cells behind. Maturation was conducted for 14 days at 12 ºC. The maturated beer was packaged in 330 mL bottles with sacarose solution (6.0 g/l) for 15 days at 0 °C. Fermentation, maturation and refermentation were monitored by taking samples and analyzing them for pH, yeast viability, apparent fermentability, alcohol and free amino nitrogen concentration.

2.3 Germination

The evaluation of germination percentage of quinoa seeds and buckwheat grains was carried out according to the methodology described by the European Brewery Convention (EBC, 2005). The samples were collected in duplicate. In the control test, the Petri dishes had their bottom lined with filter paper (Whatman, nº 1), and then 4 mL of water were added so that all the paper absorbed the water. After this, 100 whole buckwheat grains and 100 quinoa seeds placed in closed Petri dishes and taken to a germination chamber at a temperature of 15 ± 1.5 ° C for 120 hours. At the end of 120 hours the germinated grains and seeds were counted, and the germination capacity was the value obtained (%). An optical and morphological analysis of malt was performed with Nikon Eclipse microscope model E200 used with the aid of a stereoscopic magnifier with the magnification of 7x. With the help of a ruler and spatula, images were obtained. Using

74 ImageJ software it was possible to measure the size of the rootlet.

2.4 Physical-chemical analysis

2.4.1 Real extract, ethanol extract and real degree of fermentation

Samples (30 mL) of beer were poured from bottle into beakers to be degassed in a sonicator (UNIQUE® Ultra-Sonic Cell Disruptor). Beer analyzer DMA 4500 M (ANTON PAAR®) was used to measure the physical-chemical parameters and was calibrated with distilled water. Samples were placed in autosampler vials, then measured automatically. The Anton Paar's analyzer is an equipment based on a near infrared spectrometer to measure alcohol content of beverages. Together with a density meter, it automatically determines original and real extract (Furusho, 2009). After the analysis, real extract (% w/w), original extract (% w/w), ethanol content and real degree of fermentation (RDF) (%) were obtained.

2.4.2 Color

Color of beer samples was measured following the procedure described in Callemien and Collin, 2007). Beer samples were centrifuged (Eppendorf centrifuge 5810 R; Hamburg, Germany) until free of turbidity. Absorbance readings were performed in a UV-Visible spectrophotometer (Shimadzu UV-1650PC, Japan, Kyoto) at 430 nm. A 1 cm cuvette was used and, therefore, a correction factor was applied to the path of ½ inch light. The equation below was used to calculate color, and the value is expressed in SRM (Standard Research Method) (Eq. 1).

푆푅푀 = 10 푥 퐴 푥 퐹푐 (1) with, A as Absorbance of the sample at 430 nm and Fc as correction factor for the ½ inch cuvette (for the cuvette of 10 mm the factor is 1.27).

2.4.3 Bitterness

Bitterness was determined by the International Method of Unity (Bitterness Units, BU) from The American Society of Brewing Chemists (ASBC, 2009). Ten milliliters of beer were transferred with a drop of octyl alcohol into a 50 mL centrifuge tube. Then, 1 mL of 3 N hydrochloric acid and 20 mL of isooctane (2,2,4-trimethylpentane) were added to the samples. Extraction was performed under stirring for 15 minutes on horizontal platform (Tecnal, TE-420, Brasil, São Paulo). After that, the samples were centrifuged

75 for phase separation and the upper iso-octane layer was collected. The absorbance at 275 nm was obtained in a UV-visible spectrophotometer. The bitterness was calculated according to the Equation 2.

퐵푈 = 퐴푥50 (2) with, A as Absorbance at wavelength of 275 nm.

2.4.4 pH

The pH of beer samples and wort was measured in a pHmeter digimed DM-22, Brasil, São Paulo, at room temperature (25 ºC).

2.4.5 Free amino nitrogen (FAN)

The ninhydrin method, or international method for measuring FAN of ASBC (2009) was used to determine the amount of nitrogen in the wort and beer. The method quantifies amino acids, ammonia and to some degree, amine nitrogen in peptides and proteins. The color reagent was obtained with ninhydrin and glycine standard was obtained with exactly 2 mg of amine nitrogen per liter. Samples of the wort were collected during brewing. For analysis, the wort was filtered and diluted 100 times and the beer was degassed, filtered and diluted 50 times. Two milliliters of the samples were added in test tubes, as well as distilled water (zero) or glycine standard. Then, 1 mL of the colored reagent was added, and the tubes were incubated at 100 °C for 16 minutes. After cooling, 5 mL of the diluted sample was added to each tube and the absorbance was measured in a UV-Visible spectrophotometer at 570 nm. FAN was obtained by the following Equation 3.

푚푔 푎푏푠표푟푏푎푛푐푒 표푓 푡ℎ푒 푠푎푚푝푙푒 퐹퐴푁 ( ) = 푥2푥퐹 (3) 퐿 푎푏푠표푟푏푎푛푐푒 표푓 푡ℎ푒 푔푙푦푐𝑖푛푒 푠푡푎푛푑푎푟푑 with, F as dilution factor of beer or wort.

2.5 Phenolic profile

Beer samples were analyzed for phenolic compounds concentration as described by Perrone, Farah, and Donangelo (2012) with adaptations. Briefly, 1 mL of sample was mixed to 10 µL of each Carrez’s solutions (Carrez’s solution I: 0.3 M K2Fe (CN)6; Carrez’s solution II: 1.0 M zinc acetate). After centrifugation (Microspin, Eppendorf AG,

76 Hamburg, Germany), the supernatant stored at -20 °C until analysis. Phenolic compounds were analyzed by High-performance liquid chromatography with diode array detector (HPLC-DAD). The LC (liquid Chromatography) system (Shimadzu, Kyoto, Japan) comprised a LC-10ADvp quaternary pump, a CTO-10A Svp column oven, a manual injector (Rheodyne) with a 20 µL loop and an SPD-M10Avp DAD. Chromatographic separations were achieved using a Kromasil® C18 column (5 μm, 250 mm × 4.6 mm) coupled to a Kromasil® C-18 pre-column (5 μm, 10mm × 3 mm) maintained at a constant temperature of 40 °C. The LC mobile system consisted of a gradient of water with 0.3% formic acid (eluent A), methanol (eluent B) and acetonitrile (eluent C, kept at 1% during the whole run), with a constant flow rate of 1.0 mL/min. Prior to injection, the column was equilibrated with 81% A. After injection of sample, this proportion was decreased to 79% in 1 min, 56% in 18 min and 14% in 23 min and kept constant until the end of the 30 min run. Between injections, 10 min intervals were used to re-equilibrate the column with 81% A. Phenolic compounds were monitored by DAD between 190 to 370 nm and identified by comparison of their retention times and UV spectra with those of commercial standards. Quantification was performed by external standardization. Data were acquired by LCMS solution software (Shimadzu Corp., version 2.00, 2000). Results were expressed as mg of compound per 100 g.

2.6 Centesimal composition of buckwheat and quinoa

Buckwheat grains and quinoa seeds were analyzed to obtain its centesimal composition. Moisture content was measured by direct oven drying at 105ºC (IAL, 1985 and AOAC, 1996). Mineral matter was determined by incinerating the product at a temperature of 500-550 °C in a muffle (IAL, 1985 and AOAC, 1996). Lipids were measured according to the Soxhlet method (IAL, 1985 and AOAC, 1995). The method used for protein determination was that of Micro-Kjeldhal (AOAC, 1995). The NIFEXT fraction of the English "Nitrogen free extract" comprises the most digestible carbohydrates, that is, that are not included in the fiber fraction. For the calculation, the five determinations of moisture (%), real extract (%), protein (%), crude fiber (%) and ash (%) were added. The result was subtracted from 100 (RCD 360/2003). For the calculation of the caloric energy, the coefficients of Atwater were used, where for proteins, 4.0, carbohydrates, 4.0, lipids, 9.0 (RDC 360/2003).

2.7 Enzyme immunoassay for the qualitative determination of gliadins

77 RIDASCREEN® Gliadin is a sandwich enzyme immunoassay for the qualitative analysis of contaminations by prolamins from wheat (gliadin), rye (secalin), and barley (hordein) in raw products like flours (buckwheat, rice, corn, oats, teff) and spices as well as in processed food like beverages. All samples were extracted with the Cocktail (patented) (R7006/R7016, official R5-Mendez method) (r-Biopharm, 2009).

2.8 Statistics

All of experiments were carried out in triplicate. Data were reported as means ± standard deviation (SD) for triplicate determinations. Analysis of variance and significant difference tests were conducted to identify differences among means by one-way ANOVA using Statistica software (version 7.0, StatSoft Inc., Tulsa, OK). Differences were considered significant when p<0.05. Analyzes of variance (ANOVA) and the Tukey test were performed to identify significant differences among the means, using the Statistica 7.0 program. Differences between means at the 5% level (P <0.05) were considered significant.

3 RESULTS AND DISCUSSION

3.1 Quinoa and buckwheat characterization

Centesimal composition of quinoa seed and buckwheat grains was determined and results are shown in Table 1. Buckwheat grains presented higher values of moisture and carbohydrates than quinoa and barley and superior contents of lipids and proteins in relation to barley. Similar moisture content was found for buckwheat (13.06 %) and quinoa (9.60 %) by Mateo et al. (2011). The higher moisture content of buckwheat impacts in less water demand for germination (Rubio-Flores and Serna-Saldivar, 2016). Higher protein content of quinoa is depicted in Table 1, which has been already documented before, as well as its nutritional value. This seed proteins are rich in amino acids like lysine, threonine and methionine that are deficient in cereals (Stikic et al. 2012). The higher carbohydrate content of buckwheat and quinoa in relation to barley could be an advantage for fermentation. However, because of low diastatic power of buckwheat and quinoa (Rubio-Flores and Serna-Saldivar, 2016), the addition of enzymes is necessary during mashing to convert this carbohydrate in fermentable sugar.

78 Table 1.12Centesimal composition of quinoa seed and buckwheat grains compared to barley

Buckwheat Quinoa Barley (Fugita et al., 2003) Parameters Average± 푆퐷 Average ± 푆퐷 Average Moisture (%) 14.15 ± 0.10 10.50 ± 0.40 10.65 Lipids (%) 4.08 ± 1.93 5.34 ± 1.80 2.71 Proteins (%) 10.97 ± 0.48 15.47 ± 2.27 9.29 Carbohydrates (%) 69.12 ± 0.29 66.66 ± 3.42 58.92 Ash (%) 1.68 ± 1.25 2.03 ± 0.05 1.74 Energy value 357 ± 14 376 ± 11 297 (Kcal/100g)

SD: standard deviation.

3.2 Buckwheat and quinoa malting process

Eighty seven percent of quinoa seeds were germinated after 120 hours at 15 °C, close to what is found in the literature for this seed, which is around 90%, according to Kunze (1996). The germination percentage of buckwheat was 70%, being lower than quinoa, but compatible with that found in the literature for this grain, which was 75% (Phirais, 2006). According to Hamman et al. (2003) 86 to 98 % of barley grains germinates. Therefore, quinoa seeds shows great potential for beer production. Throughout the malting process samples were collected and their moisture measured (Figure 1). During the steeping stage, buckwheat grains and quinoa seeds shows a swelling. Then, during germination the buckwheat had a slight decrease in moisture, while quinoa showed an increase, but not significant. In the kilning stage both showed a decrease in moisture.

79 Moisture x Time 100%

75% Germination Steeping Kilning

50% Moisture (%) Moisture

25%

0% 0 24 48 72 96 120 Time (h)

Quinoa Buckwheat

Figure 1. Moisture evolution during malting process for quinoa seeds and buckwheat grains.

During germination the length of the quinoa (Fig 2a) and buckwheat (Figure 2b) rootlet were measured. For quinoa, 32.25 ± 2.2 mm, was measured in average, which is similar to that obtained by Hager et al. (2014) for this seeds rootlet (35.0 ± 6.3 mm) after 72 h of germination. The radicles of buckwheat attained a length of 4.07 ± 1.3 mm corroborating with Mathur (1989) (5 mm).

(a) (b) Figure 2. Germinated quinoa (a) and germinated buckwheat (b).

80 3.3 Physical-chemical parameters of gluten free beer

Physical-chemical parameters obtained for the three types of beer (Q beer, B beer and QB beer) were compared to two commercial gluten-free beers (a Pilsen type and an American Lager type), obtained from barley with the enzymatic hydrolysis of gluten during fermentation (Table 2). For alcohol content, significant differences were observed for all samples. However, they were all in range of 3.0 to 5.0 % wt, which is required for beers (Glenn, 2000), pointing out an adequate amount of fermentable sugars in buckwheat and quinoa malts. For original extract all samples were statistically equivalent, except for B beer, which was inferior to all. Original extract estimates soluble sugar concentration in the wort prior to fermentation (Llario et al., 2006). Barley beer usually presents an original extract of circa 12 % (w/w) (Nakatani, 1991), similar to that of Q beer and QB beer. Therefore, the result indicates that quinoa malt presents more soluble sugar than buckwheat malt, in spite of the lower carbohydrate content of the seed in relation to this grain (Table 1). However, real extract of buckwheat beer (B beer), that is the amount of sugar that did not underwent fermentation and remains in the beer (Llario et al., 2006), was significantly lower than for quinoa beer (Q beer) and quinoa and buckwheat beer (QB beer), which resulted in a higher RDF (real degree of fermentation) for B beer. According to ASBC Methods of Analysis, real degree of fermentation (RDF) is considered to be more reliable to indicate fermentable components depletion in wort, namely fermentability (Huerta-Zurita et al., 2019). This means that in spite of lower soluble sugar content in buckwheat malt, it contained more fermentable sugar than quinoa malt. This results in a less sweet beer with lower energetic value, as Llario et al. (2006) points out. Therefore, Q beer and QB beer are more like commercial beer B in terms of sweetness and B beer would taste more like commercial beer A.

81 Table 2.13Mean values of alcohol (% w/w), real extract (% w/w),original extract (% w/w), real degree of fermentation (RDF) (%), pH, Bitterness units (BU) and color for beers produced from buckwheat (B beer), quinoa (Q beer) or buckwheat and quinoa (QB beer) malts comparing with two commercial gluten-free beers.

Parameters

Type of beers Alcohol Real Extract Original Extract RDF (%) pH BU Color (% w/w) (% w/w) (% w/w) B beer1 4.27 ± 0.02 AB 1.97 ± 0.01 C 8.63 ± 0.01 B 77.96 ± 0.81 A 5.82 ± 0.02 A 10.67 ± 0.38 A 4.58 ± 0.02 C Q beer2 4.07 ± 0.61 B 5.58 ± 1.21 A 11.61 ± 0.03 A 53.47 ± 8.27 C 4.86 ± 0.004 D 4.18 ± 0.75 D 9.41 ± 0.4 A QB beer3 4.02 ± 0.24 AB 6.10 ± 0.91 AB 12.51 ± 1.21 A 51.15 ± 2.22 BC 5.33 ± 0.01 C 11.33 ± 0.05 A 7.98± 0.95 AB Commercial beer 3.66 ± 0.04 A 3.09 ± 0.75 BC 10.19 ± 0.64 AB 67.24 ± 0.01 AB 5.64 ± 0.02 B 6.59 ± 0.01 B 7.56 ± 0.06 B A4 Commercial beer 3.55 ± 0.01 AB 4.24 ± 0.01 AB 11.52 ± 0.01 A 64.66 ± 0.01 B 5.34 ± 0.14 C 8.91 ± 0.01 C 6.34 ± 0.14 B B5

1B beer: beer made from malted buckwheat grains; 2Q beer: beer made from malted quinoa seeds; 3QB beer: beer made from malted quinoa seeds and buckwheat grains; 4Commercial beer A is a Pilsen type beer with moderate bitterness made from barley, with an enzymatic gluten degradation process during fermentation; 5Commercial beer B is a Premium American Lager type beer made from barley, with an enzymatic gluten degradation process during fermentation Different superscript letters in the same line indicate significant difference between B beer, Q beer, QB beer, Commercial beers A and B (ANOVA followed by Tukey’s multiple comparison post hoc test, p < 0.05).

82 In relation to pH, only QB beer was similar to a commercial gluten-free beer. Commercial beers pH usually range from 3 to 5 (Guyot-Declerck, 2005). In this sense only Q beer would be adequate (Table 2). However, both commercial beers tested (commercial beer A and commercial beer B) presented values over 5. pH of B beer was the highest, which could impact in the sensory analysis of this beverage. BU (Bitterness Unit) is the international unit expressing the concentration of hops bitter α-acids, dissolved and isomerized during boiling. The major part (80%) of beer bitterness comes from hops addition at the boiling stage, during which α- and β-acids present in this material thermally isomerize into iso- α-acids, which is the main bitter compound in beer (Haseleu et al., 2010). It is an important quality parameter of beer. BU was similar for B beer and QB beer, which was higher than commercial beers and Q beer (Table 2). It should be noted that all worts (quinoa, buckwheat and the mixture of both) were identically hopped. However, the perceived bitterness can also be affected by polyphenols present in the beer, that is why this parameter cannot be analyzed alone (Velić et al., 2018). Another important quality parameter is the beer color that cause a deep impact on the perceived beer flavor and quality. The color comes mainly from products of Maillard reactions originating in malting, during mashing in boiling of wort and from the oxidation polyphenols originating from grains (Shellhammer and Bamforth, 2008). In Table 2, color value of B beer was the only one that was statistically different from all other samples, with a low value. Taking into account the fact that the color of the beer is closely associated with the products of the Maillard (non-enzymatic redness reaction) reaction, affected by the heat treatment and high concentrations of sugar, this might be due to the lower content of sugar present in buckwheat, as detected in original extract parameter (Table 2). QB beer was the most similar to commercial beers in relation to color. All beers are within color range para o style American/European light lager with color units between 4-8 ° SRM, except Q beer which has a higher value, but less than British Pale Ale 20-30 ° SRM (Shellhammer, 2008). Free amino nitrogen (FAN) analysis is of great importance for beer quality because a low amount of FAN can have a negative influence on yeast growth affecting the fermentation. However, an excessive concentration of FAN in wort can increase the synthesis of higher alcohols, deteriorating beer flavor (Kordialik-Bogacka et al., 2018). Figure 2 shows the results for FAN concentration in commercial beers and for wort and beer produced with quinoa (Q beer), buckwheat (B beer) and both (QB beer).

83

Wort

Beer commercial beer A

commercial beer B

QB beer

Q beer

B beer

0 50 100 150 200 Free amino nitrogen (mg L-1)

Figure 3. FAN for beers and wort: B beer: beer made from malted buckwheat grains; Q beer: beer made from malted quinoa seeds; QB beer: beer made from malted quinoa seeds and buckwheat grains; Commercial beer A is a Pilsen type beer with moderate bitterness made from barley, with an enzymatic gluten degradation process during fermentation; Commercial beer B is a Premium American Lager type beer made from barley, with an enzymatic gluten degradation process during fermentation. Values are expressed as mean ± standard deviation of six independent experiments (n=6). Figure 3 shows that FAN reduced from wort to final beer, which might have happened during fermentation and maturation. Annemüller et al. (2011) recommend initial concentrations of 150 - 200 mg FAN/L in the wort and suggest a consumption of 100 - 140 mg FAN/L during the fermentation, indicating a residual value of 20 - 40 mg FAN/L. Residual values in this range mean that the FAN were totally consumed, since usually residual values cannot be consumed by the yeast. In this sense, all three beers produced (Q beer, B beer and QB beer) can be considered adequate in relation to FAN. Wijngaard and Arendt (2008) found FAN concentrations in buckwheat wort varying from 95 to 159 mg / L, which was the result of different infusion temperatures used, and higher infusion temperatures resulted in lower FAN. Deželak et al. (2014) detected a much higher amount of FAN in quinoa beer, which might be related to deficiency of other nutrients impacting in FAN consumption be the yeast. Kordialik- Bogacka et al. (2018) detected less than 140 mg/L of FAN for the wort produced with

84 40% quinoa, which is considered the minimal limit for all worts.

3.4 Phenolics of gluten free beer

Phenolic compounds extracted from malt and hop during beer production can contribute to antioxidant properties of this beverage, which is a good characteristic for human consumption (Callemien and Collin, 2009). Furthermore, antioxidants are generally thought to play a significant role in malting and brewing due to their ability to delay or prevent oxidation reactions and oxygen free radical reactions (Zhao et al., 2008). Coffee, tea, chocolate, wines, fruits, oils and certain types of grains are known for its high phenolic content (Waterhouse, 2003). Fumi et al. (2011) included beer in this list with results ranging from 84.4 - 515.8 mg gallic acid equivalents per liter. Table 3 shows the results of the phenolic compounds detected for Q beer, B beer and QB beer in comparison to the two commercial gluten-free beers. Higher phenolic content was found for the beers produced in the present work in comparison to the commercial ones. Q beer presented higher phenolic content of all. Zhao et al. (2010) analyzed twenty seven Chinese lager beers, all made mainly from barley malt, and reported that phenolic contents ranged from 4.47 mg/l (Reeb beer) to 15.50 mg/l (Blue Lion beer). They attributed the great variations in phenolic profiles for different beers to the differences in raw materials, brewing process and original gravity.

85 Table 3.14Phenolic compounds (mg/L) of beer produced with buckwheat (B beer), quinoa (Q beer) or buckwheat and quinoa (QB beer) comparing with two commercial gluten-free beers

QB beer1 B beer2 Q beer3 Commercial beer A4 Commercial beer B5 Phenolic Compounds mg/L mg/L mg/L mg/L mg/L Gallic Acid 127.72 ± 17.51 73 ± 1 164.22 ± 2.04 79.01 ± 2.03 37.48 ± 1.01 Caffeic Acid 227.63 ± 27.81 293 ± 1 nd nd nd Quercetin 117.42 ± 18.54 36 ± 0.4 126.48 ± 10.51 nd nd Rutin 136.99 ± 16.48 nd 71.50 ± 1.53 nd nd 3,4-Dihydroxybenzoic acid 99.91 ± 2.78 18 ± 1 nd nd nd 3,4-Dihidroxyphenylacetic acid nd* 300.2 ± 0.1 98.94 ± 1.02 24.31 ± 0.41 246.16 ± 2.07 p-Hidroxybenzoic Acid nd nd 86.7 ± 6.12 nd 56.73 ± 1.01 2,4-Dihydroxybenzoic acid nd nd 12.1 ± 0.4 nd nd Vanillic Acid nd nd 123.42 ± 4.08 nd nd Benzoic Acid nd nd 34.68 ± 0.20 12.16 ± 1.01 nd Ferulic Acid nd nd 89.76 ± 7.14 nd nd Syringic acid nd nd nd 28.36 ± 1.01 nd 4-Hydroxyphenylacetic acid nd nd nd 111.43 ± 1.01 80.03 ± 6.08 Total 709.67a ± 83.12 720.2a ± 3.5 862b ± 33.05 255.27d ± 5.47 420.4c ± 10.17 1B beer: beer made from malted buckwheat grains; 2Q beer: beer made from malted quinoa seeds; 1 QB beer: beer made from malted quinoa seeds and buckwheat grains; 4Commercial beer A is a Pilsen type beer with moderate bitterness made from barley, with an enzymatic gluten degradation process during fermentation; 5Commercial beer B is a Premium American Lager type beer made from barley, with an enzymatic gluten degradation process during fermentation Different superscript letters in the same line indicate significant difference between B beer, Q beer, QB beer, Commercial beers A and

86 B (ANOVA followed by Tukey’s multiple comparison post hoc test, p < 0.05). * nd: Not detected.

87 Baiano and Terracone (2013) found results ranging from 279 mg of gallic acid equivalents per liter to a gluten-free beer. Therefore, comparing the results presented in Table 3 with those in the literature, the values in the table were higher except for commercial beer B, being the Q beer that who had the highest percentage of phenolic compounds, among the other produced beers and commercial samples. The gallic acid was the most abundant phenolic compound in this beer and 3,4-Dihydroxybenzoic acid and caffeic acid was not found in this sample, but in the B beers and QB beer this phenolic compound appears. Gallic acid have the potential of a protective effect contrary to some neurological disorders (Khan et al. 2012). Studies have demonstrate that modest consumption of beer can be related with reduced risk of coronary heart disease (Bamforth, 2002) even as an inhibitory effect on pancreatic cancer growth (Gerloff et al. 2010). In the study by Carciochi et al. (2016), p-coumaric acid was the predominant in quinoa, but the p-OH-benzoic acid, Vanillic acid, Ferulic acid and Quercetin was also founded. These results are in agreement with Tang et al. (2015), who found that vanillic acid was the most abundant of identified phenolic acids in raw quinoa followed by ferulic acid.

3.5 Enzyme immunoassay for the qualitative determination of gliadins

Figure 4 presents the enzyme immunoassay for the qualitative determination of gliadin. As expected, beers Q, QB and B, from materials such as buckwheat and quinoa presented negative result for the presence of gliadin, while beers with gluten showed the positive result. Corroborating the results of Guerdrum & Bamforth (2001), beers brewed with gluten free raw material contain very low levels of gliadin, while those beers that overtly contain wheat as a principle grist component contain the highest levels of gliadin.

88

Figure 4. Enzyme immunoassay for the qualitative determination of gliadins in triplicates of B beer, Q beer, QB beer and commercial gluten beer.

4 CONCLUSIONS

Beer physical-chemical parameters are key economic concern for brewers and it is the subject of a considerable amount of research. All three beers produced (Q beer, B beer and QB beer) showed mean values of original extract, real extract, RDF close to the expected values, considering the barley malt parameters. FAN values for wort and beer were adequate as well. This indicates the possibility of obtaining a fermented product

89 from other cereals sources with physical-chemical properties close to traditional beer. The beer produced from buckwheat (B beer) presented higher pH and low color values. On the other side, the quinoa beer showed low BU values. In this sense, QB beer was more adequate and might have a better acceptability in sensory analysis. Phenolic content of these beers were higher than commercial barley beers, indicating a better nutritional property.

90 FINAL CONSIDERATIONS

The production conditions of alginate beads with wine industry by-product

(WIBP) is influenced by WIBP and CaCl2 concentrations as well as complexation time (CT) when erosion degree of the beads immersed by 15 days is considered. When the beads are immersed for longer periods (90 days) only calcium chloride concentration and CT influence erosion. Considering 15 to 90 immersion days, the encapsulation conditions to reduce the degree of erosion in wheat beer are lower CaCl2 concentrations, higher

WIBP amounts and reduced CT. To avoid excessive swelling, CaCl2 should be used in lower concentrations with high sodium alginate and medium amount of WIBP and medium CT. Best conditions to obtain beads with simultaneously lower swelling (606.7

%) and erosion degree (7.6 %) are: 1.5% sodium alginate, 4% WIBP, 0.26 M CaCl2 and 26 min CT. Beads containing the residue of the wine industry are potential additives for wheat beer increasing the consumption of antioxidants by the final consumer. The incorporation of encapsulated phenolic compounds into beer was observed for the first time in the present study. The red grape pomace (WIBP) used as core material for the beads was rich in antioxidants with high concentration of phenolic compounds. These enriched beads were added to a craft wheat beer and the phenolic compounds were not released during the 60 days of storage, protecting the phenolic content and the antioxidant activity of the core material. The release of phenolic compounds from the beads during simulated digestion, specially of WIBP components that were not present in beer, shows the advantage of using these beads as enrichment material to beverages in general. Physical-chemical parameters are key economic concern for brewers and it is the subject of a considerable amount of research. All three beverage compositions had shown mean values near the expected, considering the barley malt parameters. This indicates the possibility of obtaining a similar fermented product from other cereals sources with physical-chemical properties close to traditional beer. In overall, quinoa had a great significance in the final composition parameters, since considerable differences were observed between compositions comprising quinoa to others without it. All three types of beer had alcohol content similar to barley beer (alcohol = 3.92 % wt). The beer produced with buckwheat was the one real degree of fermentation (RDF = 77.96%) and closer to a barley beer (RDF = 80.05%), showing the potential of using this raw material to produce a gluten-free beer.

91 SUGGESTIONS FOR FUTURE WORK

• Sensory analysis of gluten-free beer added with wine industry by-product beads; • Application of beads in beer flavor development; • Use of other pseudo-cereals for the manufacture of gluten-free beer; • Analyze the gluten-free beer in Elisa technique.

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