Universidade do Minho Escola de Engenharia
Maria Teresa Bonifácio Viana Lopes
July 2022 Maria Teresa Bonifácio Viana Lopes UMinho|2022
DEVELOPMENT AND CHARACTERIZATION OF FUNCTIONAL INGREDIENTS THROUGH
VALORIZATION FROM BREWER'S SPENT GRAIN
DEVELOPMENT AND CHARACTERIZATION OF FUNCTIONAL INGREDIENTS THROUGH VALORIZATION FROM BREWER'S SPENT GRAIN
This work was financially supported by Co-promoção, POCI-01-0145 -FEDER-006684, NORTE- 08-5369-FSE-000036. We would also like to thank the scientific collaboration of CBQF under the FCT – Fundação para a Ciência e a Tecnologia through project Multibiorefinery –_Multi-purpose strategies for the valorization of a wide range of agroforestry by-products and fisheries: A step forward in the creation of an integrated biorefinery, (POCI-01-0145 -FEDER-0068) and the project UID/Multi/50016/2020, IID/BIO/04469/2020and thePhD grant UMINHO/BD/11/2016 to Teresa Bonifácio-Lopes.
Universidade do Minho Escola de Engenharia
Maria Teresa Bonifácio Viana Lopes
Supervisors:
Professor José António Teixeira
Professora Maria Manuela Estevez Pintado
Thesis submitted to Universidade do Minho, Universidade de Aveiro e Universidade Católica Portuguesa to attain the degree of PhD in Food Science and Technology and Nutrition
Universidade do Minho Escola de Engenharia
DEVELOPMENT AND CHARACTERIZATION OF FUNCTIONAL INGREDIENTS THROUGH
VALORIZATION FROM BREWER'S SPENT GRAIN
DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS
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Acknowledgments
I would like to acknowledge the role of Universidade do Minho, Escola Superior de Biotecnologia of the Universidade Católica and of Universidade de Aveiro for accepting me as a Ph. D. student and to Escola Superior de Biotecnologia of the Universidade Católica for supplying the necessary conditions to carry out this work.
Firstly, I would like to thank my supervisors who accepted me and guided me through this journey.
To Professor Manuela Pintado, for the continuous support, for your patience, motivation, knowledge and for always believing in me. Thank you for all the trust you deposited in me as researcher. To Professor José Teixeira, thank you for all your comments and support. To my labmates for helping and supporting me and for all the fun and joy. A special thanks to all the IDIOTS. To Débora Campos, thank you so much for all your support, for all your patience, for always believing in me and for all the help you gave me throughout the writing of this thesis. To Ana Vilas-Boas, my roommate, thank too for all the support you gave me, all the friendship and for helping me in the final stage of my thesis writing. To Emília for all the talks we had at lunch and all the walks we had, you helped me putting my head on its place! To Manuela, thank you for all the lunches we had. To Eduardo, Sara and Tânia for always offering their help whenever I needed it. To Eze, Miguel and Paloma for always checking on me and for all the love and support. To Ariana, Clarisse, Pedro, Xana and Ana Lino, thank you so much for your friendship and love. To Juca, you are the best cousin I could have! Your support during this time helped me more than you can imagine it. To Maria João, thank you for all your friendship and love, our drinks at the weekends and your support helped me a lot. To Joana Carolina, thank you for all your friendship. You are the best friend I could have during this phase of my life.
Thank you for all the support, love, patience.
To Sparky, I needed you the most, I had you on my side and you loved me always. To Pixie and Elis – our babies and the most spoiled cats in the world. To Texugo, Fluffy and Misty, Kurica and Nikita. To my aunt Margarida and my uncle Rogério. Thank you for all you support, love, patience and for making me feel more like a daughter than a niece. Thank you for receiving me in Rome every time I needed to clear my head. To my family. To my grandparents, mom, dad, Rui and Daniela. Your support was more important to me than you can imagine and you were always by my side no matter what, no matter what did or said. Your love got me here. And last but not least, to Carlos. You appeared in my life in the worse phase possible and you stayed by my side always. Even in the bad days you were there. All your patience and support were essential for me. All the nights you stood there watching me struggling and never turned your back on me, just being there for me. Your love got me here and was the most important thing to get me here.I am so grateful for everyone I have in my life. Your support means the world to me.
DECLARAÇÃO DE INTEGRIDADE
Declaro ter atuado com integridade na elaboração do presente trabalho académico e confirmo que não recorri à prática de plágio nem a qualquer forma de utilização indevida ou falsificação de informações ou resultados em nenhuma das etapas conducente à sua elaboração.
Mais declaro que conheço e que respeitei o Código de Conduta Ética da Universidade do Minho.
Resumo DESENVOLVIMENTO E CARACTERIZAÇÃO DE INGREDIENTES FUNCIONAIS ATRAVÉS DA
VALORIZAÇÃO DE DRÊCHE Resumo
O principal objetivo desta tese foi explorar o valor do subproduto da produção de cerveja, a drêche, promovendo o princípio do desenvolvimento sustentável com a obtenção de ingredientes de valor acrescentado.
Assim, a partir destes resíduos, foram realizadas extrações utilizando a técnica sólido-líquido, aquecimento óhmico e solventes utilizados na indústria alimentar (água e etanol) (para garantir a manutenção da integridade composicional, funcionalidade e segurança dos extratos bioativos obtidos).
Todos os extratos hidroetanólicos obtidos pela extração sólido-líquido apresentaram alta capacidade antioxidante, e os maiores valores foram obtidos para o extrato 60% etanol:água (v/v). A identificação por HPLC mostrou que a catequina e a vanilina foram os principais compostos identificados com maior concentração (223.6 e 109.2 g/g drêche, respetivamente) usando 60% etanol:água (v/v). Nos ensaios de atividade biológica os extratos mostraram-se multifuncionais (capacidade anti-hipertensiva, atividade antibacteriana e antibiofilme).
Todos foram não genotóxicos, mas a citotoxicidade foi dependente da concentração do extrato, com aplicação totalmente segura até 1 mg/mL.
Nos extratos antioxidantes obtidos por aquecimento óhmico o ácido 4-hidroxibenzóico foi o composto que apresentou maior concentração (125.86 g/ g drêche) e o extrato de etanol 60%:água (v/v) foi o que apresentou maior atividade antioxidante. No entanto, ambos os extratos demonstraram não ter atividade mutagénica, e ambos foram capazes de inibir 50% da enzima conversora de angiotensina-I (ECA), sendo o extrato com melhor resultado o 80% etanol:água (v/v). Os extratos de drêche apresentaram efeito inibitório contra Bacillus cereus e em menor grau contra Listeria monocytogenes, a 5,00 e 2,50 mg/mL, para 80%
etanol:água (v/v) e 60% etanol:água (v/v) respetivamente, e também, as mesmas concentrações inibiram a formação de biofilme.
A análise do impacto do trato gastrointestinal (TGI) mostrou que o método sólido-líquido (60%
etanol:água (v/v)) gerou extratos com maior atividade antioxidante e maior teor de fenólicos totais. No entanto, os compostos presentes no extrato obtidos por aquecimento óhmico com 80% etanol:água (v/v) apresentaram maiores índices de bioacessibilidade dos polifenóis. Todos os extratos aumentaram o crescimento dos microrganismos probióticos testados.
As farinhas resultantes da extração foram estudadas ao longo do TGI, mostrando que após uma diminuição inicial, existe um aumento na atividade antioxidante e fenólicos totais até final do TGI. Além disso, também modularam positivamente o crescimento de bactérias probióticas bem como do seu metabolismo, com a produção de ácidos gordo de cadeia curta.
Palavras-chave: aquecimento óhmico; atividade antioxidante; extração solido-líquido; microbiota; trato gastrointestinal
Abstract
DEVELOPMENT AND CHARACTERIZATION OF FUNCTIONAL INGREDIENTS THROUGH VALORIZATION FROM BREWER’S SPENT GRAIN
Abstract
The main objective of this thesis was to explore the value of the by-product of beer production, BSG, to promote the principle of sustainable development by obtaining value-added ingredients. So, from this residue, extractions were carried out using solid-liquid and ohmic heating extractions and solvents used in the food industry (ethanol and water) (to guarantee the maintenance of compositional integrity, functionality and safety of the bioactive extracts obtained).
All hydroethanolic extracts showed high antioxidant capacity, and the highest values were obtained for the extract 60% ethanol:water (v/v). Identification by HPLC showed that catechin and vanillin were the main compounds identified with the highest concentration (223.6 e 109.2 g/g BSG respectively) obtained by extraction using 60% ethanol:water (v/v). In the biological activity assays, the extracts proved to be multifunctional (antihypertensive capacity, antibacterial and antibiofilm activity). All were non-genotoxic, but cytotoxicity was dependent on extract concentration, with totally safe application for all extracts up to 1 mg/mL.
In the antioxidant extracts obtained by ohmic heating 4-hydroxybenzoic acid was the compound that showed the highest concentration (125.86 g/ g drêche) and the 60% ethanol extract:water (v/v) was the one that showed the highest antioxidant activity. However, both extracts showed no mutagenic activity, and both were able to inhibit 50% of the angiotensin-I converting enzyme (ACE), with the extract with the best result being 80%
ethanol:water (v/v). The BSG extracts showed an inhibitory effect against Bacillus cereus and to a lesser extent against Listeria monocytogenes, at 5.00 and 2.50 mg/mL, for 80% ethanol:water (v/v) and 60% ethanol:water (v/v). v) respectively, and also, the same concentrations inhibited biofilm formation.
The gastrointestinal tract (GID) analysis showed that the solid-liquid method containing 60%
ethanol:water (v/v) was the extraction with the highest antioxidant activity and the highest content of total phenolics. However, the compounds present in the extract obtained by ohmic heating with 80% ethanol:water (v/v) showed higher values of bioaccessibility indexes for polyphenols. All extracts increased the growth of the probiotic microorganisms tested
The flours resulting from solid-liquid extraction and ohmic heating were studied along the GID showing that after an initial decrease there was an increase in antioxidant activity and total phenolic compounds along the GID These flours also positively modulated the growth of probiotic bacteria as well as their metabolism with the production of short chain fatty acids
Keywords: antioxidant activity; gastrointestinal tract; microbiota; ohmic heating; solid-liquid extraction
List of Contents Table of contents
ACKNOWLEDGMENTS ... III RESUMO ... V ABSTRACT ... VI TABLE OF CONTENTS ... VII LIST OF TABLES ... XIII LIST OF FIGURES ... XV ABBREVIATIONS: ... XVII SCOPE AND OUTLINE ... XIX
PART I – Bibliographic Survey ... 1
CHAPTER 1 - CURRENT EXTRACTION TECHNIQUES TOWARDS BIOACTIVE COMPOUNDS FROM BREWER’S SPENT GRAIN - A REVIEW ... 2
Abstract ... 2
Key words ... 2
Highlights ... 3
1.1.Introduction ... 3
1.2.BSG composition... 4
1.3.BSG applications ... 6
1.3.1. BSG in foods and health ... 9
1.4.Extraction of added-value compounds ... 12
1.4.1. Pretreatment advantages ... 12
1.4.2. Extraction of phenolic compounds ... 13
1.4.2.1. Supercritical carbon dioxide ... 13
1.4.2.2. Autohydrolysis ... 14
1.4.2.3. Alkaline hydrolysis ... 14
1.4.2.4. Solvent Extraction ... 15
1.4.3. Extraction of carbohydrates... 16
1.4.3.1. Autohydrolysis ... 16
1.4.3.2. Ultrasound assisted extraction ... 17
1.4.3.3. Dilute acid hydrolysis ... 19
1.4.3.4. Enzymatic hydrolysis ... 20
1.4.3.5. Microwave assisted extraction ... 20
List of Contents
1.4.4. Others – proteins ... 21
1.4.4.1. Ultrafiltration ... 23
1.4.4.2. Ultrasound assisted extraction ... 23
1.4.5. Electric-field based technologies ... 23
1.5.Conclusions ... 24
PART II – Brewer’s spent grain valorization using solid-liquid extraction ... 26
CHAPTER 2 - BIOACTIVE EXTRACTS FROM BREWER’S SPENT GRAIN ... 27
Abstract ... 27
Keywords ... 27
Highlights ... 28
2.1. Introduction ... 28
2.2. Materials ... 30
2.2.1. Raw Material ... 30
2.2.2. Extraction procedure ... 30
2.2.3. Chemical characterization of BSG ... 30
2.2.4. Chemical characterization of BSG extracts ... 31
2.2.4.1. Phenolic content ... 31
2.2.4.2. Determination of phenolics profile and composition ... 31
2.2.4.3. ABTS radical cation assay ... 32
2.2.4.4. Oxygen Radical Absorbance Capacity (ORAC) ... 32
2.2.4.5. Agarose gel electrophoresis for DNA protection ... 33
2.2.5. Biological activity ... 34
2.2.5.1. Microorganisms and culture conditions ... 34
2.2.5.2. Growth inhibition curves assay ... 34
2.2.5.3. Biofilm formation assay ... 34
2.2.5.4. Genotoxicity ... 35
2.2.5.5. Antihypertensive Activity: Angiotensin-I converting enzyme (ACE) ... 35
2.2.6. Biocompatibility assay ... 36
2.2.6.1. Cell line growth conditions ... 36
2.2.6.2. Presto Blue assay ... 36
2.2.7. Statistical analysis ... 37
List of Contents
2.3.1. Composition of BSG and BSG extracts ... 40
2.3.2. Chemical characterization of BSG extracts ... 41
2.3.2.1. Phenolic content of BSG extracts and antioxidant capacity of BSG extracts 41 2.3.2.2. Phenolic compounds ... 44
2.3.2.3. Agarose gel electrophoresis for DNA protection ... 46
2.3.3. Biological activity ... 49
2.3.3.1. Growth inhibition curves assay ... 49
2.3.3.2. Biofilm formation assay ... 51
2.3.3.3. Genotoxicity ... 53
2.3.3.4. Antihypertensive Activity: Angiotensin-I converting enzyme ... 54
2.3.4. Biocompatibility assays ... 55
2.3.4.1. Presto Blue assay ... 55
2.4. Conclusion ...58
PART III – Brewer’s spent grain valorization using ohmic heating extraction ... 59
CHAPTER 3 - EXPLORING THE BIOACTIVE POTENTIAL OF BREWERS SPENT GRAIN OHMIC EXTRACTS ... 60
Abstract ...60
Keywords ...60
Highlights ...61
3.1. Introduction ...61
3.2. Materials ...63
3.2.1. Raw Material ...63
3.2.2. Extraction procedure ...63
3.2.3. Chemical composition of the solid residue ...64
3.2.4. Characterization of BSG extracts ...65
3.2.4.1. Total polyphenolic content ...65
3.2.4.2. Determination of phenolic profile and composition ...65
3.2.4.3. ABTS radical cation assay ...66
3.2.4.4. Oxygen Radical Absorbance Capacity (ORAC) ...66
3.2.4.5. Agarose gel electrophoresis for DNA protection ...67
3.2.5. Biological activity ...68
3.2.5.1. Microorganisms and culture conditions ...68
List of Contents
3.2.5.2. Inhibition curves assay ...68
3.2.5.3. Biofilm formation assay ...68
3.2.5.4. Genotoxicity ...69
3.2.5.5. Anti-hypertensive Activity: Angiotensin-I converting enzyme (ACE) ...69
3.2.6. Biocompatibility assay ...70
3.2.6.1. Cell line growth conditions ...70
3.2.6.2. Biocompatibility assays ...70
3.2.7. Statistical analysis ...71
3.3. Results and discussion ...71
3.3.1. Chemical composition of the solid residue ...71
3.3.2. Chemical characterization of BSG extracts ...74
3.3.2.1. Polyphenolic content and compounds of BSG extracts ...74
3.3.2.2. Antioxidant capacity of BSG extracts ...78
3.3.2.3. Agarose gel electrophoresis for DNA protection ...79
3.3.3. Biological activity ...82
3.3.3.1. Growth inhibition curves assay ...82
3.3.3.2. Biofilm formation assay ...83
3.3.3.3. Genotoxicity ...86
3.3.3.4. Antihypertensive Activity: Angiotensin-I converting enzyme ...87
3.3.4. Biocompatibility assays ...88
3.3.4.1. Presto Blue assay ...88
3.4. Conclusion ...91
Part IV: Bioactivity valorization ... 92
CHAPTER 4 - IMPACT OF GASTROINTESTINAL DIGESTION SIMULATION ON BREWER’S SPENT GRAIN GREEN EXTRACTS AND THEIR PREBIOTIC ACTIVITY... 93
Abstract ...93
Keywords ...94
Highlights ...94
4.1. Introduction ...94
4.2. Materials ...97
4.2.1. Raw Material ...97
List of Contents
4.2.3. In vitro gastrointestinal digestion ...98
4.2.4. Bioaccesibility index ...98
4.2.5. Antioxidant activity, phenolic content, identification and characterization ...98
4.2.5.1. ABTS radical cation assay ...98
4.2.5.2. Oxygen Radical Absorbance Capacity (ORAC) ...99
4.2.5.3. Total phenolic content ...99
4.2.5.4. Determination of polyphenols profile by HPLC ... 100
4.2.6. Prebiotic activity ... 100
4.2.7. Statistical Analysis ... 101
4.3. Results and discussion ... 102
4.3.1. Antioxidant activity ... 102
4.3.2. Total phenolic content: ... 107
4.3.2.1. Folin-Ciocalteu ... 107
4.3.2.2. Identification and quantification of phenolic compounds... 108
4.3.3. Prebiotic activity ... 112
4.4. Conclusion ... 116
CHAPTER 5 - IMPACT OF CIRCULAR BREWER’S SPENT GRAIN FLOUR AFTER IN VITRO GASTROINTESTINAL DIGESTION ON HUMAN GUT MICROBIOTA ... 117
Abstract ... 117
Keywords ... 118
Highlights ... 118
5.1. Introduction ... 118
5.2. Materials ... 121
5.2.1. Raw Material ... 121
5.2.2. Extraction procedure ... 121
5.2.3. Simulated in vitro gastrointestinal tract... 122
5.2.3.1. Oral digestion... 122
5.2.3.2. Gastric digestion ... 122
5.2.3.3. Duodenal digestion ... 122
5.2.4. Antioxidant activity ... 122
5.2.4.1. ABTS radical cation assay ... 122
5.2.4.2. Oxygen Radical Absorbance Capacity (ORAC) ... 123
List of Contents
5.2.4.3. Total phenolic content ... 123
5.2.4.4. Determination of polyphenolic profile and composition ... 123
5.2.4.5. Bioaccesibility index ... 124
5.2.5. Prebiotic activity ... 124
5.2.6. In vitro fermentation assays ... 125
5.2.7. Gut microbiota evaluation ... 125
5.2.7.1. DNA extraction ... 125
5.2.7.2. Real-time PCR for microbial analysis at stool ... 126
5.2.7.3. Determination of organic acids ... 128
5.2.8. Statistical analysis ... 128
5.3. Results and discussion ... 130
5.3.1. Content and profile of phenolic compounds ... 130
5.3.1.1. Total phenolic content ... 130
5.3.1.2. Individual phenolic compounds ... 132
5.3.2. Antioxidant activity ... 135
5.3.2.1. ABTS and ORAC... 135
5.3.3. Prebiotic activity ... 140
5.3.4. Evolution of the gut microbiota profile groups ... 144
5.3.5. Organic acids and sugar profiles during gut microbiota fermentation ... 149
5.3.6. Polyphenols profile during gut microbiota fermentation... 153
5.4. Conclusion ... 156
Part V: Conclusions and Future Perspectives ... 157
CHAPTER 6 – CONCLUSIONS ... 158
CHAPTER 7 – FUTURE PERSPECTIVES ... 161
Part IV: Bibliography ... 162
CHAPTER 8 – BIBLIOGRAPHY ... 163
List of Tables List of Tables
Table 1 – BSG composition (adapted from Mussatto, 2014 and Lynch et al., 2016) ... 5 Table 2 – Summary of different phenolic compounds extraction methodologies and possible applications of BSG ...11 Table 3 - Summary of different carbohydrates extraction methodologies and possible applications of BSG...18 Table 4 – Summary of the advantages and disadvantages of the different BSG’s extraction methodologies ...22 Table 5 – Proximate composition of BSG sample and derived solid residues obtained after extraction.
Results are expressed in % of dry weight ...39 Table 6 – Total phenolic content and antioxidant capacity of the BSG extracts (Mean±S.D). ...42 Table 7 – Phenolic compounds identified in BSG by HPLC (Mean±S.D). ...44 Table 8 – Growth inhibition (%) of different concentrations of the BSG extracts powders dissolved in culture media, against MSSA, S. enteritidis, E. coli, B. cereus and L. monocytogenes bacterial strains (Mean ± S.D.). Results in average inhibition percentage relative to the control. (NI – No Inhibition) ...50 Table 9 – Genotoxicity of different concentrations (10, 5, 2.5, 1 and 0.2 mg/mL) of BSG extracts against S. typhimurium TA98 (His-) bacterial strains (Mean±S.D.). ...54 Table 10 – Antihypertensive of the different BSG extracts. ...55 Table 11 – Proximate composition of Brewer’s Spent Grain (BSG) sample and derived solid residues obtained after ohmic heating extraction. Results are expressed in % of dry weight...73 Table 12 – Total polyphenolic content (Folin-Ciocalteau) and antioxidant capacity (ABTS and ORAC) of the Brewer’s Spent Grain (BSG) extracts for ohmic heating extraction (OHE) and solid-liquid extraction (SLE) (the control values for conventional extraction). ...77 Table 13 – Phenolic compounds identified in Brewer’s Spent Grain (BSG) ohmic heating extraction by HPLC analysis. ...77 Table 14 – Growth inhibition (%) of different concentrations of the brewer’s spent grain extracts powders dissolved in liquid culture media, against Bacillus cereus (B. cereus) and Listeria monocytogenes (L. monocytogenes) bacterial strains (initial inoculum concentration 2% (v/v)).83 Table 15 – Genotoxicity of different concentrations (10, 5, 2.5, 1 and 0.2 mg/mL) of Brewer’s Spent
Grain extracts against Salmonella Typhimurium TA98 (His-) bacterial strains. ...86 Table 16 – Antihypertensive of the different Brewer’s Spent Grain (BSG) extracts. ...88
List of Tables
Table 17 – Percentages of differences between the different phases of the gastrointestinal tract (GID) simulation of brewer’s spent grain (BSG) extracts. ... 106 Table 18 – Concentration of polyphenols for 60% ethanol:water and 80% ethanol:water (solid-heating extraction); and 60% ethanol:water and 80% ethanol:water (ohmic heating extraction) of brewer’s spent grain (BSG), for the non-digested (initial) and for the three stages of the GID (oral, stomach and duodenum) expressed as mg/100g of BSG (mean standard deviation) and bioaccessibility index. ... 111 Table 19 - Primer sequences and real-time PCR conditions used for gut microbiota analysis. ... 127 Table 20 – Concentration of total phenolic compounds and individual phenolic compounds present in BSG flours obtained after extraction using 60% ethanol:water and 80% ethanol:water (SLE) and 60% ethanol:water and 80% ethanol:water (OHE) of, for the non-digested (initial) and for the three stages of the GID (oral, stomach and intestine) expressed as mg/ 100g BSG as well as the total phenolic compounds (mg gallic/g BSG) and bioaccessibility index (means.d.).134 Table 21 – Percentages of differences between the different phases of the gastrointestinal tract (GID)
simulation of brewer’s spent grain (BSG) flours. ... 139 Table 22 - Feacal microbiota composition of volunteer participants. ... 144 Table 23 - Concentration of organic acids (succinic, lactic, acetic, propionic and butyric) and sugars (saccharose and glucose) throughout fermentation of FOS and BSG extraction residues with human microbiota (w/v). ... 152 Table 24 - Concentration of polyphenols throughout fermentation of BSG flours with human microbiota (mg/100 g). ... 155
List of figures List of Figures
Figure 1 – Summary of BSG applications ... 8 Figure 2 – Prevention of DNA oxidation (by H2O2 (a1) and H2O2/FeCl3 system (a2)) and pro-oxidant effect (in the absence (b1) and presence (b2) of iron cations) of the different concentrations (% (v/v)) of the BSG extracts (Water - ■, 100% ethanol - , 80% ethanol:water – ▲and 60%
ethanol:water - ). ...48 Figure 3 – Biofilm formation inhibition of different concentrations (5.00, 2.50, 1.25 and 0.625 (mg/mL) of the BSG extracts (water, 100% ethanol, 80% ethanol and 60% ethanol) against MSSA (a), S. enteritidis (b), E. coli (c), B. cereus (d) and L. monocytogenes (e) bacterial strains ...52 Figure 4 – Presto blue assay results. a – cell line HT29-MTX was used to evaluate potential toxicity caused by different concentrations (20, 10, 5, 1 and 0.5 mg/mL) of the extracts used (water, 80% ethanol:water, 60% ethanol:water); b - cell line Caco-2 was used to evaluate potential toxicity caused by different concentration of the extracts used (water, 80% ethanol:water, 60%
ethanol:water). ...57 Figure 5 – Prevention of DNA oxidation (by H2O2 (a1) and H2O2/FeCl3 system (a2)) and pro-oxidant effect (in the absence (b1) and presence (b2) of iron cations) of the different concentrations (% (v/v)) of the BSG extracts (80% ethanol:water – ▲and 60% ethanol:water - ). ...81 Figure 6 – Biofilm formation inhibition of different concentrations (5.000, 2.500, 1.250 and 0.625 (mg/mL) of the BSG extracts (80% ethanol and 60% ethanol) against MSSA (a), Salmonella Enteritidis (b), Escherichia coli (c), Bacillus cereus (d) and Listeria monocytogenes (e) bacterial strains ...85 Figure 7 – Presto blue assay results. a – cell line HT29-MTX was used to evaluate potential toxicity caused by different concentrations (10, 5, 1 and 0.5 mg/mL) of the extracts used (80%
ethanol:water, 60% ethanol:water); b - cell line Caco-2 was used to evaluate potential toxicity caused by different concentration of the extracts used (80% ethanol:water, 60%
ethanol:water). ...90 Figure 8 – Antioxidant activity through ABTS, ORAC values, and total phenolic compounds through Folin-Ciocalteau of different brewer’s spent grain extracts (from solid-liquid extraction and ohmic heating extraction) with the simulation through gastrointestinal digestion. ... 105 Figure 9 – Growth curves of probiotic microorganisms Bifidobacterium animalis B0 (a and b) and Bifidobacterium animalis spp. lactis BB12 (c and d) after applying brewer’s spent grain (BSG)
List of Figures
solid residues of solid-liquid extraction (SLE) (a and c) and ohmic heating extraction (OHE) (b and d) using three concentrations (2, 1.5 and 1%). ... 114 Figure 10 – Growth curves of probiotic microorganisms Lactobacillus acidophilus LA-5 (a and b) and Lacticaseibacillus casei 01 (c and d) after applying brewer’s spent grain (BSG) solid residues of solid-liquid extraction (SLE) (a and c) and ohmic heating extraction (OHE) (b and d) using three concentrations (2, 1.5 and 1%). ... 115 Figure 11 – ABTS and ORAC values of brewer’s spent grain solid residues (from solid-liquid extraction (SLE) and ohmic heating extraction (OHE)) throughout GID. ... 138 Figure 12 – Growth curves of probiotic microorganisms Bifidobacterium animalis B0 (a and b) and Bifidobacterium animalis spp. lactis BB12 (c and d) after applying BSG solid residues of SLE (a and c) and OHE (b and d) using three concentrations (2, 1.5 and 1% (v/w). x Positive control – Glucose; Positive control – FOS; + 60% ethanol:water SLE or OHE_2% (v/w); - 60%
ethanol:water SLE or OHE_1.5% (v/w); 60% ethanol:water SLE or OHE_1% (v/w); -80%
ethanol:water SLE or OHE_2% (v/w); ⧫ 80% ethanol:water SLE or OHE_1.5% (v/w); 80%
ethanol:water SLE or OHE_1% (v/w) ... 142 Figure 13 – Growth curves of probiotic microorganisms Lacticaseibacillus casei 01 (a and b) and Lactobacillus acidophilus LA-5 (c and d) after applying BSG solid residues of SLE (a and c) and OHE (b and d) using three concentrations (2, 1.5 and 1% (v/w)). x Positive control – Glucose; Positive control – FOS; + 60% ethanol:water SLE or OHE_2% (v/w); - 60%
ethanol:water SLE or OHE_1.5% (v/w); 60% ethanol:water SLE or OHE_1% (v/w); -80%
ethanol:water SLE or OHE_2% (v/w); ⧫ 80% ethanol:water SLE or OHE_1.5% (v/w); 80%
ethanol:water SLE or OHE_1% (v/w). ... 143 Figure 14 – Evolution of the gut microbiota groups (relative differences to the negative control in %) . ■ -
FOS; ■ – 60% ethanol:water SLE; ■ – 80% ethanol:water SLE; ■ – 60% ethanol:water OHE; ■ – 80% ethanol:water OHE. ... 147 Figure 15 - Firmicutes:Bacteroidetes (F:B) (a) ratio and variation of the pH (b) throughout fermentation of FOS and BSG extraction residues with human microbiota. ... 148
Abbreviations:
Abbreviations:
AAE – Ascorbic acid equivalent
AAPH – 2,2’-azo-bis-(2-methylpropionamidine)-dihydrochloride ABTS – 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) AOAC – Association of Official Analysis Chemists International AX – Arabinoxylans
AXOS – arabinoxylooligosacharides BI – Bioaccessibility index
BSG – Brewer’s spent grain DW – Dry weight
EFSA – European Food Safety Agency EU – European Union
FDA – Food and Drug Administration FOS - Fructooligosaccharides GAE – Gallic acid equivalent GalOS – Galactooligosaccharides GID – Gastrointestinal tract GOS - galactooligosaccharides His- - Histidine negative His+ - Histidine positive
HPLC – High-Performance Liquid Chromatography iACE – Angiotensin converting enzyme
MSSA – Methicillin sensitive Staphylococcus aureus MW – Molecular weight
n.d. – Non detected n. i. – no inhibition n.q. – Non quantified OD – Optical density OH – Ohmic heating
OHE – Ohmic heating extraction
ORAC – Oxygen radical absorbance capacity
Abbreviations:
SCFAs – Short-chain fatty acids SD – Standard deviation
SDG – Sustainable development goal SLE – Solid-to-liquid extraction TE – Trolox equivalent
TPC – Total phenolic content UAE - Ultrasound assisted extraction
Scope and Outline Scope and Outline
The main objective of this thesis was to extract, using liquid solid extraction with or without the application of ohmic heating, and to characterize bioactive compounds from hydroethanolic extracts of a beer by-product – brewer’s spent grain (BSG) and also to evaluate how these extracts and their solid residues behaved throughout the gastrointestinal tract simulation and how the solid residues of the extractions stimulated the microbiota of feacal human donors.
This thesis is organized in four parts which include seven chapters to describe how the research evolved throughout time.
Part I includes chapter 1. This chapter gives a general revision of literature of the extraction techniques used to extract bioactive compounds from BSG.
Part II includes chapter 2. This chapter describes the total characterization of BSG and BSG hydroethanolic extracts using liquid solid extraction. This characterization includes soluble and insoluble fiber, protein, sugars and lipids content of the solid residues after extractions, the total phenolic content, antioxidant activity, and quantification and characterization of the polyphenolic compounds of the extracts, antimicrobial and biofilm, antihypertensive activities, cytotoxicity and genotoxicity of the extracts.
Part III integrates chapter 3. In this chapter the total characterization of BSG hydroethanolic extracts using ohmic heating extraction is described. As in chapter 2 this characterization also includes soluble and insoluble fiber, protein, sugars and lipids of the solid residues of the extraction. The total phenolic content, antioxidant activity and quantification and characterization of the polyphenolic compounds present in the extracts and their antimicrobial and antibiofilm, antihypertensive activities, genotoxicity and cytotoxicity is also presented.
Part IV is divided into chapters 4 and 5 and is the characterization of the bioactivities of the BSG extracts and their solid residues. In chapter 4 the simulation of the gastrointestinal tract of the BSG hydroethanolic extracts is described. During the three phases of gastrointestinal tract (oral, stomachal and intestinal) the total phenolic content, antioxidant activity and quantification and identification of polyphenolic compounds was measured. At the end of the gastrointestinal tract the prebiotic activity of the extracts was measured. Chapter 5 evaluates the potential of BSG flours upon the gastrointestinal tract, their prebiotic activities and fermentation with human intestinal microbiota.
Part V encompasses chapters 6 and 7, where the conclusions of the thesis are present as well as the future perspectives.
Scope and Outline
This thesis allowed to generate as scientific publications one published review from the state of the art and four papers (two published and two submitted) in international scientific peer-reviewed journals.
Scope and Outline
Part I: Bibliographic survey
Part II: Brewer’s spent grain valorization using solid-liquid extraction
Part III: Brewer’s spent grain valorization using ohmic heating extraction Chapter 1
Current extraction techniques towards bioactive compounds from
brewer’s spent grain
Chapter 2 Bioactive extracts from
brewer’s spent grain
Chapter 3 Exploring the bioactive potential of brewers spent
grain ohmic extracts
Scope and Outline
Part IV: Bioactivity valorization
Part V: Conclusions and future perspectives Chapter 4
Impact of gastrointestinal digestion simulation on BSG
green extracts and their prebiotic activity
Chapter 5
Impact of circular brewer’s spent grain flour after in
vitro gastrointestinal digestion on human gut
microbiota
Chapter 6 Conclusions
Chapter 7 Future perspectives
Scope and Outline The information present in the seven chapters that constitute this thesis has already been published or has been submitted to international peer reviewed international scientific journals:
Chapter 1:
Teresa Bonifácio-Lopes, José A. Teixeira, Manuela Pintado, 2020. Current extraction techniques towards bioactive compounds from brewer’s spent grain - a review. Critical Reviews in Food Science and Nutrition, 60:16, 2730-2741, DOI: 10.1080/10408398.2019.1655632
Chapter 2:
Teresa Bonifácio-Lopes,Ana Vilas-Boas,Ezequiel R. Coscueta, Eduardo M. Costa,Sara Silva, Débora Campos,José A. Teixeira, Manuela Pintado, 2020. Bioactive esxtracts from brewer’s spent grain.
Food & Function, 11, 8963-8977, DOI: 10.1039/D0FO01426E
Chapter 3:
Teresa Bonifácio-Lopes, Ana Vilas-Boas, Manuela Machado, Eduardo M. Costa, Sara Silva, Ricardo N. Pereira, Débora Campos, José A. Teixeira, Manuela Pintado, 2021, Exploring the bioactive potential of brewers spent grain ohmic extracts. Innovative Food Science and Technologies, Elsevier 76, 102943, DOI: 10.1039/D0FO01426E
Chapter 5:
Teresa Bonifácio-Lopes, Luís . G. Castro, Ana Vilas-Boas, Débora Campos, José A. Teixeira, Manuela Pintado, 2021, Impact of gastrointestinal digestion simulation on brewer’s spent grain green extracts and their prebiotic activity. Submitted to Journal of Food Science and Nutrition
Chapter 6:
Teresa Bonifácio-Lopes, Marcelo, D. Catarino, Ana Vilas-Boas, Tânia B. Ribeiro, Débora Campos, José A. Teixeira, Manuela Pintado, 2021, Impact of circular brewer’s spent grain flour after in vitro gastrointestinal digestion on human gut microbiota. Submitted to Foods
“Success is not final; failure is not fatal; it is the courage to continue that counts.
Winston S. Churchill
PART I – Bibliographic Survey
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review
Chapter 1 - Current extraction techniques towards bioactive compounds from brewer’s spent grain - a review
Teresa. Bonifácio-Lopes 1,2, José A. Teixeira2, Manuela Pintado1
1 Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, 4169-005 Porto, Portugal
2 CEB - Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal This chapter has been published in:
Critical Reviews in Food Science and Nutrition (2020);
doi.org/10.1080/10408398.2019.1655632 Adapted for this dissertation
Abstract
Background: Brewer’s spent grain is one of the most abundant by-products of the brewing industry and is rich in various bioactive compounds (phenolic acids, insoluble dietary fiber and proteins). While at the present brewer’s spent grain is mainly used as animal feed its rich nutritional content makes it an interesting alternative for food applications.
Scope and approach: As the range of applications of the bioactive compounds extracted from by-products has been growing in recent years, there is the need to obtain and characterize these bioactive compounds. Extraction methods (supercritical carbon dioxide, autohydrolysis, alkaline hydrolysis, solvent extraction, ultrasound assisted extraction, dilute acid hydrolysis, enzymatic hydrolysis, microwave assisted extraction) have been developed and are always being subjected to new approaches to allow better extraction yields of the bioactive compounds.
Key findings and conclusions: This review aims to provide a better understanding of the current advantages and limitations of brewer’s spent grain extraction processes and to provide a background of brewer’s spent grain composition and applications.
Key words
Bioactive compounds; brewer’s spent grain; chemical composition; extraction techniques
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review Highlights
The re-use of agro-industrial by-products can origin new added-value products
BSG is an agro-industrial by-product and is a source of various bioactive compounds
Extraction methods have been applied to extract these compounds from BSG
It is needed to find new techniques and new possible applications to the BSG
1.1. Introduction
One of the food waste objectives to reach Sustainable Development Goal (SDG) is to halve food waste by 2030 assuming that today around 100 million tons of food are wasted every year in the European Union (EU). The action plan to reduce food waste envisioned by the EU action plan for the circular economy, which is a strategic and operational approach based on the reduction, reuse, recovery and recycling of materials and energy, enhancing the value and consequently the useful life of products, materials and resources in the economy. The re-use of agro-industrial by-products can give origin to new added-value products with functional compounds and the addition of functional compounds will benefit the industry and the consumers; the industry from an economic point of view and the consumers through the use of high valuable bioactive compounds that besides the health benefits may also replace positively the synthetic additives (Fărcaş et al., 2015, Spinelli et al., 2016).
So, developing food processes that are more efficient and sustainable should be an important step while guaranteeing food quality and safety, but, at the same time, making the energy used efficient and reducing water and gas consumption (Fasolin et al., 2019)
Beer is one of the most commonly consumed beverages in the world and global beer production was of 190.90 million kiloliters in 2017 being China the largest beer producing country (38.79 million kiloliters) followed by the United States of America (21.78 million kiloliters) and Brazil (14.00 million kiloliters) (Kirin Holdings, 2018). Brewer’s spent grain (BSG) is one of the most abundant by-product of the brewing industry. Currently, one of the most common use of BSG is animal feed but has certain characteristics (both nutritional and functional) that makes of BSG a good added-value by- product.
BSG is a source of bioactive compounds such as phenolic acids, insoluble dietary fiber or proteins, compounds with a particular interest for the industry and the elaboration of added-value products (Lynch et al., 2016, McCarthy et al., 2012, Mussatto et al., 2006, Mussatto, 2014, Steiner et al., 2015, Vieira et al., 2014). Spent grains are the solid residue separated from the beer wort by filtration after the mashing phase of the brewing process and are obtained from barley and constitute the insoluble fraction of the wort (del Río et al., 2013, Pires et al., 2012, Vieira et al., 2014).
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review
1.2.BSG composition
BSG is composed of husk, pericarp and endosperm of barley grain (Lynch et al., 2016). BSG chemical composition may vary according to the barley variety, the harvest time, the conditions of malting and mashing, and the type of other constituents added during the brewing process. However, BSG, is normally chemically composed by: 24% non-cellulosic polysaccharides (namely arabinoxylans - AX), (1–3, 1–4)-β-D- glucan), 20% lignin, 20% cellulose (glucose), 21% protein, 10% lipids and 5% of ash. Hemicellulose (xylose and arabinose) and small amounts of starch can also be present (Mussatto et al., 2006). A detailed description of BSG composition is presented in table 1.
The BSG also contain high content of diverse minerals (calcium, cobalt, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium and sulfur), vitamins (biotin, choline, folic acid, niacin, pantothenic acid, riboflavin, thiamine and pyridoxine) and free essential amino acids (leucine, valine, threonine, and lysine) and non-essential amino acids (alanine, serine, glycine, glutamic acid, aspartic acid, tyrosine, proline, arginine) that confer it relevant nutritional value.
In addition to these components, BSG contain phenolic compounds, being the most important class the hydroxycinnamic acids (ferulic and p-coumaric acids derivates being the main ones). Ferulic and p- coumaric acids have demonstrated antioxidant, antiallergenic, anti-inflammatory and antimicrobial (mainly ferulic acid) properties and the extracts with these phenolic compounds may represent a new source of functional ingredients to be applied in the food development (Wen et al., 2019). European Food Safety Authority (EFSA), has recently accepted AX (from wheat endosperm) and (1-3,1-4)-β-glucan (from oatmeal, oat bran, barley, barley bran, or mixtures of these sources) as health beneficial bioactive compounds (Comissão Europeia, 2012).
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review Table 1 – BSG composition (adapted from Mussatto, 2014 and Lynch et al., 2016)
Major components g kg−1 dry weight basis
Cellulose (glucan) 3-330
Hemicellulose 192-419
Xylan 136-206
Arabinan 56-419
Starch 10-120
Lignin 115-278
Lipids 30-106
Acetyl groups 11-14
Proteins 142-310
Ashes 11-46
Extractives 58-107
Phenolics 7-20
Minerals mg kg−1 dry weight basis
Silicon 1400-10740
Phosphorus 4600-6000
Calcium 2200-3515
Magnesium 1900-2400
Sulfur 1980-2900
Potassium 258.1-700
Sodium 100-309.3
Iron 100-193.4
Zinc 82.1-178
Aluminium 36-81.2
Manganese 40.9-51.4
Cobalt 17.8
Copper 11.4-18
Strontium 10,4-12.7
Iodine 11
Barium 8.6-13.6
Chromium <0.5-5.9
Molybdenum 1.4
Boron 3.2
Amino acids % of total protein
Non-essential
Histidine 26.27
Glutamic acid 16.59
Aspartic acid 4.81
Valine 4.61
Arginine 4.51
Alanine 4.12
Serine 3.77
Tyrosine 2.57
Glycine 1.74
Asparagine 1.47
Glutamine 0.07
Essential
Lysine 14.31
Leucine 6.12
Phenylalanine 4.64
Isoleucine 3.31
Threonine 0,71
Tryptophan 0.14
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review
1.3. BSG applications
Nowadays, BSG is mainly used in animal nutrition. The main market is dairy cattle feed but as BSG provides, among other nutrients, protein and fiber, it is starting to be used to feed other animals as pigs, fish and poultry. As it is a bioactive compound and a low-cost product can also be used in human nutrition. Some products were already developed with BSG incorporated, like flours and bakery products. By ingesting BSG the human health can benefit as BSG is proved to increase feacal weight, accelerate transit time, reduce plasma cholesterol and fat excretion and decrease gallstones incidence (Mussatto, 2014). Brewer’s spent grain has interest as a source of energy and charcoal production.
Thermochemical conversion such as combustion and pyrolysis are some of the alternative uses to BSG.
By using BSG in combustion process the resultant energy can be used to help to suppress the energetic demand of the breweries. Nonetheless, the combustion of BSG will develop the emission of toxic gases and particles that contain sulfur dioxide and nitrogen. Pyrolysis of BSG results in the formation of char, bio-oil and permanent gases, being bio-oil the most abundant fraction and contains a complex mixture of hydrocarbons (Mussatto, 2014). Gonçalves et al. (2017) studied the conversion of BSG to bio-oil and activated carbon, producing yields of 19%, 56% and 25% of charcoal, liquid (two phases – aqueous and organic (bio-oil)) and gases, respectively. Due to its high content of hemicellulose and cellulose, BSG can also be an interesting source of ethanol production (with a 86.3% conversion efficiency using dilute acid hydrolysis) (Mussatto, 2014). Because of its lower content in ash and high amount of fibrous materials, BSG, can be used in charcoal brick production. Dried BSG is pressed and carbonized in low- oxygen atmosphere and the resultant bricks have high calorific value and the burning properties are low due to the higher temperature ignition and longer burning period. The lower content in ash and high amount of fibrous materials also make BSG useful in paper manufacture (it can be used, for instance, to prepare paper towels, business cards and coasters) (Mussatto, 2014). The low cost and easily availability of BSG make it interesting as an adsorbent as it removes either organic compounds from waste gases or dyes from wastewater. BSG without any treatment is capable of adsorb a common dye used in the paper and textile industries present in effluents. Activated carbons with similar and better adsorption capacity than others can also be produced by lignin present in BSG (Mussatto, 2014, Silva et al., 2004). Mussatto et al. (2010) used ligin from BSG to prepare activated carbon, using phosphoric acid as an impregnating agent, by chemical activation. the best result was the one using 3 g of phosphoric acid/g of lignin at 600 ºC. Due to being rich in polysaccharides and in associated proteins and minerals, BSG has several applications in the biotechnological processes as: substrate for
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review cultivation of microorganisms and enzyme production; carrier for cell immobilization; additive or carrier in brewing and source of value-added products (Mussatto et al., 2006, Mussatto, 2014) (Figure 1).
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review
Figure 1 – Summary of BSG applications
Brewer's Spent Grain
Addiitive or carrier in
brewing
Anti-foaming agent
Carrier for immobilising brewer's yeast
Paper manufacture
Source of added-value
products
Polysaccharides
Phenolic compounds
Proteins
Substracte
Cultivation of microorganisms
Pleurotos
Agrocybe
Lentinus
Streptomyces
Enzyme production
Xylanase by Aspergillus awamori Alpha-amylase
by Bacillus subtilis
Nutrition
Human
Incorporation in food
Animal
Animal feed
Adsorbent Production
Energy
Direct combustion
Fermentation to produce biogas
Anaerobic fermentation
Charcoal Brick component
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review
1.3.1. BSG in foods and health
Several compounds found in BSG can be isolated and applied in food to develop new functional foods, and the protein content in BSG might be exploited for human nutrition. McCarthy et al., (2013a), described the ability of BSG protein isolates to decrease, with great significance, pro-inflammatory cytokine IFN-γ production, making these isolates useful in the treatment of inflammatory diseases (Coelho et al., 2014, McCarthy et al., 2013a, McCarthy et al., 2013b, Meneses et al., 2013, Mussatto
& Roberto, 2006, Mussatto et al., 2006, Steiner et al., 2015). Human body produces free radicals as a by-product of metabolic processes and the defense system (various enzymes such as catalase, superoxide dismutase and glutathione peroxidase take part in this system), will, normally, detoxify the free radicals produced. There are some dietary antioxidants (vitamins C, E and A) that are able to detoxify the free radicals, yet, sometimes, there is an over-production of the free radical – oxidative stress. This oxidative stress leads to oxidative damage in cellular components and biomolecules and may result in diseases. Plants and cereals are rich in phenolic compounds and are being used as natural sources of antioxidants and can be useful in detoxifying the free radicals and, thus, reduce the oxidative stress. Plant and cereals antioxidants can be also used in foods to retard the oxidative deterioration of lipids (Gangopadhyay et al., 2016, Muniandy et al., 2015).
Brewer’s spent grain are also rich in prebiotics and according to Gibson et al. (2017) prebiotics are “a substrate that is selectively utilized by host microorganisms conferring a health benefit”.
Fructooligosaccharides (FOS), galactooligosaccharides (GalOS), inulin and lactulose are known prebiotics. Additionally, β-glucan reportedly has prebiotic potential by stimulating the growth of lactobacilli and bifidobacteria in the gastrointestinal tract (Caleffi et al., 2015, Gómez et al., 2015, Mitsou et al., 2010, Wang et al., 2015). (1-3,1-4)--D-glucan and AX, are also present in BSG and have health claims associated (the intake of at least 3 g of barley (1-3,1-4)--D-glucan is needed to lower/reduce blood cholesterol). Barley -glucans were able to reduce/lower blood cholesterol.
Arabinoxylans contribute to the reduction of the glucose rise after a meal (a consumption of 8 g of AX- rich fiber (from the wheat endosperm) per 100 g of available carbohydrates is needed) (Steiner et al., 2015).
Arabinoxylans and arabinoxylooligosacharides (AXOS) are considered to be good potential prebiotics because they are nondigestible oligosaccharides by gastric or pancreatic enzymes, promote the good environment of the gastrointestinal tract and some groups of beneficial gut microflora are able to use them, conferring benefits to the health of the host. Arabinoxylooligosacharides are more resistant
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review
to heat and acidic pH when compared to AX and FOS due to their longer chain and the linkages between their constituents which makes of them interesting food ingredients (Courtin et al., 2009).
Because BSG contains polymeric/oligomeric material composed of xylose BSG could also be used as a source of AXOS. For instance, some studies state that feruloylated AXOS have good antioxidant and radical scavengers properties (Aguedo et al., 2015, Coelho et al., 2014, Gómez et al., 2015, Reis et al., 2015). McCarthy et al., (2012) explored the antioxidant activity of phenolic extracts of BSG. The phenolic extracts showed to protect against oxidative DNA damage and against genotoxic effects of hydrogen peroxide and 3-morpholinosydnonimine hydrochloride (SIN-1).
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review Table 2 – Summary of different phenolic compounds extraction methodologies and possible applications of BSG
Extraction method Extraction conditions Target compounds Foreseen applications Reference
Supercritical carbon dioxide extraction Pressure:15-35 MPa; temperature: 40- 60ºC; CO2 + ethanol: 0-60% v/v. Best conditions: 240 min. temperature of 40 ºC, pressure of 35 MPa and CO2 + 60%
ethanol (v/v). Phenolic: (0.35 ± 0.01 mg/g BSG); flavonoid: (0.22 ± 0.01
mg/g BSG); antioxidant
properties:(2.09 ± 0.04%/g BSG)
Phenolic compounds Potential re-use of this brewery by- product and the possibility to achieve a promising system for a large-scale extraction
Spinelli et al. (2016)
Autohydrolysis Solid/liquid ratios: 1/10 and 1/30 g BSG/ml water; temperature: 121 ºC;
time: 10 or 90 min.
Hydrolysate with the most pleasant aroma was obtained with 1 g BSG/ 10 ml water
Phenolic compounds The compounds extracted can be reused in the production of food or beverages.
Meneses et al. (2011)
Alkaline hydrolysis NaOH 1.0; 1.5 and 2.0% w/v;
temperature: 80, 100 and 120 ºC;
reaction times: 30, 60 and 90 min.
Best hydrolysis conditions: 2% NaOH, 120 ºC and 90 min obtaining 145.3 mg/l ferulic acid and 138.8 mg/l p- coumaric.
Ferulic and p-coumaric acids. Re-use of BSG in the food, cosmetic and/or pharmaceutical fields. Further assessments of the acceptability and safety of the phenolic acids extracted from BSG are necessary.
Mussatto et al. (2007)
Solvent extraction Water; 100%, 80%, 60%, 40% and 20%
of methanol, ethanol and acetone mixtures with water, 100% of ethyl acetate and hexane.
Most content of total phenols and antioxidant potential: acetone:water 60%.
Phenolic compounds Applications in the food, cosmetic and pharmaceutical industries, since antioxidant phenolic compounds could be used as a natural and inexpensive alternative to synthetic antioxidants.
Meneses et al. (2013)
Ohmic heating Extracts using 45% ethanol:water at 80 ºC using different times (20-90 min) and different voltages (496.0 – 840.0 V/vm). The best extract was the one using 840.0 V/cm..
Phenolic compounds Application in food, pharmaceutical or cosmetic industries
Jesus et al. (2020)
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review
1.4. Extraction of added-value compounds
Various types of extraction methods can be used to extract certain compounds from BSG like phenolic compounds, glucans or dietary fiber. The cost, time and availability of the process are variables that influence the choice of the most appropriate method. The extracts obtained can expand BSG applications, providing other uses than animal feed.
1.4.1. Pretreatment advantages
To break down the structure of the material in study it is usual to do a pretreatment stage such as acid hydrolysis, autohydrolysis, dilute acid, alkaline hydrolysis and others. The pretreatment improves the extraction efficacy allowing for an improvement of the extraction yields both in modern high-tech extraction approaches and in classic, less expensive, methodologies. Moreover, phenolic compounds are contained within the cell vacuole and are easily extracted if the solvent access to these structures is facilitated (Silva et al., 2017). Pretreatment methods like autohydrolysis, dilute acid, alkaline or enzymatic hydrolysis and others can be used to help extract phenolic and carbohydrates compounds. Selection of pre-treatment method to use will depend on what type of compound that is wanted to extract. The pretreatment helps to preserve the pentose fractions, limit the formation of degradation compounds that will prevent the development of fermentative microorganisms and minimize the energy and costs (del Campo et al., 2006).
Autohydrolysis is a pretreatment used in the extraction of hemicelluloses. In this type of method, no chemicals are used which makes of it an interesting alternative as it is eco-friendly.
Autohydrolysis has been used to remove hemicelluloses and too high temperatures decrease the amount of hemicelluloses recovered (Li et al., 2017).
Dilute acid pretreatment helps to deconstruct the cell wall of plants. Despite the fact that is low cost, it can lead to the production of acetic and formic acid and other inhibitors of enzymatic saccharification and fermentation microorganisms such as 5-hydroxymethylfurfural. Inorganic acids such as sulfuric, hydrochloric, nitric and phosphoric can be used, being the sulfuric acid the one used the most because it is the one with highest hemicellulose degradation efficiency. Dilute acid pretreatment is used in the production of bioethanol from agricultural waste (Mikulski & Kłosowski, 2018, Rajan & Carrier, 2014).
Just as autohydrolysis and dilute acid, alkaline acid pretreatment is environmentally friendly and low cost. The resulted biomass of alkaline pretreatment is enriched in cellulose. Alkaline pretreatment is normally applied to lignocellulosic materials, improving the biodegradability of the raw material by
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review Enzymatic pretreatment is also a low-cost method as it does not require an expensive equipment, however the high cost of enzymes it is still a challenge when this method is used to the production of biogas at an industrial scale. This pretreatment can also be used to disrupt cell walls using low energy. This method uses oxidative and hydrolytic enzymes and most of these enzymes are not inhibited by the final products (Hosseini et al., 2018, Zhang et al., 2018).
1.4.2. Extraction of phenolic compounds
As referenced above, some studies showed that BSG contains phenolic compounds such as ferulic and p-coumaric acids (Steiner et al., 2015). In general, to extract bioactive compounds from BSG some extraction techniques have been used, namely solid-liquid extraction, microwave-assisted extraction, hydrothermal treatment and enzymatic and alkaline reactions (table 2) (Steiner et al., 2015).
To extract the phenolic compounds with antioxidant properties solid-to-liquid extraction can be used. Still, it is necessary to take in consideration the extraction solvent as this is an important factor when recovering these compounds (Meneses et al., 2013). To extract phenolic compounds as ferulic or p-coumaric acid, different methods have been investigated, namely alkaline hydrolysis with NaOH, enzymatic extraction by adding esterease from Aspergillus niger or xylanase from Trichoderma viride (Bartolomé et al., 1997, Mussatto et al., 2007).
1.4.2.1. Supercritical carbon dioxide
In the extraction of phenolic compounds, supercritical fluid extraction can be a possible alternative. As this is a high-cost process, it is mainly used as a technique to obtain high valuable substances and is fast, selective and there’s no residual solvents. Carbon dioxide can be used as a solvent in supercritical fluid extraction. It has good solvation power when it is in the supercritical state and also it has gas-like and liquid-like qualities in this state. Carbon dioxide is cheap and is generally recognized as safe by the Food and Drug Administration (FDA) and EFSA. On the other hand, carbon dioxide has disadvantages being the main one its lower polarity. This problem can be solved with the use of co-solvents. However, the co-solvents might affect the efficiency of the extraction of antioxidants.
One of the most used co-solvents in this kind of extraction is ethanol. The phenolic compounds extracted from this method can give to BSG a re-use (Junior et al., 2014, Spinelli et al,. 2016).
Spinelli et al. (2016) used supercritical carbon dioxide extraction to extract bioactive compounds from BSG using ethanol as a co-solvent. In this study, the conditions utilized to extract bioactive compounds from BSG were: pressure (15-35 MPa), temperature (40-60 ºC) and CO2 +
Chapter 1 – Current techniques towards bioactive compounds of brewer’s spent grain – a review
ethanol (0-60% ethanol concentration v/v). The best conditions to extract antioxidant compounds were 240 min., a temperature of 40 ºC, pressure of 35 MPa and CO2 + 60% ethanol (v/v), achieving a high phenolic (0.35 ± 0.01 mg/g BSG) and flavonoid (0.22 ± 0.01 mg/g BSG) content and good antioxidant properties (2.09 ± 0.04%/g BSG).
Ferrentino et al. (2019) used supercritical carbon dioxide extraction for the extraction of oils with antioxidant activity from BSG. Ethanol was and was not used as a co-solvent (4 and 8% using 20 and 30 MPa and temperatures of 40 and 50 ºC). The best conditions to extract oils with antioxidant capacity from BSG were 30 MPa, 50 ºC and 8% ethanol (with an antioxidant activity of 14.2 ± 0.1 mg TEA/g of sample and total phenolic content of 0.3 mg GAE/g of sample).
1.4.2.2. Autohydrolysis
Autohydrolysis, also called hydrothermal treatment, is a technique that does not use chemical agents thus being a better option in comparison with the techniques that use chemical agents. This process uses liquid water under high temperature and pressure. In order to obtain the target compounds, the best reaction conditions need to be studied as these conditions are going to influence the products obtained by autohydrolysis (oligosaccharides, monosaccharides, sugar degradation products and acetic acid). Autohydrolysis is more environmentally friendly, not requiring chemical catalysts. The compounds extracted could be used in other industries such as food industries (Carvalheiro et al., 2005, Meneses et al., 2011, Ruiz et al., 2013).
Meneses et al. (2011) carried out BSG autohydrolysis (under different conditions) in order to extract aroma compounds. The autohydrolysis reactions were carried out in an autoclave under different solid/liquid ratios (1/10 and 1/30 g BSG/ml water) at 121 ºC, during 10 or 90 min. A group of untrained panelists attributed a value for the intensity of aroma perceived in each sample and additionally they also selected the sample considered having the most pleasant aroma. In this study aroma compounds were extracted and the condition to extract the most pleasant aroma was 1 g BSG/10 ml water with either 10 or 90 min of extraction.
1.4.2.3. Alkaline hydrolysis
Alkaline reagents disrupt the cell wall dissolving lignin and hemicelluloses. By removing lignin, the enzymatic hydrolysis yields improve (higher access of cellulolytic enzymes to cellulose) and lowers the probability of non-productive enzyme binding to lignin. When using alkaline hydrolysis not-bonded
