Microbiological and lipid profiles of broa: contributions for the characterization of a traditional portuguese bread

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M

ICROBIOLOGICAL AND

L

IPID

P

ROFILES OF

BROA

:

C

ONTRIBUTIONS

FOR THE

C

HARACTERIZATION OF A

T

RADITIONAL

P

ORTUGUESE

B

READ

Thesis submitted in accordance with the requirements for the degree of PhD in Food

Engineering

by João Miguel Ferreira da Rocha

under the supervision of Prof. Francisco Xavier Delgado Domingos Antunes Malcata

and the co‐supervision of Prof. Maria Luísa Duarte Martins Beirão da Costa

JURY President: Reitor da Universidade Técnica de Lisboa.

Members: Doutora Maria Luísa Duarte Martins Beirão da Costa, professora catedrática aposentada do Instituto Superior de Agronomia da Universidade Técnica de Lisboa;

Doutor Francisco Xavier Delgado Domingos Antunes Malcata, professor catedrático do Instituto Superior da Maia, Porto;

Doutora Ivonne Delgadillo Giraldo, professora associada da Universidade de Aveiro;

Doutor Manuel José de Carvalho Pimenta Malfeito Ferreira, professor auxiliar do Instituto Superior de Agronomia da Universidade Técnica de Lisboa;

Doutora Carla Maria Cadete Martins Moita Brites, investigadora auxiliar do Instituto Nacional de Recursos Biológicos, I.P..

Instituto Superior de Agronomia | Universidade Técnica de Lisboa

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M

ICROBIOLOGICAL AND

L

IPID

P

ROFILES OF

BROA

:

C

ONTRIBUTIONS

FOR THE

C

HARACTERIZATION OF A

T

RADITIONAL

P

ORTUGUESE

B

READ

Thesis submitted in accordance with the requirements for the degree of PhD in Food

Engineering

by João Miguel Ferreira da Rocha

under the supervision of Prof. Francisco Xavier Delgado Domingos Antunes Malcata

and the co‐supervision of Prof. Maria Luísa Duarte Martins Beirão da Costa

JURY President: Reitor da Universidade Técnica de Lisboa.

Members: Doutora Maria Luísa Duarte Martins Beirão da Costa, professora catedrática aposentada do Instituto Superior de Agronomia da Universidade Técnica de Lisboa;

Doutor Francisco Xavier Delgado Domingos Antunes Malcata, professor catedrático do Instituto Superior da Maia, Porto;

Doutora Ivonne Delgadillo Giraldo, professora associada da Universidade de Aveiro;

Doutor Manuel José de Carvalho Pimenta Malfeito Ferreira, professor auxiliar do Instituto Superior de Agronomia da Universidade Técnica de Lisboa;

Doutora Carla Maria Cadete Martins Moita Brites, investigadora auxiliar do Instituto Nacional de Recursos Biológicos, I.P..

Instituto Superior de Agronomia | Universidade Técnica de Lisboa

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To my family.

For their lifelong teaching, encouragement and unconditional love.

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“…a cela é o lugar ideal para nos conhecemos a nós próprios, para aprofundarmos de forma realista e regular os processos da nossa mente e dos nossos sentimentos. Ao avaliarmos a nossa evolução enquanto indivíduos tendemos a concentrar‐nos em factos externos como a posição social, o poder de influência e a popularidade, a riqueza e o nível de instrução. Estes são, de facto, factores importantes para a avaliação do sucesso individual no que se refere a aspectos materiais e é perfeitamente compreensível que muitas pessoas se empenhem em alcançá‐los. Existem no entanto factores internos que podem ser ainda mais decisivos na avaliação de uma pessoa enquanto ser humano: a honestidade, a sinceridade, a simplicidade, a humildade, a generosidade, a ausência de vaidade, a disponibilidade para ajudar os outros – qualidades ao alcance de todas as almas – constituem os alicerces da vida espiritual de cada um de nós. A evolução em matérias desta natureza é impensável sem uma introspecção séria, sem nos conhecermos as nossas fraquezas e os nossos erros. No mínimo, se não nos der mais nada, a cela proporciona‐nos a oportunidade de analisarmos todos os dias a nossa conduta na sua globalidade, de ultrapassarmos o que de mau houver em nós e desenvolvermos o que passamos ter de bom. A meditação frequente nem que seja durante 15 minutos por dia antes de adormecer, pode ser muito proveitosa a este respeito, no início pode parecer‐nos difícil identificar os aspectos negativos da nossa vida, mas com perseverança este exercício poderá revelar‐se altamente compensador. Não devemos esquecer que um santo é um pecador que não cessa de se esforçar.”. [Excerpt from a letter to Winnie Mandela in prison Koonstad, dated from February 1st, 1975. In: Nelson Mandela, 2010. Nelson Mandela – Arquivo Íntimo [Conversations with myself], Editora objectiva, Carnaxide, pp. 211‐212].

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ASTRACT

MICROBIOLOGICAL AND LIPID PROFILES OF BROA: CONTRIBUTIONS FOR

THE CHARACTERIZATION OF A TRADITIONAL PORTUGUESE BREAD

ABSTRACT

Scientific knowledge of artisanal broa contributes to a better quality and market expansion thereof. Towards this purpose, samples from fourteen artisanal producers were assayed for comprehensive microbial and lipid identification and quantification.

Fermentation played a major role upon the prevailing microflora: viable numbers of yeasts, lactobacilli, streptococci, lactococci, enterococci and leuconostocs increased, whereas those of molds, Enterobacteriaceae, Pseudomonadaceae, staphylococci and micrococci decreased. After fermentation, yeasts and lactic‐acid bacteria dominated.

Neutral‐lipids were quantified by normal phase‐high performance liquid chromatography‐ evaporative‐light scattering detection and electrospray ionization‐tandem mass‐spectrometry, after employing two independent extraction procedures – yielding non‐starch and starch lipids, as well as free, bound and starch lipids. Solid‐phase extraction separated neutral‐lipids prior to derivatization of sterol‐esters, triacylglycerol, free‐fatty acids, sterols with diacylglycerols and monoacylglycerols, permitted fatty‐acids separation by gas‐liquid chromatography. Breadmaking affected lipid extractability, and neutral‐lipid and fatty‐acid profiles. Triacylglycerols decreased during dough‐mixing and fermentation – so those of diacylglycerols and monoacylglycerols increased, whereas sterol‐esters remained constant; the effect of baking was less pronounced. Triacylglycerols dominated, but sterol‐esters, diacylglycerols and free‐fatty acid amounts were considerable. Palmitic, oleic and linoleic acids were dominant. The nutritional value of broa was apparent, owing to poly‐ and monoene dominance.

KEYWORDS

Broa, Fatty acids, Glycerides and sterols, Gram‐positive and Gram‐negative rods and cocci, Lactic‐acid bacteria, Maize and rye flours, Microflora, Neutral lipids, Sourdough, Yeasts and molds.

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ASTRACT

PERFIL MICROBIOLÓGICO E LIPÍDICO DA BROA: CONTRIBUIÇÕES PARA A

CARACTERIZAÇÃO DE UM PÃO TRADICIONAL PORTUGUÊS

RESUMO

O conhecimento científico da broa artesanal permite melhorar a sua qualidade e expandir o seu mercado; assim, identificou‐se e quantificou‐se a microflora e os lípidos em amostras de quatorze produtores artesanais.

O efeito da fermentação na microflora foi considerável: os números viáveis de viáveis de leveduras, lactobacilos, estreptococos, lactococos, enterococos e leuconostocs aumentou, enquanto os de bolores, Enterobacteriaceae, Pseudomonadaceae, estafilococos e micrococos diminuiu. Após fermentação, assistiu‐se à dominância das leveduras e bactérias do ácido láctico.

Quantificaram‐se os lípidos‐neutros por cromatografia‐líquida‐de‐alta‐resolução, com detecção por dispersão‐evaporativa‐de‐luz e electrodispersão‐espectrometria‐de‐impacto‐de‐ massa, após dois métodos de extracção independentes, originando lípidos não‐amiláceos e amiláceos, e lípidos livres, ligantes e amiláceos. Uma extracção‐em‐fase‐sólida separou os lípidos‐neutros antes de derivatizar os ésteres‐esterol, triglicerídeos, ácidos‐gordos livres, esteróis com diacilglicerídeos e monoacilglicerídeos, permitindo quantificar os ácidos‐gordos por cromatografia‐líquida‐gasosa. A panificação influenciou a extractabilidade dos lípidos e o perfil dos lípidos‐neutros e ácidos‐gordos: os triglicerídeos diminuíram durante a fermentação e, consequentemente, os diglicerídeos e monoglicerídeos aumentaram, enquanto os esterol‐ ésteres permaneceram inalterados; o efeito da cozedura foi menos pronunciado. Os triglicerídeos dominaram, mas os ésteres‐esterol, diglicerídeos e ácidos‐gordos apresentaram‐ se em quantidades consideráveis. Os ácidos palmítico, oleico e linoléico dominaram. O valor nutricional da broa tornou‐se evidente através do domínio de poli‐ e monoenos.

PALAVRAS‐CHAVE

Ácidos‐gordos, Bactérias do ácido láctico, Broa, Cocos e bacilos gram‐positivos e gram‐ negativos, Farinhas de milho e centeio, Glicerídeos e esteróis, Leveduras e bolores, Lípidos‐ neutros, Massa ácida, Microflora.

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MICROBIOLOGICAL AND LIPID PROFILES OF BROA: CONTRIBUTIONS FOR

THE CHARACTERIZATION OF A TRADITIONAL PORTUGUESE BREAD

EXPANDED ABSTRACT

Broa is a traditional sourdough bread, made of maize (Zea mays) and rye (Secale cereale) flours. It is widely manufactured at the farm level in Northern Portugal, following ancient manufacturing procedures – including use of a piece of dough from the previous batch as starter culture. Because broa plays important roles – from economic, social and cultural standpoints, scientific knowledge will be useful to rationally improve quality, and thus expand its market. Towards this goal, samples supplied by 14 artisanal producers, selected from 4 sub‐ regions were assayed for microbial quantification and identification, and also lipid qualitative and quantitative characterization. Total viable counts, as well as viable mesophilic and thermophilic microorganisms, yeasts and molds, Gram‐ rods, endospore‐forming and nonsporing Gram+ rods, and catalase+ and catalase‐ Gram+ cocci counts were obtained, during two periods of the year. Fermentation played a major effect upon the various (dominant) microbial groups: the viable counts of yeasts, lactobacilli, streptococci, lactococci, enterococci and leuconostocs increased, whereas those of molds, Enterobacteriaceae, Pseudomonadaceae, staphylococci and micrococci decreased relative to maize and rye flours; hence, the role of fermentation towards natural control of undesired microorganisms is apparent. The results unfolded a unique and rather complex wild microflora in flours and sourdoughs, but could not discriminate among sub‐regions or seasons, or type of flour for that matter. After fermentation, the dominant groups of microorganisms were yeasts and lactic acid bacteria (LAB). The most frequently identified yeasts were Saccharomyces cerevisiae and Hansenula anomala, whereas the most frequently isolated LAB were (heterofermentative) Leuconostoc spp. and (homofermentative) Lactobacillus spp.; Lactobacillus brevis, L. curvatus and L. lactis ssp. lactis were the dominant strains for the Lactobacillus genera; Lactococcus lactis ssp. lactis for lactococci; Enterococcus casseliflavus, E. durans and E. faecium for enterococci; and Streptococcus constellantus and S. equinus for streptococci.

Sourdough for broa belongs to type I group of sourdoughs: usually one day before baking, the sour ferment or mother dough (usually called “isco”) – i.e. a piece of ripened dough kept from a previous batch, is mixed to maize and rye flours, kneaded with water and let to ferment overnight. In addition, mother’s dough may be kept for several days or weeks, according to the baking frequency. Therefore, a similar approach was followed in attempts to study the microbiological profile throughout sourdough fermentation and mother dough storage: dough was prepared following the traditional protocol, and kept for several weeks under controlled temperature and relative humidity. The experimental results produced indicated a dominance of yeasts, Lactobacillus and Bacillus viable counts during most of the time and also by the late as 39 d – although a large diversity of microorganisms was noticed at the beginning of

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fermentation. The pH decreased drastically in the first 24 h – chiefly because of the metabolic activity of LAB. The effect of sourdough aeration was further studied as independent experiment for 14 d, which showed to have several effects on the microbial profile. Finally, the evolution of microflora in maize and rye flours during storage at two different temperatures (4 and 20 ºC, for 8 and 39 d, respectively) and in a mother‐dough (left at 4ºC, for 6 d) was also studied and the results unfolded a general maintenance of their microbial profile in all cases, although minor but important changes could be detected. Comprehensive qualitative and quantitative characterization of neutral lipids (NL) was carried out by normal phase‐high performance liquid chromatography‐evaporative light scattering detection (NP‐HPLC‐ELSD) and electrospray ionization tandem mass spectrometry (ESI‐ MS/MS), after employing two independent extraction procedures – yielding non‐starch (NSL) and starch lipids (SL), as well as free lipids (FL), bound lipids (BL) and SL. Furthermore, two solid‐phase extraction (SPE) procedures were used to separate the NL classes of extracts prior to independent derivatization – viz. sterol esters (SE), triacylglycerols (TAG), free fatty acids (FFA), sterols (S) with diacylglycerols (DAG), and monoacylglycerols (MAG), and quantification by gas‐liquid chromatography (GLC).

The NL were separated to baseline, via NP‐HPLC‐ELSD, on two series microbore 3 µm‐silica gel columns, in the following order: SE, high‐molecular weight (HMW)‐TAG, FFA, low‐molecular weight (LMW)‐TAG, 1,3‐DAG, S, 1,2‐DAG and MAG. All neutral lipids but MAG were eluted at the (optimum) flow rate of 0.1 mL/min, within a total running time of 76 min. Complete or partial separation of molecular species in NL permitted identification by automatic tandem mass spectrometry (MS/MS) of ammonium adducts, produced via positive electrospray ionization (ESI); moreover, NP‐HPLC‐ESI‐MS and ‐MS2 proved essential in NL identification, and further NP‐HPLC‐ELSD quantitation. Ion chromatograms and MS – and especially tandem MS, were required to accurately identify the peaks showing up in ESI‐extracted ion chromatogram (EIC) and NP‐HPLC‐ELSD chromatograms. The 1,3‐DAG, free S and 1,2(2,3)‐DAG were accordingly resolved to the baseline. Short‐ and medium‐chain TAG were fully separated, but long‐chain TAG only partially – and TAG with the same chain length, but different degrees of unsaturation were partially separated as well; conversely, FFA, DAG and MAG possessing distinct chain lengths could be separated to a limited extent.

The effect of the breadmaking process upon lipid extractability, and NL and fatty acids profile was ascertained by surveying maize and rye flours, sourdough and broa matrices – i.e. the representative breadmaking stages. NSL or FL accounted for the major portion of lipids extracted. The main NL varied according to lipid extract: TAG in NSL, FL and BL, and FFA in SL. The amounts of BL and SL (or SL solely) increased, and those of FL (or NSL) decreased throughout breadmaking (i.e. wetting and mechanical work during dough preparation, fermentation and baking) – thus anticipating their effects in lipid interactions (mainly to starch granules and proteins). The SE content remained essentially unchanged along fermentation

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and baking; TAG decreased during dough mixing and fermentation, so those of DAG and MAG increased; and the effect of baking in NL fractions tended to be less apparent. TAG were the dominant NL, but considerable amounts of SE, DAG and FFA were also found. The GLC program used was able to separate carboxyl acids ranging from 4 to 24 carbon atoms, and proved adequate for fatty acid quantitation in cereals. The fatty acid composition varied according to the type of matrix (maize and rye flours, sourdough and broa), extract and NL fraction. Palmitic, oleic and linoleic acids were the major fatty acid in all food items. The nutritional value of broa was owing to dominance of poly‐ and monoenes, and of unsaturated fatty acids in general.

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RESUMO

PERFIL MICROBIOLÓGICO E LIPÍDICO DA BROA: CONTRIBUIÇÕES PARA A

CARACTERIZAÇÃO DE UM PÃO TRADICIONAL PORTUGUÊS

RESUMO ALARGADO

A broa é um pão fermentado tradicional, feito de farinhas de milho (Zea mays) e centeio (Secale cereale). Este produto é amplamente produzido ao nível rural no Norte de Portugal, seguindo processos ancestrais – incluindo o uso de uma porção de massa ácida da produção anterior como fermento. Devido ao importante papel desempenhado – do ponto de vista económico, social e cultural, o conhecimento científico neste campo será importante para melhorar a qualidade e expandir o mercado da broa. Neste sentido, amostras fornecidas por 14 produtores artesanais, seleccionados a partir de 4 sub‐regiões, foram analisadas em termos de identificação e quantificação microbiana, e bem assim de caracterização lipídica.

Em dois períodos do ano procedeu‐se a contagens de viáveis totais, bem como de microrganismos mesófilos e termófilos, bolores e leveduras, bacilos Gram‐, bacilos Gram+ formadores e não‐formadores de esporos, e cocos Gram+ catalase+ e catalase‐. A fermentação teve um efeito preponderante sobre os principais grupos de microrganismos: as contagens de viáveis de leveduras, lactobacilos, estreptococos, lactococos, enterococos e leuconostocs aumentaram, enquanto as de bolores, Enterobacteriaceae, Pseudomonadaceae, estafilococos e micrococos diminuíram, comparativamente às farinhas de milho e de centeio. Após fermentação, as leveduras e bactérias do ácido láctico (BAL) apareceram como dominantes; foi, pois, evidente o papel de fermentação sobre o controle natural de microrganismos indesejáveis. Estes resultados também revelaram a existência de uma microflora complexa e única – embora não pudesse ser discriminada por sub‐regiões ou estações do ano, nem por tipo de farinha. Após fermentação, as leveduras e as BAL foram os grupos dominantes de microrganismos. As espécies de leveduras mais frequentes foram Saccharomyces cerevisiae e Hansenula anomala, ao passo que as BAL mais frequentemente isoladas foram Leuconostoc spp. (heterofermentativos) e Lactobacillus spp. (homofermentativos); Lactobacillus brevis, L. curvatus e L. lactis ssp. lactis para o género de Lactobacillus; Lactococcus lactis ssp. lactis para os lactococos; Enterococcus casseliflavus, E. durans e E. faecium para os enterococos; e Streptococcus constellantus e S. equinus para e os streptococos.

O fermento ácido ou azedo (conhecido por crescente) usado no fabrico de broa pertence aos fermentos tipo I: geralmente, um dia antes da cozedura, a massa‐mãe (popularmente designada por “isco”) – i.e. um pedaço de massa fermentado proveniente do lote anterior, é misturado com farinha de milho e centeio, amassado com água e deixado a fermentar durante a noite. Além disso, a massa‐mãe pode ficar guardada vários dias ou semanas, de acordo com a frequência de produção de broa. Com base nestas considerações, foi seguida uma abordagem semelhante à anterior na tentativa de estudar o perfil microbiológico ao longo da fermentação e armazenamento da massa‐mãe: a massa foi preparada seguindo o protocolo tradicional, e mantida durante várias semanas sob temperatura e humidade relativa

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RESUMO

controladas. Os resultados indicaram o domínio nas contagens de viáveis de leveduras, Lactobacillus e Bacillus durante a maior parte do tempo, e até ao final do período de 39 dias, apesar da grande diversidade de microrganismos presentes no início. O pH diminuiu drasticamente nas primeiras 24 h – devido principalmente à acção das BAL. O efeito do arejamento na fermentação foi posteriormente estudado numa experiência independente durante 14 dias, tendo os resultados demonstrado a existência de diversas diferenças no perfil microbiológico. Finalmente, foi também estudado a evolução da microflora em farinhas de milho e centeio durante o seu armazenamento a duas temperaturas diferentes (4 e 20º C, durante 8 e 39 d, respectivamente) e numa massa‐mãe (a 4ºC, durante 6 d). Em geral, os resultados demonstraram a manutenção do seu perfil microbiológico, embora alterações mínimas mas importantes puderam ser detectadas.

Procedeu‐se à caracterização qualitativa e quantitativa exaustiva dos lípidos neutros (NL), por cromatografia líquida de alta resolução em fase normal com detecção por dispersão evaporativa de luz (NP‐HPLC‐ELSD) e ionização por electrodispersão com espectromeria de massa e fragmentação por impacto (ESI‐MS/MS), após aplicar dois métodos de extracção independentes – viz. originando lípidos não‐amiláceos (NSL) e amiláceos (SL), e lípidos livres (FL), ligantes (BL) e SL. Posteriormente, foram utilizados dois procedimentos de extracção em base sólida para separar os NL, antes de se proceder à derivatização independente dos esterol‐ ésteres (SE), triglicerídeos (TAG), ácidos‐gordos livres (FFA), esteróis (S) com diacilglicerídeos (DAG), e monoacilglicerídeos (MAG) – e de se passar à quantificação dos ácidos gordos por cromatografia líquida‐gasosa (GLC).

Os NL foram separados em relação à linha‐base, por NP‐HPLC‐ELSD, com duas colunas microbore de sílica gel de 3 µm em série, na seguinte ordem: SE, triglicerídeos de elevado peso molecular (HMW‐TAG), FFA, triglicerídeos de baixo peso molecular (HMW‐TAG), 1,3‐DAG, S, 1,2‐DAG e MAG. Todos os NL, à excepção dos MAG, eluíram a um fluxo (óptimo) de 0,1 mL/min, para um tempo total de corrida de 76 min. A separação completa (ou parcial) das espécies moleculares de NL permitiu a identificação de aductos de amónio por MS/MS, produzidos por ionização por electrodispersão; para além disso, o NP‐HPLC‐ESI‐MS e –MS2 demonstrou ser essencial na identificação de NL, e posterior quantificação por NP‐HPLC‐ELSD. Os cromatogramas iónicos e os espectros de massa – em especial os relativos aos fragmentos, foram indispensáveis para identificar correctamente os picos dos cromatogramas de extracção iónica (EIC) e dos cromatogramas de NP‐HPLC‐ELSD. Os 1,3‐DAG, S livres e 1,2(2,3)‐DAG foram separados em relação à linha de base. Os TAG de cadeia curta e média foram separados completamente, mas os TAG de cadeia longa apenas o foram parcialmente – sem que os TAG com o mesmo comprimento de cadeia, mas com diferentes graus de insaturação apenas puderam ser parcialmente separados; por seu turno, os FFA, DAG e MAG com comprimentos de cadeia distintos apenas puderam ser resolvidos numa extensão limitada.

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RESUMO

O efeito do processo de panificação sobre a extracção dos lípidos, e sobre o perfil de NL e ácidos gordos foi caracterizado através do estudo das matrizes de farinhas de milho e centeio, do fermento e da broa – isto é, as etapas representativas do processo de panificação. Os NSL e os FL contabilizaram a maior porção de lípidos extraídos. Os NL predominantes variaram de acordo com o extracto lipídico: TAG nos NSL, FL e BL, e FFA nos SL. As quantidades de BL e SL (ou apenas SL) aumentaram, enquanto as de FL (ou NSL) diminuíram ao longo do processo de panificação (ou seja, a adição de água e o trabalho mecânico exercido durante a preparação da massa panar, a fermentação e a cozedura) – antecipando, então, os seus efeitos sobre as interacções dos lípidos, principalmente as lípidos‐proteínas e lípidos‐amido. O conteúdo em SE permaneceu essencialmente constante durante a fermentação e a cozedura; os TAG diminuíram durante a mistura da massa e fermentação, pelo que os DAG e MAG aumentaram; o efeito da cozedura nas fracções de NL foram menos evidentes. Os TAG foram os NL dominantes, mas também se observou quantidades consideráveis de SE, DAG e FFA. O programa de GLC usado foi capaz de separar ácidos carboxílicos com 4 a 24 átomos de carbono, e demonstrou ser adequado para a quantificação de ácidos gordos em cereais. A composição em ácidos gordos variou de acordo com o tipo de matriz (farinha de milho e de centeio, fermento e broa), de extracto e de fracção de NL. Os ácidos gordos dominantes em todos os itens alimentares estudados foram o palmítico, o oleico e o linoléico. O valor nutricional da broa tornou‐se assim evidente, devido ao domínio de poli‐ e monoenos, a par da supremacia dos ácidos gordos insaturados em geral.

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ACKNOWLEDGEMENTS

One of the most encouraging things in life is that we always have the opportunity to get what we want, provided that first we know what we really want. Moreover, we must always aim at something to stay alive: that is what it is all about our short expedition on Earth. Whenever we reach a goal, a debit automatically falls into our life’s account: the duty of expressing gratitude. This is quite a pleasant experience for me, although being aware that “as we express our gratitude, we must never forget that the highest appreciation is not to utter words, but to live by them.” (John Fitzgerald Kennedy, 1917‐1963).

First of all, I would like to express all my gratitude to my supervisor, Prof. F. Xavier Malcata, whose expertise and endless understanding greatly contributed to my unique research experience and academic track. I appreciate his vast knowledge and skills, his comprehensive assistance in writing manuscripts and preparing the thesis, and all the support he consistently gave me towards the progress of my work. He will always be the paradigm of excellence for me – of a hard worker, talented, intelligent, imaginative, pragmatic, perseverant, motivated and successful instructor and researcher. What I have learned from him goes far beyond science; his teachings and the example of his professional career shaped my way of living life and hence my personality.

To Prof. Luísa Beirão, my many thanks in accepting to be my co‐supervisor, especially given the difficult circumstances backing up her decision – in a gesture very special to me. Also my thanks for all corrections and suggestions made on this thesis – which improved substantially its quality. Thank you to have raise questions never thought so far, and for her understanding when confronted with my tight deadlines.

Special thanks to Prof. Paavo Kalo, for all the motivation and encouragement, persistence, understanding and kindness shown during and after my stays in Helsinki. Alone – and certainly involved in terabytes of information from books and journals, I am sure it would have be impossible to do the type of experiments that were performed under his supervision. With his great involvement and teachings, my professional skills grew exponentially. He embraces my definition of a “truly investigator”, and of a devoted and passionate researcher and academic. He is the confirmation that research and knowledge go along and are to be used, argued, experienced and shared. My pleasure in doing research reached dangerous levels on such great time spent in “Latokartanonkaarl 11”. By the way, the book you gently presented me (“Skiing – technique, tactics, training”) did not bring me further practical improvement. To Prof. Velimatti Ollilainen, thanks for all the support given with the LC‐MS, and the great moments of joy together.

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ACKNOWLEDGEMENTS

During the PhD, I had the privilege to work in several other laboratories. Thank you to all the collaboration and knowledge shared: with Prof. Fernando Bernardo, from Faculty of Veterinary Medicine, Technical University of Lisbon [Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa]; Prof. Manuel António Coimbra, from Department of Chemistry, University of Aveiro [Departamento de Química, Universidade de Aveiro]; and Prof. Milton Costa, from Laboratory of Microbiology, Faculty of Science and Technology, University of Coimbra [Laboratório de Microbiologia da Faculdade de Ciências e Tecnologia, Universidade de Coimbra]. My gratitude to College of Biotechnology of Portuguese Catholic University [Escola Superior de Biotecnologia, Universidade Católica Portuguesa] where I spent most of my research, as well as to Centre of Biotechnology and Fine Chemistry [Centro de Biotecnologia e Química Fina] for the possibility to perform a substantial part of the experimental work. Finally, a special acknowledgement to Agronomy Superior Institute, Technical University of Lisbon [Instituto Superior de Agronomia, Universidade Técnica de Lisboa], and especially to the Chairman of the Direction Board, Prof. Carlos José Noéme, for promptly accepting me as PhD student, and the sympathy he demonstrated from the very first contact with my supervisor.

In all the aforementioned places, I also received the gift of knowing lovely people, with who I spent fantastic working and extra‐working moments, and with whom I could build‐up strong friendships – which had a great meaning for me, and clearly enriched my life. And because “without friends no one would choose to live, though he had all other goods” (Aristotle, 384‐ 322 BC), I would like to take this chance to thank all of them. A word of gratitude to students and professors with whom I worked, for all the experience shared. And a special thanks to all my lab and office friends from College of Biotechnology: we spent so many good moments together – and, much more important, we shared almost all kinds of moments and feelings.

I recognize that this research would not have been possible without the support of several members of the Regional Directorate of Agriculture of Entre‐Douro‐e‐Minho [Direcção Regional de Agricultura de Entre‐Douro‐e‐Minho] (DRAEDM) and many local farmers. I am grateful for their cooperation, and particularly to Eng. Alda Brás from DRAEDM. Financial support for me was provided by a Ph.D. fellowship within program PRAXIS XXI, ref. PRAXIS XXI/BD/16060/98, administered by Foundation for Science and technology [Fundação para a Ciência e a Tecnologia]. Partial financial support was received within program PAMAF – IED, administered by Ministry of Agriculture, Rural Development and Fisheries [Ministério da Agricultura, Desenvolvimento Rural e Pescas], through research grant “Corn bread: characterization of the traditional production process and technological improvement” [Pão de milho: caracterização do processo tradicional de produção e melhoramento tecnológico] (ref. 1022).

I would also like to express my heartfelt thanks and gratitude to my family, for their unconditional support made available all over my life. My family is the greatest gift of my existence – a friend to my soul, meaning of my life. My family taught me everything I really

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ACKNOWLEDGEMENTS

needed to know. Looking inside myself through a mystic mirror, I quietly face out with the magic image of each member.

To Catharina, my intimate companion and my lover. Her constant smile and generosity in sharing this goal with me were essential contributions and sources of motivation to pursuit this hard (but right) track. My warm thanks for all her care and enormous patience, especially in those moments when I was absent. I am convinced that many times she gave me much, not knowing that she gave at all. The most beautiful things in world cannot be told, or even written – they must be felt with our heart and soul. You make me an entirely happy man and husband.

To my mother and father in‐law, Lylia and Delmon, my thanks for the friendship, love and support given (even with an ocean of distance). Thanks for all the moments including those of Christmas celebration. These thanks for moments of enjoy are extended to Rafael, Isabela, Adriana and Sérgio.

To my charming grandfather, Pedro, with whom I daily learned how beautiful life can be –, who is the foundation of my moral inspiration, and for whom I always feel this huge and irrational pride. The most remarkable thing about him (“my good man”) is that as more as I look at him, to his life and personality – supported on love, honesty, humbleness, optimism and courage – the more morality and character I expect from our society! To my grandparents, Sebastião and Francelina – who I hardly knew, but whose love was always present: thank you for being mine.

To my dear sister, Paula – my friend provided by Nature, who always shared with me childhood memories and grown‐up dreams – and who is always present in the right moments and in the right places: “A friend loves at all times; and a brother is born for adversity” (17:17). The best thing about having a sister is that the strongest and best friend in life is guaranteed. I just do not understand why I always felt like her guardian, a kind of “El hidalgo et bravo caballero Don Quijote de la Mancha”, defending her against all the imaginary wind‐mills – even when she was 50 cm taller than me! Sisterhood – the “religion of sisterhood”, is a powerful thing: I can be boring and tedious and you still adore me, and I know that it is not just because I had to use all second‐hand things that previously belonged to you!

My gratitude to my brother‐in‐law, Mário, who I love as a brother. In fact, if it is true that I desired a brother in my childhood (“Mom, I want a brother and older than me!”), it is not less true that I got one twenty years later (a small delay of the stork!). The size of his heart is an inexhaustible source of pleasure and inspiration. His happiness is contagious. All our great moments of joy and laughing have been important sources of energy to me. I did not choose

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ACKNOWLEDGEMENTS

my sister’s husband – but she surely made great choice! A big hug to your parents, Cândida e Amílcar, as well as to other relatives João, Graça and Joana.

To my niece and goddaughter, Mariana, I deeply wish that her dreams always become true. My desire is that her life can be an enormous and coloured balloon of happiness and success. Her happiness and constant smile fully delights any heart. How I wish she lives forever in her “Country of Wonders”, and let me always be part of it. Indeed, everything we can learn with a child is one of the sweetest gifts given by Nature to human beings. And I cannot forget to thank her for the status of “daughter‐in‐law” that she awarded me: since, then I feel a respectable gentleman!

Last but far from least, I must acknowledge my parents, Domingos and Maria Teresa, with an infinite amount of tenderness; for their encouragement and friendship, unlimited patience and love, and instilling inspiration, faith and courage – the finest and the most vital lessons that they could teach me as parents. Thanks for being a driving‐force in pursuing my PhD, and for the family time I have used up with my work and professional concerns: without their unique love, encouragement and serenity, I would never finish this thesis. This diploma is much more yours than mine! I would even dare to say it is only yours! I doubt I will ever be able to fully convey my appreciation. On the contrary, I am sure that I will eternally owe you gratitude and love. I will be always grateful for you having given me such a rich and balanced life. They are my echo and my mirror, intonating and shining back to me with a world of possibilities. They are my unconditional partners. They are the ones who know when I smile even in a dark moon night. Here, I promise I will try to always be a witness of their great personality and kindness – although, to be realistic, this is a hard task! Because of their existence, I figured out that my first basic instinct is not survival, but you: I would give my own life for your survival!

In memory of my grandmother, Primorosa – my shining star, my aureole, my soft spring fragrance, my beautiful green valley, my ocean of inspiration. She was the one who gave me the opportunity to perceive how beautiful, glorious and astonishing some human beings can be, and to experience living in such humbleness in the entire life. We will eternally be one, and the same! We will be always standing close together and forever! She is still here, because part of me is her! With all my love.

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SYMBOLS, ACRONYMS AND ABBREVIATIONS

ACN:DB, Number of acyl group carbons:Number of double bonds; AcOH, Acetic acid; AD, Anno Domini; AOP, Appéllation d'origine protégée; APCI, Atmospheric pressure chemical ionization; apo AI, Apolipoprotein AI; apo B, Apolipoprotein B; apo E, apolipoprotein E; Ar, Argon; aw,

water‐activity; B, Broa (Bread); BC, Before Christ; BL, Bound lipid(s); BLIS, Bacteriocin‐like inhibitory substances; BSTFA, N,O‐bis(trimethylsily)trifluoroacetamide; C, Cholesterol; C10:0, Capric acid; C12:0, Lauric acid; C14:0, Myristic acid; C16:0, Palmitic acid; C18:0, Stearic acid; C18:1, Oleic acid; C18:2, Linoleic acid; C18:3, Linolenic acid; C20:4 (ω‐6), Arachidonic acid; C20:5 (ω‐3), eicosapentaenoic acid; C22:6 (ω‐3), Docosahexaenoic acid; C22:i, Eicosanoids; C4:0, Butyric acid; C6:0, Caproic acid; Catalase‐, Catalase‐negative; Catalase+, Catalase‐positive; CD, Coealiac disease; CE, Cholesterol ester(s); CFU, Colony‐forming units; CH2Cl2,

Dichloromethane; CHCl3, Chloroform; CHD, Coronary heart disease(s); CI, Chemical ionization;

CID, Collision‐induced decay; CO2, Carbon dioxide; DAG, Diacylglycerol(s); DF, Dietary fiber(s);

DGDG, digalactosyl diacylglycerol(s); DY, Dough yield; EFA, Essential fatty acid(s); EI, Electron impact; EIC, Extracted ion chromatogram(s); ELS, Evaporative light scattering; ELSD, Evaporative light scattering detector/detection; EPS, Exopolysaccharide(s); ESI, Electrospray ionization; EU, European Union; F, mixture of maize and rye flours; FA, Fatty acid(s); FAME, Fatty acid methyl ester(s); FAO, Food and Agriculture Organization of the United Nations; FFA, Free fatty acid(s); FI, Flow injection; FID, Flame ionization detector/detection; Fig., Figure; FL, Free lipid(s); FN, Falling number; FQ, Fermentation quotient; FT, Fourier transform; GC, Gas‐ liquid chromatography; GI, Glycaemic index; GL, Glycolipid(s); GLC, Gas‐liquid chromatography; GM, Genetically modified; GMP, Good Manufacturing Practices; Gram‐, Gram‐negative; Gram+, Gram‐positive; GRAS, Generally Regarded As Safe; HACCP, Hazard Analysis and Critical Control

Point(s); HCl, Hydrochloric acid; HDL, High‐density lipoprotein(s); HePS,

Heteropolysaccharide(s); HMW‐TAG, High‐molecular weight triacylglycerol(s); HoPS, Homopolysaccharide(s); HPLC, High‐performance liquid chromatography; ICR, Ion cyclotron resonance; IDL, intermediate‐density lipoproteins; II, Insulin index; INE, Instituto Nacional de Estatística; ISTD, Internal standard; LAB, Lactic acid bacteria; LC, Liquid chromatography; LDL, Low‐density lipoprotein(s); LMW‐TAG, Low‐molecular weight triacylglycerol(s); LOX, Lipoxygenase(s); LP, Lysophosphatide(s); Lp[a], Lipoprotein[a]; M, Maize flour; m/z, Mass to charge ratio; MAG, Monoacylglycerol(s); MALDI, Matrix‐assisted laser desorption ionization; MAV, Minimum acceptable value; MCT, Medium‐chain triacylglycerol(s); MCFA, Medium‐chain fatty acid(s); MeOH, Methanol; MGDG, Monogalactosyldiglycerol(s); mPN, Minimum probable number; MPN, Most probable number; MRV, Minimum recommended value; MS, Mass spectrum/spectra; MS2, MS/MS, Tandem mass spectrum/spectra; MS3, MS/MS/MS, Tandem mass spectrum/spectra; MTBE, Methyl‐tert‐butyl ether; MUFA, Monounsaturated fatty acid(s); N2, Nitrogen; NaOMe, Sodium methoxide; NaOMe/MeOH, Sodium methoxide in methanol;

NH3, Ammonia; NI, Negative ion; NIP, Non‐identified peak(s); NL, Neutral lipid(s)/Simple

lipid(s); NP, Normal‐phase; NSL, Non‐starch lipid(s); Oxidase‐, Oxidase‐negative; Oxidase+, Oxidase‐positive; PA, Phytic acid; PC, Phospatidylcholine; PCA, Principal component analysis (PC, Principal component); PhL, Phospholipid(s); PL, Polar lipid(s); PTFE, Polytetrafluoroethylene (Teflon); PUFA, Polyunsaturated fatty acid(s); R, Rye flour; rac, Racemic; RCF, Response correction factor(s); RI, Refractive index; RP, reversed phase; RRT, Relative retention time; Rs, Resolution of adjacent peaks; RS, Resistant starch; RT, Retention

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SYMBOLS, ACRONYMS AND ABBREVIATIONS

time; S, Free sterol(s); sat/SFA, Saturated fatty acid(s); SCFA, Short‐chain fatty acids; SE, Steryl/Sterol ester(s); SL, Starch lipid(s); SMAFA, Saturated and monounsaturated fatty acid(s);

sn, Stereospecifically number; So, Sourdough; SPE, Solid‐phase extraction; TAG,

Triacylglycerol(s); TIC, Total ion chromatogram; TL, Total lipid(s); TNL, Total neutral lipid(s); TTA, Total titratable acidity; uns/UFA, Unsaturated fatty acid(s); USA, United States of America; UV, Ultra‐violet; VLDL, Very low‐density lipoprotein(s); W, Wheat flour; WHO, World Health Organization; Wa, Water; WSB, Water‐saturated n‐butanol.

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SCOPE OF THESIS

This thesis is just the foundations for a scientifically‐based characterization of broa – which is believed will contribute to a better knowledge of this food product, to more rationally improve its manufacture process and to more effectively support health claims associated with its consumption – both of which will help expand the market niches for it, and consequently its economic value as a food commodity. This thesis focused on addressing the following questions: ‐ Which kind of microbial ecosystem is contributed by raw‐materials – i.e. in maize and rye flours, mother‐dough and sourdough, used for breadmaking of broa? ‐ How does the microbiological system behave throughout sourdough preparation and mother‐dough storage?

‐ Does microflora of maize and rye flours vary significantly during their storage period and are they sensitive to variations of temperature?

‐ Which kind of neutral lipids are present in broa and its raw‐materials?

‐ How does the neutral lipid profile change throughout breadmaking?

‐ How does the fatty acid composition change in NL of broa?

To date, no research work had specifically tackled these topics – yet the importance of this traditional specialty bread in the Portuguese economy fully justifies allocation of resources to elucidate the effects of processing upon the final product.

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SCOPE OF THESIS

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BRIEF LIST OF TOPICS

ABSTRACT ... ix KEYWORDS ... ix

RESUMO ... xi PALAVRAS‐CHAVE ... xi EXPANDED ABSTRACT ... xiii

RESUMO ALARGADO ... xvii ACKNOWLEDGEMENTS ... xxi SYMBOLS, ACRONYMS AND ABBREVIATIONS ... xxv SCOPE OF THESIS ... xxvii LIST OF FIGURES AND SCHEMES ... 41 LIST OF TABLES ... 51 PART ONE | INTRODUCTION |59 CHAPTER 1. HISTORY OF BREADMAKING AND BROA, A PORTUGUESE TRADITIONAL SOURDOUGH BREAD ... 63 CHAPTER 2. SOURDOUGH BREAD ... 109

PART TWO | CONTRIBUTIONS TO UNDERSTANDING OF THE MICROBIAL ECOLOGY IN BROA | 171

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BRIEF LIST OF TOPICS CHAPTER 3. TAXONOMIC IDENTIFICATION OF MICROORGANISMS IN FLOURS AND SOURDOUGH ... 175 CHAPTER 4. MICROBIOLOGICAL CHARACTERIZATION OF MAIZE AND RYE FLOURS, AND SOURDOUGH FOR THE MANUFACTURE OF BROA ... 209 CHAPTER 5. MICROBIOLOGICAL PROFILE OF FLOURS, AND MOTHER‐DOUGH THROUGHOUT STORAGE TIME ... 299 CHAPTER 6. MICROBIOLOGICAL PROFILE OF BROA SOURDOUGH THROUGHOUT TIME ... 349

PART THREE | CONTRIBUTIONS TO UNDERSTANDING OF THE LIPID PROFILE IN BROA | 405 CHAPTER 7. SEPARATION AND IDENTIFICATION OF NEUTRAL LIPIDS BY NORMAL PHASE HIGH‐ PERFORMANCE LIQUID CHROMATOGRAPHY WITH EVAPORATIVE LIGHT‐SCATTERING AND ELECTROSPRAY MASS SPECTROMETRY DETECTION ... 409 CHAPTER 8. IDENTIFICATION OF MOLECULAR SPECIES OF NEUTRAL LIPIDS BY NORMAL PHASE LIQUID CHROMATOGRAPHY‐POSITIVE ELECTROSPRAY TANDEM MASS SPECTROMETRY ... 465 CHAPTER 9. COMPOSITION AND CONTENTS OF NEUTRAL LIPID CLASSES IN FREE, BOUND AND STARCH LIPIDS VIA NORMAL PHASE HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHY ... 503 CHAPTER 10. COMPOSITION AND CONTENTS OF NEUTRAL LIPID CLASSES IN NON‐STARCH AND STARCH LIPIDS VIA NORMAL PHASE HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHY ... 539 CHAPTER 11. COMPOSITION AND CONTENTS OF FATTY ACIDS IN NEUTRAL LIPID CLASSES FROM FREE, BOUND AND STARCH EXTRACTS VIA GAS‐LIQUID CHROMATOGRAPHY ... 567 CHAPTER 12. COMPOSITION AND CONTENTS OF FATTY ACIDS IN NEUTRAL LIPID CLASSES FROM NON‐STARCH AND STARCH EXTRACTS VIA GAS‐LIQUID CHROMATOGRAPHY ... 641 PART FOUR | GENERAL CONCLUSIONS AND FUTURE WORK | 699

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EXPANDED LIST OF TOPICS

ABSTRACT ... ix KEYWORDS ... ix RESUMO ... xi PALAVRAS‐CHAVE ... xi EXPANDED ABSTRACT ... xiii RESUMO ALARGADO ... xvii ACKNOWLEDGEMENTS ... xxi SYMBOLS, ACRONYMS AND ABBREVIATIONS ... xxv SCOPE OF THESIS ... xxvii LIST OF FIGURES AND SCHEMES ... 41 LIST OF TABLES ... 51 PART ONE | INTRODUCTION | 59 CHAPTER 1. HISTORY OF BREADMAKING AND BROA, A PORTUGUESE TRADITIONAL SOURDOUGH BREAD ... 63 KEYWORDS ... 65 ABBREVIATIONS ... 65 1.1. HISTORICAL BACKGROUND OF BREAD ... 67 1.1.1. Egypt ... 68 1.1.2. Greece ... 70 1.1.3. Rome ... 72

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EXPANDED LIST OF TOPICS 1.1.4. FROM THE MIDDLE AGE TOWARD TO THE PRESENT ... 74 1.2. CONSUMPTION OF CEREALS AND BREAD ... 77 1.2.1. Cereals ... 77 1.2.2. Bread ... 78 1.3. CORN, RYE AND BROA ... 80 1.3.1. Corn ... 80 1.3.2. Rye ... 83 1.3.3. Broa ... 84 1.3.3.1. Broa and its assignments ... 85 1.3.3.2. Broa and its social, cultural and economical importance ... 86 1.3.4. Broa of Avintes ... 88 1.3.4.1. Historical references ... 88 1.3.4.2. The woman baker of Avintes ... 90 1.3.4.3. Broa of Avintes: in the sequence of past and in path of future ... 91 1.4. Breadmaking process of broa ... 91 REFERENCES ... 107 CHAPTER 2. SOURDOUGH BREAD ... 109 KEYWORDS ... 111 ABBREVIATIONS ... 111 2.1. INTRODUCTION ... 113 2.2. SOURDOUGH PROCESSES ... 114 2.3. SOURDOUGH MICROFLORA ... 118 2.4. ROLE OF SOURDOUGH ... 122 2.4.1. Technological effects ... 122 2.4.2. Nutritional value ... 123 2.4.3. Anti‐bacterial and anti‐fungal effects ... 139 2.4.4. Leavening ... 143 2.4.5. Bread flavor ... 144 2.5. LIPIDS AND BREADMAKING ... 148 2.5.1. Lipids and cereals ... 148 2.5.2. Technological aspects ... 150 2.5.3. Nutritional and health aspects ... 154 REFERENCES ... 163 PART TWO | CONTRIBUTIONS TO UNDERSTANDING OF THE MICROBIAL ECOLOGY IN BROA | 171 CHAPTER 3. TAXONOMIC IDENTIFICATION OF MICROORGANISMS IN FLOURS AND SOURDOUGH ... 175 ABSTRACT ... 177 KEYWORDS ... 178 ABBREVIATIONS ... 178

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EXPANDED LIST OF TOPICS 3.2. MATERIAL AND METHODS ... 179 3.2.1. Sampling ... 179 3.2.2. (Presumptive) Microbiological quantification... 180 3.2.3. Microbiological identification ... 180 3.3. RESULTS AND DISCUSSION ... 182 3.4. CONCLUSIONS ... 204 REFERENCES ... 205

CHAPTER 4. MICROBIOLOGICAL CHARACTERIZATION OF MAIZE AND RYE FLOURS, AND SOURDOUGH FOR THE MANUFACTURE OF BROA ... 209 ABSTRACT ... 211 KEYWORDS ... 212 ABBREVIATIONS ... 212 4.1. INTRODUCTION ... 213 4.1.1. Sourdough and breadmaking ... 213 4.1.2. General considerations on the culture media for microbiology ... 215 4.1.2.1. Tryptone soy agar (TSA) ... 215 4.1.2.2. Yeast extract dextrose chloramphenicol agar (YEDCA) ... 215 4.1.2.3. Rose‐Bengal chloramphenicol agar base (RBCAB) ... 216 4.1.2.4. Violet red bile dextrose agar (VRBDA) and MacConkey agar ... 217 4.1.2.5. Pseudomonas agar base (PAB) ... 218 4.1.2.6. Herellea agar (HA) ... 218 4.1.2.7. Alcaligenes nutrient agar yeast extract (ANAYES) ... 219 4.1.2.8. Bacillus cereus medium (BCM) ... 219 4.1.2.9. Trypticase soy yeast extract starch agar (TSYES) ... 220 4.1.2.10. Reinforced clostridial medium (RCM) ... 220 4.1.2.11. Lactobacillus de Man, Rogosa and Sharp agar (MRS) ... 221 4.1.2.12. Baird‐Parker medium base (BPM) ... 221 4.1.2.13. Fermentation medium (FM) ... 222 4.1.2.14. Plate count agar with furazolidone (PCAF) ... 224 4.1.2.15. M17 agar ... 224 4.1.2.16. Lactic streak agar (LSA) ... 225 4.1.2.17. Kenner faecal streptococcal agar (KFS) ... 226 4.1.2.18. Kanamycin esculin azide agar (KEAA) ... 226 4.1.2.19. Mayeux, Sandine and Elliker agar (MSE) ... 227 4.1.3. General considerations on bacteriological and chemical analyses of water ... 228 4.2. MATERIAL AND METHODS ... 233 4.2.1. Sampling and chemical for microbiological analysis ... 233 4.2.2. Breadmaking process ... 233 4.2.3. Culture media ... 234 4.2.4 (Presumptive) Microbiological enumeration ... 236 4.2.5. Sampling and chemicals for chemical analysis ... 237 4.2.6. Microbial and chemical analysis of breadmaking water ... 241 4.2.7. Statistical analyses ... 241 4.2.7.1. Effect of region, sample type and season upon microbiological profile ... 241 4.2.7.2. Effect of production level and flour type upon microbiological profile ... 242 4.2.7.3. Effect of breadmaking water samples upon microbiological and chemical profile ... 243 4.2.7.4. Principal component analyses of microbiological data ... 243

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EXPANDED LIST OF TOPICS 4.3. RESULTS AND DISCUSSION ... 244 4.3.1. Effect of region, sample type and season upon microbiological profile ... 244 4.3.1.1. Total microorganisms ... 244 4.3.1.1.1. Study within samples ... 244 4.3.1.1.2. Study within regions ... 259 4.3.1.1.3. Study within seasons ... 260 4.3.1.2. Yeasts and molds ... 260 4.3.1.2.1. Study within samples ... 260 4.3.1.2.2. Study within regions ... 261 4.3.1.2.3. Study within seasons ... 261 4.3.1.3. Gram‐ rods ... 262 4.3.1.3.1. Study within samples ... 263 4.3.1.3.2. Study within regions ... 264 4.3.1.3.3. Study within seasons ... 265 4.3.1.4. Gram+ rods ... 265 4.3.1.4.1. Study within samples ... 266 4.3.1.4.2. Study within regions ... 268 4.3.1.4.3. Study within seasons ... 268 4.3.1.5. Gram+ cocci ... 269 4.3.1.5.1. Study within samples ... 270 4.3.1.5.2. Study within regions ... 272 4.3.1.5.3. Study within seasons ... 273 4.3.2. Effect of production level upon microbiological profile ... 274 4.3.2.1. Total microorganisms ... 274 4.3.2.2. Yeasts and molds ... 274 4.3.2.3. Gram‐ rods ... 274 4.3.2.4. Gram+ rods ... 275 4.3.2.5. Gram+ cocci ... 275 4.3.3. Effect of flour type upon microbiological profile ... 276 4.3.3.1. Total microorganisms ... 276 4.3.3.2. Yeasts and molds ... 276 4.3.3.3. Gram‐ rods ... 276 4.3.3.4. Gram+ rods ... 276 4.3.3.5. Gram+ cocci ... 277 4.3.4. Principal component analyses of microbiological data ... 277 4.3.5. Chemical characteristics ... 280 4.3.5.1. Ash ... 280 4.3.5.2. Moisture ... 281 4.3.5.3. pH and total acidity ... 281 4.3.5.4. Buffering capacity ... 283 4.3.5.5. Lactic and acetic acids ... 283 4.3.5.6. Fermentation quotient (FQ) ... 284 4.3.6. Microbiological and chemical profile of breadmaking water ... 284 4.4. CONCLUSIONS ... 288 REFERENCES ... 292 CHAPTER 5. MICROBIOLOGICAL PROFILE OF FLOURS, AND MOTHER‐DOUGH THROUGHOUT STORAGE TIME ... 299

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EXPANDED LIST OF TOPICS ABSTRACT ... 301 KEYWORDS ... 302 ABBREVIATIONS ... 302 5.1. INTRODUCTION ... 303 5.1.1. Maize and rye flours ... 303 5.1.2. Flours and mother‐dough in broa ... 304 5.2. MATERIAL AND METHODS ... 304 5.2.1. Feedstocks and chemicals ... 304 5.2.2. Sampling procedures ... 305 5.2.3. Microbiological assays ... 305 5.2.4. Statistical analyses ... 307 5.3. RESULTS AND DISCUSSION ... 308 5.3.1. Microbiological profile of maize and rye flours stored at room temperature ... 308 5.3.2. Microbiological profile of maize and rye flours stored under refrigeration ... 338 5.3.3. Microbiological profile of mother‐dough stored under refrigeration ... 344 5.4. CONCLUSIONS ... 347 REFERENCES ... 348

CHAPTER 6. MICROBIOLOGICAL PROFILE OF BROA SOURDOUGH THROUGHOUT TIME ... 349 ABSTRACT ... 351 KEYWORDS ... 352 ABBREVIATIONS ... 352 6.1. INTRODUCTION ... 353 6.1.1. Sourdough in breadmaking ... 353 6.1.1.1. Applications of sourdough ... 353 6.1.1.2. Microbial viable counts in sourdoughs ... 354 6.1.1.3. Sourdough microflora ... 356 6.1.1.3.1. Lactic acid bacteria (LAB) ... 356 6.1.1.3.2. Yeasts ... 358 6.1.1.4. Microbiological interactions in sourdough ... 360 6.1.1.5. Impact of sourdough fermentation ... 360 6.1.2. Sourdough for broa production ... 367 6.2. MATERIAL AND METHODS ... 368 6.2.1. Traditional manufacture of sourdough ... 368 6.2.2. Sampling and (presumptive) microbiological enumeration ... 369 6.2.3. Statistical analyses ... 373 6.2.3.1. Effect of time ... 373 6.2.3.2. Effect of aeration ... 373 6.2.3.3. Multivariate analyses ... 373 6.3. RESULTS AND DISCUSSION ... 373 6.3.1. pH ... 374 6.3.2. Microbiological characteristics during sourdough fermentation ... 374 6.3.2.1. Total viable counts ... 374 6.3.2.2 Yeasts and molds ... 381 6.3.2.3. Gram‐ rods ... 381 6.3.2.4. Gram+ rods ... 382 6.3.2.5. Gram+ cocci ... 384 6.3.2.6. Multivariate analyses ... 385

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EXPANDED LIST OF TOPICS 6.3.3. Microbiological characteristics during sourdough fermentation with aeration ... 388 6.3.3.1. Total viable counts ... 388 6.3.3.2 Yeasts and molds ... 388 6.3.3.3. Gram‐ rods ... 389 6.3.3.4. Gram+ rods ... 396 6.3.3.5. Gram+ cocci ... 396 6.3.3.6. Multivariate analyses ... 397 6.3.4. Final considerations ... 397 6.4. CONCLUSIONS ... 398 REFERENCES ... 400

PART THREE | CONTRIBUTIONS TO UNDERSTANDING OF THE LIPID PROFILE IN BROA | 405 CHAPTER 7. SEPARATION AND IDENTIFICATION OF NEUTRAL LIPIDS BY NORMAL PHASE HIGH‐ PERFORMANCE LIQUID CHROMATOGRAPHY WITH EVAPORATIVE LIGHT‐SCATTERING AND ELECTROSPRAY MASS SPECTROMETRY DETECTION ... 409 ABSTRACT ... 411 KEYWORDS ... 412 ABBREVIATIONS ... 412 7.1. INTRODUCTION ... 413 7.2. MATERIALS AND METHODS ... 416 7.2.1. Materials ... 416 7.2.2. Sample preparation ... 417 7.3. CHROMATOGRAPHIC METHODS ... 418 7.3.1. High performance liquid chromatography (HPLC) ... 418 7.3.1.1. Equipment ... 418 7.3.1.2. Methods ... 418 7.3.2. High performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI‐MS) ... 420 7.3.2.1. Equipment ... 420 7.3.2.2. Methods ... 420 7.4. RESULTS AND DISCUSSION ... 421 7.4.1. Optimization of chromatographic method ... 422 7.4.1.1. Effects of column combination and flow rate ... 422 7.4.1.2. Effects of binary gradient and post‐run time ... 426 7.4.2. Applications of optimized NP‐HPLC‐ELSD to some lipid extracts ... 429 7.4.3. Analyses of standard mixtures and lipid extracts using the developed HPLC‐ELSD chromatographic method, complemented with ESI‐MS detection and ESI‐MS2 identification ... 439 7.4.3.1. Sterol esters (SE) ... 439 7.4.3.2. Triacylglycerols (TAG) ... 439 7.4.3.3. Separation of triacylglycerols (TAG) and free fatty acids (FFA) ... 440 7.4.3.4. Free fatty acids (FFA) ... 441 7.4.3.5. Diacylglycerols (DAG) ... 443 7.4.3.6. Separation of diacylglycerols (DAG) and free sterols (S) ... 446

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EXPANDED LIST OF TOPICS 7.4.3.8. Monoacylglycerols (MAG) ... 447 7.4.4. Applications of optimized chromatographic methods in analyses of simple lipids (NL) from maize flour free lipid extract ... 448 7.5. CONCLUSIONS ... 457 7.5.1. Identification of neutral lipid (NL) classes and molecular species in each lipid class ... 457 7.5.2. Separation of neutral lipid (NL) classes and molecular species in each lipid class ... 457 REFERENCES ... 461

CHAPTER 8. IDENTIFICATION OF MOLECULAR SPECIES OF NEUTRAL LIPIDS BY NORMAL PHASE LIQUID CHROMATOGRAPHY‐POSITIVE ELECTROSPRAY TANDEM MASS SPECTROMETRY ... 465 ABSTRACT ... 467 KEYWORDS ... 468 ABBREVIATIONS ... 468 8.1. INTRODUCTION ... 469 8.2. MATERIAL AND METHODS ... 470 8.2.1. Materials ... 470 8.2.1.1. Standards ... 470 8.2.1.2. Solvents ... 470 8.2.2. Liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS) ... 471 8.2.2.1. High performance liquid chromatography (HPLC) ... 471 8.2.2.2. HPLC with electrospray ionization mass spectrometry (LC‐ESI‐MS) ... 471 8.2.2.3. Flow injection electrospray ionization mass spectrometry (FI‐ESI‐MS) ... 472 8.2.2.4. Calibration ... 472 8.3. RESULTS AND DISCUSSION ... 472 8.3.1. Triacylglycerols (TAG) ... 472 8.3.2. Diacylglycerols (DAG) ... 476 8.3.3. Monoacylglycerols (MAG) ... 481 8.3.4. Sterol esters (SE) ... 486 8.3.5. Free sterols (S) ... 486 8.3.6. Free fatty acids (FFA) ... 486 8.3.7. Analysis of low erucic acid rapeseed oil lipids ... 488 8.4. CONCLUSIONS ... 493 8.4.1. Triacylglycerols (TAG) ... 493 8.4.2. Diacylglycerols (DAG) ... 494 8.4.3. Monoacylglycerols (MAG) ... 494 8.4.4. Fragmentation paths of acylglycerols (TAG, DAG and MAG) ... 495 8.4.5. Sterol esters (SE) and free sterols (S) ... 495 8.4.6. Free fatty acids (FFA) ... 496 8.4.7. Analysis of low erucic acid rapeseed oil ... 496 8.4.8. Significance of the results ... 498 REFERENCES ... 499

CHAPTER 9. COMPOSITION AND CONTENTS OF NEUTRAL LIPID CLASSES IN FREE, BOUND AND STARCH LIPIDS VIA NORMAL PHASE HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHY ... 503 ABSTRACT ... 505 KEYWORDS ... 506

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EXPANDED LIST OF TOPICS ABBREVIATIONS ... 506 9.1. INTRODUCTION ... 507 9.2. MATERIAL AND METHODS ... 508 9.2.1. Feedstocks and chemicals ... 508 9.2.2. Traditional manufacture of broa ... 509 9.2.3. Extraction of lipids ... 510 9.2.3.1. Specific extraction of free lipids (FL) ... 510 9.2.3.2. Specific extraction of bound lipids (BL) ... 511 9.2.3.3. Specific extraction of starch lipids (SL) ... 511 9.2.4. Purification of lipids ... 511 9.2.5. Purification of neutral lipids (NL) ... 512 9.2.6. Separation of neutral lipid (NL) classes ... 513 9.2.6.1 Preparation of calibration curves ... 514 9.3. RESULTS AND DISCUSSION ... 515 9.3.1. Purification of lipids ... 515 9.3.2. Extraction of lipids ... 517 9.3.3. Purification of neutral lipids (NL) ... 522 9.3.4. Separation of neutral lipid (NL) classes ... 525 9.4. CONCLUSIONS ... 535 REFERENCES ... 536

CHAPTER 10. COMPOSITION AND CONTENTS OF NEUTRAL LIPID CLASSES IN NON‐STARCH AND STARCH LIPIDS VIA NORMAL PHASE HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHY ... 539 ABSTRACT ... 541 KEYWORDS ... 542 ABBREVIATIONS ... 542 10.1. INTRODUCTION ... 543 10.2. MATERIAL AND METHODS ... 546 10.2.1. Feedstocks and chemicals ... 546 10.2.2. Extraction of lipids ... 547 10.2.2.1. Extraction of non‐starch lipids (NSL) ... 547 10.2.2.2. Extraction of starch lipids (SL) ... 547 10.2.3. Separation of lipids ... 548 10.2.4. Purification of neutral lipids (NL) ... 548 10.2.5. Separation of neutral lipid (NL) classes ... 549 10.2.6. Quantification of neutral lipid (NL) classes ... 549 10.3. RESULTS AND DISCUSSION ... 550 10.3.1. Extraction and separation of lipids ... 550 10.3.2. Purification of neutral lipids (NL) ... 552 10.3.3. Quantification of neutral lipid (NL) classes ... 553 10.4. CONCLUSIONS... 561 REFERENCES ... 563

CHAPTER 11. COMPOSITION AND CONTENTS OF FATTY ACIDS IN NEUTRAL LIPID CLASSES FROM FREE, BOUND AND STARCH EXTRACTS VIA GAS‐LIQUID CHROMATOGRAPHY ... 567 ABSTRACT ... 569