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Semelhante a Influência de bactérias produtoras de levana em pães de massa lêveda de trigo sarraceno
Semelhante a Influência de bactérias produtoras de levana em pães de massa lêveda de trigo sarraceno (20)
Influência de bactérias produtoras de levana em pães de massa lêveda de trigo sarraceno
- 1. Influência de bactérias do ácido acético produtoras de levana em pães de massa lêveda de trigo sarraceno 1° CICLO DE SEMINÁRIOS DE FERMENTADOS Profa. Dra.: Wilma Aparecida Spinosa Doutoranda: Natália N. Y. Hata Londrina, Abril de 2019 Universidade Estadual de Londrina Centro de Ciências Agrárias Pós-Graduação em Ciência de Alimentos
- 2. Q – A1
- 3. Introdução Fermento Ácidos e aromas Massa Lêveda Trigo Centeio Farinha e água BAL Levedura - Acidificação; - Proteólise; - Síntese de enzimas; - Compostos antifúngicos; - Exopolissacarídeos (EPS). - Sensorial; - Física; - Valor nutricional
- 4. Amido e Farinhas isentas de glúten - ↓ Qualidade Sensorial - ↓ Qualidade do atributo textura - ↓ Qualidade nutricional EPS Goma xantana Hidroxipropil metilcelulose - ↑ Qualidade - Livre de aditivos - Produtos clean-label (Rótulos limpos e livres de aditivos)
- 5. LEVANA Polímero de frutose Resíduos de frutose unidos por ligações β-(2,6) na cadeia principal e ligações β-(2,1) na cadeia lateral Resíduo de glicose ao término de sua cadeia (KIRTEL et al., 2017; SRIKANTH et al., 2015a). Fonte: Kirtel et al. (2017), modificado. Produção por microrganismos e plantas; Aprovada para uso em alimentos: Estados Unidos; União Européia; Japão; Coréia Austrália; Nova Zelândia.
- 6. SÍNTESE DA LEVANA • Aerobacter; • Azotobacter; • Bacillus; • Corynebacterium; • Erwinia; • Mycobacterium; • Pseudomonas; • Streptococcus; • Zymomonas; • Halomonas; • Lactobacillus; • Serratia; Bacillus subtilis natto Natto Muitas bactérias produzem levana por meio da ação da levanasacarase (LS) (EC 2.4.1.10) a partir de substrato a base de sacarose Fonte: https://www.pakutaso.com/20180132 010post-14735.html BAA: Acetobacter, Gluconobacter, Gluconacetobacter, Kozakia, Asaia e Neoasaia. Fonte: ATEŞ; ONER, 2017; IDOGAWA et al., 2014; JAKOB; STEGER; VOGEL, 2012; SRIKANTH et al., 2015a, SRIKANTH et al., 2015b; SEMJONOVS et al., 2015; UA-ARAK; JAKOB; VOGEL, 2017a).
- 7. PROPRIEDADES Massa molecular varia de 2 a 100 milhões de Dalton (Da) com Grau de Polimerização (GP) >100; Biocompatibilidade; Produz soluções de baixa viscosidade; Alta solubilidade em água; Insolúveis em solventes orgânicos (exc: DMSO); Resistentes a ação de amilases e invertase de leveduras; Alta força adesiva Maior que outros polissacarídeos “Adesivo verde”.
- 8. APLICAÇÕES Antiinflamatória; Anti-diabética; Hipocolesterolêmica; Imunoestimuladora; Agente anti-AIDS; Atividade anti-viral (Herpes, Vírus H5N1, adenovírus); Aumento da absorção do cálcio; Atividade antibacteriana contra patógenos de origem alimentar; Redução do peso; Atividade anti-oxidante; Atividade anti-tumoral. ÁREA MÉDICA/FARMACÊUTICA - Hidratante eficaz; - Anti-irritante; - Neutraliza a inflamação e clareia manchas hiperpigmentadas da pele; - Eficiente fixador de cabelo. ÁREA COSMÉTICA E CUIDADOS PESSOAIS - Prebiótico (vários estudos em animais, mas sem evidências conclusivas em humanos); - Ótimo substituto de gordura e da goma arábica. - Aplicação em produtos de panificação (Melhora da textura – maciez, aumento do volume); ÁREA DE ALIMENTOS Fonte: SRIKANTH et al., (2015b); DAHECH et al., (2011); BELGHITH et al. (2012); XU et al. (2006); KANG et al. (2009); ÖNER; HERNÁNDEZ; COMBIE, (2016); Byun; Lee; Mah, (2014).
- 9. Massa Lêveda Levana produzida in situ Trigo Centeio Lactobacillus reuteri L. sanfranciscensis BAL BAA Kozakia baliensis DSM 14400 Neoasaia chiangmaiensis NBRC 101099 - Redução da dureza do miolo; - Aumento da vida de prateleira; - Melhoramento no teor de umidade; - Aumento do volume do pão
- 10. Avaliar o potencial de BAA produtoras de levana na produção de massa lêveda utilizando o melaço como fonte natural de sacarose para a melhoria da qualidade de produtos GF. OBJETIVO Fonte: SHI et al. (2014)
- 11. MATERIAIS E MÉTODO Cultivo e Produção de Levana Gluconobacter (G.) albidus TMW 2.1191 Kozakia (K.) baliensis NBRC 16680 - 20 g/L gluconato de sódio; - 3 g/L extrato de levedura; - 2 g/L peptona de caseína; - 3 g/L glicerol; - 0,2 g/L MgSO4.7H2O; - 10 g/L manitol Meio NaG (pH 6.0) 30 ºC / 200 RPM Contagem inicial: 107 UFC/g de massa Gluconobacter (G.) albidus TMW 2.1191 Meio NaG (80 g/L sacarose) Erlenmeyer de 2 L contendo 300 mL de meio 30 ºC / 200 RPM / 32 h
- 12. MATERIAIS E MÉTODO Fermentação da massa lêveda 30 ºC / 200 RPM / até 72 h Erlenmeyer de 2 L contendo 300 g de massa Pré-cultura Fabricação de pães (24, 30 ou 48 hs) / estoque -20 ºC) Farinha de Trigo Sarraceno orgânico 35% de Melaço de cana (base-farinha) Massa lêveda CONTROLE (QA- quimicamente acidificado) - 20 mg/(g de farinha) de cloranfenicol; - 10 mg/(g de farinha) de eritromicina; - Acidificação (ácido acético, ácido lático e ácido glicônico).
- 13. MATERIAIS E MÉTODO Contagem de células, pH e identificação das cepas Massa lêveda BAL BAA Diluição seriada em salina peptonada 0,1% Placa - NaG Placa – MRS (Man, Rogosa and Sharpe medium) Fermentações de até 72 h Massa lêveda pH Tempos: - 0; - 6; - 24; - 30; - 48; - 54; - 72. BAA Morfologia da colônia BAL Levedura MALDI-TOF MS Identificação
- 14. MATERIAIS E MÉTODO Determinação de metabólitos Ácido acético Ácido lático HPLC - Coluna Rezex ROA (Phenomenex, USA); - Fase móvel: H2SO4 5 mN; - Fluxo: 0,7mL/min Açúcares Ácido Glucônico
- 15. Isolamento de levana a partir da massa lêveda ou do meio Massa lêveda Meio de fermentação 13000 RPM / 4 ºC / 30 min Centrifugação 2 volumes de etanol / overnight - 20 ºC Precipitação sobrenadante Centrifugação Liofilização Quantificação Meio de fermentação / Pesagem da levana liofilizada Hidrólise (Ácido perclorico (0.5%) / 5 h HPLC - Coluna Rezex RPM (Phenomenex, USA); - Fase móvel: H2Od; - Fluxo: 0,6mL/min Massa lêveda
- 16. Preparo e assamento do pão Pão CONTROLE - 100 g de farinha de trigo Sarraceno; - 100 g de água; - 2 g de sal; - 3 g de fermento de levedura seco Pão de massa lêveda - Massa lêveda de 24, 30 ou 48 h de fermentação (40% do peso total); - Farinha de trigo Sarraceno; - Água. Pão contendo levana isolada - 0,1 a 2,0 % (base-farinha) de levana liofilizada Homogeneização dos ingredientes Distribuição em formas (50g de massa) Resfriamento / 2h Repouso 30 ºC / 80% umidade / 45 min) 230 °C / 15 min ANÁLISES
- 17. Análise sensorial dos pães de massa lêveda Controle (24, 30 e 48h) Fatias de 15 mm Codificação com números de 3 dígitos Ex: 452; 125; 321 18 Avaliadores não treinados Teste afetivo - Cor; - Aroma; - Textura; - Sabor; - Aceitação geral. Pães de Massa lêveda (fermentada por G. albidus ou K. baliensis durante 24, 30 e 48h)
- 18. Análise instrumental dos pães (Volscan Profiler 300, Stable Micro Systems, UK) Scanner a base de laser Volume específico (mL/g) Dureza do miolo Texturômetro (TA.XT.plus, Stable Micro Systems - Cilindro probe de 20 mm; - Velocidade de 0,50 mm/s; - Fatia de 7 mm; - Força exercida (N).
- 19. Análise estrutural da levana produzida do BAA Massa lêveda Meio de fermentação Tamanho e massa molecular Asymmetric flow field-flow fractionation () coupled to multi-angle laser light scattering (MALS) UV detection (Dionex Ultimate 3000, Thermo Fisher Scientific, USA)
- 20. Análise estatística - Dados analisados por ANOVA; - Teste de Tukey para descrever médias a um nível de 5% de significância (p<0,05).
- 21. RESULTADOS E DISCUSSÃO Caracterização da massa lêveda fermentada por BAA G. albidus K. baliensis Crescimento mais lento de BAA Menor taxa de transferência de oxigênio no meio de fermentação Crescimento de BAL ácido- tolerantes
- 22. RESULTADOS E DISCUSSÃO Caracterização da massa lêveda fermentada por BAA BAL Pediococcus pentosaceus (70,4%) Weissella cibaria (28,7%) Não produzem levana G. albidus 14.85 ± 3.92 g/kg farinha (48h) K. baliensis10.96 ± 2.24 g/kg farinha (54h) • Máxima produção de levana por BAA (~15 g/ (kg farinha) para G. albidus (48 h) e 11 g/~(kg farinha) K. baliensis (54 h) Maiores que aquelas produzidas por BAL (2-6,56 g/kg far.) • Diminuição da concentração da levana: • Menor taxa de produção final da fase estaciónária • Hidrólise da levana lenavases Monômeros de frutose metabolismo catabólico fonte de açúcares
- 23. Caracterização da massa lêveda fermentada por BAA Massa lêveda de BAA algumas similaridades ao de BAL pH e [ácido lático] comparáveis às de BAL; Massa lêveda de BAA ácido glicônico adicional e maior [ácido acético] Acidificação Efeitos positivos e negativos propriedade e qualidade dos pães. Composições de ácidos orgânicos Pães GF
- 24. Análise das características dos pães de massa lêveda Coloração!!! Tamanho dos poros do miolo
- 25. Análise das características dos pães de massa lêveda G. albidus K. baliensis Todos os atributos Pães de massa lêveda de 24 e 30 h Cor e sabor Controle Amargo Sabor e textura Pães de massa lêveda de 48 h Ácido
- 26. Cor • Atribuído à adição do melaço fonte de sacarose para produção de levana Pães não foram reportados como doces; Aroma e sabor • Melhoria do aroma e sabor Ácidos orgânicos + reação de Maillard + compostos voláteis produzidos e do melaço; • Esta combinação Mascaramento do sabor amargo comum em pães de trigo sarraceno degradação enzimática da rutina (polifenol) – semente e farinha; Aroma e sabor • Amargor mascarados pela acidez dos pães (48h) textura densa e odor mais ácido comparado á outros; • Acidez resultado das altas concentrações de ácido acético e lático nas massas de 48h
- 27. Análise das características dos pães de massa lêveda
- 28. • Massa lêveda fermentada 24h condições favoráveis à atividade das leveduras (produção de CO2) Maior volume com relação à outros pães; • pH e [ácido orgânico] apropriados aumentar a atividade da levedura em pães melhor desenvolvimento da massa; • Aumento do Metabolismo da levedura e produção de CO2 açúcares residuais introduzidos por meio da massa contendo melaço 24 e 30h; • Altas [ácido acético] e [ácido lático] e baixo pH massa lêveda de 48h textura densa e amargor dos pães altas concentrações de ácidos inibição de crescimento e atividade da levedura.
- 29. • Diferentes tipos de ácidos atividade da levedura diferentemente; • Níveis de ácido lático e acético recomendados melhoria das propriedades físicas e sensoriais 90 - 175 mmol/(kg farinha) e 70 – 230 mmol/(kg farinha), respectivamente.
- 30. Influência da levana adicionada na qualidade dos pães de trigo sarraceno
- 31. Tamanho das levanas produzidas por G. albidus em massas de trigo sarraceno
- 32. • Mecanismo da levana na qualidade dos pães GF não são claros tamanho e massa molecular exopolissacarídeos de maior peso molecular influência na qualidade e taxa de endurecimento do pão; • Redução da dureza do miolo interação intramolecular da água com cada molécula de levana relação com maior volume do pão. • Redução do peso molecular maiores tempos de fermentação degradação parcial da levana in situ preferível menores tempos; • Parcial degradação da levana Instabilidade da levana a baixos pH’s ou pela atividade hidrolítica da levanasacarase ou levanase secretadas
- 33. CONCLUSÃO • Fermentação de massa lêveda por BAA Tipo de cepa; tempo de fermentação e concentração de ácidos orgânicos; pH; quantidade e estrutura da levana propriedades e características dos pães • Pães de qualidade balanço do tempo de fermentação (pH e [ácidos]) quantidade e tamanho e massa molecular da levana; • Produção de levana em grande quantidade e de alto peso molecular por BAA benéfico para Pães GF requerimento de oxigênio e forte acidificação desafio para produção em grande escala
- 34. CONCLUSÃO • Fermentação de massa lêveda por BAA Melhorou as características sensoriais e físicas; • Volume específico e a dureza do miolo ácidos orgânicos e pH; • Levana a 1% propriedades estruturais dos pães de trigo sarraceno.
- 35. OBRIGADA!!!
Notas do Editor
- Frutanas são homopolissacarídeos constituídos por resíduos de frutose unidos por ligações β-(2,6) e β-(2,1). Os dois tipos de frutanas mais amplamente distribuídos na natureza são a levana, ligada predominantemente por ligações β-(2,6), e a inulina, com ligações β-(2,1) (KANG et al., 2009; HAN, 1990). Levana é um polímero de frutose composto por resíduos de D-frutofuranosil unidos por ligações β-(2,6) na cadeia principal e ligações β-(2,1) na cadeia lateral, além de apresentar um resíduo de D-glicopiranosil ao término de sua cadeia (KIRTEL et al., 2017; SRIKANTH et al., 2015a). A levana é um biopolímero atóxico, biologicamente ativo, podendo ser produzido por uma ampla gama de microrganismos e por um limitado número de espécies de plantas vários estudos científicos têm comprovado a sua segurança por meio de testes mutagênicos e toxicológicos, assim permitindo a produção industrial de levana de grau alimentício por várias companhias, das quais podemos incluir a RealBiotechCo. (Chungnam, Coréia) e a Advance Co., Ltd (Tóquio, Japão). Atualmente, a levana é aprovada como um alimento e aditivo alimentar e comercializada com nome de ―Frutana‖ em vários países como os Estados Unidos, União Européia, Japão, Austrália e Nova Zelândia Suas aplicações potenciais na indústria de alimentos são de agente estabilizante, emulsificante, auxiliar de formulação, agente finalizador de superfície, agente encapsulante, osmorregulador, crioprotetor e como carreador de aromas e sabores (ATEŞ; ONER, 2017; HAN, 1990; WU; CHOU; SHIH, 2013). Outras companhias como a Montana Biotech e a Sugar Processing Research Institute também têm desenvolvido derivados de levana, que culminaram por estender ainda mais o campo de aplicação deste biopolímero, que vai desde um útil ingrediente para produtos de beleza até a sua utilização como agente anti-AIDS (ATEŞ; ONER, 2017; KANG et al., 2009).
- Frutanas são homopolissacarídeos constituídos por resíduos de frutose unidos por ligações β-(2,6) e β-(2,1). Os dois tipos de frutanas mais amplamente distribuídos na natureza são a levana, ligada predominantemente por ligações β-(2,6), e a inulina, com ligações β-(2,1) (KANG et al., 2009; HAN, 1990). Levana é um polímero de frutose composto por resíduos de D-frutofuranosil unidos por ligações β-(2,6) na cadeia principal e ligações β-(2,1) na cadeia lateral, além de apresentar um resíduo de D-glicopiranosil ao término de sua cadeia (KIRTEL et al., 2017; SRIKANTH et al., 2015a). A levana é um biopolímero atóxico, biologicamente ativo, podendo ser produzido por uma ampla gama de microrganismos e por um limitado número de espécies de plantas vários estudos científicos têm comprovado a sua segurança por meio de testes mutagênicos e toxicológicos, assim permitindo a produção industrial de levana de grau alimentício por várias companhias, das quais podemos incluir a RealBiotechCo. (Chungnam, Coréia) e a Advance Co., Ltd (Tóquio, Japão). Atualmente, a levana é aprovada como um alimento e aditivo alimentar e comercializada com nome de ―Frutana‖ em vários países como os Estados Unidos, União Européia, Japão, Austrália e Nova Zelândia Suas aplicações potenciais na indústria de alimentos são de agente estabilizante, emulsificante, auxiliar de formulação, agente finalizador de superfície, agente encapsulante, osmorregulador, crioprotetor e como carreador de aromas e sabores (ATEŞ; ONER, 2017; HAN, 1990; WU; CHOU; SHIH, 2013). Outras companhias como a Montana Biotech e a Sugar Processing Research Institute também têm desenvolvido derivados de levana, que culminaram por estender ainda mais o campo de aplicação deste biopolímero, que vai desde um útil ingrediente para produtos de beleza até a sua utilização como agente anti-AIDS (ATEŞ; ONER, 2017; KANG et al., 2009).
- In sourdough, levan is formed naturally in situ during a classical wheat/rye sourdough fermentation by Mixed results on the effect of in situ EPS on the quality of GF sourdough breads were shown by different studies, for example, Galle et al. (2012) reported the reduction in the crumb hardness and prolongation of the shelf-life of sorghum sourdough breads in the presence of in situ fructan by L. reuteri Y2, while no positive influence was found by levan from L. reuteri LTH5448 on sorghum breads (Schwab et al., 2008) or from L. sanfranciscensis on wheat breads (Kaditzky et al., 2008). One influence of EPS on the bread quality was the structure of the EPS particles, in which the EPS with higher molar mass resulted in the most improved moisture content, baking loss and crumb firmness of the buckwheat-rice breads, as shown by the work of Rühmkorf et al. (2012b), or the higher increase of loaf volume and more reduction of crumb hardness in the wheat breads by Jakob et al. (2012).
- Since all the sourdough and sourdough bread studies have been focusing only on the LAB The aim of this study was to evaluate the potential of levan-producing AAB on sourdough production using molasses as a natural source of sucrose for the improvement of GF bread quality toward the naturality and clean labeling approach
- Gluconobacter (G.) albidus TMW 2.1191 and Kozakia (K.) baliensis NBRC 16680 were cultivated aerobically in sodium gluconate (NaG) medium modified from Adachi et al. (1979) containing 20 g/L sodium gluconate, 3 g/L yeast extract, 2 g/L peptone from casein, 3 g/L glycerol, 0.2 g/L MgSO4$7H2O and 10 g/L mannitol (pH 6.0). For agar plates, 20 g/L agar was added. Pre-cultures in liquid NaG medium were grown to the mid-exponential growth phase at 30 C, 200 rpm and used as starter cultures for subsequent sourdough fermentations (initial cell count ca. 3 107 CFU/g dough). For the production of levan in laboratory medium, 300 mL of NaG medium containing 80 g/L sucrose in 2-L Erlenmeyer flask was cultivated with G. albidus for 32 h at 30 C, 200 rpm
- 2.2. Sourdough fermentations Sourdoughs (dough yield 350) were prepared with organic buckwheat flour (Bauck GmbH & Co. KG, Rosche, Germany), tap water and 35% (flour base) of sugarcane molasses (August T€opfer & Co., Hamburg, Germany) as described in Ua-Arak et al. (2016). The 300 g doughs in 2-L Erlenmeyer flasks were inoculated with precultures and incubated at 30 C, 200 rpm for up to 72 h. To prepare sourdoughs for bread makings, buckwheat doughs were fermented for 24, 30 or 48 h and stored at 20 C. Chemically acidified control doughs were prepared by adding 20 mg/g flour chloramphenicol and 10 mg/g flour erythromycin, and were acidified with 100% acetic acid, 90% D,L-lactic acid and 50% gluconic acid before incubation without inoculation. Different amounts of organic acids were added to obtain the concentrations similar to the acids formed in the real sourdoughs by G. albidus at 24, 30 and 48 h, respectively (see Results, Table 1). All fermentations were carried out in triplicate.
- 2.3. Cell counts, pH and strain identification The bacterial cell counts of AAB and LAB were determined as described earlier (Ua-Arak et al., 2016). Sourdoughs were serially diluted with 0.1% peptone-salt solution (1 g/L peptone from casein, 8.5 g/L NaCl, pH 7.0) and subsequently plated in duplicate on NaG agar plates containing 65 mg/L penicillin G to suppress the Grampositive bacteria. In the case of K. baliensis, sucrose (40 g/L) was also added in the NaG agar plates (NaGS) to improve the cell growth and increase the colony differentiation. For LAB counts, the modified de Man, Rogosa and Sharpe medium (mMRS, pH 6.2) (Stolz et al., 1995) containing 10 g/L maltose, 5 g/L glucose, 5 g/L fructose, 15 g/L agar and 3 g/L 2-phenyl ethanol to suppress the Gramnegative bacteria (Lilley and Brewer, 1953) was used. To monitor the changes during sourdough fermentations over 72 h, cell counts, with the limit of detection of 100 CFU/mL (ca. 68 CFU/g dough), and the dough pH were determined at 0, 6, 24, 30, 48, 54 and 72 h. For the strain identification, AAB could be differentiated visually due to their distinctive colony morphologies (pale-pink colonies for G. albidus or slimy-opaque colonies for K. baliensis due to sucrose addition to the NaG formulation). In addition, the matrix-assistedlaser- desorption-ionization-time-of-flight mass spectrometer, MALDI-TOF MS, (Microflex LT, Bruker Daltonics, Germany)was used to identify LAB and yeast strains isolated from the sourdoughs. After the colony counting, single colonies were randomly picked from agar plates (3 colonies per plate), smeared on the target and extracted as described in Kern et al. (2013). The unknown strains were identified by comparing the obtained mass spectra from each colony to the Bruker and our in-house databases.
- Acetic and lactic acids were determined by high pressure liquid chromatography (HPLC) as described earlier in Ua-Arak et al. (2016). In summary, samples were first deproteinized with 5% perchloric acid before analyzing in a Rezex ROA column (Phenomenex, USA) with 5 mN H2SO4 as a mobile phase at 0.7 mL/min. For sugar and gluconic acid analysis, the supernatants were heated at 80 C for 15 min to stop the enzymatic reactions, deproteinized and centrifuged at 13000g for 10 min to collect the clear extracts. Sucrose, D-fructose and D-glucose as well as D-gluconic acid were quantified in triplicate by enzymatic assay kits (Megazyme, Ireland) in a 96-well plate at 25 C using a microplate reader (SPECTRO Star Nano, BMG Labtech GmbH, Germany).
- 2.5. Isolation of levan from buckwheat doughs or medium Levan was isolated in duplicate from the sourdoughs or from culture medium by ethanol precipitation method according to Korakli et al. (2001). In summary, cells and flour particles were first removed by centrifugation at 13000g for 30 min at 4 C, then the polysaccharides were precipitated from the supernatant with two volumes of absolute ethanol (- 20 C) overnight at 4 C. After centrifugation, the precipitates were air-dried, re-dissolved in dH2O and dialyzed against dH2O before freeze drying for at least 24 h (FreezeZone 2.5 Plus, Labconco, USA). Levans produced in laboratory medium were directly weighed after freeze drying for quantification. For the levans produced in situ in sourdoughs, which contained both levan and water soluble polysaccharides from flour, the fructose concentrations in the freeze dried samples were first determined after acid hydrolysis with 0.5% perchloric acid for 5 h by HPLC, and the levan concentrations were later calculated based on the amount of fructose using levan from laboratory medium as a standard. Fructose determinationwas measured in duplicate on the Rezex RPM column (Phenomenex, USA) using dH2O as eluent with the flow rate of 0.6 mL/min (Rühmkorf et al., 2012a). Samples used for structural analysis (Section 2.9) were dissolved, dialyzed and freeze dried for the second time to improve the EPS purity.
- 2.6. Bread preparation and baking Control breads (DY 200) were prepared by mixing 100 g of buckwheat flour with 100 g of tap water, 2 g of salt and 3 g of instant dried yeast (Fermipan® Red, UK). To prepare sourdough breads, sourdoughs from 24, 30 or 48 h fermentation were added at 40% (total weight), while the amount of flour and water in the recipe were reduced to obtain the same concentrations as in the control breads. For breads containing isolated levan, 0.1e2 g of water were replaced by the freeze dried levan (0.1e2% flour base, respectively). Doughs were mixed with a hand mixer (450W Bosch, Germany) using speed no.1 for 10 s and speed no. 5 for 1 min and 50 s before distributing 50 g into mini aluminum trays. The doughs were proofed at 30 C, 80% humidity for 45 min and then baked at 230 C for 15 min in the oven (Wachtel Piccolo, Germany). Bread loaves were cooled for 2 h before analysis. Three bread loaves of each sample were made from one individual baking and three separated bakings were performed for each type of breads.
- 2.7. Sensory evaluation of sourdough breads Breads were sliced into 15-mm thickness, coded with 3-digit numbers and presented to the untrained panels (n ¼ 18) in randomized orders. An affective test was used to determine the acceptance of consumers on 5 attributes (color, aroma, texture, taste and overall acceptance) of the bread samples: C, 24, 30, 48 (control, sourdough breads of 24, 30 and 48 h fermented by G. albidus or K baliensis). A 9-point Hedonic scale was used for the attribute rating, ranging from dislike extremely (1) to like extremely (9).
- 2.8. Instrumental analysis of breads The analysis of bread volume and crumb hardness were modified from Konitzer et al. (2013). The specific volume [mL/g] of a bread loaf was measured in triplicate by a laser-based scanner (Volscan Profiler 300, Stable Micro Systems, UK). For the crumb hardness determination, the heel of the loaf of bread (10 mmthick) was first removed and then the bread was sliced into 15 mm thickness. Texture profile analysis (TPA) of the bread crumbs were performed by a texture analyzer (TA.XT.plus, Stable Micro Systems, UK) using a 20 mm diameter cylinder probe with a test speed of 0.50 mm/s. The force that the probe required to penetrate the bread slice to 7.0 mmwas recorded and displayed as crumb hardness [N]. Four slices/bread of a total 3 breads per sample were analyzed in one individual baking.
- 2.9. Structural analyses of levans from AAB After levan isolation and quantification, the levan samples produced by G. albidus in NaGS medium (Section 2.1) or in situ in the buckwheat sourdoughs (Section 2.2) were also characterized to compare the molecular sizes and masses of levans from different conditions. The structural analyses of levans were performed by asymmetric flow field-flow fractionation () coupled to multi-angle laser light scattering (MALS) (Dawn Heleos II, Wyatt Technology, Germany) and UV detection (Dionex Ultimate 3000, Thermo Fisher Scientific, USA) as described previously (Ua-Arak et al., 2016). The freeze dried levan from Section 2.5 was re-dissolved in dH2O to 0.1e0.33 g/L and was injected into the separation channel (100 mL) using 50mMNaNO3 (aq.) as an eluent. Data were analyzed by ASTRA 6.1 software (Wyatt Technology, Germany) using a sphere model (Jakob et al., 2013). The shown data are representatives of at least two measurements.
- 3.1. Characterization of buckwheat sourdoughs fermented by AAB Buckwheat doughs that were inoculated with G. albidus or K. baliensis were monitored over 72 h (Fig. 1). For both strains, the cell count of AAB increased from ca. 3 107 CFU/g dough to approximately 2 108 CFU/g dough within 24 h and slightly reduced to 106e107 at 72 h. On the contrary, there was an increasing number of LAB from initially around 102 CFU/g dough to 107e108 at 24 h and remained in a relatively higher concentration than the AAB counts. The pH of sourdoughs reduced gradually from around 6 to the end pH at 72 h of 3.64 ± 0.09 (G. albidus) and 3.80 ± 0.03 (K. baliensis), respectively. The majority of LAB isolated from the sourdoughs were identified to be Pediococcus pentosaceus (70.4%) andWeissella cibaria (28.7%). Further HPLC analysis of these strains on mMRS medium or buckwheat dough containing sucrose did not reveal any levan or other EPS production (data not shown), confirming that AAB strains were the only EPS-producers in the sourdoughs. The amounts of in situ levan produced by AAB during the dough fermentations were different between the two strains. G. albidus produced more levan than K. baliensis throughout the fermentation process, having the highest concentrations of 14.85 ± 3.92 g/kg flour in G. albidus doughs at 48 h and 10.96 ± 2.24 g/kg flour in K. baliensis doughs at 54 h, respectively(Fig. 2). The lower growth of AAB was consequently followed by a better growth of the acid-tolerant LAB strains, which co-existed in the buckwheat flour ingredient (Moroni et al., 2011). The lower oxygen availability in the larger flasks
- 3.1. Characterization of buckwheat sourdoughs fermented by AAB Buckwheat doughs that were inoculated with G. albidus or K. baliensis were monitored over 72 h (Fig. 1). For both strains, the cell count of AAB increased from ca. 3 107 CFU/g dough to approximately 2 108 CFU/g dough within 24 h and slightly reduced to 106e107 at 72 h. On the contrary, there was an increasing number of LAB from initially around 102 CFU/g dough to 107e108 at 24 h and remained in a relatively higher concentration than the AAB counts. The pH of sourdoughs reduced gradually from around 6 to the end pH at 72 h of 3.64 ± 0.09 (G. albidus) and 3.80 ± 0.03 (K. baliensis), respectively. The majority of LAB isolated from the sourdoughs were identified to be Pediococcus pentosaceus (70.4%) andWeissella cibaria (28.7%). Further HPLC analysis of these strains on mMRS medium or buckwheat dough containing sucrose did not reveal any levan or other EPS production (data not shown), confirming that AAB strains were the only EPS-producers in the sourdoughs. The amounts of in situ levan produced by AAB during the dough fermentations were different between the two strains. G. albidus produced more levan than K. baliensis throughout the fermentation process, having the highest concentrations of 14.85 ± 3.92 g/kg flour in G. albidus doughs at 48 h and 10.96 ± 2.24 g/kg flour in K. baliensis doughs at 54 h, respectively(Fig. 2). the maximum amount of levan produced by AAB strains in buckwheat sourdoughs were still higher than those produced by LAB in other studies, being ca. 15 g/ kg flour in G. albidus doughs at 48 h and 11 g/kg flour K. baliensis doughs at 54 h, compared to around 2e6.56 g/kg flour in LAB sourdoughs The decreasing levan concentration was probably due to the slower production rate when the cells were in the late stationary phase, together with the simultaneous enzymatic hydrolyses of levan by extracellular levanases (Menendez et al., 2002) or the levansucrases itself (Kim et al., 1998; Mendez- Lorenzo et al., 2015). Additionally, the released fructose monomers from levan hydrolysis might be used in the catabolic metabolism of the cells when the other available sugar sources were depleted.
- 3.1. Characterization of buckwheat sourdoughs fermented by AAB Buckwheat doughs that were inoculated with G. albidus or K. baliensis were monitored over 72 h (Fig. 1). For both strains, the cell count of AAB increased from ca. 3 107 CFU/g dough to approximately 2 108 CFU/g dough within 24 h and slightly reduced to 106e107 at 72 h. On the contrary, there was an increasing number of LAB from initially around 102 CFU/g dough to 107e108 at 24 h and remained in a relatively higher concentration than the AAB counts. The pH of sourdoughs reduced gradually from around 6 to the end pH at 72 h of 3.64 ± 0.09 (G. albidus) and 3.80 ± 0.03 (K. baliensis), respectively. The majority of LAB isolated from the sourdoughs were identified to be Pediococcus pentosaceus (70.4%) andWeissella cibaria (28.7%). Further HPLC analysis of these strains on mMRS medium or buckwheat dough containing sucrose did not reveal any levan or other EPS production (data not shown), confirming that AAB strains were the only EPS-producers in the sourdoughs. The amounts of in situ levan produced by AAB during the dough fermentations were different between the two strains. G. albidus produced more levan than K. baliensis throughout the fermentation process, having the highest concentrations of 14.85 ± 3.92 g/kg flour in G. albidus doughs at 48 h and 10.96 ± 2.24 g/kg flour in K. baliensis doughs at 54 h, respectively(Fig. 2). The analysis of sourdoughs from AAB used in the bread baking revealed some similarities to the conventional sourdoughs from LAB reported in other studies. The pH and lactic acid concentrations were comparable to those from the traditional LAB strains, while the AAB sourdoughs contained additional gluconic acid as well as higher concentration of acetic acid, especially in 30 and 48 h doughs. For example, the wheat sourdough of the levan-producing L. sanfranciscensis TMW 1.392 had a pH of 3.5 and contained 142.05 mmol/kg flour of lactate and 55.34 mmol/kg flour of acetate after 22 h of fermentation (Kaditzky et al., 2008), while the sorghum sourdough of the levan-producing L. reuteri LTH5448 had a pH of 3.8 and contained 214 mM/g lactate and 121 mM/g acetate, respectively (Schwab et al., 2008). Since acidification has been reported for its positive (Ronda et al., 2014; Villanueva et al., 2015) and negative (Kaditzky et al., 2008) effects on the dough properties and bread quality, the different organic acid compositions in sourdoughs should generally be critically considered during GF bread development.
- 3.2. Analyses of sourdough bread characteristics Fig. 3 displays the appearance of the buckwheat sourdough breads made from the sourdoughs of G. albidus (top) and K. baliensis (bottom) at three different time points compared to the plain buckwheat breads (control). Sourdough breads had brown or lightbrown color, and were relatively different in their bread sizes and crumb pores. In both strains, sourdough breads from 24 h had slightly larger pore size than the control, while sourdough breads from 48 h had finer pore size and denser crumb than the others.
- For the sensory evaluation of breads, an affective test was performed on the sourdough breads of each AAB strain compared to the control. The 9-points Hedonic scale was used to rate the preference of consumers towards five attributes of breads (color, aroma, texture, taste and overall acceptance); and the averaged values (n ¼ 18) of each bread sample were presented in the spider diagrams in Fig. 4. The differences in the sensory quality of sourdough breads, especially at 24 and 30 h, and the control could be clearly observed. The sourdough breads of G. albidus at 24 and 30 h were significantly more accepted (p < 0.05) in the color, taste and overall acceptance than the control and 48 h sourdough breads; while the color, aroma, taste and overall acceptance of sourdough breads of K. baliensis at 24 and 30 h had significantly higher preference to the control and 48 h sourdough breads. The average ratings in all attributes of 24 and 30 h sourdough breads in both strains were in the range of 6e7, which corresponded to the “like slightly” to “like moderately” category scales. On the other hand, the ratings of the control breads, especially on the color and taste attributes, were around 3 to 4, which corresponded to the “dislike slightly” to “dislike moderately” scales. Similar to the control breads, the sourdough breads at 48 h in both strains were less preferred especially on the texture and taste attributes, in which the breads were reported to be too sour, while the control breads were too bitter. The sensory evaluations of sourdough and control breads demonstrated that the sensory quality of buckwheat breads could be significantly improved with an addition of sourdoughs made from AAB fermentation at 24 and 30 h, in particular on the taste attribute.
- The improved aroma and taste in buckwheat breads could be from the combination of organic acids, Maillard reaction, possible volatile compounds production by AAB and LAB (Czerny and Schieberle, 2002) as well as the molasses itself. These combinations masked the bitter taste normally encountered in the buckwheat breads (Campo et al., 2016; Rozylo et al., 2015; Torbica et al., 2010), which resulted from the enzymatic degradation of rutin, a polyphenol that existed in a high concentration in buckwheat seeds and flour (Costantini et al., 2014; Suzuki et al., 2015). This bitterness in buckwheat breads were also masked by sour taste in the breads from 48 h sourdoughs, which had also very dense texture and sour smell compared to the others. The sour taste in breads most likely resulted from the high concentrations of acetic and lactic acids in the sourdoughs at longer fermentation time (48 h).
- In addition to the sensory evaluation, the physical properties related to the quality of sourdough breads were also determined, including the loaf specific volume and crumb hardness. In order to observe the influence of organic acids produced by the bacteria during the fermentation on the bread characteristics, the chemically acidified (C.A.) breads prepared with buckwheat molasses doughs containing acids at the concentrations similar to sourdoughs at 24, 30 and 48 h were prepared and compared. The specific volume and crumb hardness of C.A. breads and sourdough breads of G. albiensis and K. baliensis at 24, 30 and 48 h were displayed in Fig. 5. Compared to the control breads (C), similar results were observed in the sourdough breads and the C.A. breads. The sourdough breads (G. albidus and K. baliensis) and C.A. breads of 24 h had significantly higher specific loaf volume and lower crumb hardness than the controls, while all breads from 48 h were the smallest and had the hardness crumbs (p < 0.001). No significant difference in the bread volume and hardness was observed between control and breads from 30 h.
- Sourdough fermentation by AAB at 24 h apparently presented the condition favorable to the yeast activity during the proofing process, as seen by the higher loaf volume of sourdough breads and C.A. breads at 24 h than other breads. The appropriate pH and amounts of organic acids in the sourdoughs could increase the baker’s yeast activity (higher CO2 release in shorter times), which subsequently resulted in a better dough development (Moroni et al., 2012). For example, up to 1.0% of acetic acid was found to stimulate S. cerevisiae under appropriate conditions (Garay-Arroyo et al., 2004; Pampulha and Loureiro-Dias, 2000; Taherzadeh et al., 1997). In addition, the yeast metabolism and CO2 production might be increased due to the presence of residual sugars (Galle et al., 2012), which were introduced by the addition of sourdoughs containing molasses (and remaining sugars) especially from 24 and 30 h. On the other hand, the high concentrations of acetic and lactic acids as well as very low pH in the sourdoughs at 48 h were responsible for the sourness and dense texture (small volume, hard crumbs) of breads, since too high acid concentrations negatively affected the yeast growth and activity
- Interestingly, different types of acids seemed to affect the yeast activity differently. Barber et al. (1992) reported a stronger reduction in loaf volume with acetic acid addition (dough pH 4.3) than with lactic acid (dough pH 4.1), indicating that the type of acids had more effects on the activity of baker’s yeast than the pH value
- 3.3. Influence of added levan on the buckwheat bread quality To observe the sole effects of levan on the properties of buckwheat breads, isolated levan produced by G. albidus was added in the baking recipe from 0.1 to 2% (flour base). The specific loaf volume and crumb hardness of the respective breads were illustrated in boxplots in Fig. 6. An addition of 1% isolated levan increased the specific volume of buckwheat breads significantly, from 1.993 ± 0.087 g/mL in the control breads to 2.054 ± 0.060 g/mL, but higher addition at 2% did not significantly improve the bread volumes. Gradual decreases of crumb hardness were observed when increasing amounts of isolated levan were used, in which the breads with 1 and 2% of isolated levan were significantly softer than the control breads. The addition of isolated levan in the buckwheat breads at an appropriate concentration could improve the quality of the breads both in the size and softness. From the results, 1% addition of isolated levan seemed to be a suitable amount to be used in this tested recipe.
- 3.4. Size of levans produced by G. albidus in buckwheat doughs In order to relate the influence of isolated levan on buckwheat breads to the in situ levan-containing sourdough breads, the molecular sizes and masses of levans produced by G. albidus were compared among each other. Table 2 shows the number average and weight average of geometric radius (Rn geo, Rw geo) and molar mass (Mn, Mw) of the in situ produced levans isolated from the sourdoughs used in the baking of section 3.2, in comparison to the radius and mass of isolated levan used in section 3.3. The molar size (Rw geo) of levans produced by G. albidus in the sourdoughs decreased from around 168 nm at 24 h to around 108 nm at 48 h, while the estimated molar mass (Mw) reduced from around 438 MDae30 MDa, respectively. The geometric radius of levan produced in the chemical medium (NaGS) was similar to those in the 48 h sourdoughs, while the molecular weight was slightly higher. Fig. 7 shows the distributions of the geometric radius and molar mass of in situ levans from sourdoughs at different time points and the levan from laboratory medium, in which the gradual reductions in the size and mass of levan particles during the fermentation process could be observed. All samples had similar distributions of molecular size and weight, in which the polydispersity index (Mw/Mn) of these levans were in the range from 1.131 to 1.161. From the above results, the isolated levan used in the baking of section 3.3 had the molar size and mass comparable to the in situ levan produced in the buckwheat sourdoughs at 48 h.
- The decrease in molecular weights of levan particles at longer fermentation time (Section 3.4) indicated a partial degradation of in situ levan during the fermentation, suggesting that shorter fermentation time would be preferred to the longer one. This partial degradation of EPS could be either explained by the instability of levan at lower pH (Runyon et al., 2014) or by the exo-hydrolytic activities of levansucrases or levanases secreted by AAB
- In conclusion, several factors such as strains, fermentation time, type and concentration of organic acids, pH, as well as amount and structure of levan were involved during the unconventional GF sourdough fermentation by selected AAB strains, affecting the sourdough properties, the proofing process and subsequently the sourdough breads characteristics. To successfully obtain a significantly improved bread quality, it is very important to have the right balance between the time of fermentation (consequently, the pH and organic acid concentrations) as well as the effective amount and molecular size and mass of levan. The ability to produce large quantity and high molecular weight levan by AAB is still very attractive and can be beneficial to the GF bread development, nevertheless, the high oxygen requirement for growth during sourdough fermentation as well as the strong acidification can be a challenge for a large scale production. This work demonstrated the effect of AAB strains on the sourdough fermentation and their respective GF breads, which were improved in sensory and physical quality. The specific volume and crumb hardness were mainly influenced by organic acids and pH, while a significant positive influence of levan on the structural properties of buckwheat breads could solely be proven after addition of isolated levan at 1% flour base.
- This work demonstrated the effect of AAB strains on the sourdough fermentation and their respective GF breads, which were improved in sensory and physical quality. The specific volume and crumb hardness were mainly influenced by organic acids and pH, while a significant positive influence of levan on the structural properties of buckwheat breads could solely be proven after addition of isolated levan at 1% flour base.