Accepted Manuscript Use of sourdough made with quinoa (Chenopodium quinoa) flour and autochthonous selected lactic acid bacteria for enhancing the nutritional, textural and sensory features of white bread Carlo Giuseppe Rizzello, Anna Lorusso, Marco Montemurro, Marco Gobbetti PII:
S0740-0020(15)00253-1
DOI:
10.1016/j.fm.2015.11.018
Reference:
YFMIC 2499
To appear in:
Food Microbiology
Received Date: 16 August 2015 Revised Date:
6 November 2015
Accepted Date: 27 November 2015
Please cite this article as: Rizzello, C.G., Lorusso, A., Montemurro, M., Gobbetti, M., Use of sourdough made with quinoa (Chenopodium quinoa) flour and autochthonous selected lactic acid bacteria for enhancing the nutritional, textural and sensory features of white bread, Food Microbiology (2015), doi: 10.1016/j.fm.2015.11.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Use of sourdough made with quinoa (Chenopodium quinoa) flour and
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autochthonous selected lactic acid bacteria for enhancing the nutritional,
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textural and sensory features of white bread
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Carlo Giuseppe Rizzello*, Anna Lorusso, Marco Montemurro, Marco Gobbetti
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Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70126 Bari, Italy
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*Corresponding author. Tel.: +39 0805442950; Fax: +390805442911.
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E-mail address: [email protected]
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Abbreviations
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IDF: insoluble dietary fiber; SDF: soluble dietary fiber; DY: dough yield; TTA: Total titratable
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acidity; WSE: Water/salt-soluble extract; FAA: free amino acids; QS: quinoa sourdough; QD:
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quinoa dough; FQ: fermentation quotient; ME:
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digestibility; QSB: quinoa sourdough bread; WB: wheat bread; QB: quinoa bread; TPA: Texture
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Profile Analysis; CS: Chemical Score; EAA: essential amino acid; EAAI: Essential Amino Acids
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Index; PER: Protein Efficiency Ratio; NI:Nutritional Index; HI: hydrolysis index; GI: glycemic
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index; QF: quinoa flour.
methanolic extract; IVPD: in vitro protein
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Abstract
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Lactic acid bacteria were isolated and identified from quinoa flour, spontaneously fermented
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quinoa dough, and type I quinoa sourdough. Strains were further selected based on acidification
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and proteolytic activities. Selected Lactobacillus plantarum T6B10 and Lactobacillus rossiae
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T0A16 were used as mixed starter to get quinoa sourdough. Compared to non-fermented flour,
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organic acids, free amino acids, soluble fibers, total phenols, phytase and antioxidant activities,
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and in vitro protein digestibility markedly increased during fermentation. A wheat bread was made
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using 20% (w/w) of quinoa sourdough, and compared to baker’s yeast wheat breads manufactured
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with or without quinoa flour. The use of quinoa sourdough improved the chemical, textural, and
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sensory features of wheat bread, showing better performances compared to the use of quinoa flour.
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Protein digestibility and quality, and the rate of starch hydrolysis were also nutritional features that
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markedly improved using quinoa sourdough as an ingredient.
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This study exploited the potential of quinoa flour through sourdough fermentation. A number of
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advantages encouraged the manufacture of novel and healthy leavened baked goods.
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Keywords
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Quinoa, Lactic acid bacteria, Sourdough, Bread
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1. Introduction
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Quinoa (Chenopodium quinoa Willd.) is a seed crop, which is traditionally cultivated in the
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Andean region since thousands of years. Commonly, quinoa grains and flour are used for human
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consumption and animal feeding. Quinoa has a very elevated genetic variability, which makes
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possible to select, adapt and breed cultivars for a wide range of environmental conditions (Bertero
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et al., 2004). Indeed, quinoa has the capacity of adapting to a range of agro-ecological conditions,
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showing tolerance to frost, salinity and drought, and having the potential to grow on marginal
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soils. These features, together with an undoubtedly nutritional value, determine a worldwide
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interest for this crop (Stikic et al., 2012). During the last years, the production of quinoa markedly
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increased, thus emphasizing the opportunity to cultivate this crop in various regions. Diverse
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climatic regions of USA, Canada, India, England, Denmark, Greece, Italy and other European
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countries were shown to be suitable for an extended cultivation (Stikic et al., 2012). FAO selected
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the quinoa as one of the crops that are destined to offer food security in the 21st century (Jacobsen
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et al., 2003).
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The high nutritional value of quinoa seeds is mainly due to high concentrations of proteins,
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minerals, and vitamins (Fleming and Galwey, 1995). Proteins are rich in amino acids like lysine,
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threonine and methionine, which, on the contrary, are deficient in cereals. A special interest was
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deserved to the use of quinoa for people who are affected by celiac disease. Quinoa was
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considered an efficient nutritional alternative to gluten-containing wheat, rye and barley (Jacobsen
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et al., 2003), being largely used as an ingredient for making breads, biscuits, cookies, crepes,
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muffins, pancakes and tortillas. While the nutritional value and the chemical composition of
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quinoa were characterized, several aspects concerning the technological applications have
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received less attention (Stikic et al., 2012). Overall, the baking quality is considered rather low due
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to the absence of gluten, (Stikic et al., 2012) and flavor, texture and appearance of baked goods,
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including quinoa in the recipes, were reported only as moderately acceptable (Stikic et al., 2012).
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ACCEPTED MANUSCRIPT Sourdough fermentation has the potential to exploit the technological, nutritional, functional and
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sensory features of wheat and non-wheat flours. Besides the well-known advantages documented
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for processing wheat and rye, an abundant literature showed how the sourdough may enhance
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various features of milling byproducts (Coda et al., 2015a; Rizzello et al., 2010a; Rizzello et al.,
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2010b), minor cereals (Coda et al., 2011a; Coda et al., 2010a), legumes (Curiel et al., 2015;
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Rizzello et al., 2014a), teff, and buckwheat (Coda et al., 2014). In particular, sourdough
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fermentation improves dough workability, bread structure and organoleptic and nutritional
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properties of raw flours. Furthermore, it increases the content of biogenic compounds and the
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uptake of minerals, and decreases the level of anti-nutritional factors and the value of the
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glycaemic response (Gobbetti et al., 2014).
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Nevertheless, fermentation processes depend on specific determinants, which have to be strictly
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controlled to get standardized and agreeable products (Coda et al., 2014). Among these
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determinants, the type of flour is one of the most important. It affects the technological features
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and the nutritional value of the baked goods and, more in general, the microbial fermentation
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through the level and type of fermentable carbohydrates, nitrogen sources and growth factors
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(Coda et al., 2014). To exploit the potential of particular flour matrices, the selection of adequate
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starter cultures is needed (Coda et al., 2014). Regarding this aspect, the literature shows very few
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information on the quinoa lactic acid bacteria microbiota and on the selection of suitable strains
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for industrial or artisanal baking.
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First, this study aimed at selecting autochthonous lactic acid bacteria strains to be used for quinoa
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sourdough fermentation. The quinoa sourdough made with selected starters was characterized and
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used as an ingredient to enrich white wheat bread. An integrated characterization, including
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nutritional, texture and sensory features, was carried out to show the numerous advantages of the
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process.
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2. Materials and methods 4
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Organic quinoa (Chenopodium quinoa) dehulled seeds imported from Argentina (Fundacion
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Nuevagestion, San Ignacio de Loyola, Jujuy) were used in this study. Quinoa flour (QF) was
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obtained from seeds by the laboratory mill Ika-Werke M20 (GMBH, and Co. KG, Staufen,
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Germany). Protein (total nitrogen × 5.7), lipids, ash and moisture contents were determined
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according to the AACC approved methods 46-11A, 30-10.01, 08-01, and 44-15A, respectively
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(AACC, 2010). Total carbohydrates were calculated as the difference [100- (proteins + lipids +
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ash + total dietary fibers )]. Proteins, lipids, carbohydrates and ash were expressed as % of dry
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matter (d.m.).
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The determination of insoluble (IDF) and soluble (SDF) dietary fibers was carried out by AOAC
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approved methods 991.42 and 993.19, respectively (Horwitz and Latimer, 2006).
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2.2. Microbiological analysis and isolation of lactic acid bacteria. Lactic acid bacteria were
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isolated from: quinoa flour (T0), quinoa flour dough, having dough yield (DY, dough weight x
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100/flour weight) of 160, and subjected to incubation at 30°C for 16 h (T1), and quinoa type I
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sourdough (T6). Quinoa type I sourdough was made and propagated through the traditional
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protocol commonly used for wheat flour fermentation, without using starter cultures or baker’s
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yeast. Quinoa flour was mixed with tap water at 60 rpm for 5 min, with a IM 5-8 high-speed mixer
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(Mecnosud, Flumeri, Italy), and the dough (DY 160) was incubated at 30°C for 16 h (T1). After
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this first fermentation, six back-slopping steps (refreshments) were further carried out, mixing
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30% of the previously fermented dough with flour and water (DY of 160), and incubating for 16 h
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at 30°C. After each fermentation, doughs were stored at 4˚C until the next refreshment. The pH
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value of doughs was determined by a pHmeter (Model 507, Crison, Milan, Italy) with a food
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penetration probe. Total titratable acidity (TTA) was determined after homogenization of 10 g of
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dough with 90 ml of distilled water, and expressed as the amount (ml) of 0.1 M NaOH required to
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neutralize the solution, using phenolphthalein as indicator (official AACC method 02-31.01). The
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ACCEPTED MANUSCRIPT rate of volume increase of doughs was determined as described by Minervini et al. (2011). After
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six refreshments, the acidification rate and volume increase were stable, and quinoa sourdough
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was used for microbiological analysis and isolation of lactic acid bacteria. Ten grams of quinoa
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flour (F), dough (T1) or sourdough (T6) were homogenized with 90 ml of sterile peptone water
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(1% [wt/vol] of peptone and 0.9% [wt/vol] of NaCl) solution. Presumptive lactic acid bacteria
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were enumerated using three different agar media: MRS (Oxoid, Basingstoke, Hampshire, United
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Kingdom), modified MRS (mMRS) (containing 1% [wt/vol] maltose, 5% [vol/vol] fresh yeast
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extract, pH 5.6) (Oxoid, Basingstoke, Hampshire, United Kingdom), and SDB (sourdough
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bacteria, Kline and Sugihara, 1971). Media were supplemented with cycloheximide (0.1 g liter).
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Plates were incubated at 30°C for 48 h, under anaerobiosis (AnaeroGen and AnaeroJar, Oxoid).
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Approximately ten colonies of presumptive lactic acid bacteria were randomly selected from the
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plates containing the two highest sample dilutions. Gram-positive, catalase-negative, non-motile
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rods and cocci isolates were cultivated into MRS, mMRS, or SDB broth at 30°C for 24 h and re-
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streaked onto the same agar medium. All isolates considered for further analyses were able to
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acidify the culture medium.
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2.3. Genotypic characterization by Randomly Amplified Polymorphic DNA-Polymerase
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Chain Reaction (RAPD-PCR) analysis. Genomic DNA of lactic acid bacteria was extracted
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according to De los Reyes-Gavilán et al. (1992). Three oligonucleotides, P1 5’- ACGCGCCCT-3’,
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P4 5’-CCGCAGCGTT-3’, and M13 5’-GAGGGTGGCGGTTCT-3’, with arbitrarily chosen
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sequences, were used for bio-typing of lactic acid bacteria isolates. Reaction mixture and PCR
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conditions for primers P1 and P4 were those described by Corsetti et al. (2003), whereas those
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reported by Zapparoli et al. (1998) were used for primer M13. RAPD-PCR profiles were acquired
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through the Gel Doc 2000 Documentation System and compared using the Fingerprinting II
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InformatixTM Software (Bio-Rad Laboratories). Dice coefficients of similarity and UPGMA
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algorithm were used to estimate the similarity of the electrophoretic profiles. Since RAPD profiles
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of the isolates from one batch of each type of sourdough were confirmed by analyzing isolates
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from two other batches, strains isolated from a single batch were further analyzed.
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2.4 Genotypic identification of lactic acid bacteria
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To identify presumptive lactic acid bacteria, two primer pairs (Invitrogen Life Technologies,
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Milan, Italy), LacbF/LacbR and LpCoF/LpCoR, were used for amplifying the 16S rDNA
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(Pontonio et al., 2015). Electrophoresis was carried out on agarose gel at 1.5% (wt/vol)
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(Gellyphor, EuroClone) and amplicons were purified with GFXTM PCR DNA and Gel Band
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Purification Kit (GE Healthcare).
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Sequencing electrophoregrams data were processed with Geneious (http://www.geneious.com).
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rDNA sequences alignments were carried out using the multiple sequence alignment method
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(Pontonio et al., 2015) and identification queries were fulfilled by a BLAST search in GenBank
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(http://www.ncbi.nlm.nih.gov/genbank/).
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2.5. Selection of autochthonous lactic acid bacteria and preparation of selected sourdough
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starter
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Autochthonous lactic acid bacteria strains (n.123) were cultivated into MRS, mMRS, or SDB
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broth (depending on the isolation media) at 30°C for 24 h. Cells were harvested by centrifugation
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(10,000 x g, 10 min, 4°C), washed twice in 50 mM sterile potassium phosphate buffer (pH 7.0)
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and re-suspended in tap water at the cell density of ca. 8.0 log cfu/ml. Quinoa flour (62.5 g) and
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37.5 ml of tap water, containing the above cellular suspension of each lactic acid bacterium (cell
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density in the dough of ca. log 7.0 cfu/g), were used to prepare 100 g of dough (DY of 160).
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Mixing was done manually for 5 min. Doughs were fermented at 30°C for 16 h, according to the
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optimal growth temperature of the selected lactic acid bacteria and the fermentation time allowing
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the obtaining of the proper biochemical properties (Coda et al., 2010b; Nionelli et al., 2014).
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doughs were prepared according to Weiss et al. (1993) and used to analyze free amino acids
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(FAA). FAA were analyzed by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd.,
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Cambridge Science Park, England) with a Na-cation-exchange column (20 by 0.46 cm internal
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diameter), as described by Rizzello et al. (2010a).
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Lactobacillus plantarum T6B10 and Lactobacillus rossiae T0A16 were used together as mixed
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starter to obtain a selected quinoa sourdough (QS). Cell suspensions were prepared as described
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by Rizzello et al. (2010a), the DY was 160 and the initial cell density of lactic acid bacteria was
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7.0 log cfu/g. Fermentation of QS was at 30°C for 16 h.
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2.6. Quinoa sourdough characterization
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Kinetics of growth and acidification were determined and modelled in agreement with the
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Gompertz equation, as modified by Zwietering et al. (1990): y= k + A exp{- exp[(µmax or Vmax
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e/A)(λ-t) + 1]}; where y is the growth expressed as log cfu/g/h or the acidification rate expressed
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as dpH/dt (units of pH/h) at the time t; k is the initial level of the dependent variable to be
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modelled (log cfu/g or pH units); A is the cell density or pH (units) variation (between inoculation
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and the stationary phase); µmax or Vmax is the maximum growth rate expressed as ∆log cfu/g/h or
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the maximum acidification rate expressed as dpH/h, respectively; λ is the length of the lag phase
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measured in hours. The experimental data were modelled by the non-linear regression procedure
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of the Statistica 8.0 software (Statsoft, Tulsa, USA). Moreover, the growth curve parameters were
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estimated also using the dynamic model described by Baranyi and Roberts (1994) by using the
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Excel add-in DMFit tool (version 3_5) for curve fitting.
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pH and TTA were determined as described above. Proximal composition of the sourdough
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fermented quinoa flour was determined as described above for raw quinoa flour, after milling a
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freeze-dried sourdough with the laboratory mill Ika-Werke M20 (GMBH). The WSE was used to
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analyze organic acid, and free amino acids. Organic acids were determined by High Performance
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Buckinghmshire, UK) equipped with an Aminex HPX-87H column (ion exclusion, Biorad,
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Richmond, CA), and an UV detector operating at 210 nm. Elution was at 60°C, with a flow rate of
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0.6 ml/min, using H2SO4 10 mM as mobile phase (Coda et al., 2011b). The fermentation quotient
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(FQ) was determined as the molar ratio between lactic and acetic acids. Free amino acids were
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analyzed by a Biochrom 30 series Amino Acid Analyzer as described above.
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A dough, made with quinoa flour (DY 160) and without the inoculum of starters was incubated in
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the same conditions of QS, and used as control (QD).
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2.7. Total phenols and antioxidant activity
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The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was determined on the
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methanolic extract (ME) of quinoa flour and doughs. Five grams of each sample were mixed with
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50 ml of 80% methanol to get ME. The mixture was purged with nitrogen stream for 30 min,
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under stirring condition, and centrifuged at 4,600 × g for 20 min. ME were transferred into test
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tubes, purged with nitrogen stream and stored at ca. 4°C before analysis. The concentration of
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total phenols was determined as described by Slinkard and Singleton (1997), and expressed as
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gallic acid equivalent. The free radical scavenging capacity was determined using the stable
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radical DPPH˙ (Rizzello et al., 2010a). The scavenging activity was expressed as follows: DPPH
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scavenging activity (%) = [(blank absorbance – sample absorbance) / blank absorbance] x 100.
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The value of absorbance was compared with 75 ppm butylated hydroxytoluene (BHT), which was
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used as the antioxidant reference.
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2.8. Phytase activity
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Phytase activity was determined on the WSE of doughs, by monitoring the rate of hydrolysis of p-
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nitrophenyl phosphate (p-NPP) (Sigma, 104-0). The assay mixture contained 200 µL of 1.5 mM p-
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NPP (final concentration) in 0.2 M Na-acetate, pH 5.2, and 400 µL of WSE. The mixture was 9
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incubated at 45ºC and the reaction was stopped by adding 600 µL of 0.1 M NaOH. The p-
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nitrophenol released was determined by measuring the absorbance at 405 nm (Rizzello et al.,
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2010a). One unit (U) of activity was defined as the amount of enzyme required to liberate 1
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µmol/min of p-nitrophenol under the assay conditions.
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2.9. Condensed tannins
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Condensed tannins were determined using the vanillin assay, as described by Price et al. (1978).
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Samples were extracted with HCl:methanol (1:100) for 2.5 h at room temperature and centrifuged
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at 4,000 rpm for 20 min. Extracts were covered from light and analysed promptly at 30°C.
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Vanillin reagent (equal volumes of 1% vanillin in methanol and 8% concentrated hydrochloric
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acid in methanol) was added to extracts. Blanks were prepared by adding 4% concentrated
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hydrochloric acid in methanol to extracts. The calibration curve was obtained using catechin and
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the results were expressed as catechin equivalents.
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2.10. In vitro protein digestibility
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The in vitro protein digestibility (IVPD) of QD and QS was determined by the method of Akeson
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and Stahman (1964). One gram of each sample was incubated with 1.5 mg of pepsin, in 15 ml of
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0.1 M HCl, at 37°C for 3h. After neutralization with 2 M NaOH and addition of 4 mg of
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pancreatin, in 7.5 ml of phosphate buffer (pH 8.0), 1 ml of toluene was added to prevent microbial
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growth, and the solution was incubated for 24 h at 37°C. After 24 h, the enzyme was inactivated
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by addition of 10 ml of trichloroacetic acid (20%, wt/vol), and the undigested protein was
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precipitated. The volume was made up to 100 ml with distilled water and centrifuged at 5000 rpm
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for 20 min. The concentration of protein of the supernatant was determined by the Bradford
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method (Bradford, 1976). The precipitate was subjected to protein extraction, according to Weiss
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et al. (1993), and the concentration of protein was determined. The in vitro protein digestibility
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was expressed as the percentage of the total protein, which was solubilized after enzyme
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hydrolysis.
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2.11. Bread making
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A quinoa sourdough bread (QSB, DY of 160) was manufactured at the pilot plant of the
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Department of Soil, Plant and Food Science of the University of Bari (Italy), according to the
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two-stage protocol including the production of sourdough (fermentation for 16h at 30°C, step I)
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first, and the mixing with flour, water, and baker’s yeast (1.5 h at 30°C, step II), later. QS were
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used at the percentage of 20% (w/w) (Nionelli et al., 2014; Pontonio et al., 2015). A baker’s
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yeast bread containing the same percentage of quinoa flour (12.5%) of QSB, but without the
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use of lactic acid bacteria as starters (QB), and a baker’s yeast wheat bread (WB) produced
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without the addition of quinoa flour, were manufactured and used as the controls. Baker’s yeast
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was added at the percentage of 2% w/w, corresponding to a final cell density of ca. 7 log cfu/g
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in all the breads. Doughs were mixed at 60 × g for 5 min with an IM 5-8 high-speed mixer
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(Mecnosud, Flumeri, Italy) and fermentation was at 30°C for 1.5 h. All breads were baked at
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220°C for 30 min (Combo 3, Zucchelli, Verona, Italy).
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Water activity (aw) was determined at 25ºC by the Aqualab Dew Point 4TE water activity
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meter (Decagon Devices Inc., USA). Saturated fats and sugars were determined with the
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AACC methods 58-18.01 and 80-04.01, respectively (AACC, 2010). Fermentations were
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carried out in triplicate and each bread was analyzed twice.
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2.12. Texture, image and color analyses
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Instrumental Texture Profile Analysis (TPA) was carried out with a TVT-300XP Texture
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Analyzer (TexVol Instruments, Viken, Sweden), equipped with a cylinder probe P-Cy25S. For the
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analysis, boule shaped loaves (300 g) were baked, packed in polypropylene micro perforated bags
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and stored for 24 h at room temperature. Crust was not removed. The selected settings were the 11
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was carried out (Rizzello et al., 2012), using Texture Analyzer TVT-XP 3.8.0.5 software (TexVol
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Instruments). Height, width, depth, area, and specific volume of breads were measured by the
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BVM-test system (TexVol Instruments). The following textural parameters were obtained by the
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texturometer software: hardness (maximum peak force); fracturability (the first significant peak
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force during the probe compression of the bread); and resilience (ratio of the first decompression
276
area to the first compression area).
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The chromaticity co-ordinates of the bread crust (obtained by a Minolta CR-10 camera) were also
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reported in the form of a color difference, dE*ab, as follows: dE*ab = (d) + (d) + (d )
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where dL, da, and db are the differences for L, a, and b values between sample and reference (a
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white ceramic plate having L = 93.4, a = –0.39, and b = 3.99).
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The crumb features of breads were evaluated after 24 h of storage using the image analysis
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technology with the UTHSCSA ImageTool as previously described by Rizzello et al. (2012).
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2.13. Nutritional characterization
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Energy value was calculated as reported by USDA method (IOM, 2002). The in vitro digestibility
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of breads, was determined as described by Rizzello et al. (2014b). The supernatant, which
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contained the digested protein, was freeze-dried and used for further analyses. The modified
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method AOAC 982.30a (Horwitz and Latimer, 2006) was used to determine the total amino acid
289
profile. The digested protein fraction, which derived from 1 g of sample, was added of 5.7 M HCl
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(1 ml/10 mg of proteins), under nitrogen stream, and incubated at 110°C for 24 h. Hydrolysis was
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carried out under anaerobic conditions to prevent the oxidative degradation of amino acids. After
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freeze-drying, the hydrolyzate was re-suspended (20 mg/ml) in sodium citrate buffer, pH 2.2, and
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filtered through a Millex-HA 0.22 µm pore size filter (Millipore Co.). Amino acids were analyzed
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by a Biochrom 30 series Amino Acid Analyzer as described above. Since the above procedure of
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hydrolysis does not allow the determination of tryptophan, it was estimated by the method of
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NaOH, and shaken for 30 min at room temperature. The sample was centrifuged (10,000 rpm for
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10 min), and 0.5 ml of the supernatant were mixed with 5 ml of ninhydrin reagent (1 g of
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ninhydrin in 100 ml of HCl 37% : formic acid 96%, at the ratio 2:3) and incubated for 2 h at 37°C.
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The reaction mixture was cooled at room temperature and made up to 10 ml with the addition of
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diethyl ether. The absorbance at 380 nm was measured. A standard tryptophan curve was prepared
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using a tryptophan (Sigma Chemicals Co.) solution in the range 0-100 µg/ml.
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Chemical Score (CS) estimates the amount of protein required to provide the minimal essential
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amino acid (EAA) pattern, which is present in the reference protein (hen’s egg). It was calculated
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using the equation of Block and Mitchel (1946), which compares the content of EAA of the breads
306
for the amount of the same amino acid of the reference. The sequence of limiting essential amino
307
acids corresponds to the list of EAA, having the lowest chemical score (Block and Mitchel, 1946).
308
The protein score indicates the chemical score of the most limiting EAA that is present in the test
309
protein (Block and Mitchel, 1946). Essential Amino Acids Index (EAAI) estimates the quality of
310
the test protein, using its EAA content as the criterion. EAAI was calculated according to the
311
procedure of Oser (1959). It considers the ratio between EAA of the test protein and EAA of the
312
reference protein, according to the following equation:
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=
( ∗ 100)( ∗ 100)(… )( ∗ 100) [] ( ∗ 100)( ∗ 100)(… )( ∗ 100)[ ]
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The Biological Value (BV) indicates the utilizable fraction of the test protein. BV was calculated
314
using the equation of Oser (1959): BV = ([1,09*EAAI]-11,70). The Protein Efficiency Ratio
315
(PER) estimates the protein nutritional quality based on the amino acid profile after hydrolysis.
316
PER was determined using the model developed by Ihekoronye (1981): PER = –0,468 +
317
(0,454*[Leucine]) – (0,105*[Tyrosine]). The Nutritional Index (NI) normalizes the qualitative and
318
quantitative variations of the test protein compared to its nutritional status. NI was calculated
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using the equation of Crisan and Sands (1978), which considers all the factors with an equal
320
importance: NI = (EAAI*Protein(%)/100).
321
2.14. Starch hydrolysis index and predicted glycaemic index
323
The analysis of starch hydrolysis was carried out on breads. The procedure mimicked the in vivo
324
digestion of starch (De Angelis et al., 2009). Portions of breads, containing 1 g of starch, were
325
given in randomized order to 10 volunteers. The glucose content was measured with Enzy Plus D-
326
Glucose kit (DiffchambVästraFrölunda, Sweden). The degree of starch digestion was expressed as
327
the percentage of potentially available starch hydrolyzed at different times (30, 60, 90, 120, 150,
328
and 180 min). A non-linear model (De Angelis et al., 2009) was applied to describe the kinetics of
329
starch hydrolysis. The hydrolysis curves were obtained with the equation reported below, using
330
the software Statistica 8.0. Hydrolysis curves follow a first order equation: C = C∞ (1-e-kt) where C
331
is the concentration at t time, C∞ is the equilibrium concentration, k is the kinetic constant and t is
332
the chosen time. Wheat flour bread (WB) was used as the control to estimate the hydrolysis index
333
(HI = 100). The predicted GI was calculated using the equation: GI = 0.549*HI + 39.71 (Capriles
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and Areas, 2013) with wheat bread as the reference (GI wheat bread = 100).
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2.15. Sensory analysis
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Sensory analysis of breads was carried out by 10 panellists (5 male and 5 female, mean age: 35
338
years, range: 18-54 years), according to the method described by Haglund et al (Haglund et al.,
339
1998; Rizzello et al., 2010b). Elasticity, colour of crust and crumb, acid taste, acid flavour,
340
sweetness, dryness, and taste were considered as sensory attributes using a scale from 0 to10, with
341
10 the highest score. Salty taste, previously described as another wheat sourdough bread attribute,
342
was also included (Rizzello et al., 2010b). Friedman's nonparametric test was used for the
343
statistical treatment of the results.
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ACCEPTED MANUSCRIPT 2.16. Statistical Analysis
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Fermentations were carried out in triplicate and each analysis was repeated twice. Data were
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subjected to one-way ANOVA; pair-comparison of treatment means was obtained by Tukey’s
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procedure at P
S0740-0020(15)00253-1
DOI:
10.1016/j.fm.2015.11.018
Reference:
YFMIC 2499
To appear in:
Food Microbiology
Received Date: 16 August 2015 Revised Date:
6 November 2015
Accepted Date: 27 November 2015
Please cite this article as: Rizzello, C.G., Lorusso, A., Montemurro, M., Gobbetti, M., Use of sourdough made with quinoa (Chenopodium quinoa) flour and autochthonous selected lactic acid bacteria for enhancing the nutritional, textural and sensory features of white bread, Food Microbiology (2015), doi: 10.1016/j.fm.2015.11.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Use of sourdough made with quinoa (Chenopodium quinoa) flour and
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autochthonous selected lactic acid bacteria for enhancing the nutritional,
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textural and sensory features of white bread
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Carlo Giuseppe Rizzello*, Anna Lorusso, Marco Montemurro, Marco Gobbetti
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Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70126 Bari, Italy
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*Corresponding author. Tel.: +39 0805442950; Fax: +390805442911.
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E-mail address: [email protected]
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Abbreviations
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IDF: insoluble dietary fiber; SDF: soluble dietary fiber; DY: dough yield; TTA: Total titratable
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acidity; WSE: Water/salt-soluble extract; FAA: free amino acids; QS: quinoa sourdough; QD:
16
quinoa dough; FQ: fermentation quotient; ME:
17
digestibility; QSB: quinoa sourdough bread; WB: wheat bread; QB: quinoa bread; TPA: Texture
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Profile Analysis; CS: Chemical Score; EAA: essential amino acid; EAAI: Essential Amino Acids
19
Index; PER: Protein Efficiency Ratio; NI:Nutritional Index; HI: hydrolysis index; GI: glycemic
20
index; QF: quinoa flour.
methanolic extract; IVPD: in vitro protein
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Abstract
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Lactic acid bacteria were isolated and identified from quinoa flour, spontaneously fermented
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quinoa dough, and type I quinoa sourdough. Strains were further selected based on acidification
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and proteolytic activities. Selected Lactobacillus plantarum T6B10 and Lactobacillus rossiae
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T0A16 were used as mixed starter to get quinoa sourdough. Compared to non-fermented flour,
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organic acids, free amino acids, soluble fibers, total phenols, phytase and antioxidant activities,
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and in vitro protein digestibility markedly increased during fermentation. A wheat bread was made
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using 20% (w/w) of quinoa sourdough, and compared to baker’s yeast wheat breads manufactured
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with or without quinoa flour. The use of quinoa sourdough improved the chemical, textural, and
30
sensory features of wheat bread, showing better performances compared to the use of quinoa flour.
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Protein digestibility and quality, and the rate of starch hydrolysis were also nutritional features that
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markedly improved using quinoa sourdough as an ingredient.
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This study exploited the potential of quinoa flour through sourdough fermentation. A number of
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advantages encouraged the manufacture of novel and healthy leavened baked goods.
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Keywords
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Quinoa, Lactic acid bacteria, Sourdough, Bread
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1. Introduction
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Quinoa (Chenopodium quinoa Willd.) is a seed crop, which is traditionally cultivated in the
41
Andean region since thousands of years. Commonly, quinoa grains and flour are used for human
42
consumption and animal feeding. Quinoa has a very elevated genetic variability, which makes
43
possible to select, adapt and breed cultivars for a wide range of environmental conditions (Bertero
44
et al., 2004). Indeed, quinoa has the capacity of adapting to a range of agro-ecological conditions,
45
showing tolerance to frost, salinity and drought, and having the potential to grow on marginal
46
soils. These features, together with an undoubtedly nutritional value, determine a worldwide
47
interest for this crop (Stikic et al., 2012). During the last years, the production of quinoa markedly
48
increased, thus emphasizing the opportunity to cultivate this crop in various regions. Diverse
49
climatic regions of USA, Canada, India, England, Denmark, Greece, Italy and other European
50
countries were shown to be suitable for an extended cultivation (Stikic et al., 2012). FAO selected
51
the quinoa as one of the crops that are destined to offer food security in the 21st century (Jacobsen
52
et al., 2003).
53
The high nutritional value of quinoa seeds is mainly due to high concentrations of proteins,
54
minerals, and vitamins (Fleming and Galwey, 1995). Proteins are rich in amino acids like lysine,
55
threonine and methionine, which, on the contrary, are deficient in cereals. A special interest was
56
deserved to the use of quinoa for people who are affected by celiac disease. Quinoa was
57
considered an efficient nutritional alternative to gluten-containing wheat, rye and barley (Jacobsen
58
et al., 2003), being largely used as an ingredient for making breads, biscuits, cookies, crepes,
59
muffins, pancakes and tortillas. While the nutritional value and the chemical composition of
60
quinoa were characterized, several aspects concerning the technological applications have
61
received less attention (Stikic et al., 2012). Overall, the baking quality is considered rather low due
62
to the absence of gluten, (Stikic et al., 2012) and flavor, texture and appearance of baked goods,
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including quinoa in the recipes, were reported only as moderately acceptable (Stikic et al., 2012).
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ACCEPTED MANUSCRIPT Sourdough fermentation has the potential to exploit the technological, nutritional, functional and
65
sensory features of wheat and non-wheat flours. Besides the well-known advantages documented
66
for processing wheat and rye, an abundant literature showed how the sourdough may enhance
67
various features of milling byproducts (Coda et al., 2015a; Rizzello et al., 2010a; Rizzello et al.,
68
2010b), minor cereals (Coda et al., 2011a; Coda et al., 2010a), legumes (Curiel et al., 2015;
69
Rizzello et al., 2014a), teff, and buckwheat (Coda et al., 2014). In particular, sourdough
70
fermentation improves dough workability, bread structure and organoleptic and nutritional
71
properties of raw flours. Furthermore, it increases the content of biogenic compounds and the
72
uptake of minerals, and decreases the level of anti-nutritional factors and the value of the
73
glycaemic response (Gobbetti et al., 2014).
74
Nevertheless, fermentation processes depend on specific determinants, which have to be strictly
75
controlled to get standardized and agreeable products (Coda et al., 2014). Among these
76
determinants, the type of flour is one of the most important. It affects the technological features
77
and the nutritional value of the baked goods and, more in general, the microbial fermentation
78
through the level and type of fermentable carbohydrates, nitrogen sources and growth factors
79
(Coda et al., 2014). To exploit the potential of particular flour matrices, the selection of adequate
80
starter cultures is needed (Coda et al., 2014). Regarding this aspect, the literature shows very few
81
information on the quinoa lactic acid bacteria microbiota and on the selection of suitable strains
82
for industrial or artisanal baking.
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First, this study aimed at selecting autochthonous lactic acid bacteria strains to be used for quinoa
84
sourdough fermentation. The quinoa sourdough made with selected starters was characterized and
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used as an ingredient to enrich white wheat bread. An integrated characterization, including
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nutritional, texture and sensory features, was carried out to show the numerous advantages of the
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process.
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2. Materials and methods 4
ACCEPTED MANUSCRIPT 2.1. Quinoa flour
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Organic quinoa (Chenopodium quinoa) dehulled seeds imported from Argentina (Fundacion
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Nuevagestion, San Ignacio de Loyola, Jujuy) were used in this study. Quinoa flour (QF) was
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obtained from seeds by the laboratory mill Ika-Werke M20 (GMBH, and Co. KG, Staufen,
94
Germany). Protein (total nitrogen × 5.7), lipids, ash and moisture contents were determined
95
according to the AACC approved methods 46-11A, 30-10.01, 08-01, and 44-15A, respectively
96
(AACC, 2010). Total carbohydrates were calculated as the difference [100- (proteins + lipids +
97
ash + total dietary fibers )]. Proteins, lipids, carbohydrates and ash were expressed as % of dry
98
matter (d.m.).
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The determination of insoluble (IDF) and soluble (SDF) dietary fibers was carried out by AOAC
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approved methods 991.42 and 993.19, respectively (Horwitz and Latimer, 2006).
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2.2. Microbiological analysis and isolation of lactic acid bacteria. Lactic acid bacteria were
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isolated from: quinoa flour (T0), quinoa flour dough, having dough yield (DY, dough weight x
104
100/flour weight) of 160, and subjected to incubation at 30°C for 16 h (T1), and quinoa type I
105
sourdough (T6). Quinoa type I sourdough was made and propagated through the traditional
106
protocol commonly used for wheat flour fermentation, without using starter cultures or baker’s
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yeast. Quinoa flour was mixed with tap water at 60 rpm for 5 min, with a IM 5-8 high-speed mixer
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(Mecnosud, Flumeri, Italy), and the dough (DY 160) was incubated at 30°C for 16 h (T1). After
109
this first fermentation, six back-slopping steps (refreshments) were further carried out, mixing
110
30% of the previously fermented dough with flour and water (DY of 160), and incubating for 16 h
111
at 30°C. After each fermentation, doughs were stored at 4˚C until the next refreshment. The pH
112
value of doughs was determined by a pHmeter (Model 507, Crison, Milan, Italy) with a food
113
penetration probe. Total titratable acidity (TTA) was determined after homogenization of 10 g of
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dough with 90 ml of distilled water, and expressed as the amount (ml) of 0.1 M NaOH required to
115
neutralize the solution, using phenolphthalein as indicator (official AACC method 02-31.01). The
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ACCEPTED MANUSCRIPT rate of volume increase of doughs was determined as described by Minervini et al. (2011). After
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six refreshments, the acidification rate and volume increase were stable, and quinoa sourdough
118
was used for microbiological analysis and isolation of lactic acid bacteria. Ten grams of quinoa
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flour (F), dough (T1) or sourdough (T6) were homogenized with 90 ml of sterile peptone water
120
(1% [wt/vol] of peptone and 0.9% [wt/vol] of NaCl) solution. Presumptive lactic acid bacteria
121
were enumerated using three different agar media: MRS (Oxoid, Basingstoke, Hampshire, United
122
Kingdom), modified MRS (mMRS) (containing 1% [wt/vol] maltose, 5% [vol/vol] fresh yeast
123
extract, pH 5.6) (Oxoid, Basingstoke, Hampshire, United Kingdom), and SDB (sourdough
124
bacteria, Kline and Sugihara, 1971). Media were supplemented with cycloheximide (0.1 g liter).
125
Plates were incubated at 30°C for 48 h, under anaerobiosis (AnaeroGen and AnaeroJar, Oxoid).
126
Approximately ten colonies of presumptive lactic acid bacteria were randomly selected from the
127
plates containing the two highest sample dilutions. Gram-positive, catalase-negative, non-motile
128
rods and cocci isolates were cultivated into MRS, mMRS, or SDB broth at 30°C for 24 h and re-
129
streaked onto the same agar medium. All isolates considered for further analyses were able to
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acidify the culture medium.
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2.3. Genotypic characterization by Randomly Amplified Polymorphic DNA-Polymerase
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Chain Reaction (RAPD-PCR) analysis. Genomic DNA of lactic acid bacteria was extracted
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according to De los Reyes-Gavilán et al. (1992). Three oligonucleotides, P1 5’- ACGCGCCCT-3’,
135
P4 5’-CCGCAGCGTT-3’, and M13 5’-GAGGGTGGCGGTTCT-3’, with arbitrarily chosen
136
sequences, were used for bio-typing of lactic acid bacteria isolates. Reaction mixture and PCR
137
conditions for primers P1 and P4 were those described by Corsetti et al. (2003), whereas those
138
reported by Zapparoli et al. (1998) were used for primer M13. RAPD-PCR profiles were acquired
139
through the Gel Doc 2000 Documentation System and compared using the Fingerprinting II
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InformatixTM Software (Bio-Rad Laboratories). Dice coefficients of similarity and UPGMA
141
algorithm were used to estimate the similarity of the electrophoretic profiles. Since RAPD profiles
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of the isolates from one batch of each type of sourdough were confirmed by analyzing isolates
143
from two other batches, strains isolated from a single batch were further analyzed.
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2.4 Genotypic identification of lactic acid bacteria
146
To identify presumptive lactic acid bacteria, two primer pairs (Invitrogen Life Technologies,
147
Milan, Italy), LacbF/LacbR and LpCoF/LpCoR, were used for amplifying the 16S rDNA
148
(Pontonio et al., 2015). Electrophoresis was carried out on agarose gel at 1.5% (wt/vol)
149
(Gellyphor, EuroClone) and amplicons were purified with GFXTM PCR DNA and Gel Band
150
Purification Kit (GE Healthcare).
151
Sequencing electrophoregrams data were processed with Geneious (http://www.geneious.com).
152
rDNA sequences alignments were carried out using the multiple sequence alignment method
153
(Pontonio et al., 2015) and identification queries were fulfilled by a BLAST search in GenBank
154
(http://www.ncbi.nlm.nih.gov/genbank/).
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2.5. Selection of autochthonous lactic acid bacteria and preparation of selected sourdough
157
starter
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Autochthonous lactic acid bacteria strains (n.123) were cultivated into MRS, mMRS, or SDB
159
broth (depending on the isolation media) at 30°C for 24 h. Cells were harvested by centrifugation
160
(10,000 x g, 10 min, 4°C), washed twice in 50 mM sterile potassium phosphate buffer (pH 7.0)
161
and re-suspended in tap water at the cell density of ca. 8.0 log cfu/ml. Quinoa flour (62.5 g) and
162
37.5 ml of tap water, containing the above cellular suspension of each lactic acid bacterium (cell
163
density in the dough of ca. log 7.0 cfu/g), were used to prepare 100 g of dough (DY of 160).
164
Mixing was done manually for 5 min. Doughs were fermented at 30°C for 16 h, according to the
165
optimal growth temperature of the selected lactic acid bacteria and the fermentation time allowing
166
the obtaining of the proper biochemical properties (Coda et al., 2010b; Nionelli et al., 2014).
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168
doughs were prepared according to Weiss et al. (1993) and used to analyze free amino acids
169
(FAA). FAA were analyzed by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd.,
170
Cambridge Science Park, England) with a Na-cation-exchange column (20 by 0.46 cm internal
171
diameter), as described by Rizzello et al. (2010a).
172
Lactobacillus plantarum T6B10 and Lactobacillus rossiae T0A16 were used together as mixed
173
starter to obtain a selected quinoa sourdough (QS). Cell suspensions were prepared as described
174
by Rizzello et al. (2010a), the DY was 160 and the initial cell density of lactic acid bacteria was
175
7.0 log cfu/g. Fermentation of QS was at 30°C for 16 h.
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2.6. Quinoa sourdough characterization
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Kinetics of growth and acidification were determined and modelled in agreement with the
179
Gompertz equation, as modified by Zwietering et al. (1990): y= k + A exp{- exp[(µmax or Vmax
180
e/A)(λ-t) + 1]}; where y is the growth expressed as log cfu/g/h or the acidification rate expressed
181
as dpH/dt (units of pH/h) at the time t; k is the initial level of the dependent variable to be
182
modelled (log cfu/g or pH units); A is the cell density or pH (units) variation (between inoculation
183
and the stationary phase); µmax or Vmax is the maximum growth rate expressed as ∆log cfu/g/h or
184
the maximum acidification rate expressed as dpH/h, respectively; λ is the length of the lag phase
185
measured in hours. The experimental data were modelled by the non-linear regression procedure
186
of the Statistica 8.0 software (Statsoft, Tulsa, USA). Moreover, the growth curve parameters were
187
estimated also using the dynamic model described by Baranyi and Roberts (1994) by using the
188
Excel add-in DMFit tool (version 3_5) for curve fitting.
189
pH and TTA were determined as described above. Proximal composition of the sourdough
190
fermented quinoa flour was determined as described above for raw quinoa flour, after milling a
191
freeze-dried sourdough with the laboratory mill Ika-Werke M20 (GMBH). The WSE was used to
192
analyze organic acid, and free amino acids. Organic acids were determined by High Performance
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Buckinghmshire, UK) equipped with an Aminex HPX-87H column (ion exclusion, Biorad,
195
Richmond, CA), and an UV detector operating at 210 nm. Elution was at 60°C, with a flow rate of
196
0.6 ml/min, using H2SO4 10 mM as mobile phase (Coda et al., 2011b). The fermentation quotient
197
(FQ) was determined as the molar ratio between lactic and acetic acids. Free amino acids were
198
analyzed by a Biochrom 30 series Amino Acid Analyzer as described above.
199
A dough, made with quinoa flour (DY 160) and without the inoculum of starters was incubated in
200
the same conditions of QS, and used as control (QD).
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2.7. Total phenols and antioxidant activity
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The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was determined on the
204
methanolic extract (ME) of quinoa flour and doughs. Five grams of each sample were mixed with
205
50 ml of 80% methanol to get ME. The mixture was purged with nitrogen stream for 30 min,
206
under stirring condition, and centrifuged at 4,600 × g for 20 min. ME were transferred into test
207
tubes, purged with nitrogen stream and stored at ca. 4°C before analysis. The concentration of
208
total phenols was determined as described by Slinkard and Singleton (1997), and expressed as
209
gallic acid equivalent. The free radical scavenging capacity was determined using the stable
210
radical DPPH˙ (Rizzello et al., 2010a). The scavenging activity was expressed as follows: DPPH
211
scavenging activity (%) = [(blank absorbance – sample absorbance) / blank absorbance] x 100.
212
The value of absorbance was compared with 75 ppm butylated hydroxytoluene (BHT), which was
213
used as the antioxidant reference.
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2.8. Phytase activity
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Phytase activity was determined on the WSE of doughs, by monitoring the rate of hydrolysis of p-
217
nitrophenyl phosphate (p-NPP) (Sigma, 104-0). The assay mixture contained 200 µL of 1.5 mM p-
218
NPP (final concentration) in 0.2 M Na-acetate, pH 5.2, and 400 µL of WSE. The mixture was 9
ACCEPTED MANUSCRIPT 219
incubated at 45ºC and the reaction was stopped by adding 600 µL of 0.1 M NaOH. The p-
220
nitrophenol released was determined by measuring the absorbance at 405 nm (Rizzello et al.,
221
2010a). One unit (U) of activity was defined as the amount of enzyme required to liberate 1
222
µmol/min of p-nitrophenol under the assay conditions.
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2.9. Condensed tannins
225
Condensed tannins were determined using the vanillin assay, as described by Price et al. (1978).
226
Samples were extracted with HCl:methanol (1:100) for 2.5 h at room temperature and centrifuged
227
at 4,000 rpm for 20 min. Extracts were covered from light and analysed promptly at 30°C.
228
Vanillin reagent (equal volumes of 1% vanillin in methanol and 8% concentrated hydrochloric
229
acid in methanol) was added to extracts. Blanks were prepared by adding 4% concentrated
230
hydrochloric acid in methanol to extracts. The calibration curve was obtained using catechin and
231
the results were expressed as catechin equivalents.
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2.10. In vitro protein digestibility
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The in vitro protein digestibility (IVPD) of QD and QS was determined by the method of Akeson
235
and Stahman (1964). One gram of each sample was incubated with 1.5 mg of pepsin, in 15 ml of
236
0.1 M HCl, at 37°C for 3h. After neutralization with 2 M NaOH and addition of 4 mg of
237
pancreatin, in 7.5 ml of phosphate buffer (pH 8.0), 1 ml of toluene was added to prevent microbial
238
growth, and the solution was incubated for 24 h at 37°C. After 24 h, the enzyme was inactivated
239
by addition of 10 ml of trichloroacetic acid (20%, wt/vol), and the undigested protein was
240
precipitated. The volume was made up to 100 ml with distilled water and centrifuged at 5000 rpm
241
for 20 min. The concentration of protein of the supernatant was determined by the Bradford
242
method (Bradford, 1976). The precipitate was subjected to protein extraction, according to Weiss
243
et al. (1993), and the concentration of protein was determined. The in vitro protein digestibility
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was expressed as the percentage of the total protein, which was solubilized after enzyme
245
hydrolysis.
246
2.11. Bread making
248
A quinoa sourdough bread (QSB, DY of 160) was manufactured at the pilot plant of the
249
Department of Soil, Plant and Food Science of the University of Bari (Italy), according to the
250
two-stage protocol including the production of sourdough (fermentation for 16h at 30°C, step I)
251
first, and the mixing with flour, water, and baker’s yeast (1.5 h at 30°C, step II), later. QS were
252
used at the percentage of 20% (w/w) (Nionelli et al., 2014; Pontonio et al., 2015). A baker’s
253
yeast bread containing the same percentage of quinoa flour (12.5%) of QSB, but without the
254
use of lactic acid bacteria as starters (QB), and a baker’s yeast wheat bread (WB) produced
255
without the addition of quinoa flour, were manufactured and used as the controls. Baker’s yeast
256
was added at the percentage of 2% w/w, corresponding to a final cell density of ca. 7 log cfu/g
257
in all the breads. Doughs were mixed at 60 × g for 5 min with an IM 5-8 high-speed mixer
258
(Mecnosud, Flumeri, Italy) and fermentation was at 30°C for 1.5 h. All breads were baked at
259
220°C for 30 min (Combo 3, Zucchelli, Verona, Italy).
260
Water activity (aw) was determined at 25ºC by the Aqualab Dew Point 4TE water activity
261
meter (Decagon Devices Inc., USA). Saturated fats and sugars were determined with the
262
AACC methods 58-18.01 and 80-04.01, respectively (AACC, 2010). Fermentations were
263
carried out in triplicate and each bread was analyzed twice.
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2.12. Texture, image and color analyses
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Instrumental Texture Profile Analysis (TPA) was carried out with a TVT-300XP Texture
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Analyzer (TexVol Instruments, Viken, Sweden), equipped with a cylinder probe P-Cy25S. For the
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analysis, boule shaped loaves (300 g) were baked, packed in polypropylene micro perforated bags
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and stored for 24 h at room temperature. Crust was not removed. The selected settings were the 11
ACCEPTED MANUSCRIPT following: test speed 1 mm/s, 30% deformation of the sample and one compression cycle. TPA
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was carried out (Rizzello et al., 2012), using Texture Analyzer TVT-XP 3.8.0.5 software (TexVol
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Instruments). Height, width, depth, area, and specific volume of breads were measured by the
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BVM-test system (TexVol Instruments). The following textural parameters were obtained by the
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texturometer software: hardness (maximum peak force); fracturability (the first significant peak
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force during the probe compression of the bread); and resilience (ratio of the first decompression
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area to the first compression area).
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The chromaticity co-ordinates of the bread crust (obtained by a Minolta CR-10 camera) were also
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reported in the form of a color difference, dE*ab, as follows: dE*ab = (d) + (d) + (d )
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where dL, da, and db are the differences for L, a, and b values between sample and reference (a
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white ceramic plate having L = 93.4, a = –0.39, and b = 3.99).
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The crumb features of breads were evaluated after 24 h of storage using the image analysis
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technology with the UTHSCSA ImageTool as previously described by Rizzello et al. (2012).
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2.13. Nutritional characterization
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Energy value was calculated as reported by USDA method (IOM, 2002). The in vitro digestibility
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of breads, was determined as described by Rizzello et al. (2014b). The supernatant, which
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contained the digested protein, was freeze-dried and used for further analyses. The modified
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method AOAC 982.30a (Horwitz and Latimer, 2006) was used to determine the total amino acid
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profile. The digested protein fraction, which derived from 1 g of sample, was added of 5.7 M HCl
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(1 ml/10 mg of proteins), under nitrogen stream, and incubated at 110°C for 24 h. Hydrolysis was
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carried out under anaerobic conditions to prevent the oxidative degradation of amino acids. After
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freeze-drying, the hydrolyzate was re-suspended (20 mg/ml) in sodium citrate buffer, pH 2.2, and
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filtered through a Millex-HA 0.22 µm pore size filter (Millipore Co.). Amino acids were analyzed
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by a Biochrom 30 series Amino Acid Analyzer as described above. Since the above procedure of
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hydrolysis does not allow the determination of tryptophan, it was estimated by the method of
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12
ACCEPTED MANUSCRIPT Pintér-Szakács & Molnán-Perl (1990). One gram of sample was suspended in 10 ml of 75 mM
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NaOH, and shaken for 30 min at room temperature. The sample was centrifuged (10,000 rpm for
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10 min), and 0.5 ml of the supernatant were mixed with 5 ml of ninhydrin reagent (1 g of
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ninhydrin in 100 ml of HCl 37% : formic acid 96%, at the ratio 2:3) and incubated for 2 h at 37°C.
300
The reaction mixture was cooled at room temperature and made up to 10 ml with the addition of
301
diethyl ether. The absorbance at 380 nm was measured. A standard tryptophan curve was prepared
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using a tryptophan (Sigma Chemicals Co.) solution in the range 0-100 µg/ml.
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Chemical Score (CS) estimates the amount of protein required to provide the minimal essential
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amino acid (EAA) pattern, which is present in the reference protein (hen’s egg). It was calculated
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using the equation of Block and Mitchel (1946), which compares the content of EAA of the breads
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for the amount of the same amino acid of the reference. The sequence of limiting essential amino
307
acids corresponds to the list of EAA, having the lowest chemical score (Block and Mitchel, 1946).
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The protein score indicates the chemical score of the most limiting EAA that is present in the test
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protein (Block and Mitchel, 1946). Essential Amino Acids Index (EAAI) estimates the quality of
310
the test protein, using its EAA content as the criterion. EAAI was calculated according to the
311
procedure of Oser (1959). It considers the ratio between EAA of the test protein and EAA of the
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reference protein, according to the following equation:
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=
( ∗ 100)( ∗ 100)(… )( ∗ 100) [] ( ∗ 100)( ∗ 100)(… )( ∗ 100)[ ]
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The Biological Value (BV) indicates the utilizable fraction of the test protein. BV was calculated
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using the equation of Oser (1959): BV = ([1,09*EAAI]-11,70). The Protein Efficiency Ratio
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(PER) estimates the protein nutritional quality based on the amino acid profile after hydrolysis.
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PER was determined using the model developed by Ihekoronye (1981): PER = –0,468 +
317
(0,454*[Leucine]) – (0,105*[Tyrosine]). The Nutritional Index (NI) normalizes the qualitative and
318
quantitative variations of the test protein compared to its nutritional status. NI was calculated
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using the equation of Crisan and Sands (1978), which considers all the factors with an equal
320
importance: NI = (EAAI*Protein(%)/100).
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2.14. Starch hydrolysis index and predicted glycaemic index
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The analysis of starch hydrolysis was carried out on breads. The procedure mimicked the in vivo
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digestion of starch (De Angelis et al., 2009). Portions of breads, containing 1 g of starch, were
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given in randomized order to 10 volunteers. The glucose content was measured with Enzy Plus D-
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Glucose kit (DiffchambVästraFrölunda, Sweden). The degree of starch digestion was expressed as
327
the percentage of potentially available starch hydrolyzed at different times (30, 60, 90, 120, 150,
328
and 180 min). A non-linear model (De Angelis et al., 2009) was applied to describe the kinetics of
329
starch hydrolysis. The hydrolysis curves were obtained with the equation reported below, using
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the software Statistica 8.0. Hydrolysis curves follow a first order equation: C = C∞ (1-e-kt) where C
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is the concentration at t time, C∞ is the equilibrium concentration, k is the kinetic constant and t is
332
the chosen time. Wheat flour bread (WB) was used as the control to estimate the hydrolysis index
333
(HI = 100). The predicted GI was calculated using the equation: GI = 0.549*HI + 39.71 (Capriles
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and Areas, 2013) with wheat bread as the reference (GI wheat bread = 100).
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2.15. Sensory analysis
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Sensory analysis of breads was carried out by 10 panellists (5 male and 5 female, mean age: 35
338
years, range: 18-54 years), according to the method described by Haglund et al (Haglund et al.,
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1998; Rizzello et al., 2010b). Elasticity, colour of crust and crumb, acid taste, acid flavour,
340
sweetness, dryness, and taste were considered as sensory attributes using a scale from 0 to10, with
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10 the highest score. Salty taste, previously described as another wheat sourdough bread attribute,
342
was also included (Rizzello et al., 2010b). Friedman's nonparametric test was used for the
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statistical treatment of the results.
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ACCEPTED MANUSCRIPT 2.16. Statistical Analysis
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Fermentations were carried out in triplicate and each analysis was repeated twice. Data were
347
subjected to one-way ANOVA; pair-comparison of treatment means was obtained by Tukey’s
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procedure at P
