Impact of sourdough on sensory properties and consumers' preference of gluten-free breads enriched with teff flour

Accepted Manuscript Impact of sourdough on sensory properties and consumers’ preference of gluten-free breads enriched with teff flour Eva Campo, Lis del Arco, Leyre Urtasun, Rosa Oria, Ana Ferrer-Mairal PII:

S0733-5210(15)30066-7

DOI:

10.1016/j.jcs.2015.09.010

Reference:

YJCRS 2047

To appear in:

Journal of Cereal Science

Received Date: 15 June 2015 Revised Date:

28 September 2015

Accepted Date: 30 September 2015

Please cite this article as: Campo, E., del Arco, L., Urtasun, L., Oria, R., Ferrer-Mairal, A., Impact of sourdough on sensory properties and consumers’ preference of gluten-free breads enriched with teff flour, Journal of Cereal Science (2015), doi: 10.1016/j.jcs.2015.09.010. 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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Impact of sourdough on sensory properties and consumers’ preference

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of gluten-free breads enriched with teff flour

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Plant Food Research Laboratory, Faculty of Veterinary, University of Zaragoza C/Miguel Servet 177, CP: 50013, Zaragoza (Spain)

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Eva Campo, Lis del Arco, Leyre Urtasun, Rosa Oria & Ana Ferrer-Mairal*

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Phone: +34 976-761584

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Fax: + 34 976 76 15 90

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Email: [email protected]

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RUNNING TITLE: Sensory profile of enriched GF breads

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ACCEPTED MANUSCRIPT Abstract

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This work studies the influence of the addition of teff flour (5, 10 and 20 %) and

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different dried (buckwheat or rice) or fresh (with Lb. helveticus) sourdoughs on the

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sensory quality and consumer preference of GF breads. A set of 10 GF breads

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combining these ingredients was submitted to sensory descriptive analysis performed

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by a trained panel. The four breads with the most promising sensory profile were

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evaluated by celiac consumers to look for attributes driving product acceptability.

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The combination of teff (10 %) with cereal sourdough (rice or buckwheat) enhanced

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bread aroma, increasing the fruity, cereal and toasty notes. High levels of teff (20%)

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and Lb. Helveticus sourdough induced a decrease on the loaf area. The visual

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appearance of breads with 20 % teff was highly appreciated by consumers, while bread

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combining 10 % teff and rice sourdough was preferred in terms of flavour. The bitter

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taste of buckwheat sourdough was generally considered as a negative attribute.

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However, a group of consumers liked bitter bread as they associated it to a traditional,

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artisan, “malty-like” product. This work highlights the great potential of combining teff

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and selected sourdoughs to obtain GF breads with target attributes and improved

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sensory profile.

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Keywords: Gluten-free bread, teff, sourdough, sensory properties

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1. INTRODUCTION Recent studies have stated the need and opportunity for the improvement of sensory

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properties in gluten free breads designed to celiac population. Celiac disease is an

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immune-mediated enteropathy triggered by the ingestion of gluten in genetically

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susceptible individuals, and it affects 1% of the world population (Catasi and Fasano,

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2008). Nowadays, the exclusion of gluten from the diet is the only effective treatment

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for celiac disease. Among celiac consumers the sensory characteristics of gluten free-

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bread has been reported as the most important variables considered for purchase

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decision (Do Nascimento et al., 2014). GF breads are generally described as products

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with flat appearance, pale crust, crumbly texture, high staling rate and bland flavour

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(O’Shea et al., 2014).

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The use of sourdough has been described as a useful tool to improve the quality of

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baked products due to the superior quality and prolonged shelf-life of the resulting

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products (Hammes and Ganzel, 1998). This aspect gains special importance in the case

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of gluten-free bakery products, for which fermentation with sourdough has been shown

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to improve overall bread quality, enhancing the textural properties and prolonging shelf

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life (Moroni et al., 2009; Schober et al., 2007; Wolter et al., 2014; Moore et al., 2008).

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The positive contribution of sourdough could be exploited for the design of high quality

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GF bread from various GF cereals and pseudocereals. For example, the effect of adding

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fresh and freeze-dried amaranth and buckwheat sourdoughs in gluten free bread

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production has been recently explored by Rozylo and co-workers (2015a; 2015b). The

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authors conclude that moderate additions of freeze-dried sourdoughs from these

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ingredients may be a good option to substitute the fresh sourdough. Besides, sourdough

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fermentation also seems to be a promising approach to improve the aroma quality of GF

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breads, as breads made from different GF flours exhibit a flat and undesirable aroma

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ACCEPTED MANUSCRIPT (Hager et al., 2012). Several studies demonstrated that selected lactic acid bacteria

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strains are able to generate very specific aroma profiles and odorant compositions

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(Czerny and Schieberle, 2002; Wolter et al. 2014). Some other bacteria such as

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Lactobacillus helveticus, have been recently evaluated as starters cultures for sourdough

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breadmaking, showing promising results (Plessas et al., 2008).

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Teff (Eragrostis tef) flour has been studied as a valuable ingredient to improve the

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quality of GF products in several studies, mainly due to its superior nutritional quality.

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Teff is rich in carbohydrates, fibre and has a complete set of essential amino acids. Teff

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is also particularly high in iron and has more calcium, copper and zinc than other cereal

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grains (Abebe et al., 2007). The technological properties of teff indicate that there is a

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great potential to be used in different food applications, particularly in baked products.

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However, the level of addition of this cereal to the blend can be critical from a sensory

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viewpoint. Several authors (Ben-Fayed et al., 2008; Mohammed et al., 2009; Alaunyte

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et al., 2012) have stated that addition levels between 5% and 10% are accepted by

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consumers, whereas levels between 20% and 30% are less appreciated, mainly as a

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consequence of the bitterness and undesirable aftertaste. With respect to the aroma

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properties, it has been pointed out that breads elaborated with a 95% of teff flour

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reduced the intensities of yeast, dough-like, malty and buttery notes, which have been

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reported as positive attributes in wheat crumb (Hager et al. 2012).

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The main aim of the present work is to study if the use of sourdough in combination

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with teff flour at different percentages could provide GF breads with improved sensory

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quality. For this purpose, several bread formulations combining teff flour (5%, 10% and

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10%) with different fresh and dried sourdoughs were evaluated by a trained sensory

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panel. Additionally, in order to identify attributes driving product preference, a

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consumer test of four selected bread formulations was carried by a group of celiac

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participants.

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2. MATERIALS AND METHODS

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2.1. Materials The raw materials common to all GF breads were rice flour (protein 7.5%,

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carbohydrates 66,7% and fat 3% from Nomen, Spain), maize starch (protein 0,26%,

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carbohydrates 76,5% and fat 0,05% content) from Tereos-Syral (Spain), as well as salt,

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sugar and olive oil purchased from a local supermarket. All formulations also included

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hydroxipropylmethycellulose (HPMC; metolose SFE 4000), supplied by ShinEtsu

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(Germany), which was characterized by a viscosity of 4000 mPas according to the

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manufacturer specifications.

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White teff flour (Baukhof, Germany) (protein 10%, carbohydrates 66,7% and fat 3.0%

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content) was sourced from Baukhof, (Germany).

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The dried sourdoughs, rice based (Bio Reis 25) and buckwheat based (Bio Buchweizen

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80) were sourced by Böcker (Germany).

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A dried culture of Lb. Helveticus (Abiasa, Spain) was used for the preparation of fresh

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sourdough. It was prepared by mixing flour (50% rice flour + 50 % maize starch) and

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water (1:1), 1% of dry yeast and 2% of Lb. Helveticus. An incubation period of 16 h at

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35 ºC and 80% relative humidity was applied as described in previous studies (Plessas

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et al., 2008).

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A commercial GF bread was used for reproducibility screening during sensory

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descriptive evaluation. The ingredients of this bread were maize starch, water, eggs,

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maize flour, sugar, vegetal oil, yeast, salt, jelling agent (xanthan and guar gum) and

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ascorbic acid as preserver according to the manufacturer label (Ricardera, Spain).

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2.2. Bread elaboration

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ACCEPTED MANUSCRIPT The formulas of gluten free breads (Table 1) were produced as previously described

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(Hager et al., 2012) with slight modifications. Standard GF bread (Control) was

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formulated using 100% flour (60% rice flour +40% maize starch), 3% yeast, 2% salt,

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2% olive oil, 0,75% HPMC and 90% water (based on flour, BF). Teff enriched breads

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were prepared substituting different percentages (5%, 10% and 20%) of the flour

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components with teff flour. Buckwheat and rice based sourdough breads (SDBu and

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SDRi) were elaborated by substituting 15 % of the flour component by the same

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percentage of each commercial dried sourdough and Lb. Helveticus sourdough (SDLh)

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breads by substituting 15% of the flour component by the prepared fresh sourdough.

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Combinations of teff (10%) and sourdough (15%, FB) were elaborated (Te-SDRi, Te-

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SDBu and Te-SDLh). For all the samples, ingredients were mixed in a high speed

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mixer (Titanium Chef KMC010, Kenwood, United Kingdom) for 1 min at speed 2 and

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for 1.5 min at speed 3. The dough was scaled into 50 g portions, placed in aluminium

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moulds and proofed at 30ºC and 85% relative humidity for 75 min in a fermentation

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chamber (Iverpan FC-22, Salva, Spain). The breads were allowed to cool for 1.5 hours

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at room temperature until different analysis were performed.

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2.3. Digital analysis of images Images were obtained using a digital camera (PANASONIC LUMIX DMC-FZ7).

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Samples were illuminated by two day light lamps (colour temperature 5400K), set at

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45º degree angles and placed at a distance of 32,5 cm. Images were obtained in a TIFF

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format and processed with the Matrox 8.0 (Matrox Electronic Systems Ltd, Canada).

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The parameters yielded were slice volume and crust and crumb characteristics.

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2.4. Sensory descriptive analysis

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2.4.1. Panel selection Fifteen candidates were recruited from staff and graduate students of the Food Science

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Faculty at the University of Zaragoza. They were all non-celiac participants that

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showed availability and commitment to attend the panel sessions over a period of two

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months. Their sensory aptitude (aroma recognition and identification of basic tastes)

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was determined according to ISO 8586-1 (1993).

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In the first session, 20 reference standards from general aroma families (floral, fruity,

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spicy…) and specific to bread flavour (yeasty, butter, cereal…) were provided to

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panellists. These were either from the collection “Le Nez du Vin, The Masterkit”, from

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Editions Jean Lenoir (Carnoux-en-Provence, France) or prepared from natural products.

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Candidates were asked to smell each sample and to choose one of the fourth proposed

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answers. Correct responses were summed up to assign a total score to each candidate.

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In the second session, panellists were asked to identify the taste of eight aqueous

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solutions containing basic taste compounds at two concentration levels (w/w): NaCl

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(0.15%, 0.25 %), saccharose (0.3%, 0.5%), caffeic acid (0.05%, 0.07%) and citric acid

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(0.1 %, 0.2 %). Two or one points, respectively, were assigned to correct responses

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obtained for taste recognition at the lowest and the highest concentrations. Scores were

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added to provide a global mark to each candidate. The final panel was formed by the

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eleven members with the highest scores, 5 men and 6 women ranging from 23 to 65

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years old (average =33).

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2.4.2. Panel training

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General training in non GF bread. Two 2-hour sessions were devoted to general

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training in bread evaluation. The aim of this general training was twofold: a) to explore

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and get familiar with the sensory space of a variety of regular commercial breads and b)

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ACCEPTED MANUSCRIPT to provide panellists a list of vocabulary with the most common attributes generally

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employed to describe breads. During both sessions, a total of 20 breads (10 per session)

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from very different styles and elaboration processes (baguette, ciabatta, flute, Vienna,

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rustic-style (“hogaza”), toast bread, rye bread, barley bread, multicereals, crackers-

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style, and white tin loaf) were presented to the panellists. This assortment provided a

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large and varied palette of stimulus easily recognisable with respect to aspect, flavour

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and texture. A list of terms (data not shown) compiled from previous works available in

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the literature (Heeman et al., 2008; Elia, 2011) were provided to the panellists. They

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were asked to examine the products with respect to the attributes present in the list, and

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to evaluate them in an intensity scale ranging from 0 (not perceived) to 10 (very

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intense). Individual and averaged scores were commented and discussed among

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panellists in order to point out those breads that better exhibited the attributes of the list

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and could be eventually employed as good examples/prototypes of target descriptors.

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Specific training in GF bread. Panellists participated in four 1-hour sessions of specific

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training in bread sensory analysis following the standard procedure described in ISO

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NORM 11035 (1994).

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Samples. The panel worked with an assortment of commercial and “laboratory-

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prepared” GF breads with marked sensory differences to achieve a broad representation

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of sensory attributes. Commercial breads were purchased in a local grocery store

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specialized in gluten-free products in Zaragoza (Spain). Non-commercial breads were

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prepared at the beginning of the day in the Pilot Plant of the University of Zaragoza.

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A quarter of each piece of bread (including crust and crumb) was presented (in

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randomized order) on a plastic plate encoded with a three digit number. Mineral water

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and plain crackers were available to clear the palate between samples.

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ACCEPTED MANUSCRIPT Procedure. In the first session, 12 GF breads were presented to generate the list of

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descriptors. This initial list (35 descriptors) was reduced by eliminating redundant terms

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or those evoking intensity. In session 2, panellists assessed six breads by rating the

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attributes of the list from 0 (not present) to 10 (very intense). Principal Component

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Analysis (PCA) was run on the mean sensory scores to look for correlations between

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terms. The attributes were then reduced to 22 after the elimination of non-pertinent

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terms and grouping of synonyms displayed close together in the PCA map. The final

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list is presented in Table 2. This includes descriptors related to the visual appearance,

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orto- and retronasal aroma, taste and textural properties, together with a definition and

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the reference standards used for training. During session 3, eight breads with

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distinguishing sensory features were presented. The aim of this session was to identify

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samples exhibiting maximum intensities of the selected terms on the list. In the case of

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disagreements, a discussion was organized until a consensus was reached. The fourth

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session was devoted to intensity rating training. Four GF breads were evaluated in

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duplicate by scoring the terms in the list from 0 (not present) to 10 (very intense). After

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completion of the session, judges could compare their individual scores with the

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average of the group and re-evaluate the breads to help in concept alignment. Panellists’

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performance was checked regarding the ability to discriminate between breads,

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reproducibility and homogeneity of the panel in scoring by univariate (Analysis of

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Variance (ANOVA) and multivariate statistical methods (Principal Component

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Analysis (PCA)) as detailed by Campo and co-workers (Campo et al. 2010). Based on

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these indicators, the panel was deemed successfully trained.

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2.4.3. Formal evaluation of GF breads

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ACCEPTED MANUSCRIPT Samples. Twelve breads were evaluated by the panel. Ten of them were prepared in the

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laboratory as described in the Bread Elaboration section. One GF commercial bread

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was evaluated in duplicate for reproducibility control purposes.

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Procedure. Panellists participated in three 1-hour sessions to evaluate the 12 samples (4

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per session). Breads were prepared and presented to panellists as previously explained.

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Each session was divided in two 30-min parts. First, panellists evaluated the non-visual

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properties under red light in a sensory laboratory equipped with individual testing

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booths and serving hatches. After this, panellists evaluated the visual properties in a

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meeting room under white light. Intensity ratings for each of the descriptive terms were

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scored using a 10-point scale ranging from 0 (not present) to 10 (very intense). Texture

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was evaluated from manual examination and mastication.

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Univariate analysis. A one way analysis of variance (ANOVA) in which bread was the

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factor and judges were considered as repetitions was performed on data derived from

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descriptive analysis. All analyses were performed with the software SPSS 15.0 (SPSS

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Inc., Chicago, IL, USA).

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Multivariate analysis. Standardized Principal Component Analysis (PCA) was

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performed on the mean ratings among the judges for significant terms derived from

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ANOVA and each type of bread (correlation matrix). All analyses were carried out with

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SPAD software (version 5.5, CISIA-CERESTIA, Montreuil, France).

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2.5.

Consumers’ preference test

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Participants. Celiac candidates were recruited through an informative mail from the

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“Aragonese Celiac Association”. Participants could be selected only if they were above

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14 year-old and had been diagnosed with celiac intolerance for, at least, one year. A

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group of 39 celiac consumers participated in the test; 23 women and 16 men between

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ACCEPTED MANUSCRIPT 15 and 70 year-old. In a screening questionnaire, 82 % stated they consumed bread

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daily, while the other 18 % consumed bread at least 2 or 3 times per week.

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Samples. A subset of four breads were selected to be assessed by consumers (control +

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3 breads) on the basis of descriptive data. Breads were prepared in the morning, and

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tasted during the afternoon. They were presented to consumers as previously explained.

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Ranking test. Following the standard procedure ISO 8587:2006 samples were presented

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simultaneously to the consumers. They were asked to rank them from “least liked”

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(left) to “most liked” according to both a) visual appearance and b) overall taste. They

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were also asked to provide a few terms that described the tasted products.

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Data analysis. Scores of 1 to 4 were assigned from the “least liked” to the “most liked”

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ranks of the breads, and added to obtain a total score for each bread (rank-sum). Data

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was analysed by Friedman’s test. If significance was observed, the least significance

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difference (LSD) test was run to establish differences between means.

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3. RESULTS AND DISCUSSION

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3.1.Sensory descriptive analysis

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3.1.1.

Univariate analysis

According to one-way ANOVA (Table 3), the following attributes varied significantly

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among breads: alveolar homogeneity, maize, cereal, toasty, butter, synthetic, fruity,

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sweetness, saltiness, sourness, bitterness, crumb elasticity, crumbliness, crustiness and

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hardness. These attributes were therefore useful in characterizing differences among the

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breads.

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Visual appearance. Control sample was perceived as the most homogeneous. The rest

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of samples varied in homogeneity, as reflected in Figure 1. The cross section images of

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the breads studied by digital image analysis (data not shown) also reflects the influence

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of the formulation on the slice volume, being the 20 % teff addition and the Lb.

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Helveticus sourdough the ingredients causing a major decrease on the loaf area.

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Aroma. Six attributes varied significantly either in the ortho- or retronasal way: maize,

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cereal, toasty, butter, synthetic and fruity. The other terms were not significantly

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different due to: a) judges rated these terms very low and/or b) the variability among

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judges intra samples was higher than the variability inter samples. A summary of the

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most important observations is listed below:

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Maize. Variations of this attribute were more important when evaluated retronasally

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than orthonasally, as indicated by the P-value (0.010 vs. <0.001, respectively). Breads

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with the highest intensities on this note were from rice and Lb. Helveticus sourdough

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(SDRi and SDLh).

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Cereal. Te-SDRi and Te-SDBu breads, containing rice and buckwheat, presented the

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highest intensities for this attribute, both in ortho- and retronasal perception.

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ACCEPTED MANUSCRIPT Toasty. Similarly to the cereal note, this attribute was most intense in the Te-SDRi and

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Te-SDBu formulations, especially retronasally.

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Butter. This was very intense on the commercial bread, being clearly perceived by all

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judges in both replicates. On the contrary, this attribute was not noticed in the breads

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prepared in the laboratory.

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Fruity. This term varied significantly among samples when perceived orthonasally

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(P=0.009), but not retronasally (P=0.196). This could be explained because the fruity

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aromas are associated to highly volatile molecules (mainly ethyl esters) that escape

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from the product and interact with the pituitary by orthonasal perception. The fruity

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aromas could be related to teff content, as suggested by the high intensities of 20 % Te.

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The combination of teff and rice dough could also act in a synergic way to boost the

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perception of the fruity note in this sample (10% Te-SDRi).

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Taste. Commercial breads were perceived as the most sweet and least salty. Rice and

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buckwheat doughs supplemented with teff (Te-SDRi and Te-SDBu) were perceived as

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the most bitter. The latter was also characterized by a high acidity, which is in

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accordance with the lowest pH of this sample (data not shown).

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Texture. Four attributes (elasticity, crumbliness, crustiness and hardness) varied

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significantly (P<0.001), suggesting a great influence of formulation. The main

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differences appeared between the laboratory made breads and the commercial one,

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which presented the lowest values for all these attributes. Major differences were

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observed for the sample with a 20% teff content; as it presented the highest value for

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crumb elasticity.

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3.1.2. Multivariate analysis (PCA)

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ACCEPTED MANUSCRIPT Principal Component Analysis (PCA) was run with all samples and significant terms

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derived from ANOVA. Aroma terms showing significance in both ortho- and retronasal

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mode (cereal, toasty and synthetic) were not included twice. On these cases, only the

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retronasal term was kept as it was more intensely perceived.

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The two replicates of the commercial sample were similarly described in independent

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tasting sessions, and were clustered together in the PCA plot (figure not included),

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which indicates a good global reproducibility of the panel. Commercial replicates were

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projected separately from the rest of the breads, mainly as a result of their strong butter

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and synthetic aromas. A second PCA was run without these samples (and without the

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butter term) in order to achieve a better interpretation of non-commercial bread

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properties. The projection of the sensory variables and samples on the PCA graph is

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presented in Figures 2a and 2b, respectively. The first component (38%) is mainly

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defined by the aroma and taste properties (highlighted in black and red, respectively);

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whereas the second dimension (28 %) underlined the textural properties (in blue). The

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third PC (10 % of explained variance) did not provide any additional information. High

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correlations appeared between the fruity, cereal and toasty aromas, and the sour and

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bitter attributes. Breads combining 10% teff with buckwheat and rice sourdough were

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the richest in these attributes. According to Figure 2b, 10% teff addition strongly

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influenced the global profile of the bread elaborated from cereal dough (buckwheat or

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rice). This can be inferred from the projection of samples without teff in the PCA graph,

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as breads SDBu and SDRi shifted from the almost zero coordinate of the first

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component, to the right side of the chart for samples with an additional 10 % teff

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content in the composition. The magnitude of the changes observed in the sensory

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profile supports the idea of a synergic effect between the teff and cereal (buckwheat or

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rice) dough. Nevertheless, the teff effect seems highly dependent on the dough

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ACCEPTED MANUSCRIPT employed as a raw ingredient. Looking Figure 2b into detail it can be seen that the

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same 10 % teff addition on the Lb. Helveticus dough did not induce any change with

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respect to the aroma or taste profile (no changes with respect to the projection of both

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samples onto the PC1 component were observed). These results can be explained by the

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fact that commercial dry sourdoughs are the result of several steps of fermentation,

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which involves a significant increase in the number and profile of bacteria and yeast.

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However, the addition of teff to the SDLh bread had a great impact on its textural

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properties. The SDLh sample was projected on the botton of PC2, whereas 10% Te-

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SDLh is projected on the top of PC2. These results show that SDLh breads

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supplemented with teff at 10 % increased on the perceived elasticity and alveolar

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homogeneity.

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3.2. Consumers’ preference test

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On the basis of descriptive results, four breads were selected to be assessed by

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consumers (control + three additional samples). Two of them (10% Te-SDBu and 10%

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Te-SDRi) presented large differences in the overall sensory profile with respect to the

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control, as displayed Figure 2b. The fourth sample (20% teff) was selected as it was

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projected half-way (with respect to PC1) between the control and the other two

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samples. The consumers test was performed by a group of 39 celiac consumers. Due to

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the difficulty to recruit celiac consumers, this is a number below the recommended by

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the standard norm ISO 8587:2006. Therefore, results will be taken as an exploratory

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approach in order to get a general idea of consumers’ perception about the presented

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products.

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Product preference was very different depending on the properties examined; visual

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appearance or overall taste. The control bread was significantly least attractive (P<0.05;

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ACCEPTED MANUSCRIPT score=55), although it was quite appreciated when evaluated in mouth (score=100).

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Many consumers defined it as the “most similar” to the regular bread they consume on

353

every day basis. Bread with 20% of teff was highly appreciated with respect to the

354

visual aspects (score=102), reaching a similar score to control bread and not differing

355

significantly from the most appreciated sample in mouth (10% Te-SDRi; score=109).

356

This means that additions of 20 % teff flour provide acceptable breads in terms of

357

visual properties and with good sensory attributes. This last observation is in

358

disagreement with results obtained by other authors (Mohammed et al., 2009) which

359

stated that this same percentage of teff flour employed as wheat flour replacement

360

resulted in breads with negative sensory characteristics. Sample 10% Te-SDRi was

361

more tasty (score=109) than appealing (score=66). On the contrary, the same addition

362

on buckwheat sourdough (10% Te-SDBu) was less liked in mouth (score=79) but much

363

liked visually (score=100). Consumers highlighted the appealing colour of this sample,

364

which reminded them of “cereal-like” or “traditional” breads. They also pointed out its

365

intense bitter flavor.

366

To examine taste data in more detail, the number of consumers that ranked each bread

367

in the fourth possible positions (from least liked to most liked) was compiled (Figure

368

3). Results show that around 50 % of consumers choose 10% Te-SDBu as the least

369

liked. However, it can be observed that there is a group of consumers (around 20%) that

370

really appreciated this bread. According to their written comments, they found this

371

sample complex in flavor, with an intense bitter taste that reminded them of

372

“malty/traditional/old-style” breads. Both trends - consumers either rejecting or loving

373

10% Te-SDBu - can be due to large differences on bitter sensitivity (Kalmus, 1971)

374

aspect that strongly determines acceptability (Glanville and Kaplan, 1965). So, even if

375

most of population did not like 10% Te-SDBu, there is still a percentage that actually

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ACCEPTED MANUSCRIPT enjoyed this kind of product and could represent a target consumer group. Acceptance

377

of novel products such 10% Te-SDBu, which is far from the general GF bread standard,

378

could increase through:

379

a) exposure: humans have an innate aversion to bitter tastes that can be overcome by

380

consumption and earned experience (Steiner, 1974). A key factor would be the time

381

since celiac diagnosis (from young child to elderly), and whether these individuals

382

enjoyed bitter taste before diagnosis.

383

b) health benefits information: several studies prove the enhanced liking of consumers

384

by baked, non GF products labelled as “healthy”, either by a rich fibre content (Baixauli

385

et al. 2008), or low cholesterol (Kihlberg et al. 2005). Make population aware of the

386

benefits of novel healthy ingredients in GF products could help in increasing their

387

acceptance.

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4. CONCLUSIONS

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This work provides meaningful information with respect to the sensory properties of

391

GF breads elaborated from teff flour in combination with different sourdoughs. Teff

392

had a great impact on the sensory profile that was highly dependent on the sourdough

393

used as a raw material. Teff addition to cereal-based sourdoughs (rice and buckwheat)

394

modified the aroma profile of the breads, increasing the fruity, toasty and cereal notes.

395

However, the same addition of teff to a fresh sourdough with Lb. Helveticus bread did

396

not change the aromatic profile, but the textural properties, increasing the perceived

397

elasticity. The combination of teff flour and selected sourdoughs therefore allows

398

elaborating GF breads with target sensory attributes, able to fulfil celiac consumer

399

desires and demands. Indeed, some of the breads presented large heterogeneity in visual

400

or in-mouth properties that were differently appreciated by consumers. This work also

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ACCEPTED MANUSCRIPT highlights some sensory attributes driving preference. Breads with a dark colour or with

402

a flavour similar to regularly consumed breads are highly appreciated, whereas bitter

403

samples are rejected by most of the consumers. However, and within the limited scope

404

of this study, bitter breads may have a specific target consumer segment that look for

405

“malty/traditional/old-style” products differing from regular GF breads, which may

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represent a new consumer’s trend in the GF market.

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Acknowledgments

409

This research was supported by the Department of Industry and Innovation from the

410

Aragon Government & European Social Fund (Project SGI 229367) & Universidad de

411

Zaragoza (Project JIUZ2-012-CIE-04). Authors are grateful to Böcker and to

412

Asociación Celiaca Aragonesa for their collaboration.

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Abebe, Y., Bogale, A., Hambidge, K.M., Stoecker, B.J., Bailey, K., Gibson, R.S., 2007.

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consumed in rural Sidama, Southern Ethiopia, 395 and implications for bioavailability.

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Alaunyte, .I, Stojceska, V., Plunkett, A., Ainsworth, P., Derbyshire, E., 2012.

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enzymes in straight dough and sourdough breadmaking. Journal of Cereal Science, 55,

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Campo, E., Ballester, J., Langlois, J., Dacremont, C., Valentin, D., 2010. Comparison

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of conventional descriptive analysis and a citation frequency-based descriptive method

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sourdough fermentation. Journal of Agricultural and Food Chemistry 50, 6835-6840.

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Elia, M., 2011. A procedure for sensory evaluation of bread: protocol developed by a

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trained panel. Journal of Sensory Studies 26, 269-277.

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Glanville, E.V., Kaplan AR., 1965. Food preference and sensitivity of taste for bitter

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compounds. Nature 27, 851-853.

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Investigation of product quality, sensory profile and ultrastructure of breads made from

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a range of commercial gluten-free flours compared to their wheat counterparts.

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Hammes, W.P., Ganzel, M.G., 1998. Sourdough breads and related products. In:Woods,

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199-216.

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sensory quality of fresh bread: Descriptive attributes and consumer perceptions. Food

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research international 41, 989-997.

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monitoring of assessors. Part 1: Selected assessors. International Organization for

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Standardization (Geneva, Switzerland).

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establishing a sensory profile by a multidimensional approach. International

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Organization for Standardization (Geneva, Switzerland).

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Kalmus, H. Genetics of taste. 1971. Handbook of sensory physiology. Springer Beidler,

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Berlin.

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Kihlberg, I., Johansson, L., Langsrud, O., Risvik, E., 2005. Effects of information on

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liking of bread. Food quality and preference 16, 25-35.

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Mohammed, M.I.O., Mustafa, A.I., Osman, G.A.M., 2009. Evaluation of wheat breads

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supplemented with teff Grain flour. Australian Journal of Crop Science 3, 207-212.

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Moore, M., Dal, B.F., Arendt, E., 2008. Sourdough fermented by Lactobacillus

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plantarum FST 1.7 improves the quality and shelf life of gluten-free bread. European

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Food Research and Technology 226, 1309–1316.

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Moroni, A.V., Dal Bello, F., Arendt, E.K., 2009. Sourdough in gluten-free bread

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making: an ancient technology to solve a novel issue? Food Microbiology 26, 676–684.

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O'Shea, N., Arendt, E., Gallagher, E., 2014. State of the art in gluten-free research.

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Journal of Food Science 79, R1067–R1076.

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Plessas, S., Fisher, A., Koureta, K., Psarianos, C., Nigam, P., Koutinas, A.A., 2008.

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Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus

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and L. helveticus for sourdough bread making. Food Chemistry 106, 985–990.

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Rózylo, R., Rudy, S., Krzykowski, A., Dziki, D., 2015a. Novel application of freeze-

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dried amaranth sourdough in gluten-free bread production. Journal of Food Process

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Engeneering 38, 135-143.

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Rózylo, R., Rudy, S., Krzykowski, A., Dziki, D., Gawlik-Dziki, U., Rozylo, K.,

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Skonecki, S., 2015b. Effect of adding fresh and freeze-dried buckwheat sourdough on

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gluten-free bread quality. International Journal of Food Science and Technology 50,

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313-322.

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Schober, T.J., Bean, S.R., Boyle, D.L., 2007. Gluten-free sorghum bread improved by

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sourdough fermentation: biochemical, rheological, and microstructural background.

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Journal of Agricultural and Food Chemistry 55, 5137–5146.

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Steiner, J.E., 1974. Innate, discriminative human facial expressions to taste and smell

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stimulation. Annals of the New York Academy of Sciences 237, 229-233.

515 Wolter, A., Hager, A.S., Zannini, E., Czerny, M., Arendt, E.K., 2014. Impact of

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sourdough fermented with Lactobacillus plantarum FST 1.7 on baking and sensory

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properties of gluten-free breads. European Food Research and Technology 239, 1-12.

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GF: gluten-free HPMC: hidroxypropylmethylcelullose BF: based on flour SDBu: buckwheat based sourdough SDRi: rice based sourdough SDLh: sourdough with Lactobacillus helveticus Te-SDBu: teff + buckwheat based sourdough Te-SDRi: teff + rice based sourdough Te-SDLh: teff + sourdough with Lactobacillus helveticus PCA: Principal component analysis ANOVA: Analysis of variance LSD: Least significance difference PC1: Principal component 1 PC2: Principal component 2 O-: Orthonasal R-: Retronasal

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List of abbreviations

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CLEAN VERSION

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Impact of sourdough on sensory properties and consumers’ preference

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of gluten-free breads enriched with teff flour

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Plant Food Research Laboratory, Faculty of Veterinary, University of Zaragoza C/Miguel Servet 177, CP: 50013, Zaragoza (Spain)

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Eva Campo, Lis del Arco, Leyre Urtasun, Rosa Oria & Ana Ferrer-Mairal*

550 (*) Corresponding author

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Phone: +34 976-761584

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Fax: + 34 976 76 15 90

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Email: [email protected]

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RUNNING TITLE: Sensory profile of enriched GF breads

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ACCEPTED MANUSCRIPT Abstract

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This work studies the influence of the addition of teff flour (5, 10 and 20 %) and

560

different dried (buckwheat or rice) or fresh (with Lb. helveticus) sourdoughs on the

561

sensory quality and consumer preference of GF breads. A set of 10 GF breads

562

combining these ingredients was submitted to sensory descriptive analysis performed

563

by a trained panel. The four breads with the most promising sensory profile were

564

evaluated by celiac consumers to look for attributes driving product acceptability.

565

The combination of teff (10 %) with cereal sourdough (rice or buckwheat) enhanced

566

bread aroma, increasing the fruity, cereal and toasty notes. High levels of teff (20%)

567

and Lb. Helveticus sourdough induced a decrease on the loaf area. The visual

568

appearance of breads with 20 % teff was highly appreciated by consumers, while bread

569

combining 10 % teff and rice sourdough was preferred in terms of flavour. The bitter

570

taste of buckwheat sourdough was generally considered as a negative attribute.

571

However, a group of consumers liked bitter bread as they associated it to a traditional,

572

artisan, “malty-like” product. This work highlights the great potential of combining teff

573

and selected sourdoughs to obtain GF breads with target attributes and improved

574

sensory profile.

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Keywords: Gluten-free bread, teff, sourdough, sensory properties

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ACCEPTED MANUSCRIPT 578

1. INTRODUCTION Recent studies have stated the need and opportunity for the improvement of sensory

580

properties in gluten free breads designed to celiac population. Celiac disease is an

581

immune-mediated enteropathy triggered by the ingestion of gluten in genetically

582

susceptible individuals, and it affects 1% of the world population (Catasi and Fasano,

583

2008). Nowadays, the exclusion of gluten from the diet is the only effective treatment

584

for celiac disease. Among celiac consumers the sensory characteristics of gluten free-

585

bread has been reported as the most important variables considered for purchase

586

decision (Do Nascimento et al., 2014). GF breads are generally described as products

587

with flat appearance, pale crust, crumbly texture, high staling rate and bland flavour

588

(O’Shea et al., 2014).

589

The use of sourdough has been described as a useful tool to improve the quality of

590

baked products due to the superior quality and prolonged shelf-life of the resulting

591

products (Hammes and Ganzel, 1998). This aspect gains special importance in the case

592

of gluten-free bakery products, for which fermentation with sourdough has been shown

593

to improve overall bread quality, enhancing the textural properties and prolonging shelf

594

life (Moroni et al., 2009; Schober et al., 2007; Wolter et al., 2014; Moore et al., 2008).

595

The positive contribution of sourdough could be exploited for the design of high quality

596

GF bread from various GF cereals and pseudocereals. For example, the effect of adding

597

fresh and freeze-dried amaranth and buckwheat sourdoughs in gluten free bread

598

production has been recently explored by Rozylo and co-workers (2015a; 2015b). The

599

authors conclude that moderate additions of freeze-dried sourdoughs from these

600

ingredients may be a good option to substitute the fresh sourdough. Besides, sourdough

601

fermentation also seems to be a promising approach to improve the aroma quality of GF

602

breads, as breads made from different GF flours exhibit a flat and undesirable aroma

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ACCEPTED MANUSCRIPT (Hager et al., 2012). Several studies demonstrated that selected lactic acid bacteria

604

strains are able to generate very specific aroma profiles and odorant compositions

605

(Czerny and Schieberle, 2002; Wolter et al. 2014). Some other bacteria such as

606

Lactobacillus helveticus, have been recently evaluated as starters cultures for sourdough

607

breadmaking, showing promising results (Plessas et al., 2008).

608

Teff (Eragrostis tef) flour has been studied as a valuable ingredient to improve the

609

quality of GF products in several studies, mainly due to its superior nutritional quality.

610

Teff is rich in carbohydrates, fibre and has a complete set of essential amino acids. Teff

611

is also particularly high in iron and has more calcium, copper and zinc than other cereal

612

grains (Abebe et al., 2007). The technological properties of teff indicate that there is a

613

great potential to be used in different food applications, particularly in baked products.

614

However, the level of addition of this cereal to the blend can be critical from a sensory

615

viewpoint. Several authors (Ben-Fayed et al., 2008; Mohammed et al., 2009; Alaunyte

616

et al., 2012) have stated that addition levels between 5% and 10% are accepted by

617

consumers, whereas levels between 20% and 30% are less appreciated, mainly as a

618

consequence of the bitterness and undesirable aftertaste. With respect to the aroma

619

properties, it has been pointed out that breads elaborated with a 95% of teff flour

620

reduced the intensities of yeast, dough-like, malty and buttery notes, which have been

621

reported as positive attributes in wheat crumb (Hager et al. 2012).

622

The main aim of the present work is to study if the use of sourdough in combination

623

with teff flour at different percentages could provide GF breads with improved sensory

624

quality. For this purpose, several bread formulations combining teff flour (5%, 10% and

625

10%) with different fresh and dried sourdoughs were evaluated by a trained sensory

626

panel. Additionally, in order to identify attributes driving product preference, a

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ACCEPTED MANUSCRIPT 627

consumer test of four selected bread formulations was carried by a group of celiac

628

participants.

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ACCEPTED MANUSCRIPT 630

2. MATERIALS AND METHODS

631

2.1. Materials The raw materials common to all GF breads were rice flour (protein 7.5%,

633

carbohydrates 66,7% and fat 3% from Nomen, Spain), maize starch (protein 0,26%,

634

carbohydrates 76,5% and fat 0,05% content) from Tereos-Syral (Spain), as well as salt,

635

sugar and olive oil purchased from a local supermarket. All formulations also included

636

hydroxipropylmethycellulose (HPMC; metolose SFE 4000), supplied by ShinEtsu

637

(Germany), which was characterized by a viscosity of 4000 mPas according to the

638

manufacturer specifications.

639

White teff flour (Baukhof, Germany) (protein 10%, carbohydrates 66,7% and fat 3.0%

640

content) was sourced from Baukhof, (Germany).

641

The dried sourdoughs, rice based (Bio Reis 25) and buckwheat based (Bio Buchweizen

642

80) were sourced by Böcker (Germany).

643

A dried culture of Lb. Helveticus (Abiasa, Spain) was used for the preparation of fresh

644

sourdough. It was prepared by mixing flour (50% rice flour + 50 % maize starch) and

645

water (1:1), 1% of dry yeast and 2% of Lb. Helveticus. An incubation period of 16 h at

646

35 ºC and 80% relative humidity was applied as described in previous studies (Plessas

647

et al., 2008).

648

A commercial GF bread was used for reproducibility screening during sensory

649

descriptive evaluation. The ingredients of this bread were maize starch, water, eggs,

650

maize flour, sugar, vegetal oil, yeast, salt, jelling agent (xanthan and guar gum) and

651

ascorbic acid as preserver according to the manufacturer label (Ricardera, Spain).

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2.2. Bread elaboration

30

ACCEPTED MANUSCRIPT The formulas of gluten free breads (Table 1) were produced as previously described

655

(Hager et al., 2012) with slight modifications. Standard GF bread (Control) was

656

formulated using 100% flour (60% rice flour +40% maize starch), 3% yeast, 2% salt,

657

2% olive oil, 0,75% HPMC and 90% water (based on flour, BF). Teff enriched breads

658

were prepared substituting different percentages (5%, 10% and 20%) of the flour

659

components with teff flour. Buckwheat and rice based sourdough breads (SDBu and

660

SDRi) were elaborated by substituting 15 % of the flour component by the same

661

percentage of each commercial dried sourdough and Lb. Helveticus sourdough (SDLh)

662

breads by substituting 15% of the flour component by the prepared fresh sourdough.

663

Combinations of teff (10%) and sourdough (15%, FB) were elaborated (Te-SDRi, Te-

664

SDBu and Te-SDLh). For all the samples, ingredients were mixed in a high speed

665

mixer (Titanium Chef KMC010, Kenwood, United Kingdom) for 1 min at speed 2 and

666

for 1.5 min at speed 3. The dough was scaled into 50 g portions, placed in aluminium

667

moulds and proofed at 30ºC and 85% relative humidity for 75 min in a fermentation

668

chamber (Iverpan FC-22, Salva, Spain). The breads were allowed to cool for 1.5 hours

669

at room temperature until different analysis were performed.

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2.3. Digital analysis of images Images were obtained using a digital camera (PANASONIC LUMIX DMC-FZ7).

673

Samples were illuminated by two day light lamps (colour temperature 5400K), set at

674

45º degree angles and placed at a distance of 32,5 cm. Images were obtained in a TIFF

675

format and processed with the Matrox 8.0 (Matrox Electronic Systems Ltd, Canada).

676

The parameters yielded were slice volume and crust and crumb characteristics.

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2.4. Sensory descriptive analysis

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2.4.1. Panel selection Fifteen candidates were recruited from staff and graduate students of the Food Science

681

Faculty at the University of Zaragoza. They were all non-celiac participants that

682

showed availability and commitment to attend the panel sessions over a period of two

683

months. Their sensory aptitude (aroma recognition and identification of basic tastes)

684

was determined according to ISO 8586-1 (1993).

685

In the first session, 20 reference standards from general aroma families (floral, fruity,

686

spicy…) and specific to bread flavour (yeasty, butter, cereal…) were provided to

687

panellists. These were either from the collection “Le Nez du Vin, The Masterkit”, from

688

Editions Jean Lenoir (Carnoux-en-Provence, France) or prepared from natural products.

689

Candidates were asked to smell each sample and to choose one of the fourth proposed

690

answers. Correct responses were summed up to assign a total score to each candidate.

691

In the second session, panellists were asked to identify the taste of eight aqueous

692

solutions containing basic taste compounds at two concentration levels (w/w): NaCl

693

(0.15%, 0.25 %), saccharose (0.3%, 0.5%), caffeic acid (0.05%, 0.07%) and citric acid

694

(0.1 %, 0.2 %). Two or one points, respectively, were assigned to correct responses

695

obtained for taste recognition at the lowest and the highest concentrations. Scores were

696

added to provide a global mark to each candidate. The final panel was formed by the

697

eleven members with the highest scores, 5 men and 6 women ranging from 23 to 65

698

years old (average =33).

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2.4.2. Panel training

701

General training in non GF bread. Two 2-hour sessions were devoted to general

702

training in bread evaluation. The aim of this general training was twofold: a) to explore

703

and get familiar with the sensory space of a variety of regular commercial breads and b)

32

ACCEPTED MANUSCRIPT to provide panellists a list of vocabulary with the most common attributes generally

705

employed to describe breads. During both sessions, a total of 20 breads (10 per session)

706

from very different styles and elaboration processes (baguette, ciabatta, flute, Vienna,

707

rustic-style (“hogaza”), toast bread, rye bread, barley bread, multicereals, crackers-

708

style, and white tin loaf) were presented to the panellists. This assortment provided a

709

large and varied palette of stimulus easily recognisable with respect to aspect, flavour

710

and texture. A list of terms (data not shown) compiled from previous works available in

711

the literature (Heeman et al., 2008; Elia, 2011) were provided to the panellists. They

712

were asked to examine the products with respect to the attributes present in the list, and

713

to evaluate them in an intensity scale ranging from 0 (not perceived) to 10 (very

714

intense). Individual and averaged scores were commented and discussed among

715

panellists in order to point out those breads that better exhibited the attributes of the list

716

and could be eventually employed as good examples/prototypes of target descriptors.

717

Specific training in GF bread. Panellists participated in four 1-hour sessions of specific

718

training in bread sensory analysis following the standard procedure described in ISO

719

NORM 11035 (1994).

720

Samples. The panel worked with an assortment of commercial and “laboratory-

721

prepared” GF breads with marked sensory differences to achieve a broad representation

722

of sensory attributes. Commercial breads were purchased in a local grocery store

723

specialized in gluten-free products in Zaragoza (Spain). Non-commercial breads were

724

prepared at the beginning of the day in the Pilot Plant of the University of Zaragoza.

725

A quarter of each piece of bread (including crust and crumb) was presented (in

726

randomized order) on a plastic plate encoded with a three digit number. Mineral water

727

and plain crackers were available to clear the palate between samples.

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ACCEPTED MANUSCRIPT Procedure. In the first session, 12 GF breads were presented to generate the list of

729

descriptors. This initial list (35 descriptors) was reduced by eliminating redundant terms

730

or those evoking intensity. In session 2, panellists assessed six breads by rating the

731

attributes of the list from 0 (not present) to 10 (very intense). Principal Component

732

Analysis (PCA) was run on the mean sensory scores to look for correlations between

733

terms. The attributes were then reduced to 22 after the elimination of non-pertinent

734

terms and grouping of synonyms displayed close together in the PCA map. The final

735

list is presented in Table 2. This includes descriptors related to the visual appearance,

736

orto- and retronasal aroma, taste and textural properties, together with a definition and

737

the reference standards used for training. During session 3, eight breads with

738

distinguishing sensory features were presented. The aim of this session was to identify

739

samples exhibiting maximum intensities of the selected terms on the list. In the case of

740

disagreements, a discussion was organized until a consensus was reached. The fourth

741

session was devoted to intensity rating training. Four GF breads were evaluated in

742

duplicate by scoring the terms in the list from 0 (not present) to 10 (very intense). After

743

completion of the session, judges could compare their individual scores with the

744

average of the group and re-evaluate the breads to help in concept alignment. Panellists’

745

performance was checked regarding the ability to discriminate between breads,

746

reproducibility and homogeneity of the panel in scoring by univariate (Analysis of

747

Variance (ANOVA) and multivariate statistical methods (Principal Component

748

Analysis (PCA)) as detailed by Campo and co-workers (Campo et al. 2010). Based on

749

these indicators, the panel was deemed successfully trained.

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2.4.3. Formal evaluation of GF breads

34

ACCEPTED MANUSCRIPT Samples. Twelve breads were evaluated by the panel. Ten of them were prepared in the

753

laboratory as described in the Bread Elaboration section. One GF commercial bread

754

was evaluated in duplicate for reproducibility control purposes.

755

Procedure. Panellists participated in three 1-hour sessions to evaluate the 12 samples (4

756

per session). Breads were prepared and presented to panellists as previously explained.

757

Each session was divided in two 30-min parts. First, panellists evaluated the non-visual

758

properties under red light in a sensory laboratory equipped with individual testing

759

booths and serving hatches. After this, panellists evaluated the visual properties in a

760

meeting room under white light. Intensity ratings for each of the descriptive terms were

761

scored using a 10-point scale ranging from 0 (not present) to 10 (very intense). Texture

762

was evaluated from manual examination and mastication.

763

Univariate analysis. A one way analysis of variance (ANOVA) in which bread was the

764

factor and judges were considered as repetitions was performed on data derived from

765

descriptive analysis. All analyses were performed with the software SPSS 15.0 (SPSS

766

Inc., Chicago, IL, USA).

767

Multivariate analysis. Standardized Principal Component Analysis (PCA) was

768

performed on the mean ratings among the judges for significant terms derived from

769

ANOVA and each type of bread (correlation matrix). All analyses were carried out with

770

SPAD software (version 5.5, CISIA-CERESTIA, Montreuil, France).

772

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2.5.

Consumers’ preference test

773

Participants. Celiac candidates were recruited through an informative mail from the

774

“Aragonese Celiac Association”. Participants could be selected only if they were above

775

14 year-old and had been diagnosed with celiac intolerance for, at least, one year. A

776

group of 39 celiac consumers participated in the test; 23 women and 16 men between

35

ACCEPTED MANUSCRIPT 15 and 70 year-old. In a screening questionnaire, 82 % stated they consumed bread

778

daily, while the other 18 % consumed bread at least 2 or 3 times per week.

779

Samples. A subset of four breads were selected to be assessed by consumers (control +

780

3 breads) on the basis of descriptive data. Breads were prepared in the morning, and

781

tasted during the afternoon. They were presented to consumers as previously explained.

782

Ranking test. Following the standard procedure ISO 8587:2006 samples were presented

783

simultaneously to the consumers. They were asked to rank them from “least liked”

784

(left) to “most liked” according to both a) visual appearance and b) overall taste. They

785

were also asked to provide a few terms that described the tasted products.

786

Data analysis. Scores of 1 to 4 were assigned from the “least liked” to the “most liked”

787

ranks of the breads, and added to obtain a total score for each bread (rank-sum). Data

788

was analysed by Friedman’s test. If significance was observed, the least significance

789

difference (LSD) test was run to establish differences between means.

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ACCEPTED MANUSCRIPT 791

3. RESULTS AND DISCUSSION

792

3.1.Sensory descriptive analysis

793

3.1.1.

Univariate analysis

According to one-way ANOVA (Table 3), the following attributes varied significantly

795

among breads: alveolar homogeneity, maize, cereal, toasty, butter, synthetic, fruity,

796

sweetness, saltiness, sourness, bitterness, crumb elasticity, crumbliness, crustiness and

797

hardness. These attributes were therefore useful in characterizing differences among the

798

breads.

799

Visual appearance. Control sample was perceived as the most homogeneous. The rest

800

of samples varied in homogeneity, as reflected in Figure 1. The cross section images of

801

the breads studied by digital image analysis (data not shown) also reflects the influence

802

of the formulation on the slice volume, being the 20 % teff addition and the Lb.

803

Helveticus sourdough the ingredients causing a major decrease on the loaf area.

804

Aroma. Six attributes varied significantly either in the ortho- or retronasal way: maize,

805

cereal, toasty, butter, synthetic and fruity. The other terms were not significantly

806

different due to: a) judges rated these terms very low and/or b) the variability among

807

judges intra samples was higher than the variability inter samples. A summary of the

808

most important observations is listed below:

809

Maize. Variations of this attribute were more important when evaluated retronasally

810

than orthonasally, as indicated by the P-value (0.010 vs. <0.001, respectively). Breads

811

with the highest intensities on this note were from rice and Lb. Helveticus sourdough

812

(SDRi and SDLh).

813

Cereal. Te-SDRi and Te-SDBu breads, containing rice and buckwheat, presented the

814

highest intensities for this attribute, both in ortho- and retronasal perception.

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37

ACCEPTED MANUSCRIPT Toasty. Similarly to the cereal note, this attribute was most intense in the Te-SDRi and

816

Te-SDBu formulations, especially retronasally.

817

Butter. This was very intense on the commercial bread, being clearly perceived by all

818

judges in both replicates. On the contrary, this attribute was not noticed in the breads

819

prepared in the laboratory.

820

Fruity. This term varied significantly among samples when perceived orthonasally

821

(P=0.009), but not retronasally (P=0.196). This could be explained because the fruity

822

aromas are associated to highly volatile molecules (mainly ethyl esters) that escape

823

from the product and interact with the pituitary by orthonasal perception. The fruity

824

aromas could be related to teff content, as suggested by the high intensities of 20 % Te.

825

The combination of teff and rice dough could also act in a synergic way to boost the

826

perception of the fruity note in this sample (10% Te-SDRi).

827

Taste. Commercial breads were perceived as the most sweet and least salty. Rice and

828

buckwheat doughs supplemented with teff (Te-SDRi and Te-SDBu) were perceived as

829

the most bitter. The latter was also characterized by a high acidity, which is in

830

accordance with the lowest pH of this sample (data not shown).

831

Texture. Four attributes (elasticity, crumbliness, crustiness and hardness) varied

832

significantly (P<0.001), suggesting a great influence of formulation. The main

833

differences appeared between the laboratory made breads and the commercial one,

834

which presented the lowest values for all these attributes. Major differences were

835

observed for the sample with a 20% teff content; as it presented the highest value for

836

crumb elasticity.

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837 838

3.1.2. Multivariate analysis (PCA)

38

ACCEPTED MANUSCRIPT Principal Component Analysis (PCA) was run with all samples and significant terms

840

derived from ANOVA. Aroma terms showing significance in both ortho- and retronasal

841

mode (cereal, toasty and synthetic) were not included twice. On these cases, only the

842

retronasal term was kept as it was more intensely perceived.

843

The two replicates of the commercial sample were similarly described in independent

844

tasting sessions, and were clustered together in the PCA plot (figure not included),

845

which indicates a good global reproducibility of the panel. Commercial replicates were

846

projected separately from the rest of the breads, mainly as a result of their strong butter

847

and synthetic aromas. A second PCA was run without these samples (and without the

848

butter term) in order to achieve a better interpretation of non-commercial bread

849

properties. The projection of the sensory variables and samples on the PCA graph is

850

presented in Figures 2a and 2b, respectively. The first component (38%) is mainly

851

defined by the aroma and taste properties (highlighted in black and red, respectively);

852

whereas the second dimension (28 %) underlined the textural properties (in blue). The

853

third PC (10 % of explained variance) did not provide any additional information. High

854

correlations appeared between the fruity, cereal and toasty aromas, and the sour and

855

bitter attributes. Breads combining 10% teff with buckwheat and rice sourdough were

856

the richest in these attributes. According to Figure 2b, 10% teff addition strongly

857

influenced the global profile of the bread elaborated from cereal dough (buckwheat or

858

rice). This can be inferred from the projection of samples without teff in the PCA graph,

859

as breads SDBu and SDRi shifted from the almost zero coordinate of the first

860

component, to the right side of the chart for samples with an additional 10 % teff

861

content in the composition. The magnitude of the changes observed in the sensory

862

profile supports the idea of a synergic effect between the teff and cereal (buckwheat or

863

rice) dough. Nevertheless, the teff effect seems highly dependent on the dough

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39

ACCEPTED MANUSCRIPT employed as a raw ingredient. Looking Figure 2b into detail it can be seen that the

865

same 10 % teff addition on the Lb. Helveticus dough did not induce any change with

866

respect to the aroma or taste profile (no changes with respect to the projection of both

867

samples onto the PC1 component were observed). These results can be explained by the

868

fact that commercial dry sourdoughs are the result of several steps of fermentation,

869

which involves a significant increase in the number and profile of bacteria and yeast.

870

However, the addition of teff to the SDLh bread had a great impact on its textural

871

properties. The SDLh sample was projected on the botton of PC2, whereas 10% Te-

872

SDLh is projected on the top of PC2. These results show that SDLh breads

873

supplemented with teff at 10 % increased on the perceived elasticity and alveolar

874

homogeneity.

875 876

3.2. Consumers’ preference test

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On the basis of descriptive results, four breads were selected to be assessed by

878

consumers (control + three additional samples). Two of them (10% Te-SDBu and 10%

879

Te-SDRi) presented large differences in the overall sensory profile with respect to the

880

control, as displayed Figure 2b. The fourth sample (20% teff) was selected as it was

881

projected half-way (with respect to PC1) between the control and the other two

882

samples. The consumers test was performed by a group of 39 celiac consumers. Due to

883

the difficulty to recruit celiac consumers, this is a number below the recommended by

884

the standard norm ISO 8587:2006. Therefore, results will be taken as an exploratory

885

approach in order to get a general idea of consumers’ perception about the presented

886

products.

887

Product preference was very different depending on the properties examined; visual

888

appearance or overall taste. The control bread was significantly least attractive (P<0.05;

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ACCEPTED MANUSCRIPT score=55), although it was quite appreciated when evaluated in mouth (score=100).

890

Many consumers defined it as the “most similar” to the regular bread they consume on

891

every day basis. Bread with 20% of teff was highly appreciated with respect to the

892

visual aspects (score=102), reaching a similar score to control bread and not differing

893

significantly from the most appreciated sample in mouth (10% Te-SDRi; score=109).

894

This means that additions of 20 % teff flour provide acceptable breads in terms of

895

visual properties and with good sensory attributes. This last observation is in

896

disagreement with results obtained by other authors (Mohammed et al., 2009) which

897

stated that this same percentage of teff flour employed as wheat flour replacement

898

resulted in breads with negative sensory characteristics. Sample 10% Te-SDRi was

899

more tasty (score=109) than appealing (score=66). On the contrary, the same addition

900

on buckwheat sourdough (10% Te-SDBu) was less liked in mouth (score=79) but much

901

liked visually (score=100). Consumers highlighted the appealing colour of this sample,

902

which reminded them of “cereal-like” or “traditional” breads. They also pointed out its

903

intense bitter flavor.

904

To examine taste data in more detail, the number of consumers that ranked each bread

905

in the fourth possible positions (from least liked to most liked) was compiled (Figure

906

3). Results show that around 50 % of consumers choose 10% Te-SDBu as the least

907

liked. However, it can be observed that there is a group of consumers (around 20%) that

908

really appreciated this bread. According to their written comments, they found this

909

sample complex in flavor, with an intense bitter taste that reminded them of

910

“malty/traditional/old-style” breads. Both trends - consumers either rejecting or loving

911

10% Te-SDBu - can be due to large differences on bitter sensitivity (Kalmus, 1971)

912

aspect that strongly determines acceptability (Glanville and Kaplan, 1965). So, even if

913

most of population did not like 10% Te-SDBu, there is still a percentage that actually

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41

ACCEPTED MANUSCRIPT enjoyed this kind of product and could represent a target consumer group. Acceptance

915

of novel products such 10% Te-SDBu, which is far from the general GF bread standard,

916

could increase through:

917

a) exposure: humans have an innate aversion to bitter tastes that can be overcome by

918

consumption and earned experience (Steiner, 1974). A key factor would be the time

919

since celiac diagnosis (from young child to elderly), and whether these individuals

920

enjoyed bitter taste before diagnosis.

921

b) health benefits information: several studies prove the enhanced liking of consumers

922

by baked, non GF products labelled as “healthy”, either by a rich fibre content (Baixauli

923

et al. 2008), or low cholesterol (Kihlberg et al. 2005). Make population aware of the

924

benefits of novel healthy ingredients in GF products could help in increasing their

925

acceptance.

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This work provides meaningful information with respect to the sensory properties of

929

GF breads elaborated from teff flour in combination with different sourdoughs. Teff

930

had a great impact on the sensory profile that was highly dependent on the sourdough

931

used as a raw material. Teff addition to cereal-based sourdoughs (rice and buckwheat)

932

modified the aroma profile of the breads, increasing the fruity, toasty and cereal notes.

933

However, the same addition of teff to a fresh sourdough with Lb. Helveticus bread did

934

not change the aromatic profile, but the textural properties, increasing the perceived

935

elasticity. The combination of teff flour and selected sourdoughs therefore allows

936

elaborating GF breads with target sensory attributes, able to fulfil celiac consumer

937

desires and demands. Indeed, some of the breads presented large heterogeneity in visual

938

or in-mouth properties that were differently appreciated by consumers. This work also

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ACCEPTED MANUSCRIPT highlights some sensory attributes driving preference. Breads with a dark colour or with

940

a flavour similar to regularly consumed breads are highly appreciated, whereas bitter

941

samples are rejected by most of the consumers. However, and within the limited scope

942

of this study, bitter breads may have a specific target consumer segment that look for

943

“malty/traditional/old-style” products differing from regular GF breads, which may

944

represent a new consumer’s trend in the GF market.

945

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Acknowledgments

947

This research was supported by the Department of Industry and Innovation from the

948

Aragon Government & European Social Fund (Project SGI 229367) & Universidad de

949

Zaragoza (Project JIUZ2-012-CIE-04). Authors are grateful to Böcker and to

950

Asociación Celiaca Aragonesa for their collaboration.

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ACCEPTED MANUSCRIPT 952

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Moore, M., Dal, B.F., Arendt, E., 2008. Sourdough fermented by Lactobacillus

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Food Research and Technology 226, 1309–1316.

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Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus

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Rózylo, R., Rudy, S., Krzykowski, A., Dziki, D., 2015a. Novel application of freeze-

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dried amaranth sourdough in gluten-free bread production. Journal of Food Process

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Rózylo, R., Rudy, S., Krzykowski, A., Dziki, D., Gawlik-Dziki, U., Rozylo, K.,

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Schober, T.J., Bean, S.R., Boyle, D.L., 2007. Gluten-free sorghum bread improved by

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sourdough fermentation: biochemical, rheological, and microstructural background.

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Journal of Agricultural and Food Chemistry 55, 5137–5146.

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Steiner, J.E., 1974. Innate, discriminative human facial expressions to taste and smell

1052

stimulation. Annals of the New York Academy of Sciences 237, 229-233.

1053 Wolter, A., Hager, A.S., Zannini, E., Czerny, M., Arendt, E.K., 2014. Impact of

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properties of gluten-free breads. European Food Research and Technology 239, 1-12.

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ACCEPTED MANUSCRIPT

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GF: gluten-free HPMC: hidroxypropylmethylcelullose BF: based on flour SDBu: buckwheat based sourdough SDRi: rice based sourdough SDLh: sourdough with Lactobacillus helveticus Te-SDBu: teff + buckwheat based sourdough Te-SDRi: teff + rice based sourdough Te-SDLh: teff + sourdough with Lactobacillus helveticus PCA: Principal component analysis ANOVA: Analysis of variance LSD: Least significance difference PC1: Principal component 1 PC2: Principal component 2 O-: Orthonasal R-: Retronasal

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List of abbreviations

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48

ACCEPTED MANUSCRIPT Table 1. Sample recipes (% based on flour, BF) % Flour basis

Control

Teff samples

SD samples

Teff/SD formulations

Sample code

Control

5%Teff

10%Teff

20%Teff

SDRi

SDBu

SDLh

Rice flour Maize flour Teff flour Rice SD Buckwheat SD Lb. Helveticus SD Yeast Salt Olive oil HPMC Water

60 40

57 38 5

54 36 10

48 32 20

51 34

51 34

51 34

15

3 2 2 0.75 90

3 2 2 0.75 90

3 2 2 0.75 90

3 2 2 0.75 90

3 2 2 0.75 90

10% Te-SDLh 45 30 10

15

15 3 2 2 0.75 90

3 2 2 0.75 90

15 3 2 2 0.75 90

3 2 2 0.75 90

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3 2 2 0.75 90

10% Te-SDBu 45 30 10

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10% Te-SDRi 45 30 10 15

1

ACCEPTED MANUSCRIPT

Table 2. Descriptive vocabulary, definitions and evaluation mode used by trained assessor to evaluate GF bread. Reference standard

Visual appearance Alveolar homogeneity

Regularity of the pores in the crumb surface

High: White tin loaf; Low: Ciabatta bread

Aroma Odour associated to cornmeal Odour associated to rice Odour associated to cereal derived products like malt Odour associated to bread after baking Fermented yeast-like odour Odour associated to lactic acid Odour associated to acetic acid Odour associated to croissant Odour associated to glue Odour associated to rubber Odour associated to fried oil Odour associated to fruits like apple or pineapple

Taste

Crumb elasticity Crumbliness Crustiness Hardness Adhesiveness

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EP

Texture

Taste sensation evoked by sugar Taste sensation evoked by salt (NaCl) Taste sensation evoked by acid, e.g., tartaric acid Taste sensation typical of tonic water (quinine)

Sample recovery after hand pressing Ease with which the sample is broken into smaller particles during hand manipulation Noise made in the first bite of the sample between the molars Force required to bite completely through sample placed between the incisors Force required to remove sample completely from the palate, using the tongue during consumption

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Sweetness Saltiness Sourness Bitterness

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Maize Rice Cereal Toasty Yeasty Lactic Acetic Butter Adhesive Synthetic Rancid Fruity

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Definition

Maize crackers “Biocentury” Rice crackers “Biocentury” Barley bread Crust of rustic-style bread Baker yeast “Levital” Solution (lactic) “Firmenich White vinegar (Diluted 1/10) Fresh made Parisian croissant Ethyl acetate (1 mL in a 60 mL glass flask) Xantana powder (1/2 coffee spoon in a 60 mL flask) E,E-2,4-nonadienal (100 µL in 60 mL glass flask) Blend of apple (2/3) and pinapple juice (1/3)

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Saccharose solution (0.5 %) NaCl solution (0.2 %) Citric acid (0.2 %) Caffeic acid solution (0.05 %)

High: Fresh-baked white bread / Low: Low temperature stored white bread High: Marbel cake: Low: fresh white tin loaf High: Maize crackers “Biocentury”; Low: fresh white tin loaf High: Rye bread; Low: white tin loaf High: Muffin “La Pasión”; Low: toasted bread “Ortiz”

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Table 3. Significance of the factor ‘‘bread formulation” according to one-way ANOVA (judges as repetitions). Different letters indicate the existence of a significant difference between samples (Duncan test, 5% confidence level).

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0.010 0.581 <0,001 <0,001 0.358 0.514 0.117 <0,001 0.173 0.029 0.078 0.009

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<0.001 0.968 <0.001 <0.001 0.661 0.507 0.070 <0.001 0.190 0.002 0.087 0.196

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Descriptor Visual appearance Alveolar homogeneity Orthonasal aroma O-maize O-rice O-cereal O-toasty O-yeasty O-lactic O-acetic O-butter O-adhesive O-synthetic O-rancid O-fruity Retronasal aroma R-maize R-rice R-cereal R-toasty R-yeasty R-lactic R-acetic R-butter R-adhesive R-synthetic R-rancid R-fruity Taste Sweetness Saltiness Sourness Bitterness Texture Crumb elasticity Crumblyness Crustiness Hardness Adhesiveness

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Figure 1. Cross-section images of breads elaborated in the laboratory.

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Control (control bread); 5% teff (control + 5% teff); 10% teff (control + 10% teff); 20% teff (control + 20% teff) SDRi (rice sourdough); SDBu (buckwheat sourdough); SDLh (sourdough with Lb. Helveticus) 10% Te-SDRi (SDRi + 10% teff); 10 % Te-SDBu (SDBu + 10% teff); 10% Te-SDLh (SDLh +10% teff)

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Figure 3. Sample distribution (in %) according to ranking position (from least to most liked) in the overall taste preference test.

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Teff added to rice and buckwheat sourdoughs increases the fruity and toasty notes Teff added to Lb. Helveticus increases the perceived elasticity

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Teff added to selected cereal sourdoughs provides distinguishing GF breads

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Bitterness was perceived as a negative feature by most of the celiac consumers

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However, a market seems to exist for traditional-like breads from these

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ingredients