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Comparative study of rice bran protein concentrate and egg albumin on gluten-free bread properties
Accepted Manuscript Comparative Study of Rice Bran Protein Concentrate and Egg Albumin on GlutenFree Bread Properties
Suphat Phongthai, Stefano D'Amico, Regine Schoenlechner, Saroat Rawdkuen PII:
S0733-5210(16)30297-1
DOI:
10.1016/j.jcs.2016.09.015
Reference:
YJCRS 2223
To appear in:
Journal of Cereal Science
Received Date:
27 May 2016
Revised Date:
19 September 2016
Accepted Date:
25 September 2016
Please cite this article as: Suphat Phongthai, Stefano D'Amico, Regine Schoenlechner, Saroat Rawdkuen, Comparative Study of Rice Bran Protein Concentrate and Egg Albumin on Gluten-Free Bread Properties, Journal of Cereal Science (2016), doi: 10.1016/j.jcs.2016.09.015
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ACCEPTED MANUSCRIPT Highlights: Specific volume and crumb porosity of GF bread was improved by rice bran protein
Rice bran protein concentrate possesses the desirable sensory attributes of GF bread
The 2% enrichment of rice bran protein concentrate in GF bread reduced staling rate
The advantages of rice bran protein concentrate over egg albumin were proposed
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Comparative Study of Rice Bran Protein Concentrate and Egg Albumin on
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Gluten-Free Bread Properties
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To be submitted to
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Journal of Cereal Science
Program of Food Technology, School of Agro-Industry, Mae Fah Luang University,
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Suphat Phongthaia, Stefano D'Amicob, Regine Schoenlechnerb, and Saroat Rawdkuena*
Chiang Rai 57100, Thailand
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Institute of Food Technology, Department of Food Science and Technology,
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BOKU - University of Natural Resources and Life Sciences, Vienna, Austria
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*To whom correspondence should be addressed. Tel: +66-53-916752. Fax: +66-53-916737. E-mail: [email protected]
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Abstract Gluten-free (GF) based products have been studying for several years especially for
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quality improvement by enriching with proteins. A common protein source is egg albumin,
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but it has limitation for using causes of allergenic character. So, aim of this study was to
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replace egg albumin by rice bran protein concentrates which is a non-allergy protein in order
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to improve the quality of GF bread. Rice bran, a by-product, was used as starting material for
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rice bran protein concentrate (RBPC) preparation under alkaline-acid extraction technique.
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The obtained RBPC was composed of 68.07±0.54% protein (dry basis). For baking trials, the
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addition of RBPC had strongly influenced the rheological properties, especially elastic
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modulus (G') of GF batters during oscillation, and the relative elasticity of final GF breads.
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Breads enriched with 2% RBPC, and a combination of 1% egg albumin and 1% RBPC had
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the highest specific volume (P<0.05). Additionally, crumb porosity and sensory attributes
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were improved. RBPC also showed higher efficacy to inhibit bread staling than egg albumin.
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This study suggested that RBPC could be used as a protein source for GF bread.
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Keywords: Egg albumin, gluten-free bread, rice bran protein, rheology
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1. Introduction Bread is one of the most popular baked products, consumed as staple food in many countries. The demand for bread is increasing rapidly in developing regions of the world such
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as South East Asia and Africa (Taylor and Rosell, 2015). Traditionally, it is made of wheat or
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other flours containing gluten. Gluten in wheat flour comprises of glutenin and gliadin that
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are responsible for dough structure in terms of elasticity and strength, and extensibility and
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viscosity, respectively (Kittisuban et al., 2014). However, gluten must be eliminated from the
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diet for consumer who suffers from celiac disease (CD). CD is an inflammatory disease of
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upper small intestine (duodenum, jejunum) which could lead to intestinal mucosal damage
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and malabsorption of several important nutrients, due to the toxicity of certain protein
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sequences in the gliadin fraction (Shin et al., 2010). The only effective treatment is to exclude
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gluten from the diet throughout the life-span. For this reason, several gluten-free products
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have been developed and produced for celiac patients. In case of gluten-free bread, the major
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problem for manufacturer is to improve the quality of bread from gluten-free ingredients.
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Apparently, many commercial gluten-free breads are made of refined flours or starches which
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have lower nutrition value compared to original wheat bread (Tsatsaragkou et al., 2014),
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moreover; other characteristics are often present like poor quality with poor color, low
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volume, and crumbly texture.
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In recent years, various plant-based flours and starches such as buckwheat flour (Wronkowska et al., 2013), cassava starch (Crockett et al., 2011), corn and potato starch
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(Ziobro et al., 2013), tapioca starch (Pongjaruvat et al., 2014), and rice flour (Cornejo and
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Rosell, 2015; Shin et al., 2010) have been used for gluten-free bread preparation. Rice flour is
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one of the most suitable cereal flours for the production of gluten-free products due to its
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unique attributes such as bland taste, white color, high digestibility, and hypoallergenic
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properties (Shin et al., 2010). In order to replace the missing gluten, many substances which
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plasma protein (Rodriguez Furlán et al., 2015), egg albumin (Schoenlechner et al., 2010), soy
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protein (Ziobro et al., 2013), but also dietary fibre, hydroxypropyl methylcellulose (HPMC;
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Phimolsiripol et al., 2012), yeast β-glucan (Kittisuban et al., 2014), or transglutaminase
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(Cornejo and Rosell, 2015).
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Proteins are typically incorporated to gluten-free systems to increase the elastic
modulus by cross-linking, to improve the perceived quality by enhancing Maillard browning
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and flavor, to improve structure by gelation and to support foaming (Crockett et al., 2011;
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Matos et al., 2014; Taylor et al., 2015). In addition, interaction between emulsifier and
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protein can also improve dough strength and allow better retention of CO2 (Demirkesen et al.,
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2010). Due to the low protein content of rice flour, supplementation with other protein
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sources is necessary for improvement of gluten-free bread quality. Egg albumin is an allergic
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protein, often used as protein source in baked products. Meanwhile, other proteins including
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whey protein isolates, bovine plasma protein, and lupine protein have been reported for their
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abilities to enhance desired properties of GF bread such as specific volume and cohesiveness
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(Ziobro et al, 2013; Kittisuban et al., 2014; Rodriguez Furlán et al., 2015). The incorporation
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of vegetable proteins such as soy and pea protein isolates in order to improve quality of rice
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based gluten free muffins was also investigated by Matos et al. (2014). Rice bran protein is an
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economical protein source and has attracted great attention for application in healthy and
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non-allergic foods due to its hypoallergenic properties. Furthermore, it possesses good
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functional properties including oil and water binding capacity, foaming and emulsifying
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ability (Fabian and Ju, 2011) that may be useful for the development of gluten-free bread.
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These properties offer a good potential of using rice bran protein as quality improver of
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gluten-free bread and has up to now not been investigated. The aim of this study was
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therefore to evaluate the effect of rice bran protein enrichment to gluten-free bread, in
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comparison to egg albumin. The influence on the rheological properties of the gluten-free
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batter was investigated as well as the final bread quality.
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2.1. Raw materials
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Organic rice bran (Thai Jasmine rice, KDML 105) was supplied by Urmatt Ltd.
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(Chiang Rai, Thailand). Rice flour was obtained from StroblCaj. NaturmuehleGesmb.H
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(Linz-Ebelsberg, Austria), egg albumin powder from Enthoven-BouwhuisEiproducten B.V.
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(Raalte, Netherlands), vegetable fat powder (REVEL*-BEP) from LodersCroklaan B.V.,
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Wormerveer, Netherlands. Hydroxypropyl methylcellulose (HPMC, Metolose® Shin-Etsu
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Chemical Co., Ltd., Tokyo, Japan) was donated by HARKE Pharma GmbH, Muelheiman der
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Ruhr, Germany. Instant yeast (S.I.Lesaffre, France) was purchased from the market. The
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emulsifier was a mixture of 3 partsdiacetyl tartaric acid ester of monoglycerides (DATEM,
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Panodan M2020, Danisco®Copenhegen, Denmark) and 5 parts distilled monoglyceride
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(DMG, Dimodan PH 10, NS/B, Danisco®Copenhegen, Denmark).
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2.2. Rice bran protein preparation
Organic rice bran was defatted by mixing rice bran with 95% ethanol (1:5, w/v), and
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stirring for 1 hr. The slurry was centrifuged (Avanti J-30I, Beckman Coulter, USA) at
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5,500xg for 5 min. The precipitate was collected and extracted a second time. The final rice
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bran fraction was dried overnight in an oven at 30°C, and then passed through a 50-mesh
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sieve. Defatted rice bran (DFRB, 14.13±0.07% protein) was kept in aluminum foil zip lock
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bag at -18°C.DFRB was dispersed in distilled water (1:10, w/v), the pH adjusted to 10 using
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3M sodium carbonate. The mixture was stirred using a pilot-scale stirrer (DT-50, FRYMA-
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Maschinen AG, Switzerland) at a temperature of 50°C for 1 hr. After centrifugation (KB 6-
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collected and adjusted to pH 4.5 using 3M citric acid and centrifuged at the same conditions.
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The precipitate was adjusted to pH 7.0 and then freeze dried. The dried powder referred to
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rice bran protein concentrate (RBPC).
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2.3. Proximate analyses of RBPC
RBPC was analyzed for moisture, ash, and protein content by AOAC Official Method (2000). Dietary fibre including total, insoluble and soluble dietary fibre were determined
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following the standard method of AACC No.32-07 (2000) (Megazyme test kit, Megazyme
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International Ireland Ltd., Wicklow, Ireland).Starch content was determined by using the
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standard method of AACC No.76-13.01 (2000) (Megazyme test kit, Megazyme International
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Ireland Ltd., Wicklow, Ireland).
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2.4. Preparation of rice flour-based gluten-free bread (GF) The basic GF bread recipe was based on the study of Phimolsiripol et al. (2012) with
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some modifications, consisting of 100 g rice flour, 1 g HPMC, 1 g emulsifier, 1 g vegetable
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fat powder, 2 g salt, 3 g yeast and105 g water. Protein source was either RBPC or egg
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albumin and were incorporated at 2 or 4% (based on flour). Additionally, its mixtures (1%
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egg albumin + 1% RBPC; 2% egg albumin + 2% RBPC) were also studied. The enrichment
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with RBPC was based on protein content. Control bread was produced without any protein
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addition. In sum, this experimental design resulted in 7 different recipes.
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Rice flour and all dry ingredients (HPMC, emulsifier, vegetable fat powder, and salt)
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were homogenized in a mixer (RN10/VL2 Planetar mixer, A/S Wodschow & Co., Denmark)
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for 1 minute (speed 2). Then dry instant yeast was added, followed by addition of water (25 ±
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0.5°C) within one minute. The mixing was continued for 6 min at speed 2. Afterwards the 6/22
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fermented at 30°C, 85% RH for 30 min in a fermentation chamber (G66W, MANZ
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Backtechnik GmbH, Creglingen, Germany),then divided into two portions (400g each),
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which were proofed at 30 °C, 85% RH for another 30 min. After that, breads were baked at
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180°C for 50 min (60/rW, MANZ Backtechnik GmbH, Creglingen, Germany). The final
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bread was cooled at room temperature for 2 hr before bread quality determinations were
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carried out. The baking trials with 7 different recipes were done in duplicate, resulting in 4
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loaves of each recipe were prepared.
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2.5. Dynamic rheological measurement
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Viscoelastic properties of GF batter enriched with RBPC or egg albumin were
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performed following the method of Pongjaruvat et al. (2014) with some modifications, using
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a rheometer (KNX2100, Malvern Instruments Limited, UK) equipped with plate-plate
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geometry (20 mm in diameter) and a gap size of 2 mm. Each batter was investigated without
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the addition of yeast. After sample loading, the excess batter was carefully removed with a
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spatula. First, strain sweep experiments between 0.1 and 100% were run at a fixed frequency
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of 1 Hz in order to determine the linear viscoelastic region (LVER). Afterwards, frequency
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sweep experiments between 0.01 and 10 Hz were conducted within LEVR at a constant strain
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of 0.5 %. All measurements were performed in duplicate at 25°C.
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2.6. Bread quality determinations The specific volume of each loaf of bread was determined by the rapeseed
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displacement method according to the AACC Approved Method 55-50 (AACC, 2000).
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Specific volume was calculated as cm3/g bread. Percentage of weight loss for each loaf was
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calculated by dividing the GF bread weight with GF batter weight, multiplying by 100. All
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measurements were done in duplicate, giving 8 values for each recipe.
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Crumb firmness and relative elasticity were determined using a Texture analyzer (TAXT2i, Stable Micro SystemTM Co., Godalming, Surrey, UK). A 5-kg load cell with an SMS
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100 mm diameter compression probe (P100) was used. Each loaf of bread was sliced into
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cubes with dimensions of 4 × 4 × 3 cm (L × W × H) from the center part using a sharp saw to
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prevent structure damage. The cube was subjected to an uni-axial compression test (30%
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compression) followed by a relaxation phase for 120s. The maximum force (Fmax) of
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compression was considered as crumb firmness. The relative elasticity (REL, %) was
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calculated by dividing the residual force (Fres) at the end of the holding time by the maximum
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force, multiplying with 100. Two measurements of each loaf of bread were done, giving 8
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values for each recipe.
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Colour of crust and crumb were measured by using a DigiEye System (VeriVide Limited, UK). The instrument was calibrated with a colour calibration chart before
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determination. The controlled illumination cabinet was used to take high resolution images of
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the bread crust and crumb. The colour parameters (L*, a*, and b*) were interpreted according
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to the CIELAB definition by DigiPix software. The measurements were done in duplicate,
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giving 8 values for each recipe.
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Photos of sliced GF breads were taken using digital camera (Nikon D90, Nikon Corporation, Thailand) within the DigiEye System. The crumb bread structure of each recipe
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was analyzed using ImageJ software (https://rsb.info.nih.gov/ij/). Each image was cropped
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into 20×20 mm (actual scale) at the center of bread, and then converted to 16-bit grayscale.
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The results were evaluated with respect to number of pores, average pore size (mm), pore
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size uniformity, and area of gas cell (%). The pore size uniformity was interpreted from the
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standard deviation (SD) of the average pore size (the lower SD of pore size, the higher
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uniformity). All measurements were done in duplicate, giving 8 values for each recipe.
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2.7. Sensory evaluation and shelf life of GF breads
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Four selected GF breads, which had desired qualities, were studied for sensory
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properties and shelf life. Six attributes of GF breads including appearance, colour, smell,
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taste, texture, and overall liking were evaluated by a panel of 8 trained persons using
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quantitative descriptive analysis.
For shelf life testing, GF breads were packed in paper bags and stored under
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controlled conditions (20°C, 50% RH) for 9 days. At day 1, 3, 5, 7 and 9, crumb firmness of
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the samples was determined. The staling rate of GF breads was evaluated by using Avrami
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equation (1) described by Armero and Collar (1998). The two unknown parameters (k and n)
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were estimated by fitting the experimental data into the equation model using a non-linear
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regression method.
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where T0 is the crumb firmness at fresh bread, Tt is the crumb firmness at ‘t’ time, T∞ is the final crumb firmness, k is the constant rate of bread staling, and n is the Avrami
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exponent, which is a characteristic related to the type of crystals growth.
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(1)
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(T∞ - Tt) / (T∞ - T0) = exp(-ktn)
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2.8. Statistical analysis All experimental results were expressed as means ± SD, and statistically analyzed by
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analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) using SPSS
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statistic software. Statistical significance was accepted at a level of P<0.05. 9/22
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3. Results and discussion
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3.1 Proximate composition of RBPC The pilot scale gained RBPC showed a yield of 7.44 g/100g based on DFRB weight.
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Compared to lab scale extraction the used pilot scale procedure gained much higher yields,
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1.6-2.7 folds more protein (Chittapalo et al., 2009). The main composition of RBPC was
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protein with 68.07±0.54%dm, which can be labeled as protein concentrate due to the protein
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content of more than 60%. The moisture, starch, and ash contents were 6.85±0.26%dm,
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6.37±0.18%dm, and 3.08±0.23%dm, respectively. The total dietary fibre including soluble
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(6.37±0.45%) and insoluble dietary fibre (0.55±0.02%) was 6.91±0.46%. RBPC was not a
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100%-purified protein; some other components were co-extracted due to their solubility
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under alkaline conditions. Especially dietary fibres like arabinoxylans are commonly
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extracted at high pH levels (Mansberger et al., 2014). Because of hydrolysis also remarkable
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amounts of starch were extracted. These impurities may have some effects on GF batter and
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bread properties, especially dietary fibre and starch have been reported to have positive
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effects on GF breads by Phimolsiripol et al. (2012) and Pongjaruvat et al. (2014). Since these
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polymers have no negative impact on gluten-free bread, further purification by amylases or
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xylanases was not carried out due to economic aspects. In terms of the feasibility to produce
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RBPC in an industrial scale, allowing proteins precipitate after pH adjustment under cold
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condition instead of centrifugation could be possible to reduce the cost of operation and
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power consumption, in addition, to assemble the centrifuge machine was much complicated.
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However, the product yield may be less than the yield reported in this study.
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3.2 Effect of RBPC and egg albumin on viscoelastic properties of GF batter
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Rheological behavior of GF batters enriched with RBPC and egg albumin was examined by a rheometer in the oscillation mode. Viscoelastic properties of GF batters are
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shown in Fig. 1 in term of their strain sweep (a) and frequency sweep (b) response. The
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enrichment of proteins affected the LVER. The length of the LVER was used to determine
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the stability of a sample's structure, since structural properties are strongly related to
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elasticity. The results revealed that LVER of GF batters enriched with RBPC was shorter
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than that of enriched with egg albumin and was extended with increasing amount of egg
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albumin, indicating higher elasticity and resistance of GF batters to shear strain. At high
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strain (more than 1%), a reduction of G' (beginning of non-linear curve) was observed in the
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egg albumin enriched batters, relating to a breakdown of the dough network (Lazaridou et al.,
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2007). Compared to the control recipe (no protein), the elastic modulus or shear modulus (G')
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of GF batters was improved by the incorporation of both, RBPC and egg albumin. With
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RBPC, the elastic properties of GF batters were not affected at strains below 0.5%, at higher
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shear forces dough structure was damaged and G' decreased. However, egg albumin retained
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higher elasticity of GF batters than RBPC at applied strain over 0.5%.
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The frequency sweep test (Fig. 1b) was used as measurement of viscoelastic response that performs in LVER, where the structure integrity in the material is not affected. This
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result provides a micro-structural fingerprint of a material including viscoelastic properties of
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solid, gel like structure and viscoelastic properties of liquid. It was clearly seen that the G' of
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all GF batters slightly increased with higher frequencies in the whole range from 0.01 to 10
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Hz which matched the viscoelastic solid behaviour. In addition, the tanδ or loss tangent (data
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not shown), which is a viscous modulus to elastic modulus ratio (G''/G'), of GF batters was
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lower than 1 (higher elasticity than viscous), confirming these GF batters were more solid-
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like material than liquid-like material. Furthermore, it was also found that the enrichment
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with higher percentage of RBPC and egg albumin reduced the tanδ by increasing G', which
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could happen due to protein aggregation within the medium (Crokett et al., 2011).
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The enrichment of 4% RBPC showed highest G' followed by 2%RBPC, whereas egg albumin increased G' only to a small degree. The modification of the elastic property of GF
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batters by addition of proteins was previously noted by Crokett et al. (2011), Ziobro et al.
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(2013), and Matos et al. (2014) who applied soy and pea proteins to GF batters based on rice
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flour, and a mixture of corn and potato starch. However, there was no difference of G' for GF
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batters enriched with 2% and 4% egg albumin, indicating the maximum effect on dough
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properties was reached already by 2% enrichment.
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So to conclude shortly, the enrichment of combined proteins from RBPC and egg
albumin increased slightly the value of G', but it stabilized the GF batters structure greatly
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with respect to LVER. This observation confirmed that the major positive effect on batter
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elasticity related to the function of egg albumin.
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3.3 Effects of RBPC and egg albumin on GF bread quality
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3.3.1 Specific volume
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The effects of RBPC and egg albumin on specific volume, weight loss and texture of
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GF breads are shown in Table 1. The enrichment with 2% RBPC and the combination of 1%
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egg albumin and 1% RBPC into GF bread were comparable, they provided the highest
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specific volume among 7 recipes of GF breads (P<0.05). These enrichments could improve
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the specific volume of the control (0% protein) for about 11%. Also the use of 2% egg
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albumin improved the specific volume of GF bread. The increase of specific volume could be
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attributed to the levels of enriched proteins, because the amounts of other ingredients were
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fixed. During baking, the protein unfolds and protein-protein interactions as well as
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interactions with other ingredients of the batter formulation may occur, leading to improved
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emulsification property of proteins were prerequisite for structure forming, thus supporting
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starch and hydrocolloids in batter system (Ziobroet al., 2013). A similar result was reported
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by Rodriguez Furlán et al. (2015), the incorporation of bovine plasma protein with maximum
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level of 3.5% increased the volume of gluten-free bread significantly. In contrast, the result
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showed that the enrichment with RBPC or egg albumin at 4% had no or a negative effect on
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specific volume of GF bread. The lowest specific volume was found with 4% RBPC
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enrichment. It may be due to too high concentration of protein, which might provide high
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resistance and consistency of GF batters, resulting in a limited elasticity and less expansion
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during proofing. Also Mezaize et al. (2009) reported that a supplementation of 5% whey
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protein in rice starch based gluten-free bread caused a reduction of bread specific volume.
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Similarly, the enrichment with high levels of albumin, casein, and soybean protein exhibited
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low specific volume of GF breads (Macro and Rosell, 2008; Storck et al., 2013).
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Additionally, the fixed water content of 105 % could be responsible for the low volume of
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recipes with high protein enrichment, because of high water absorption of proteins. However,
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compared to the water uptake of hydrocolloids like HPMC this effect should be quite low and
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thus was not considered in the experimental design.
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3.3.2 Weight loss and crumb texture Weight losses of GF breads were minimized by incorporation of RBPC and egg
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albumin into the control GF-recipe (Table 1). The GF breads enriched with 4% RBPC and the
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combination of 2% egg albumin and 2% RBPC were the best recipes that could keep weight
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loss low during baking (P<0.05). The control recipe exhibited the highest weight loss, due to
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the lack of protein molecules that can hold the water within its own structure. The result
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agreed with those reported by Jiamyangyuen et al. (2004), who found that enrichment with
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loss. Except for the protein, the fibre in RBPC might be another factor that minimizes water
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loss. Fibres have high water holding capacity, as they form a porous structure composed of
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polysaccharide chains, which can hold high amount of water through hydrogen bonds or
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within their capillary structures through surface absorption (Schleißinger et al., 2013).
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Crumb firmness of control GF-bread had the lowest values (P<0.05) (Table 1). These values tended to increase with increasing the amounts of RBPC or egg albumin. The
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enrichment with 2% RBPC, 2% egg albumin, and a combination of 1% RBPC and 1% egg
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albumin into GF breads displayed comparable crumb firmness of about 14-15 N. Obviously,
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the enrichment with the excessive amounts of proteins (4%) influenced crumb firmness. This
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may be caused by high water absorptive properties of proteins, which lead to a finer and
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denser crumb structure (Kittisuban et al., 2014). Additionally, an increase of crumb firmness
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upon enrichment of proteins in GF breads might be attributed to the thickening of the gas cell
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walls within the bread crumb (Rodriguez Furlán et al., 2015). Phimolsiripol et al. (2012)
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indicated that 2% enrichment of egg albumin was sufficient for gluten-free rice bread,
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whereas further protein enrichment obviously adversely increased crumb firmness.
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Schoenlechner et al. (2010) and Crockett et al. (2011) also reported that 4% egg albumin and
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3% soy protein isolates (SPI) resulted in an increase of crumb firmness. Moreover,
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Demirkesen et al. (2010) suggested that higher modulus values of dough samples resulted in
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lower firmness values of bread samples, which was in accordance with this study.
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The relative elasticity (REL) of GF breads is an important criterion for consumer
355
acceptance, and is presented in Table 1. The enrichment with egg albumin showed significant
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positive influences on REL of GF breads. The enrichment with 2% and 4% egg albumin
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provided the highest REL (62-63%), which was about 18.86% higher than the control recipe.
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The combinations of RBPC and egg albumin (2% and 4%) also increased the REL of the GF 14/22
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360
in agreement with the study of Phimolsiripol et al. (2012) that previously mentioned the role
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of egg albumin on improvement of REL. However, GF bread enriched with 4% RBPC had a
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negative effect on REL, exhibiting the lowest value of 50.09%. Obviously, this low value of
363
elasticity in GF bread enriched with 4% RBPC was responsible for its low specific volume.
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The GF batter must be sufficiently elastic to allow the bubbles to expand during baking
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(Tsatsaragkou et al., 2014). As a result of rheological behavior of the GF batters, it could be
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indicated that the higher elasticity (G') and stability (longer LVER) of GF batters enriched
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with egg albumin went along with the higher elasticity of final GF bread.
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3.3.3 Crumb porosity
The crumb porosity of GF breads which was characterized by digital image analysis is
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presented in Table 1. The number of pores in GF breads crumb was found to be in the same
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range between 28 and 33.The smallest average pore size of GF bread crumb belonged to the
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recipe added with 4% egg albumin (P<0.05). The reduction of average pore size is probably
374
due to a structure disruption by the increased egg albumin content, providing impairment in
375
gas retention (Schoenlechner et al., 2010). In contrast, the pore size seemed to be increased
376
by addition of 2% RBPC (not significant). The control GF breads and those enriched with 2%
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egg albumin, as well as the combination of egg albumin and RBPC at 4% showed a non-
378
continuous surface with many different pores size in crumb structure (Fig. 2), resulting in low
379
pore uniformity (higher value of pore uniformity means higher variation of pore size).
380
Obviously, the pore size in bread crumb of GF bread enriched with 2% RBPC, and the
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combination of 1% egg albumin and 1% RBPC were highest among GF breads, leading to a
382
higher total area of gas cells. The formation of heteropolymers between these two types of
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protein may support this effect. Similarly, the enrichment with 3.5% bovine plasma protein
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and inulin could increase the air cell and pores uniformity in rice flour-based bread
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(Rodriguez Furlán et al., 2015). Ziobro et al. (2013) also mentioned that pea and lupin protein
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could be used to increase the pore size of corn and potato starch-based bread.
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The L*, a*, and b* values of crust and crumb of prepared GF breads are summarized
390
in Table 2. When compared to the control recipe, it was clearly seen that the addition of 2%
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and 4% egg albumin did not change the L*, a*, and b* values of bread crust (P>0.05). In case
392
of RBPC, it was found that the lightness of bread crust was significantly decreased according
393
to the amounts of enriched RBPC. Four percent enrichment with RBPC provided the lowest
394
L* (P<0.05). Furthermore, enrichment with 2% and 4% RBPC increased a* to a maximum
395
value of 11.89. This is attributed to the darker color of RBPC itself and/or maybe due to the
396
formation of pigmented products from Maillard reaction and caramelization during baking.
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GF bread enriched with a combination of RBPC and egg albumin appeared in the same range
398
of color like GF bread enriched with RBPC. It can be concluded that RBPC was the main
399
ingredient to modify the color of GF bread. This observation was confirmed by Gallagher et
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al. (2003), who found that the darker color of GF breads was caused by dairy powder
401
addition. However, not only protein affected Maillard reaction, but also reducing sugars in
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RBPC and diary powder.
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For crumb color, the changing tendency of color parameters of GF breads enriched
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with RBPC and egg albumin was similar to the crust color. However, bread crumb seemed to
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be lighter than crust with higher L* values. Obviously, the addition of RBPC could improve
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the color of GF breads crumb to be more yellowish (b*), which was a desirable color of
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wheat breads. These color parameters were in the ranges of GF breads colours reported by
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Kittisuban et al. (2014), Phimolsiripol et al. (2012), Rodriguez Furlán et al. (2015) and
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Ziobro et al. (2013).
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3.3.5 Sensory attributes and shelf life
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Sensory attributes of selected GF breads are shown in Fig. 3. Compared to the control,
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appearance, colour, smell, and overall liking were highly improved by the enrichment with
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2% RBPC. Also the texture of GF breads enriched with 2% RBPC was extremely accepted
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by panels, which is related to the firmness and relative elasticity values of 15.00±0.87 N and
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54.11±1.87%, respectively. Taste was not significantly different between the breads
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containing egg albumin or RBPC, but they were all better than the bread without protein.
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Staling is the main factor to restrict the shelf life of breads. To monitor the staling rate of GF breads, experimental data was analysed separately by fitting Avrami model in which
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all mathematical parameters (n and k) were estimated (Table 3). It was clearly seen that the
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addition of 2% RBPC displayed an important anti-staling effect in GF bread, as it had the
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lowest staling rate constant (k = 0.043), while the n value was the highest. This observation
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was in agreement with crumb firming kinetic; higher n values were often associated with
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lower k values (Armero and Collar, 1998). The GF bread containing 1% RBPC and 1% egg
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albumin had an intermediate staling rate during storage, indicating a slower rate for
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development of crumb hardness than GF breads enriched with 2% egg albumin and the
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control recipe. This might be a result of the embedding of the starch granules within the rice
428
bran protein matrices generated upon baking, which hinders starch retrogradation (Nunes et
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al., 2009b). Also lupine protein was found to act as an anti-staling agent in GF bread, as
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described by Ziobro et al. (2013). Thus, this result suggested that RBPC has a potential to be
431
applied in GF breads for extending its shelf life.
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4. Conclusion Rice flour-based GF breads were enriched with RBPC and egg albumin and their combinations. The elastic modulus (G') of GF batters was highly improved by addition of
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RBPC, while addition of egg albumin enhanced the stability of batters structure (longer
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LVER). The viscoelastic behavior of GF batters and its final bread elasticity had a positive
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correlation. The properties of GF bread including specific volume, pore size and uniformity,
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gas retention, and shelf life was improved by addition of 2% RBPC; in addition, the sensory
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attributes were well accepted by trained panels. This would partially compensate the poor
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quality of GF bread due to lack of gluten. This study suggested that egg albumin could be
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successfully replaced by RBPC in GF bread making.
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Acknowledgements
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This study was financially supported by Research and Researcher for Industry (RRI) under the Thailand Research Fund (TRF), Mae Fah Luang University, and the Technology
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Grants 2014 under Thai-Austrian Cooperation in Science, Technology and Arts. The authors
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thank Urmatt Ltd (Chiang Rai, Thailand) for providing the Thai Jasmine organic rice bran.
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Shin, M., Gang, D.O., Song, J.Y., 2010. Effects of protein and transglutaminase on the
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Tsatsaragkou, K., Gounaropoulos, G., Mandala, I., 2014. Development of gluten free bread containing carob flour and resistant starch. LWT - Food Science and Technology 58,
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Wronkowska, M.,Haros, M., Soral-Śmietana, M., 2013.Effect of starch substitution by
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Ziobro, R., Witczak, T., Juszczak, L., Korus, J., 2013. Supplementation of gluten-free bread
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Table 1 Specific volume, weight loss, bread crumb texture and porosity properties
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Table 2 Color of crust and crumb
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Table 3 Estimated values of staling kinetic parameters of selected GF breads during
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9 days storage
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Figure Captions
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Fig. 1. Viscoelastic properties of GF batters enriched with RBPC and egg albumin; (a) strain
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sweep and (b) frequency sweep response (◊ : control,
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albumin, ○: 2% RBPC, ●: 4% RBPC, + : 1% RBPC and 1% egg albumin, × : 2% RBPC and
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2% egg albumin)
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Fig. 2. GF breads enriched with different levels of egg albumin and RBPC
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Fig. 3. Sensory attributes of GF breads
: 4% egg
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(b)
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Fig. 1. Viscoelastic properties of GF batters enriched with RBPC and egg albumin; (a) strain sweep and (b) frequency sweep response (◊ : control, : 2% egg albumin, : 4% egg albumin, ○: 2% RBPC, ●: 4% RBPC, + : 1% RBPC and 1% egg albumin, × : 2% RBPC and 2% egg albumin)
Fig.2. GF breads enriched with different levels of egg albumin and RBPC
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Fig.3. Sensory attributes of GF breads
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Table 1 Specific volume, weight loss, bread crumb texture and porosity properties GF bread recipes
Specific
Weight loss
Firmness
Relative
volume
(%)
(N)
elasticity
(cm3/g)
(%)
1.99±0.07cd
15.91±0.23a
11.23±0.82d
53.73±0.91d
2% Egg albumin
2.08±0.04b
15.32±0.43b
15.07±1.34c
62.80±0.76a
4% Egg albumin
1.99±0.07cd
15.16±0.48bc
16.53±1.50b
63.51±0.57a
2% RBPC
2.18±0.03a
14.69±0.21cd
15.00±0.87c
54.11±1.87d
4% RBPC
1.95±0.06d
14.12±0.34e
18.23±2.50a
50.09±0.85e
1% Egg albumin + 1% RBPC
2.17±0.03a
15.01±0.15bc
13.99±1.35c
57.44±1.11b
2% Egg albumin + 2% RBPC
2.01±0.02c
14.25±0.23de
18.35±0.69a
55.88±0.56c
Number
Average pore
Pore size
Gas cell area
of pores
size (mm)
uniformity
(%)
2.49±0.43a
10.82±1.83bc
19.54±1.84abc
GF bread recipes
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Control (0% Protein)
33±2.75a
2% Egg albumin
30±2.12ab
2.40±0.39a
9.88 ±0.60b
17.40±3.57cd
4% Egg albumin
33±4.19a
1.89±0.32b
7.89±0.48a
15.19±2.63d
2% RBPC
33±3.49a
2.63±0.22a
6.42±0.22a
21.47±1.35a
4% RBPC
31±3.69ab
2.34±0.36a
7.49±0.24a
17.66±2.28bcd
31±2.62ab
2.59±0.21a
6.46±0.23a
20.02±1.39ab
28±2.39b
2.31±0.46a
11.77±1.41c
16.69±2.27d
1% Egg albumin + 1% RBPC
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Table 2 Color of crust and crumb GF bread recipes
Crust L*
Crumb
a*
b*
L*
a*
b*
27.65±2.11ab 87.02±0.60ab 2.31±0.16e 16.81±1.01cd
2% Egg albumin
80.92±2.35a 5.09±0.45e
26.00±1.61c
85.72±0.86b
2.14±0.12e 16.67±0.24cd
4% Egg albumin
80.74±1.09a 5.64±1.10e
26.13±1.30c
87.68±2.44a
2.17±0.17e 16.99±0.36c
2% RBPC
63.45±1.06d 10.35±0.38c 26.51±0.45bc 73.87±2.17d
6.19±0.31b 18.93±0.48b
4% RBPC
56.71±0.98e 11.89±0.73a 26.04±1.51c
63.77±3.92e
7.69±0.42a 19.56±0.98a
1% Egg albumin
76.32±1.70b 7.82±0.73d
79.51±1.51c
3.12±0.76d 16.40±0.66d
69.54±0.88c 11.00±0.47b 27.02±0.49bc 74.30±0.22d
4.85±0.06c 16.32±0.19d
(0% Protein)
28.33±1.28a
+ 1% RBPC 2% Egg albumin
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81.93±1.02a 5.42±0.65e
Control
+ 2% RBPC a
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Table 3 Estimated values of staling kinetic parameters of selected GF breads during 9 days storage Recipes
T0 (N)
T∞ (N)
n
k
Adjust R2
8.86±0.41c
52.00±4.43a 1.75 0.065
97.82
2% Egg albumin
12.22±0.15a
47.02±1.53b 1.77 0.075
98.05
2% RBPC
11.59±0.23ab 36.22±0.19c 1.96 0.043
98.17
1% Egg albumin + 1% RBPC 11.09±0.76b a
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Control (0% protein)
37.92±0.52c 1.82 0.056
98.16
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Suphat Phongthai, Stefano D'Amico, Regine Schoenlechner, Saroat Rawdkuen PII:
S0733-5210(16)30297-1
DOI:
10.1016/j.jcs.2016.09.015
Reference:
YJCRS 2223
To appear in:
Journal of Cereal Science
Received Date:
27 May 2016
Revised Date:
19 September 2016
Accepted Date:
25 September 2016
Please cite this article as: Suphat Phongthai, Stefano D'Amico, Regine Schoenlechner, Saroat Rawdkuen, Comparative Study of Rice Bran Protein Concentrate and Egg Albumin on Gluten-Free Bread Properties, Journal of Cereal Science (2016), doi: 10.1016/j.jcs.2016.09.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Highlights: Specific volume and crumb porosity of GF bread was improved by rice bran protein
Rice bran protein concentrate possesses the desirable sensory attributes of GF bread
The 2% enrichment of rice bran protein concentrate in GF bread reduced staling rate
The advantages of rice bran protein concentrate over egg albumin were proposed
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Comparative Study of Rice Bran Protein Concentrate and Egg Albumin on
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Gluten-Free Bread Properties
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To be submitted to
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Journal of Cereal Science
Program of Food Technology, School of Agro-Industry, Mae Fah Luang University,
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Suphat Phongthaia, Stefano D'Amicob, Regine Schoenlechnerb, and Saroat Rawdkuena*
Chiang Rai 57100, Thailand
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Institute of Food Technology, Department of Food Science and Technology,
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BOKU - University of Natural Resources and Life Sciences, Vienna, Austria
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*To whom correspondence should be addressed. Tel: +66-53-916752. Fax: +66-53-916737. E-mail: [email protected]
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Abstract Gluten-free (GF) based products have been studying for several years especially for
40
quality improvement by enriching with proteins. A common protein source is egg albumin,
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but it has limitation for using causes of allergenic character. So, aim of this study was to
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replace egg albumin by rice bran protein concentrates which is a non-allergy protein in order
43
to improve the quality of GF bread. Rice bran, a by-product, was used as starting material for
44
rice bran protein concentrate (RBPC) preparation under alkaline-acid extraction technique.
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The obtained RBPC was composed of 68.07±0.54% protein (dry basis). For baking trials, the
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addition of RBPC had strongly influenced the rheological properties, especially elastic
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modulus (G') of GF batters during oscillation, and the relative elasticity of final GF breads.
48
Breads enriched with 2% RBPC, and a combination of 1% egg albumin and 1% RBPC had
49
the highest specific volume (P<0.05). Additionally, crumb porosity and sensory attributes
50
were improved. RBPC also showed higher efficacy to inhibit bread staling than egg albumin.
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This study suggested that RBPC could be used as a protein source for GF bread.
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Keywords: Egg albumin, gluten-free bread, rice bran protein, rheology
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1. Introduction Bread is one of the most popular baked products, consumed as staple food in many countries. The demand for bread is increasing rapidly in developing regions of the world such
66
as South East Asia and Africa (Taylor and Rosell, 2015). Traditionally, it is made of wheat or
67
other flours containing gluten. Gluten in wheat flour comprises of glutenin and gliadin that
68
are responsible for dough structure in terms of elasticity and strength, and extensibility and
69
viscosity, respectively (Kittisuban et al., 2014). However, gluten must be eliminated from the
70
diet for consumer who suffers from celiac disease (CD). CD is an inflammatory disease of
71
upper small intestine (duodenum, jejunum) which could lead to intestinal mucosal damage
72
and malabsorption of several important nutrients, due to the toxicity of certain protein
73
sequences in the gliadin fraction (Shin et al., 2010). The only effective treatment is to exclude
74
gluten from the diet throughout the life-span. For this reason, several gluten-free products
75
have been developed and produced for celiac patients. In case of gluten-free bread, the major
76
problem for manufacturer is to improve the quality of bread from gluten-free ingredients.
77
Apparently, many commercial gluten-free breads are made of refined flours or starches which
78
have lower nutrition value compared to original wheat bread (Tsatsaragkou et al., 2014),
79
moreover; other characteristics are often present like poor quality with poor color, low
80
volume, and crumbly texture.
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In recent years, various plant-based flours and starches such as buckwheat flour (Wronkowska et al., 2013), cassava starch (Crockett et al., 2011), corn and potato starch
83
(Ziobro et al., 2013), tapioca starch (Pongjaruvat et al., 2014), and rice flour (Cornejo and
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Rosell, 2015; Shin et al., 2010) have been used for gluten-free bread preparation. Rice flour is
85
one of the most suitable cereal flours for the production of gluten-free products due to its
86
unique attributes such as bland taste, white color, high digestibility, and hypoallergenic
87
properties (Shin et al., 2010). In order to replace the missing gluten, many substances which
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89
plasma protein (Rodriguez Furlán et al., 2015), egg albumin (Schoenlechner et al., 2010), soy
90
protein (Ziobro et al., 2013), but also dietary fibre, hydroxypropyl methylcellulose (HPMC;
91
Phimolsiripol et al., 2012), yeast β-glucan (Kittisuban et al., 2014), or transglutaminase
92
(Cornejo and Rosell, 2015).
93
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Proteins are typically incorporated to gluten-free systems to increase the elastic
modulus by cross-linking, to improve the perceived quality by enhancing Maillard browning
95
and flavor, to improve structure by gelation and to support foaming (Crockett et al., 2011;
96
Matos et al., 2014; Taylor et al., 2015). In addition, interaction between emulsifier and
97
protein can also improve dough strength and allow better retention of CO2 (Demirkesen et al.,
98
2010). Due to the low protein content of rice flour, supplementation with other protein
99
sources is necessary for improvement of gluten-free bread quality. Egg albumin is an allergic
100
protein, often used as protein source in baked products. Meanwhile, other proteins including
101
whey protein isolates, bovine plasma protein, and lupine protein have been reported for their
102
abilities to enhance desired properties of GF bread such as specific volume and cohesiveness
103
(Ziobro et al, 2013; Kittisuban et al., 2014; Rodriguez Furlán et al., 2015). The incorporation
104
of vegetable proteins such as soy and pea protein isolates in order to improve quality of rice
105
based gluten free muffins was also investigated by Matos et al. (2014). Rice bran protein is an
106
economical protein source and has attracted great attention for application in healthy and
107
non-allergic foods due to its hypoallergenic properties. Furthermore, it possesses good
108
functional properties including oil and water binding capacity, foaming and emulsifying
109
ability (Fabian and Ju, 2011) that may be useful for the development of gluten-free bread.
110
These properties offer a good potential of using rice bran protein as quality improver of
111
gluten-free bread and has up to now not been investigated. The aim of this study was
112
therefore to evaluate the effect of rice bran protein enrichment to gluten-free bread, in
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comparison to egg albumin. The influence on the rheological properties of the gluten-free
114
batter was investigated as well as the final bread quality.
115 2. Materials and Methods
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2.1. Raw materials
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Organic rice bran (Thai Jasmine rice, KDML 105) was supplied by Urmatt Ltd.
119
(Chiang Rai, Thailand). Rice flour was obtained from StroblCaj. NaturmuehleGesmb.H
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(Linz-Ebelsberg, Austria), egg albumin powder from Enthoven-BouwhuisEiproducten B.V.
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(Raalte, Netherlands), vegetable fat powder (REVEL*-BEP) from LodersCroklaan B.V.,
122
Wormerveer, Netherlands. Hydroxypropyl methylcellulose (HPMC, Metolose® Shin-Etsu
123
Chemical Co., Ltd., Tokyo, Japan) was donated by HARKE Pharma GmbH, Muelheiman der
124
Ruhr, Germany. Instant yeast (S.I.Lesaffre, France) was purchased from the market. The
125
emulsifier was a mixture of 3 partsdiacetyl tartaric acid ester of monoglycerides (DATEM,
126
Panodan M2020, Danisco®Copenhegen, Denmark) and 5 parts distilled monoglyceride
127
(DMG, Dimodan PH 10, NS/B, Danisco®Copenhegen, Denmark).
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2.2. Rice bran protein preparation
Organic rice bran was defatted by mixing rice bran with 95% ethanol (1:5, w/v), and
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stirring for 1 hr. The slurry was centrifuged (Avanti J-30I, Beckman Coulter, USA) at
132
5,500xg for 5 min. The precipitate was collected and extracted a second time. The final rice
133
bran fraction was dried overnight in an oven at 30°C, and then passed through a 50-mesh
134
sieve. Defatted rice bran (DFRB, 14.13±0.07% protein) was kept in aluminum foil zip lock
135
bag at -18°C.DFRB was dispersed in distilled water (1:10, w/v), the pH adjusted to 10 using
136
3M sodium carbonate. The mixture was stirred using a pilot-scale stirrer (DT-50, FRYMA-
137
Maschinen AG, Switzerland) at a temperature of 50°C for 1 hr. After centrifugation (KB 6-
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139
collected and adjusted to pH 4.5 using 3M citric acid and centrifuged at the same conditions.
140
The precipitate was adjusted to pH 7.0 and then freeze dried. The dried powder referred to
141
rice bran protein concentrate (RBPC).
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2.3. Proximate analyses of RBPC
RBPC was analyzed for moisture, ash, and protein content by AOAC Official Method (2000). Dietary fibre including total, insoluble and soluble dietary fibre were determined
146
following the standard method of AACC No.32-07 (2000) (Megazyme test kit, Megazyme
147
International Ireland Ltd., Wicklow, Ireland).Starch content was determined by using the
148
standard method of AACC No.76-13.01 (2000) (Megazyme test kit, Megazyme International
149
Ireland Ltd., Wicklow, Ireland).
150 151
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2.4. Preparation of rice flour-based gluten-free bread (GF) The basic GF bread recipe was based on the study of Phimolsiripol et al. (2012) with
153
some modifications, consisting of 100 g rice flour, 1 g HPMC, 1 g emulsifier, 1 g vegetable
154
fat powder, 2 g salt, 3 g yeast and105 g water. Protein source was either RBPC or egg
155
albumin and were incorporated at 2 or 4% (based on flour). Additionally, its mixtures (1%
156
egg albumin + 1% RBPC; 2% egg albumin + 2% RBPC) were also studied. The enrichment
157
with RBPC was based on protein content. Control bread was produced without any protein
158
addition. In sum, this experimental design resulted in 7 different recipes.
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Rice flour and all dry ingredients (HPMC, emulsifier, vegetable fat powder, and salt)
160
were homogenized in a mixer (RN10/VL2 Planetar mixer, A/S Wodschow & Co., Denmark)
161
for 1 minute (speed 2). Then dry instant yeast was added, followed by addition of water (25 ±
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0.5°C) within one minute. The mixing was continued for 6 min at speed 2. Afterwards the 6/22
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fermented at 30°C, 85% RH for 30 min in a fermentation chamber (G66W, MANZ
165
Backtechnik GmbH, Creglingen, Germany),then divided into two portions (400g each),
166
which were proofed at 30 °C, 85% RH for another 30 min. After that, breads were baked at
167
180°C for 50 min (60/rW, MANZ Backtechnik GmbH, Creglingen, Germany). The final
168
bread was cooled at room temperature for 2 hr before bread quality determinations were
169
carried out. The baking trials with 7 different recipes were done in duplicate, resulting in 4
170
loaves of each recipe were prepared.
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2.5. Dynamic rheological measurement
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Viscoelastic properties of GF batter enriched with RBPC or egg albumin were
174
performed following the method of Pongjaruvat et al. (2014) with some modifications, using
175
a rheometer (KNX2100, Malvern Instruments Limited, UK) equipped with plate-plate
176
geometry (20 mm in diameter) and a gap size of 2 mm. Each batter was investigated without
177
the addition of yeast. After sample loading, the excess batter was carefully removed with a
178
spatula. First, strain sweep experiments between 0.1 and 100% were run at a fixed frequency
179
of 1 Hz in order to determine the linear viscoelastic region (LVER). Afterwards, frequency
180
sweep experiments between 0.01 and 10 Hz were conducted within LEVR at a constant strain
181
of 0.5 %. All measurements were performed in duplicate at 25°C.
183 184
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2.6. Bread quality determinations The specific volume of each loaf of bread was determined by the rapeseed
185
displacement method according to the AACC Approved Method 55-50 (AACC, 2000).
186
Specific volume was calculated as cm3/g bread. Percentage of weight loss for each loaf was
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calculated by dividing the GF bread weight with GF batter weight, multiplying by 100. All
188
measurements were done in duplicate, giving 8 values for each recipe.
189
Crumb firmness and relative elasticity were determined using a Texture analyzer (TAXT2i, Stable Micro SystemTM Co., Godalming, Surrey, UK). A 5-kg load cell with an SMS
191
100 mm diameter compression probe (P100) was used. Each loaf of bread was sliced into
192
cubes with dimensions of 4 × 4 × 3 cm (L × W × H) from the center part using a sharp saw to
193
prevent structure damage. The cube was subjected to an uni-axial compression test (30%
194
compression) followed by a relaxation phase for 120s. The maximum force (Fmax) of
195
compression was considered as crumb firmness. The relative elasticity (REL, %) was
196
calculated by dividing the residual force (Fres) at the end of the holding time by the maximum
197
force, multiplying with 100. Two measurements of each loaf of bread were done, giving 8
198
values for each recipe.
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Colour of crust and crumb were measured by using a DigiEye System (VeriVide Limited, UK). The instrument was calibrated with a colour calibration chart before
201
determination. The controlled illumination cabinet was used to take high resolution images of
202
the bread crust and crumb. The colour parameters (L*, a*, and b*) were interpreted according
203
to the CIELAB definition by DigiPix software. The measurements were done in duplicate,
204
giving 8 values for each recipe.
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Photos of sliced GF breads were taken using digital camera (Nikon D90, Nikon Corporation, Thailand) within the DigiEye System. The crumb bread structure of each recipe
207
was analyzed using ImageJ software (https://rsb.info.nih.gov/ij/). Each image was cropped
208
into 20×20 mm (actual scale) at the center of bread, and then converted to 16-bit grayscale.
209
The results were evaluated with respect to number of pores, average pore size (mm), pore
210
size uniformity, and area of gas cell (%). The pore size uniformity was interpreted from the
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standard deviation (SD) of the average pore size (the lower SD of pore size, the higher
212
uniformity). All measurements were done in duplicate, giving 8 values for each recipe.
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2.7. Sensory evaluation and shelf life of GF breads
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Four selected GF breads, which had desired qualities, were studied for sensory
216
properties and shelf life. Six attributes of GF breads including appearance, colour, smell,
217
taste, texture, and overall liking were evaluated by a panel of 8 trained persons using
218
quantitative descriptive analysis.
For shelf life testing, GF breads were packed in paper bags and stored under
220
controlled conditions (20°C, 50% RH) for 9 days. At day 1, 3, 5, 7 and 9, crumb firmness of
221
the samples was determined. The staling rate of GF breads was evaluated by using Avrami
222
equation (1) described by Armero and Collar (1998). The two unknown parameters (k and n)
223
were estimated by fitting the experimental data into the equation model using a non-linear
224
regression method.
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where T0 is the crumb firmness at fresh bread, Tt is the crumb firmness at ‘t’ time, T∞ is the final crumb firmness, k is the constant rate of bread staling, and n is the Avrami
230
exponent, which is a characteristic related to the type of crystals growth.
232 233
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(1)
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(T∞ - Tt) / (T∞ - T0) = exp(-ktn)
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2.8. Statistical analysis All experimental results were expressed as means ± SD, and statistically analyzed by
234
analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) using SPSS
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statistic software. Statistical significance was accepted at a level of P<0.05. 9/22
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3. Results and discussion
238
3.1 Proximate composition of RBPC The pilot scale gained RBPC showed a yield of 7.44 g/100g based on DFRB weight.
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Compared to lab scale extraction the used pilot scale procedure gained much higher yields,
241
1.6-2.7 folds more protein (Chittapalo et al., 2009). The main composition of RBPC was
242
protein with 68.07±0.54%dm, which can be labeled as protein concentrate due to the protein
243
content of more than 60%. The moisture, starch, and ash contents were 6.85±0.26%dm,
244
6.37±0.18%dm, and 3.08±0.23%dm, respectively. The total dietary fibre including soluble
245
(6.37±0.45%) and insoluble dietary fibre (0.55±0.02%) was 6.91±0.46%. RBPC was not a
246
100%-purified protein; some other components were co-extracted due to their solubility
247
under alkaline conditions. Especially dietary fibres like arabinoxylans are commonly
248
extracted at high pH levels (Mansberger et al., 2014). Because of hydrolysis also remarkable
249
amounts of starch were extracted. These impurities may have some effects on GF batter and
250
bread properties, especially dietary fibre and starch have been reported to have positive
251
effects on GF breads by Phimolsiripol et al. (2012) and Pongjaruvat et al. (2014). Since these
252
polymers have no negative impact on gluten-free bread, further purification by amylases or
253
xylanases was not carried out due to economic aspects. In terms of the feasibility to produce
254
RBPC in an industrial scale, allowing proteins precipitate after pH adjustment under cold
255
condition instead of centrifugation could be possible to reduce the cost of operation and
256
power consumption, in addition, to assemble the centrifuge machine was much complicated.
257
However, the product yield may be less than the yield reported in this study.
259
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3.2 Effect of RBPC and egg albumin on viscoelastic properties of GF batter
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Rheological behavior of GF batters enriched with RBPC and egg albumin was examined by a rheometer in the oscillation mode. Viscoelastic properties of GF batters are
262
shown in Fig. 1 in term of their strain sweep (a) and frequency sweep (b) response. The
263
enrichment of proteins affected the LVER. The length of the LVER was used to determine
264
the stability of a sample's structure, since structural properties are strongly related to
265
elasticity. The results revealed that LVER of GF batters enriched with RBPC was shorter
266
than that of enriched with egg albumin and was extended with increasing amount of egg
267
albumin, indicating higher elasticity and resistance of GF batters to shear strain. At high
268
strain (more than 1%), a reduction of G' (beginning of non-linear curve) was observed in the
269
egg albumin enriched batters, relating to a breakdown of the dough network (Lazaridou et al.,
270
2007). Compared to the control recipe (no protein), the elastic modulus or shear modulus (G')
271
of GF batters was improved by the incorporation of both, RBPC and egg albumin. With
272
RBPC, the elastic properties of GF batters were not affected at strains below 0.5%, at higher
273
shear forces dough structure was damaged and G' decreased. However, egg albumin retained
274
higher elasticity of GF batters than RBPC at applied strain over 0.5%.
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The frequency sweep test (Fig. 1b) was used as measurement of viscoelastic response that performs in LVER, where the structure integrity in the material is not affected. This
277
result provides a micro-structural fingerprint of a material including viscoelastic properties of
278
solid, gel like structure and viscoelastic properties of liquid. It was clearly seen that the G' of
279
all GF batters slightly increased with higher frequencies in the whole range from 0.01 to 10
280
Hz which matched the viscoelastic solid behaviour. In addition, the tanδ or loss tangent (data
281
not shown), which is a viscous modulus to elastic modulus ratio (G''/G'), of GF batters was
282
lower than 1 (higher elasticity than viscous), confirming these GF batters were more solid-
283
like material than liquid-like material. Furthermore, it was also found that the enrichment
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with higher percentage of RBPC and egg albumin reduced the tanδ by increasing G', which
285
could happen due to protein aggregation within the medium (Crokett et al., 2011).
286
The enrichment of 4% RBPC showed highest G' followed by 2%RBPC, whereas egg albumin increased G' only to a small degree. The modification of the elastic property of GF
288
batters by addition of proteins was previously noted by Crokett et al. (2011), Ziobro et al.
289
(2013), and Matos et al. (2014) who applied soy and pea proteins to GF batters based on rice
290
flour, and a mixture of corn and potato starch. However, there was no difference of G' for GF
291
batters enriched with 2% and 4% egg albumin, indicating the maximum effect on dough
292
properties was reached already by 2% enrichment.
293
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So to conclude shortly, the enrichment of combined proteins from RBPC and egg
albumin increased slightly the value of G', but it stabilized the GF batters structure greatly
295
with respect to LVER. This observation confirmed that the major positive effect on batter
296
elasticity related to the function of egg albumin.
298
3.3 Effects of RBPC and egg albumin on GF bread quality
299
3.3.1 Specific volume
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The effects of RBPC and egg albumin on specific volume, weight loss and texture of
301
GF breads are shown in Table 1. The enrichment with 2% RBPC and the combination of 1%
302
egg albumin and 1% RBPC into GF bread were comparable, they provided the highest
303
specific volume among 7 recipes of GF breads (P<0.05). These enrichments could improve
304
the specific volume of the control (0% protein) for about 11%. Also the use of 2% egg
305
albumin improved the specific volume of GF bread. The increase of specific volume could be
306
attributed to the levels of enriched proteins, because the amounts of other ingredients were
307
fixed. During baking, the protein unfolds and protein-protein interactions as well as
308
interactions with other ingredients of the batter formulation may occur, leading to improved
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310
emulsification property of proteins were prerequisite for structure forming, thus supporting
311
starch and hydrocolloids in batter system (Ziobroet al., 2013). A similar result was reported
312
by Rodriguez Furlán et al. (2015), the incorporation of bovine plasma protein with maximum
313
level of 3.5% increased the volume of gluten-free bread significantly. In contrast, the result
314
showed that the enrichment with RBPC or egg albumin at 4% had no or a negative effect on
315
specific volume of GF bread. The lowest specific volume was found with 4% RBPC
316
enrichment. It may be due to too high concentration of protein, which might provide high
317
resistance and consistency of GF batters, resulting in a limited elasticity and less expansion
318
during proofing. Also Mezaize et al. (2009) reported that a supplementation of 5% whey
319
protein in rice starch based gluten-free bread caused a reduction of bread specific volume.
320
Similarly, the enrichment with high levels of albumin, casein, and soybean protein exhibited
321
low specific volume of GF breads (Macro and Rosell, 2008; Storck et al., 2013).
322
Additionally, the fixed water content of 105 % could be responsible for the low volume of
323
recipes with high protein enrichment, because of high water absorption of proteins. However,
324
compared to the water uptake of hydrocolloids like HPMC this effect should be quite low and
325
thus was not considered in the experimental design.
327
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3.3.2 Weight loss and crumb texture Weight losses of GF breads were minimized by incorporation of RBPC and egg
329
albumin into the control GF-recipe (Table 1). The GF breads enriched with 4% RBPC and the
330
combination of 2% egg albumin and 2% RBPC were the best recipes that could keep weight
331
loss low during baking (P<0.05). The control recipe exhibited the highest weight loss, due to
332
the lack of protein molecules that can hold the water within its own structure. The result
333
agreed with those reported by Jiamyangyuen et al. (2004), who found that enrichment with
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335
loss. Except for the protein, the fibre in RBPC might be another factor that minimizes water
336
loss. Fibres have high water holding capacity, as they form a porous structure composed of
337
polysaccharide chains, which can hold high amount of water through hydrogen bonds or
338
within their capillary structures through surface absorption (Schleißinger et al., 2013).
339
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Crumb firmness of control GF-bread had the lowest values (P<0.05) (Table 1). These values tended to increase with increasing the amounts of RBPC or egg albumin. The
341
enrichment with 2% RBPC, 2% egg albumin, and a combination of 1% RBPC and 1% egg
342
albumin into GF breads displayed comparable crumb firmness of about 14-15 N. Obviously,
343
the enrichment with the excessive amounts of proteins (4%) influenced crumb firmness. This
344
may be caused by high water absorptive properties of proteins, which lead to a finer and
345
denser crumb structure (Kittisuban et al., 2014). Additionally, an increase of crumb firmness
346
upon enrichment of proteins in GF breads might be attributed to the thickening of the gas cell
347
walls within the bread crumb (Rodriguez Furlán et al., 2015). Phimolsiripol et al. (2012)
348
indicated that 2% enrichment of egg albumin was sufficient for gluten-free rice bread,
349
whereas further protein enrichment obviously adversely increased crumb firmness.
350
Schoenlechner et al. (2010) and Crockett et al. (2011) also reported that 4% egg albumin and
351
3% soy protein isolates (SPI) resulted in an increase of crumb firmness. Moreover,
352
Demirkesen et al. (2010) suggested that higher modulus values of dough samples resulted in
353
lower firmness values of bread samples, which was in accordance with this study.
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The relative elasticity (REL) of GF breads is an important criterion for consumer
355
acceptance, and is presented in Table 1. The enrichment with egg albumin showed significant
356
positive influences on REL of GF breads. The enrichment with 2% and 4% egg albumin
357
provided the highest REL (62-63%), which was about 18.86% higher than the control recipe.
358
The combinations of RBPC and egg albumin (2% and 4%) also increased the REL of the GF 14/22
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360
in agreement with the study of Phimolsiripol et al. (2012) that previously mentioned the role
361
of egg albumin on improvement of REL. However, GF bread enriched with 4% RBPC had a
362
negative effect on REL, exhibiting the lowest value of 50.09%. Obviously, this low value of
363
elasticity in GF bread enriched with 4% RBPC was responsible for its low specific volume.
364
The GF batter must be sufficiently elastic to allow the bubbles to expand during baking
365
(Tsatsaragkou et al., 2014). As a result of rheological behavior of the GF batters, it could be
366
indicated that the higher elasticity (G') and stability (longer LVER) of GF batters enriched
367
with egg albumin went along with the higher elasticity of final GF bread.
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370
3.3.3 Crumb porosity
The crumb porosity of GF breads which was characterized by digital image analysis is
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presented in Table 1. The number of pores in GF breads crumb was found to be in the same
372
range between 28 and 33.The smallest average pore size of GF bread crumb belonged to the
373
recipe added with 4% egg albumin (P<0.05). The reduction of average pore size is probably
374
due to a structure disruption by the increased egg albumin content, providing impairment in
375
gas retention (Schoenlechner et al., 2010). In contrast, the pore size seemed to be increased
376
by addition of 2% RBPC (not significant). The control GF breads and those enriched with 2%
377
egg albumin, as well as the combination of egg albumin and RBPC at 4% showed a non-
378
continuous surface with many different pores size in crumb structure (Fig. 2), resulting in low
379
pore uniformity (higher value of pore uniformity means higher variation of pore size).
380
Obviously, the pore size in bread crumb of GF bread enriched with 2% RBPC, and the
381
combination of 1% egg albumin and 1% RBPC were highest among GF breads, leading to a
382
higher total area of gas cells. The formation of heteropolymers between these two types of
383
protein may support this effect. Similarly, the enrichment with 3.5% bovine plasma protein
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and inulin could increase the air cell and pores uniformity in rice flour-based bread
385
(Rodriguez Furlán et al., 2015). Ziobro et al. (2013) also mentioned that pea and lupin protein
386
could be used to increase the pore size of corn and potato starch-based bread.
388
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The L*, a*, and b* values of crust and crumb of prepared GF breads are summarized
390
in Table 2. When compared to the control recipe, it was clearly seen that the addition of 2%
391
and 4% egg albumin did not change the L*, a*, and b* values of bread crust (P>0.05). In case
392
of RBPC, it was found that the lightness of bread crust was significantly decreased according
393
to the amounts of enriched RBPC. Four percent enrichment with RBPC provided the lowest
394
L* (P<0.05). Furthermore, enrichment with 2% and 4% RBPC increased a* to a maximum
395
value of 11.89. This is attributed to the darker color of RBPC itself and/or maybe due to the
396
formation of pigmented products from Maillard reaction and caramelization during baking.
397
GF bread enriched with a combination of RBPC and egg albumin appeared in the same range
398
of color like GF bread enriched with RBPC. It can be concluded that RBPC was the main
399
ingredient to modify the color of GF bread. This observation was confirmed by Gallagher et
400
al. (2003), who found that the darker color of GF breads was caused by dairy powder
401
addition. However, not only protein affected Maillard reaction, but also reducing sugars in
402
RBPC and diary powder.
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For crumb color, the changing tendency of color parameters of GF breads enriched
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404
with RBPC and egg albumin was similar to the crust color. However, bread crumb seemed to
405
be lighter than crust with higher L* values. Obviously, the addition of RBPC could improve
406
the color of GF breads crumb to be more yellowish (b*), which was a desirable color of
407
wheat breads. These color parameters were in the ranges of GF breads colours reported by
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Kittisuban et al. (2014), Phimolsiripol et al. (2012), Rodriguez Furlán et al. (2015) and
409
Ziobro et al. (2013).
410
412
3.3.5 Sensory attributes and shelf life
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Sensory attributes of selected GF breads are shown in Fig. 3. Compared to the control,
413
appearance, colour, smell, and overall liking were highly improved by the enrichment with
414
2% RBPC. Also the texture of GF breads enriched with 2% RBPC was extremely accepted
415
by panels, which is related to the firmness and relative elasticity values of 15.00±0.87 N and
416
54.11±1.87%, respectively. Taste was not significantly different between the breads
417
containing egg albumin or RBPC, but they were all better than the bread without protein.
418
Staling is the main factor to restrict the shelf life of breads. To monitor the staling rate of GF breads, experimental data was analysed separately by fitting Avrami model in which
420
all mathematical parameters (n and k) were estimated (Table 3). It was clearly seen that the
421
addition of 2% RBPC displayed an important anti-staling effect in GF bread, as it had the
422
lowest staling rate constant (k = 0.043), while the n value was the highest. This observation
423
was in agreement with crumb firming kinetic; higher n values were often associated with
424
lower k values (Armero and Collar, 1998). The GF bread containing 1% RBPC and 1% egg
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albumin had an intermediate staling rate during storage, indicating a slower rate for
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development of crumb hardness than GF breads enriched with 2% egg albumin and the
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control recipe. This might be a result of the embedding of the starch granules within the rice
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bran protein matrices generated upon baking, which hinders starch retrogradation (Nunes et
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al., 2009b). Also lupine protein was found to act as an anti-staling agent in GF bread, as
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described by Ziobro et al. (2013). Thus, this result suggested that RBPC has a potential to be
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applied in GF breads for extending its shelf life.
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4. Conclusion Rice flour-based GF breads were enriched with RBPC and egg albumin and their combinations. The elastic modulus (G') of GF batters was highly improved by addition of
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RBPC, while addition of egg albumin enhanced the stability of batters structure (longer
437
LVER). The viscoelastic behavior of GF batters and its final bread elasticity had a positive
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correlation. The properties of GF bread including specific volume, pore size and uniformity,
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gas retention, and shelf life was improved by addition of 2% RBPC; in addition, the sensory
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attributes were well accepted by trained panels. This would partially compensate the poor
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quality of GF bread due to lack of gluten. This study suggested that egg albumin could be
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successfully replaced by RBPC in GF bread making.
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Acknowledgements
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This study was financially supported by Research and Researcher for Industry (RRI) under the Thailand Research Fund (TRF), Mae Fah Luang University, and the Technology
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Grants 2014 under Thai-Austrian Cooperation in Science, Technology and Arts. The authors
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thank Urmatt Ltd (Chiang Rai, Thailand) for providing the Thai Jasmine organic rice bran.
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Table 1 Specific volume, weight loss, bread crumb texture and porosity properties
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Table 2 Color of crust and crumb
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Table 3 Estimated values of staling kinetic parameters of selected GF breads during
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9 days storage
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Figure Captions
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Fig. 1. Viscoelastic properties of GF batters enriched with RBPC and egg albumin; (a) strain
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sweep and (b) frequency sweep response (◊ : control,
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albumin, ○: 2% RBPC, ●: 4% RBPC, + : 1% RBPC and 1% egg albumin, × : 2% RBPC and
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2% egg albumin)
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Fig. 2. GF breads enriched with different levels of egg albumin and RBPC
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Fig. 3. Sensory attributes of GF breads
: 4% egg
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(b)
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Fig. 1. Viscoelastic properties of GF batters enriched with RBPC and egg albumin; (a) strain sweep and (b) frequency sweep response (◊ : control, : 2% egg albumin, : 4% egg albumin, ○: 2% RBPC, ●: 4% RBPC, + : 1% RBPC and 1% egg albumin, × : 2% RBPC and 2% egg albumin)
Fig.2. GF breads enriched with different levels of egg albumin and RBPC
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Table 1 Specific volume, weight loss, bread crumb texture and porosity properties GF bread recipes
Specific
Weight loss
Firmness
Relative
volume
(%)
(N)
elasticity
(cm3/g)
(%)
1.99±0.07cd
15.91±0.23a
11.23±0.82d
53.73±0.91d
2% Egg albumin
2.08±0.04b
15.32±0.43b
15.07±1.34c
62.80±0.76a
4% Egg albumin
1.99±0.07cd
15.16±0.48bc
16.53±1.50b
63.51±0.57a
2% RBPC
2.18±0.03a
14.69±0.21cd
15.00±0.87c
54.11±1.87d
4% RBPC
1.95±0.06d
14.12±0.34e
18.23±2.50a
50.09±0.85e
1% Egg albumin + 1% RBPC
2.17±0.03a
15.01±0.15bc
13.99±1.35c
57.44±1.11b
2% Egg albumin + 2% RBPC
2.01±0.02c
14.25±0.23de
18.35±0.69a
55.88±0.56c
Number
Average pore
Pore size
Gas cell area
of pores
size (mm)
uniformity
(%)
2.49±0.43a
10.82±1.83bc
19.54±1.84abc
GF bread recipes
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Control (0% Protein)
33±2.75a
2% Egg albumin
30±2.12ab
2.40±0.39a
9.88 ±0.60b
17.40±3.57cd
4% Egg albumin
33±4.19a
1.89±0.32b
7.89±0.48a
15.19±2.63d
2% RBPC
33±3.49a
2.63±0.22a
6.42±0.22a
21.47±1.35a
4% RBPC
31±3.69ab
2.34±0.36a
7.49±0.24a
17.66±2.28bcd
31±2.62ab
2.59±0.21a
6.46±0.23a
20.02±1.39ab
28±2.39b
2.31±0.46a
11.77±1.41c
16.69±2.27d
1% Egg albumin + 1% RBPC
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Table 2 Color of crust and crumb GF bread recipes
Crust L*
Crumb
a*
b*
L*
a*
b*
27.65±2.11ab 87.02±0.60ab 2.31±0.16e 16.81±1.01cd
2% Egg albumin
80.92±2.35a 5.09±0.45e
26.00±1.61c
85.72±0.86b
2.14±0.12e 16.67±0.24cd
4% Egg albumin
80.74±1.09a 5.64±1.10e
26.13±1.30c
87.68±2.44a
2.17±0.17e 16.99±0.36c
2% RBPC
63.45±1.06d 10.35±0.38c 26.51±0.45bc 73.87±2.17d
6.19±0.31b 18.93±0.48b
4% RBPC
56.71±0.98e 11.89±0.73a 26.04±1.51c
63.77±3.92e
7.69±0.42a 19.56±0.98a
1% Egg albumin
76.32±1.70b 7.82±0.73d
79.51±1.51c
3.12±0.76d 16.40±0.66d
69.54±0.88c 11.00±0.47b 27.02±0.49bc 74.30±0.22d
4.85±0.06c 16.32±0.19d
(0% Protein)
28.33±1.28a
+ 1% RBPC 2% Egg albumin
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81.93±1.02a 5.42±0.65e
Control
+ 2% RBPC a
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Table 3 Estimated values of staling kinetic parameters of selected GF breads during 9 days storage Recipes
T0 (N)
T∞ (N)
n
k
Adjust R2
8.86±0.41c
52.00±4.43a 1.75 0.065
97.82
2% Egg albumin
12.22±0.15a
47.02±1.53b 1.77 0.075
98.05
2% RBPC
11.59±0.23ab 36.22±0.19c 1.96 0.043
98.17
1% Egg albumin + 1% RBPC 11.09±0.76b a
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37.92±0.52c 1.82 0.056
98.16
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