Effect of Sodium Chloride and Sodium Bicarbonate on the

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Food and Beverage Chemistry/Biochemistry

Effect of sodium chloride and sodium bicarbonate on the physicochemical properties of soft wheat flour doughs and gluten polymerization Gengjun Chen, Laura Ehmke, Rebecca Miller, Pierre Faa, Gordon Smith, and Yonghui Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01197 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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Journal of Agricultural and Food Chemistry

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Effect of sodium chloride and sodium bicarbonate on the

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physicochemical properties of soft wheat flour doughs and gluten

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polymerization

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Gengjun Chen1, Laura Ehmke1, Rebecca Miller1, Pierre Faa2, Gordon Smith1,

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Yonghui Li1*

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66506

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13 14 15 16 17 18 19 20 21 22

Department of Grain Science and Industry, Kansas State University, Manhattan, KS

Frito-Lay North America, Plano, TX 75024

*Correspondence to: Yonghui Li, E-mail: [email protected], Ph: 785-532-4061, fax: 785-532-7010

Submit to Journal of Agricultural and Food Chemistry

1

ACS Paragon Plus Environment


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Abstract: Soft wheat flour doughs were prepared with different levels of salt (NaCl)

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and/or baking soda (NaHCO3). Oscillation rheology, elongational viscosity, and

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extensibility of doughs were tested to evaluate the effect of salt and/or baking soda

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on the physical properties of doughs. Furthermore, a series of physical-biochemical

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analytical techniques were used to investigate gluten polymerization in doughs,

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including Zeta potential analyzer, Fourier transform infrared spectroscopy (FTIR),

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spectrophotometer, and reversed phase high performance liquid chromatography

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(RP-HPLC). The addition of high levels of NaHCO3 (1.0 % fwb), either by itself or in

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combination with NaCl, increased dough strength, elongational viscosity, and

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viscoelasticity. RP-HPLC results demonstrated macromolecular aggregation of gluten

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proteins in the presence of NaCl and/or NaHCO3. The addition of NaHCO3 or NaCl

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also decreased both free sulfhydryl content and random coil structure of gluten

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isolated from the doughs. Overall, NaCl and/or NaHCO3 induced the changes of

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molecular conformation of gluten, which impacted the physicochemical qualities of

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soft wheat flour dough. This study provides a better understanding of salt and baking

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soda functionality in the formation of soft flour dough, which will support the

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searching of feasible sodium reduction strategies in soft flour bakery products.

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Keywords: sodium chloride, sodium bicarbonate, soft wheat flour doughs, gluten,

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gliadin, glutenin, secondary structure, hydrophobicity, molecular interaction

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Introduction

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Although worldwide dietary patterns are different, wheat-based products are still the

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major food source for most of the population. Soft wheat, accounting for about 15%

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of total US wheat production, is widely used in various chemically leavened products

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such as cookies, pretzels, cakes, crackers, etc. Baking soda (or sodium bicarbonate,

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NaHCO3) is by far the most popular chemical leavening agent and offers many

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advantages, such as versatility, easy of handling, nontoxicity, tastelessness and low

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cost. The addition of NaHCO3 in bakery goods is typically around 0.5-2% (flour basis),

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depending on the specific type of food products. Salt (or sodium chloride, NaCl) is

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another indispensable ingredient for nearly all bakery products and cereal snacks. It

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provides not only crucial sensory characteristics to food but also technological

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functions that influence the processing and quality characteristics of dough and

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cereal goods. Previous studies showed that salt increases dough resistance, elasticity,

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extensibility, and mixing stability.1,2 Salt also contributes to the products coloring,

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improves food texture, and inhibits microbial growth. The typical addition of salt in

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bakery products is about 1-2% total flour weight basis (fwb). Despite the fact that

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sodium is an essential nutrient for humans, it is only needed by the body in relatively

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small quantities. Excessive sodium intake significantly increases blood pressure,3,4

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which has been the major cause of cardiovascular diseases (CVDs) such as strokes,

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heart failure and coronary heart disease (CHD). CVDs alone account for one third of

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global deaths.5 Currently, Americans consume an average of 3,440 mg sodium per

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day, which is 50% more than the upper limit of 2,300 mg per day recommended by

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the 2015 Dietary Guidelines for Americans,6 thus sodium intake should be

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significantly reduced. However, as discussed above, salt and baking soda are still the

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two largest dietary sodium sources in baked products. For example, these products

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contribute to 30% of our daily salt intake.7,8 Therefore, reducing salt and baking soda

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in bakery products is an essential step for the overall sodium reduction goal.

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Wheat doughs possess unique viscoelastic properties attributed to the gluten

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proteins. Network formation of wheat gluten is essential for many cereal and bakery

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products. When the flour is mixed with water and other ingredients, wheat protein is 3



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hydrated, interacts with other additives such as salt and/or baking soda, and

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develops a unique polymeric network, which is associated with dough properties.

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According to different solubility, wheat flour proteins are traditionally classified as

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albumins (soluble in water), globulins (soluble in NaCl solution), prolamins (soluble in

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ethanol solution), and glutelins (soluble in acid or alkali solution), which are also

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defined as Osborne fractions.9,10 More than 80% of gluten consist of gliadins

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(prolamins fraction) and glutenins (glutelins fraction).11 The gliadins act as a

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plasticizer to influence the viscous properties; while the glutenins determine the

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elasticity and cohesiveness of the dough.12,13 Goesaert et al.14 reported the gliadins

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are noncross-linked proteins with molecular mass (Mr) of 30,000-75,000 (ω-, α- and

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γ-type). The glutenins are divided into the predominant low molecular weight

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glutenin subunits (LMW-GS, Mr of 40,000-70,000), and high molecular weight

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glutenin subunits (HMW-GS, Mr of 60,000-90,000).15

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Salt and baking soda provide many technological functions in bakery food

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systems in addition to the saltiness and leavening action. It is still a major challenge

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to remove them or reduce their amount without any negative effect on dough

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rheological properties or product quality. One major reason is that there is no clear

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understanding of how salt and/or baking soda contribute to dough properties,

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especially the interactions with gluten. Although some research studies have been

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carried out during the past,15-17 the fundamental basis of salt and baking soda in

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dough systems have yet to be well understood. The lack of foundational knowledge

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in the field has been a limiting factor in developing effective strategies to reduce

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sodium, and an in-depth knowledge of the function of salt and/or baking soda in

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doughs at a molecular level is crucial. This study was conducted to investigate the

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influences of salt and/or baking soda on the rheological properties and physical

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processibility of soft wheat flour dough and gluten macromolecular characteristics

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and interaction during dough development.

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Materials and Methods

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Materials 4


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All chemicals, solvents, and reagents used in the experiments were of analytical

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grade, and were purchased from Fisher Scientific (Fairlawn, NJ, USA) or Sigma-Aldrich

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(St. Louis, MO, USA), unless specified otherwise. Soft wheat flour (pretzel flour,

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protein content 8.0%, moisture content 12.7%, fwb) was provide by Frito-Lay North

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America.

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Mixing properties

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Dough samples were prepared with different levels of NaCl and/or NaHCO3 (%

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fwb) are shown in Table 1. The treatment includes four levels of NaCl (0, 0.5, 0.7, and

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1.0%, fwb) alone, three levels of NaHCO3 (0, 0.3, and 1.0%) alone, and four

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combinations of NaCl and NaHCO3. Mixing properties were measured using

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Mixograph following AACCI Approved Method 54-40.02.18 The optimum water

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absorption of the control flour was determined and that absorption was used for all

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treatments. All analyses were carried out in triplicate. Farinograph was tested and

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developed for each treatment according to AACCI Approved Method 54-21.01,18 and

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mixing parameters including water absorption, mix time, and dough stability were

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obtained.

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Dough extensibility

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Dough extensibility and resistance to extension were determined via Kieffer test

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using a TA-XTPlus texture analyzer (Stable Micro Systems, Godalming, UK) with

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SMS/Kieffer rig (Stable Micro Systems). Doughs containing 100 g flour, 59 g water,

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and predetermined amount of NaCl and/or NaHCO3 was mixed in a pin mixer with

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the mixing time determined from mixograph as shown in Table 1. The prepared

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dough was equilibrated for 30 min in a closed container before the measurement,

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and then it was gently molded into a rectangle with minimal handling and set on the

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grooved section of the Teflon former lubricated with the mineral oil. A lametta strip

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was placed in each groove to aid the removal of dough strip. The cover block was

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placed on top of the former and a clamp compressed the blocks together. Excess

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dough forced out the sides of the former was trimmed off. The full-length dough

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strips were selected and tested after resting for 30 mins. The following settings were

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used: pre-test speed of 2 mm/s, test speed of 3.3 mm/s, post-test speed of 10 mm/s, 5



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a distance of 75 mm and trigger force of 5 g. The dough was molded and rested in a

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clamping tool for 30 min, and full-length dough strips were tested. The output

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quantities were the resistance to extension (g) and the extensibility (mm). The

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analysis was performed in triplicate.

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Dynamic oscillatory rheology

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A Bohlin CVOR 150 rheometer (Malvern Instruments, Southborough, MA)

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equipped with a PP 20 parallel plate (gap size of 1200 μm) was utilized to evaluate

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the oscillatory rheological properties of dough. Doughs were freshly prepared in the

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pin mixer as described previously, and the dough was rested for 30 min in a closed

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container before loading on the dynamic oscillatory rheometer. Excessive dough

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sample outside the plate edge was trimmed and the paraffin oil was used on the

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lateral surface to prevent moisture evaporating or drying. Amplitude scan was first

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performed at strain values of 0.01-10% at a constant frequency of 1Hz at 25 °C to

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establish the linear viscoelastic range. Frequency sweep was then performed from

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0.1 to 100 Hz within the strain amplitude set at 0.1% (within linear viscoelasticity) at

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25 °C. Storage modulus (G’), loss modulus (G”) and tan δ (G”/G’) as a function of

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amplitude or frequency were recorded. Each treatment was analyzed in duplicate.

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Elongational viscosity property

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The TA.XTPlus (Texture Technologies, Scarsdale, NY, USA; and Stable Micro

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Systems, Godalming, UK) was used to measure the elongational viscosity of the

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doughs. Doughs were prepared similarly as previously described. The doughs were

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sheeted, covered and rested for 10 min. Three 1 inch diameter disks were cut from

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the dough and covered with mineral oil for lubricating and to prevent adhesion to

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the probe. Lubricated uniaxial compression was performed with a 2 inch diameter

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probe at 50% strain deformation at a speed of 0.4 mm/s. Three separate doughs with

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3 disks per dough were tested for each treatment.

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Extraction of gluten

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Gluten fractions were extracted from soft wheat flour dough systems prepared

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with the different levels of NaCl and NaHCO3 following AACCI Approved Method

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38-10.01.18 Gluten samples were lyophilized, ground to powder, and stored in -20 °C 6


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freezer until further analysis. Protein content was determined using a LECO TruMac

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nitrogen analyzer (LECO Corp., St Joseph, MI, USA) according to AACCI Approved

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Method 46-19.01,18 and a conversion factor of 5.7 was utilized for calculation from

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nitrogen content.

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RP-HPLC analysis

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RP-HPLC measurement of gluten extracts was conducted according to a previous

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method with some modification.19 Gluten (100 mg) was extracted stepwise twice

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with 1.0 mL of 60% (v/v) aqueous ethanol for 12 min at room temperature to obtain

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the gliadin fraction; and twice with 1.0 mL of a solution containing 50% (v/v)

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propan-1-ol, 2 M urea, 1% (w/v) dithioerythritol and 0.05 M Tris-HCl for 45 min at

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60 °C to obtain the glutenin fraction. Each extraction step started with vortex mixing

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for 1 min at room temperature. All extracts were centrifuged (15,000 xg, 10 min,

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20 °C), and corresponding extracts were combined and filtered with a 0.45 μm

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membrane (Phenex™ filter membranes, Phenomenex, Torrance, CA, USA). A HP1050

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Series HPLC (Agilent Technologies, Santa Clara, CA, USA) coupled with a diode array

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detector (DAD) was used to analyze the extracts. Two fractions of extracts were

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loaded (10 μL) onto a reversed-phase Aeris WIDEPORE XB-C18 column (3.6 μm, 150 x

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4.6 mm, Phenomenex, Torrance, CA, USA). The elution solvents were water

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containing 0.1% trifluoroacetic acid (A), and acetonitrile containing 0.1%

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trifluoroacetic acid (B) with a linear gradient from 0 min 24 % B to 30 min 56 % B,

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with flow rate of 0.6 mL/min, and temperature of 60 °C with detection at 210 nm.

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The gliadins and glutenins were identified by comparison of their retention times and

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spectra with protein standards while quantitative analysis was based on their peak

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areas from the chromatograms. Extraction and measurements were performed in

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duplicate.

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Zeta potential

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Zeta potential of samples was tested on a ZetaPALS Zeta potential analyzer

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(Brookhaven Instruments Co., Holtsville, NY, USA) with hydrodynamic light scattering

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and laser doppler electrophoresis. Zeta potential value was determined by the

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method described by Chen et al.20 Gluten suspensions were prepared with a solid 7



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content of 0.1%. Five runs comprising of 2 cycles were carried out for each

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measurement. Each sample was tested in duplicate.

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Sulfhydryl (SH) and disulfide (SS) group determination

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The concentration of free SH was determined by Ellman’s reagent

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(5’5-dithiobis(2-nirtobenzoic acid), DTNB) according to the method reported by

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Rombouts et al.21 with some modifications. Briefly, gluten (30 mg) was dispersed in

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3.0 mL of reaction buffer A (8 M urea, 3 mM EDTA, 1% SDS, 0.2 M Tris-HCl, and pH

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8.0), and sample was vortexed for 30 s and shaken at 25 °C for 60 min. Afterwards,

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0.3 mL of buffer B (10 mM DTNB in 0.2 M Tris-HCl, pH 8.0) was added to the sample

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and mixed for another 60 min. The solution was then centrifuged at 13,600 xg for 15

207

min at room temperature, and the absorbance of the supernatant was detected at

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412 nm using a double beam spectrophotometer (VWR UV-6300PC, Radnor, PA,

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USA).

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The concentration of total SH group was determined according to a previous

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methods with some modification.22,23 Briefly, 1 mL of reaction buffer (3mM EDTA, 1%

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SDS,

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2-nitro-5-thiosulfobenzoate (NTSB)) was mixed with 10 mg of gluten . The solution

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was vortexed for 30 s and mixed in a shaker for 60 min in the dark, and then

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centrifuged at 13,600 xg for 15 min followed by diluting the supernatant (0.3 mL)

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with 2.7 mL reaction buffer without NTSB. Absorbance was then measured at 412 nm

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using a double beam spectrophotometer (VWR UV-6300PC, Radnor, PA, USA). The SH

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content (CSH) was calculated according to the equation: CSH =

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absorbance, ε is the extinction coefficient of 13,600 M-1cm-1, and b is the cell path

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length. The SS content (Css) was calculated according to the equation: Css =

221 222

0.2M

Tris-HCl,

0.1M

sodium

sulfite,

pH

9.5,

and

0.5mM

, where A is the

, where CTSH is the total SH content, and CFSH is the free SH content.

Secondary structure of gluten

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Secondary structures of gluten were analyzed using a PerkinElmer Spectrum 400

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FT-IR/FT-NIR Spectrometer (PerkinElmer, Inc., Waltham, MA, USA) equipped with an

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ATR accessory. A total of 64 scans were taken at a resolution of 4 cm-1 in the range of 8


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400-4000 cm-1. Gluten secondary structures were deconvoluted from the amide I

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region (1600-1700 cm-1) via a secondary derivation. The spectra were analyzed using

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OriginPro 2016 software to obtain the relative areas of the selected amide I region.

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Surface hydrophobicity

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The sodium dodecyl sulfate (SDS) binding capacity was used to measure protein

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total surface hydrophobicity according to a previous method with some

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modification.24 The SDS solution (0.1 mmol/L, 40 mL) was used to dissolve 10 mg

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gluten , and was shaken for 60 min. After being dialyzed in distilled water for 48 h,

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one milligram of inner dialyzate was mixed with 20 mL CHCl3, and then 5 mL of

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methylene blue (0.024 g/L) was added into the CHCl3 layer. The mixture was

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centrifuged at 2500 xg for 15 min, and then the absorbance of the mixture in the

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lower layer was detected at 655 nm (VWR UV-6300PC, Radnor, PA, USA). A linearity

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(R2 > 0.995) for the SDS standard solution within the range of 0.01–0.1 mmol/L was

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obtained in the calibration data. The hydrophobicity value (H) = × 20 ×

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where c is the concentration of SDS calculated by absorbance and standard curve.

241

Statistical analysis

.

,

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Data were analyzed using the Statistical Analysis System software v9.4 (SAS

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Institute, Cary, NC, USA). Significant differences were found at the statistical presence

244

of a P value of 0.05.

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Results and Discussion

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Mixing properties of doughs

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In the Mixograph study, water absorption was held constant at 59% (optimal for

249

the control) to investigate the effect of the NaCl and NaHCO3 on dough mixing

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properties (Table 1, Figure 1). Mixing time of the control doughs was 3.50 min, and it

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increased to different extents for other treatment groups (Table 1). Addition of

252

NaHCO3 and/or NaCl increased the mixing time compared to the control; while the

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combination of 1.0% NaCl plus 1.0% NaHCO3, or 1.0% NaHCO3 alone contributed the

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most to the increased mixing time (6.2 or 6.0 min). Moreover, NaCl had relatively less

9



255

effect on mixing time than NaHCO3 when each ingredient was added individually.

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Farinograph absorption slightly decreased with the addition of NaHCO3 or in the

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combination groups (Table 1). Salt or baking soda influenced the mixing time and

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stability of all treatment groups. The addition of 1% NaHCO3 exhibited the largest

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strengthening effect, increasing the mixing time by 4 min and the stability by 9.2 min

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compared with the control. Dough stability was affected more by NaHCO3 than NaCl;

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for instance, the Farinogram curves of doughs containing 1.0% NaHCO3 or 0.5%

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NaCl/0.3% NaHCO3 became much stronger; whereas 0.7% NaCl did not have a large

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impact on the shape of the Farinogram curve (Figure 1). The results were consistent

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with our finding from Mixograph experiments.

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Extensibility and viscosity of doughs

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A previous study suggested that dough strain hardening behavior was correlated

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with the gluten network.25 Although 0.5% NaCl/0.3% NaHCO3, or 0.7% NaCl had

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relatively minor effects on the dough rupture forces, dough strength was significantly

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increased (P < 0.05) in the other treatment groups compared with the control (7.4 g)

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(Table 2). The rupture force of dough with 0.5% NaCl/1.0% NaHCO3 was the largest

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(20.3 g). Increased dough extensibility was achieved with salt alone at all the studied

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levels, as well as with lower amount of NaHCO3 (0.3%), or combination of salt with

273

0.3% NaHCO3. As shown in Table 2, the addition of 1.0% NaHCO3 produced doughs

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with the highest elongational viscosity (P > 0.05); while those groups with the

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combination of NaCl/NaHCO3 also showed increased values compared to control

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group. Furthermore, NaCl alone did not change dough viscosity even when the level

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was 1.0% fwb. Overall, the presence of NaHCO3 in soft wheat flour doughs greatly

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enhanced the strength and viscosity of dough, and it may be due to the stronger

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polymeric interaction of gluten in these systems, which will be further discussed in

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the next sections.

281

Dynamic rheometry of doughs

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Oscillational rheometry measurement was carried out to investigate the

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rheological properties of the dough. Strain and frequency sweeps curves are shown

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in Figure 2. Loss modulus (G”), storage modulus (G’), and tan δ (G”/G’) of the 10


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samples are the basic parameters used to describe the viscoelastic behavior of the

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dough systems. Although the doughs treated with NaCl at high levels (1.0% of fwb) or

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NaHCO3 all exhibited higher G´ and G” compared with the control, the difference of

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the modulus between NaHCO3 containing doughs and control was much more

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significant. It is noticeable that the combined addition of salt and baking soda

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markedly changed the G” and G’ of doughs. These results were in accordance with

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the previous reports.1,25,26 For instance, it was reported that adding more salt

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enhanced the strength and elasticity of wheat flour dough.25 The tan δ (G’’/G’) is

293

referred to as a proper measure of viscoelasticity, and Yiannopoulos et al.27

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suggested that wheat dough can be classified as a viscoelastic solid if the tan δ was

295

less than one. In our study, the tan δ values were decreased with the addition of

296

NaHCO3 or combinations among the groups at 0.1% strain (Figure 2E), which

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indicated that the dough structure became relatively more elastic in the presence of

298

NaHCO3. A previous study discovered alkali enhanced dough strength;28 in the

299

meantime, the elasticity of buckwheat dough could be increased with the addition of

300

alkali.29 The reason salt or baking soda impacted the rheological properties of doughs

301

may be attributed to the structural changes of macromolecules (i.e. cross-links of

302

gluten polymers). However, other authors did not find any change in tan δ with NaCl

303

addition.30,31 The lack of consistency on the rheology tests might be due to the

304

different wheat gluten protein quality and quantity or dough systems.

305

Gluten fractions analysis

306

After extracting gluten from soft wheat flour dough systems with different levels

307

of NaCl and NaHCO3, the fractions of gliadin and glutenin were separated and

308

identified by RP-HPLC (Figure 3), and the results are presented in Table 3. The

309

linearity of the standard calibration curve ranged from 0.065 to 1.05 mg, in which

310

the correlation coefficient indicated a good linearity (R2 > 0.98). The limit of

311

detection (LOD) was 0.016 mg/mL, and the limit of quantification (LOQ) was 0.053

312

mg/mL. According to results of recovery experiments, the average recovery rate was

313

88%, which was calculated by spiking the control samples with 1% gliadins standard

314

(n=3); while the coefficient of variation for repeatability of determinations of 11



315

samples was ±5.3% (average of five determinations within two weeks), which

316

compared favorably with the previous study.19 The gluten samples were extracted

317

step wise and further analyzed through RP-HPLC into several elution ranges including

318

gliadin fraction (ω-, α-, and γ-gliadin), and glutenin fraction of bound gliadin

319

(ωb-gliadin), HMW-GS, and LMW-GS, respectively.13,32

320

Although ω-gliadin amount remained almost constant in all the groups, the

321

extractable α- and γ-gliadin decreased gradually with the addition of NaCl and/or

322

NaHCO3; therefore, the overall extractable gliadins also decreased. In the meantime,

323

the apparent HMW-GS and LMW-GS amounts both increased when adding NaHCO3

324

into the dough system. For instance, the HMW-GS extractability concentration was

325

increased significantly from 30 to 36 μg/mg in the group within NaCl 1.0%/NaHCO3

326

1.0% (P < 0.05). The total glutenin content was increased by up to 19% with the

327

addition of NaCl 1.0%/NaHCO3 1.0%. The present results suggested that

328

gliadin-glutenin polymeric interaction and cross-linking were enhanced via the

329

addition of salt and/or baking soda, which led to a reduced amount of extractable

330

gliadins and increased amount of extractable glutenins. NaHCO3 had a stronger

331

effect than NaCl, which was consistent with Wu et al.33 who also reported the

332

alkaline reagent impact association of gluten. Lagrain et al.34 pointed out that the

333

aggregation of gluten might be through oxidation of SH groups or SH-SS exchange

334

reactions in bread dough systems. The amount of extractable ω-gliadin was not

335

affected by the addition of salt and NaHCO3, because the ω-gliadin lack cysteine

336

groups involved in the interaction with other proteins.19 SS bonds, hydrophobic, or

337

electrostatic interactions might change the microstructure of gluten protein as

338

well.33,35,36 The investigation of the possible interaction of gluten will be discussed in

339

the next sections.

340

The ratio between gliadins to glutenins (gli/glu) influences the formation of

341

dough since the gliadins are responsible for the viscosity and extensibility of dough,

342

whereas glutenins play a key role in the elasticity of dough through polymeric

343

network formation.2,35 An appropriate ratio of gli/glu is required for the viscoelastic

344

properties of doughs and the final bakery goods quality.13,37 It is noteworthy that the 12


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ratio significantly decreased with high levels of NaHCO3 or combined addition (P
0.05). A

394

significant change of β-turn structure was not observed (P > 0.05). However, it was

395

found that the formation of β-sheet was at the expense of β-turn during gluten

396

deformation in another study.42,43 An increase of β-sheet structure in the previous

397

studies was attributed to the effect of hydrogen bonding between the glutenin

398

molecules.16,42 Based on a proposed mechanism,9 the hydrogen bonds contribute to

399

the formation of more loop structure of the gluten when the molecules are hydrated,

400

and consequently the gluten aggregation are more pronounced. Therefore, NaCl

401

and/or NaHCO3 may influence the secondary conformation of gluten via inducing

402

hydrogen bonding formation and contribute to the development of gluten network

403

appearing as protein aggregation in the study.

404

Surface hydrophobicity 14


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Page 15 of 33


405

Gluten is a biomacromolecule with a 3-dimensional structure, in which

406

hydrophobic amino acid residues contribute to the tertiary conformation through

407

hydrophobic interaction. In the study, SDS binding was used to explain the degree of

408

hydrophobicity of gluten samples from soft wheat flour dough systems. This method

409

has been applied to determine the hydrophobicity of the insoluble proteins such as

410

ovalbumins.24 No significant differences of the degree of hydrophobicity of glutens as

411

affected by NaCl and/or NaHCO3 were found (P > 0.05); thus, the addition of salt or

412

baking soda at present levels does not influence the total surface hydrophobicity of

413

gluten in the current dough systems.

414

In the present study, the ratio of extractable gliadins to glutenins significantly

415

decreased within the high level of baking soda, implying that more aggregated and

416

less soluble protein network was formed. On other hand, the absolute values of zeta

417

potential were changed, which, might confirm the molecular interactions and

418

aggregations. In terms of the FTIR results, the secondary structure distributions were

419

altered, where both salt and baking soda could contribute to the reduced amount of

420

random coil structure. To further elucidate the mechanism of polymeric network

421

formation in dough systems, it was reported as a result of SS bonds formation by

422

oxidation of SH or interchange of SH-SS,29,44 which was also partially demonstrated

423

by our HPLC results of the aggregation of gliadins. The free SH content of gluten

424

reduced in the doughs treated with 1.0% NaHCO3 or combination, which suggested

425

that NaHCO3 may lead to more intense SH reactions during dough production.

426

However, there are other non-redox cross-linking worth studying, which may cause

427

the polymeric interaction of dough.

428

The inclusion of NaCl and NaHCO3 in soft flour doughs improved dough mixing

429

stability, strength, extensibility, and elasticity (to some extent), which was a result of

430

synergistic inter- and intra-molecular interactions. There are several conceptual

431

models proposed to explain how the interaction of ingredients affected dough

432

rheological behavior. It was believed that the hydration of gluten results in the

433

cohesion and adhesion properties of dough, as the mobility of hydrated gluten

434

increases in the network.16 Thus, adding NaCl or/and NaHCO3 could mitigate the 15



435

stickiness and enhance the strength of dough via the interaction of ions and water to

436

delay the hydration, which was confirmed by the longer mixing time during the

437

development of dough. Meanwhile, as a result of the interchange of SH-SS or

438

non-covalent interaction (hydrogen bonds or hydrophobic) on the molecules level,

439

the gluten polymerization could contribute to the change of dough rheological

440

behavior, which was partially consistent with our observation. However, the change

441

of the surface hydrophobic interactions was not significantly in our study. In summary,

442

the presence of NaHCO3 and/or NaCl induced the macromolecular aggregation via SH

443

related cross-linking or other non-redox reaction and altered secondary structure of

444

gluten, which resulted in the changes of physical and rheological properties of dough.

445

In order to achieve a significant amount of sodium reduction in wheat products and

446

develop high quality bakery foods, more in-depth studies are needed to address

447

protein polymerization and aggregation during the dough development.

448 449

Acknowledgement

450

This is contribution no. 18-316-J from the Kansas Agricultural Experimental Station.

451

Financial support was provided by Frito-Lay North America and the Kansas State

452

University, Department of Grain Science and Industry.

453 454

16


Page 16 of 33

Page 17 of 33


455

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456

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yeast-leavened products. I. Rheological attributes. J. Sci. Food Agric. 2012, 92

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459 460

2. He, H.; Roach, R. R.; Hoseney, R. C. Effect of Nonchaotropic Salts on Flour Bread-Making Properties. Cereal Chem. 1992, 69 (4), 366-371.

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3. Luft, F. C.; Zemel, M. B.; Sowers, J. A.; Fineberg, N. S.; Weinberger, M. H. Sodium

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bicarbonate and sodium chloride: effects on blood pressure and electrolyte

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homeostasis in normal and hypertensive man. J. Hypertens. 1990, 8 (7),

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4. Sacks, F. M.; Svetkey, L. P.; Vollmer, W. M.; Appel, L. J.; Bray, G. A.; Harsha, D.;

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Lin, P. H.; Grp, D. A.-S. C. R., Effects on blood pressure of reduced dietary

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5. Roth, G. A.; Johnson, C.; Abajobir, A.; Abd-Allah, F.; Abera, S. F.; Abyu, G.; Ahmed,

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8. Angus, F. Dietary salt intake: sources and targets for reduction. Reducing salt in

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Structural Characteristics of Wheat Gluten Polypeptide Films.

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Cereal Sci. 2009, 50 (1), 11-21.

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13. Wieser, H. Chemistry of gluten proteins. Food Microbiol, 2007, 24(2), 115-119.

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17. Ukai, T.; Matsumura, Y.; Urade, R. Disaggregation and reaggregation of gluten proteins by sodium chloride. J. Agric. Food. Chem. 2008, 56 (3), 1122-1130. 18. AACC. Approved methods of the American Association of Cereal Chemists; The Association: St. Paul, MN, 2000. 19. Wieser, H.; Antes, S.; Seilmeier, W. Quantitative determination of gluten protein

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chromatography. Cereal Chem. 1998, 75(5), 644-650.

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20. Chen, P.; Zhang, L. Interaction and properties of highly exfoliated soy

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protein/montmorillonite nanocomposites. Biomacromolecules. 2006, 7(6),

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disulfide bonds in suspensions of solubilized and particulate cereal proteins.

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23. Thannhauser, T. W.; Konishi, Y.; Scheraga, H. A. Analysis for disulfide bonds in peptides and proteins. Methods Enzymol. 1987, 143, 115-119. 24. Kato, A., Matsuda, T.; Matsudomi, N.; Kobayashi, K. Determination of protein

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Chem. 1984, 32(2), 284-288.

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25. McCann, T. H.; Day, L. Effect of sodium chloride on gluten network formation,

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26. Larsson, H. Effect of pH and sodium chloride on wheat flour dough properties:

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Ultracentrifugation and rheological measurements. Cereal Chem, 2002, 79(4),

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544-545.

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27. Yiannopoulos, S.; Kontogiorgi, A.; Poulli, E.; Krokida, M. Effect of Carob Flour

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Addition on the Rheological Properties of Gluten-Free Breads. Food Bioproc

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Tech. 2014, 7(3), 868-876.

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28. Ong, Y. L.; Ross, A. S.; Engle, D. A. Glutenin Macropolymer in Salted and Alkaline Noodle Doughs. Cereal Chem. 2010, 87(1), 79-85. 29. Shiau, S. Y.; Yeh, A. I. Effects of alkali and acid on dough rheological properties and characteristics of extruded noodles. J Cereal Sci. 2001, 33(1), 27-37.

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30. Lynch, E. J.; Dal Bello, F.; Sheehan, E. M.; Cashman, K. D.; Arendt, E. K.

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Fundamental studies on the reduction of salt on dough and bread

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characteristics. Food Res Int. 2009, 42(7), 885-891.

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31. Salvador, A.; Sanz, T.; Fiszman, S. M. Dynamic rheological characteristics of wheat

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flour-water doughs. Effect of adding NaCl, sucrose and yeast. Food

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Hydrocolloid. 2006, 20(6), 780-786.

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32. Shewry, P. R.; Halford, N. G.; Belton, P. S.; Tatham, A. S. The structure and

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properties of gluten: an elastic protein from wheat grain. Philos Trans R Soc

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Lond B Biol Sci. 2002, 357(1418), 133-142.

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33. Wu, J. P.; Beta, T.; Corke, H., Effects of salt and alkaline reagents on dynamic

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rheological properties of raw oriental wheat noodles. Cereal Chem. 2006, 83

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(2), 211-217. 19



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34. Lagrain, B.; Thewissen, B. G.; Brijs, K.; Delcour, J. A. Impact of redox agents on the

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extractability of gluten proteins during bread making. J. Agric. Food. Chem.

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2007, 55(13), 5320-5325.

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35. Butow, B. J.; Gras, P. W.; Haraszi, R.; Bekes, F., Effects of different salts on mixing

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and extension parameters on a diverse group of wheat cultivars using 2-g

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mixograph and extensigraph methods. Cereal Chem. 2002, 79 (6), 826-833.

551 552 553

36. Letang, C.; Piau, M.; Verdier, C. Characterization of wheat flour-water doughs. Part I: Rheometry and microstructure. J. Food Eng. 1999, 41(2), 121-132. 37. Barak, S.; Mudgil, D.; Khatkar, B. S. Relationship of gliadin and glutenin proteins

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with dough rheology, flour pasting and bread making performance of wheat

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varieties. LWT-Food Sci Technol. 2013, 51(1), 211-217.

556 557 558

38. Doane, T. L.; Chuang, C. H.; Hill, R. J.; Burda, C., Nanoparticle zeta-Potentials. Acc. Chem. Res. 2012, 45 (3), 317-326. 39. Bruneel, C.; Lagrain, B.; Brijs, K.; Delcour, J. A. Redox agents and

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N-ethylmaleimide affect the extractability of gluten proteins during fresh

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pasta processing. Food Chem. 2011, 127(3), 905-911.

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40. Wagner, M.; Morel, M. H.; Bonicel, J.; Cuq, B. Mechanisms of Heat-Mediated

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Aggregation of Wheat Gluten Protein upon Pasta Processing. J. Agric. Food.

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Chem. 2011, 59(7), 3146-3154.

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41. Netto, L. E. S.; de Oliveira, M. A.; Monteiro, G.; Demasi, A. P. D.; Cussiol, J. R. R.;

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Discola, K. F.; Demasi, M.; Silva, G. M.; Alves, S. V.; Faria, V. G.; Horta, B. B.,

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Reactive cysteine in proteins: Protein folding, antioxidant. defense, redox

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signaling and more. Comp Biochem Physiol C Toxicol Pharmacol. 2007, 146

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(1-2), 180-193.

569

42. Wellner, N.; Bianchini, D.; Mills, E. N. C.; Belton, P. S., Effect of selected

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Hofmeister anions on the secondary structure and dynamics of wheat

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prolamins in gluten. Cereal Chem. 2003, 80 (5), 596-600.

572

43. Wellner, N.; Mills, E. N. C.; Brownsey, G.; Wilson, R. H.; Brown, N.; Freeman, J.;

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Halford, N. G.; Shewry, P. R.; Belton, P. S., Changes in protein secondary

574

structure during gluten deformation studied by dynamic Fourier transform 20


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575 576

infrared spectroscopy. Biomacromolecules. 2005, 6 (1), 255-261. 44. Lagrain, B.; Brijs, K.; Delcour, J. A. Reaction Kinetics of Gliadin-Glutenin

577

Cross-Linking in Model Systems and in Bread Making. J. Agric. Food. Chem.

578

2008, 56(22), 10660-10666.

579

45. Georget, D. M. R.; Belton, P. S. Effects of temperature and water content on the

580

secondary structure of wheat gluten studied by FTIR spectroscopy.

581

Biomacromolecules. 2006, 7(2), 469-475.

582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 21



605 606

Figure Captions

607

Figure 1. Typical Mixograms and Farinograms of soft wheat flour doughs with (P0) 0%

608

NaCl, (P1) 1.0% NaHCO3, (P2) 0.5% NaCl/0.3% NaHCO3, and (P3) 0.7% NaCl.

609 610

Figure 2. Dynamic rheological properties of the soft wheat flour doughs with

611

addition of salt/baking soda, (A) loss modulus G” as a function of frequency at a

612

strain of 0.1%, (B) storage modulus G’ as a function of frequency at a strain of 0.1%,

613

(C) loss modulus G” as a function of strain at a frequency of 1Hz, (D) storage modulus

614

G’ as a function of strain at a frequency of 1Hz, and (E) tan δ as a function of

615

frequency at a strain of 0.1%.

616 617

Figure 3. Typical RP-HPLC chromatographs of gluten extract separated from soft

618

wheat flour dough samples of (A) gliadins in control; and (B) glutenins in control,

619

using a Aeris WIDEPORE XB-C18 column (3.6 μm, 150 x 4.6 mm) with UV detection at

620

a wavelength of 210 nm. The flow rate was 0.6 mL/min at 60 °C and injection volume

621

was 10 µL. The mobile phases were water containing 0.1% trifluoroacetic acid

622

(solvent A), and acetonitrile containing 0.1% trifluoroacetic acid (solvent B).

623 624

Figure 4. Zeta potential of glutens isolated from soft wheat flour dough added by

625

salt/baking soda in deionized water with solid content of 0.1%.

626 627

Figure 5. Typical FTIR scan of gluten extracted from soft wheat flour dough showing

628

the different bands: (A) (C=O lipid vibration at around 1750 cm-1), (B) (amide I

629

1600-1700 cm-1), and (C) (amide II 1500-1580 cm-1).

630 631

Figure 6. FTIR deconvolution and second-derivative spectra of amide I region for

632

gluten extracted from soft wheat flour dough. A full discussion of the assignment is

633

given as extended chains (1600-1615 cm-1), β-Sheet (1624-1640, 1681 cm-1), random

634

coil (1640-1650 cm-1), α-helix (1650-1660 cm-1), β-turn (1660-1670, 1694 cm-1).45 22


Page 22 of 33

Page 23 of 33


635 636

Table 1. Mixing properties of soft wheat flour dough samples.

NaCl (% fwb)

NaHCO3 (% fwb)

0 0.5 0.7 1.0 0 0 0.5 0.5 1.0 1.0

0 0 0 0 0.3 1.0 0.3 1.0 0.3 1.0

Mixograph Water Abs (% fwb) 59 59 59 59 59 59 59 59 59 59

Mix Time (min) 3.50a 5.00c 4.25b 4.25b 6.25d 6.00d 5.00c 5.75d 5.25c 6.25d

Farinograph Water Abs (% fwb) 55.0 54.0 53.4 52.8 53.8 54.7 54.0 54.8 54.3 54.8

Mix Time (min) 3.0 3.3 3.0 3.6 3.2 7.0 4.3 4.3 4.2 4.1

Stability (min) 6.3 7.0 10.5 8.5 14.2 15.5 13.0 11.4 9.3 11.6

637

abcd

638

different at P < 0.05. Statistics were not run on farinograph data as there was only

639

one replicate.

Means with different superscripts within the same column are significantly

640

23



641

642 643

Page 24 of 33

Table 2. Dough properties with different NaCl and/or NaHCO3 addition. NaCl (% fwb)

NaHCO3 (% fwb)

0 0.5 0.7 1.0 0 0 0.5 0.5 1.0 1.0

0 0 0 0 0.3 1.0 0.3 1.0 0.3 1.0

Keiffer Test Force (g) 7.4f 12.4cde 8.5ef 12.7bcde 13.7bcd 15.0bc 9.9def 20.3a 14.3bcd 17.3ab

Distance (mm) 37.2bc 47.9ab 54.1a 49.9abc 57.0a 29.6c 55.7a 30.8c 51.7ab 32.7c

abcdef

Elongational Viscosity (Pa.s) 6477cd 4433e 6307cde 5046de 6403cd 12110a 8943b 9821b 6858c 9798b

Means with different superscripts within the same column are significantly different at P < 0.05.

24


Page 25 of 33


644 645

Table 3. Gluten contents of RP-HPLC fractions isolated from soft wheat flour doughs at the different levels of salt and baking soda addition.

Sample

HPLC elution range concentration (μg/mg) ωgliadin

αgliadin

γgliadin

Total gliadins

ωbgliadin

HMW- LMWGS GS

Ratio Total glutenins (gli:glu)

Control

15.0a

107.1a

91.5ab

213.7a

3.1a

30.7b

73.1ab

106.9c

2.0a

NaCl 0.5%

14.5a

106.4ab

90.3abc

211.3ab

3.2a

32.5ab

73.7ab

112.6bc

1.9a

NaCl 0.7%

15.1a

106.5a

88.0abc

209.7abc

3.5a

34.3ab

74.2ab

118.5abc

1.8abc

NaCl 1.0%

14.4a

103.2abc

83.0bc

200.6bcde

4.1a

34.4ab

80.6ab

125.7ab

1.6bc

NaHCO3 0.3%

14.3a

102.5abc

94.7a

211.4ab

3.2a

31.1ab

72.8ab

113.7abc

1.9ab

NaHCO3 1.0%

13.7a

99.5abcd

85.0bc

198.1cde

3.9a

33.0ab

81.3a

124.8ab

1.6bc

NaCl 0.5%/NaHCO3 0.3%

14.7a

100.2abcd

90.1a

205.0abcd

3.8a

30.8ab

71.9b

113.1bc

1.8ab


14.2a

97.3cd

85.1bc

196.6de

4.3a

33.8ab

79.5ab

124.2ab

1.6c


13.7a

98.3bcd

84.6bc

196.7de

4.3a

36.7a

79.3ab

126.9a

1.6c


14.3a

93.9d

82.2c

190.4e

4.3a

36.2ab

80.2ab

127.2a

1.5c

646 647 648 649 650 651 652 653 654 655

abcde

Means with different superscripts within the same column are significantly different at P < 0.05.

25



Page 26 of 33

Table 4. The effect of baking soda or salt on the SH and SS content, secondary structure and surface hydrophobicity of glutens separated from soft wheat flour doughs.

Total SH contentA Free SH contentA SS contentA Extended Chains (%)B β-Sheet (%)B Random coil (%)B α-helix (%)B β-turn (%)B Hydrophobicity

control

salt 0.5%

salt 0.7%

salt 1.0%

soda 0.3%

soda 1.0%

56.8±1.5a 6.1±0.1a 25.4±0.5a 5.2±0.6a 19.7±1.4a 35.2±0.1a 31.4±0.8a 7.6±0.7a 13.4±1.5a

55.6±2.0a 5.2±0.1abc 25.2±0.7a 7.9±0.4a 22.0±1.8a 22.1±1.4b 36.6±0.2a 7.6±0.4a 13.1±1.1a

57.2±2.9a 5.3±0.1abc 26.2±0.9a 6.5±1.2a 23.2±1.0a 24.5±1.9b 32.9±1.6a 8.8±0.5a 13.4±1.0a

58.5±1.5a 5.0±0.2bc 26.8±0.5a 8.5±0.4a 24.3±2.1a 25.2±1.6b 32.9±5.1a 8.9±1.0a 13.3±0.7a

56.5±1.3a 5.5±0.1ab 25.5±0.9a 5.6±1.2a 22.6±0.4a 24.9±3.0b 28.8±0.9a 8.0±0.1a 13.8±0.9a

59.9±1.8a 3.9±0.2d 27.9±0.8a 5.8±0.5a 25.2±0.1a 23.3±1.0b 33.8±1.8a 7.6±1.0a 13.7±0.7a

abcd

salt 0.5% /soda 0.3% 56.3±3.3a 5.4±0.1ab 25.4±1.1a 8.4±0.4a 22.6±0.3a 23.2±0.4b 37.6±0.4a 8.1±0.1a 12.9±0.8a

salt 1.0% /soda 0.3% 58.2±3.1a 5.3±0.3abc 26.4±1.3a 7.9±0.1a 23.1±0.4a 28.9±0.4ab 39.4±1.0a 5.9±0.6a 13.4±1.1a

salt 0.5% /soda 1.0% 56.3±4.8a 4.4±0.1cd 25.9±1.7a 6.2±0.5a 21.9±2.4a 28.0±2.1ab 30.4±1.4a 6.7±1.0a 13.1±1.4a

salt 1.0% /soda 1.0% 57.1±4.6a 4.4±0.3cd 26.3±1.7a 6.8±1.4a 24.2±3.0a 28.1±0.2ab 31.8±1.5a 5.7±1.4a 13.5±1.1a

Means with different superscripts within the same row are significantly different at P < 0.05. Value is represented as the mean ± standard deviation (n=2). A SH and SS contents were expressed by nmoles/mg sample. B Gluten secondary structures were assignment as extended chains (1600-1615 cm-1), β-Sheet (1624-1640, 1681 cm-1), random coil (1640-1650 cm-1), α-helix (1650-1660 cm-1), β-turn (1660-1670, 1694 cm-1).45

26


Page 27 of 33


Figure 1.

27



control salt 0.7% soda 0.3% salt 0.5%/soda 0.3% salt 1.0%/soda 0.3%

A 3.2E+04

salt 0.5% salt 1.0% soda 1.0% salt 0.5%/soda 1.0% salt 1.0%/soda 1.0%

Page 28 of 33


B 6.5E+04

salt 0.5% salt 1.0% soda 1.0% salt 0.5%/soda 1.0% salt 1.0%/soda 1.0%

2.2E+04 G'(Pa)

G'' (Pa)

4.5E+04

1.2E+04

2.5E+04

5.0E+03

2.0E+03 0.1

1 Frequency (Hz) soda 1.0% salt 0.5% salt 1.0% salt 0.5%/soda 0.3% salt 1.0%/soda 0.3%

C

0.1

10 control salt 0.7% soda 0.3% salt 0.5%/soda 1.0% salt 1.0%/soda 1.0%

1

Frequency (Hz)


D 4.2E+04

10 salt 0.5% salt 1.0% soda 1.0% salt 0.5%/soda 1.0% salt 1.0%/soda 1.0%

1.3E+04

G' (Pa)

9.0E+03

2.2E+04

5.0E+03 1.2E+04

1.0E+03 0.10%

1.00% Strain

10.00%

2.0E+03 0.10%

1.00%

Strain

E

0.4 tan δ

G'' (Pa)

3.2E+04

control

salt 0.5%

salt 0.7%

salt 1.0%

soda 0.3%

soda 1.0%

salt 0.5%/soda 0.3%

salt 0.5%/soda 1.0%

salt 1.0%/soda 0.3%

salt 1.0%/soda 1.0%

0.2 1

Frequency (Hz)

10

Figure 2. 28


10.00%

Page 29 of 33


Figure 3.

29



Page 30 of 33

8 5.57 4

3.92

Zeta potential (mV)

1.13

1.93

0

-3.69

-4 -5.73

-5.86 -8 -9.53 -12

-12.88 -16

Figure 4.

30


-11.46

Page 31 of 33


Figure 5.

31



Figure 6.

32


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Enhanced dough physical and rheological properties with NaCl and/or NaHCO3 were a result of synergistic inter- and intra-molecular interactions 279x157mm (300 x 300 DPI)


Effect of Sodium Chloride and Sodium Bicarbonate on the

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