Sensory characteristics and functionality of sonicated whey

Food Research International 49 (2012) 694–701

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Sensory characteristics and functionality of sonicated whey S. Martini, M.K. Walsh ⁎ Department of Nutrition, Dietetics, and Food Sciences, Utah State University, 8700 Old Main Hill, 750N 1200E, 84322‐8700, Logan, UT, USA

a r t i c l e

i n f o

Article history: Received 3 July 2012 Accepted 14 September 2012 Keywords: Power ultrasound Whey protein Turbidity Protein solubility Sensory

a b s t r a c t This research employed power ultrasound (US) on a whey suspension containing 28.2% solids (10% total protein) and characterized the US treated whey with respect to descriptive sensory evaluation, thermal stability, particle size, emulsification activity, and viscosity. The sensory attributes of sonicated and control whey were determined at three pH levels (3.5, 4.5 and 7.5) with and without nose clips using a trained panel. Nose clips were used to avoid the detection of aromatics and to evaluate whey quality in terms of taste only. There were pH dependent differences in 7 of 21 attributes tested with nose clips and there were no significant differences between the control and US samples when comparing the same pH levels, with the exception of cardboard which was found at a higher level in the pH 7.5 control sample than in the US pH 7.5 sample. There were 9 pH dependent differences in attributes when tested without nose clips and there were no significant differences between the control and US samples of the same pH, with the exception of cardboard being higher in the control pH 4.5 sample, and malty being higher in the control pH 7.5 sample compared to their respective US samples. Thermal stability was monitored via transmission of samples at 600 nm and transmissions of US and control whey were significantly different based on temperature (72 or 85 °C), concentration (5 or 10% solids) and pH (3.5, 4.5 and 7.5). Transmissions were higher in all US samples compared to the controls indicating less turbidity in US samples. Transmissions were higher in samples with the lower heating treatment of 72 °C, at pH 7.5, and at 5% solids. US whey resulted in a more stable emulsion, lower particle size, and a higher viscosity compared to control whey. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Ultrasound (US) is used in food processing to induce lipid, ice, and sugar crystallization (Chow, Blindt, Chivers, & Povey, 2003, 2005; Martini, Suzuki, & Hartel, 2008), form emulsions, and inactivate microorganisms (Chandrapala, Oliver, Kentish, & Ashokkumar, 2012a; Chemat & Zill-e-Huma, 2011). High intensity sonication waves (know as power ultrasound) (20 kHz with sound intensities above 1 W cm −2) generate acoustic cavitation in liquids, where micro gas bubbles grow and implode to generate localized hot spots and increased pressure. The conditions within these collapsing bubbles generate localized temperatures exceeding 5500 °C and pressures of up to 50 MPa (reviewed in Chandrapala et al., 2012a; Chandrapala, Oliver, Kentish, & Ashokkumar, 2012b). There has been recent research on the use of US to improve the functionality of whey protein (Martini, Potter, & Walsh, 2010). Whey proteins can have many functionalities (solubility, emulsification, gelation, viscosity, and foaming) that depend on both intrinsic (amino acid composition, protein size, and protein flexibility) and extrinsic (pH, ionic strength, temperature, and concentration) conditions. In ⁎ Corresponding author at: Department of Nutrition, Dietetics, and Food Sciences, Utah State University, 8700 Old Main Hill, 750 N 1200 E, 84322‐8700, Logan, UT, USA. Tel.: + 1 435 797 2177; fax: + 1 435 797 2379. E-mail address: [email protected] (M.K. Walsh). 0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2012.09.018

general, whey proteins are the least soluble at pH 4.5–5.2 with an increase in solubility at acidic and basic pH values. There is usually a decrease in whey protein solubility with an increase in temperature. Pelegrine and Gasparetto (2005) showed that whey treated at 60 °C had the lowest solubility at pH values of 4.5 and 6.8 but much higher solubilities at pH values of 5.5, 7.8 and 3.5. Several studies showed that US can change the functional properties of whey proteins. Recently, Martini et al. (2010) used US on commercially available whey samples at different steps in the whey processing process. They found that using US at 20 kHz and 15 W for 15 min on a sample that contained 28% solids (10% final protein) resulted in a 90% decrease in turbidity compared to the control. Ashokkumar et al. (2009) showed that whey proteins (6% protein solution) sonicated after heat treatment had reduced viscosity and reduced heat-induced protein aggregates. Zisu, Bhaskaracharya, Kentish, and Ashokkumar (2010) found that US decreased whey protein particle size and viscosity and increased whey protein gel strength. They also found that these improvements in functional properties were retained after ultrafiltration and drying. Kresic, Lelas, Jambrak, Herceg, and Brncic (2008) showed an increase in water solubility of whey proteins treated with US. They suggested that US enhanced protein solubility by changing protein conformation. Jambrak, Mason, Lelas, Herceg, and Herceg (2008) evaluated the effect of US on the solubility and foaming properties of whey protein suspensions. They found that both functional properties were

S. Martini, M.K. Walsh / Food Research International 49 (2012) 694–701 Table 1 Taste concentrations in aqueous phase used for screening of panelists and during panel training on specific intensities.

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Table 2 Mean values of descriptive sensory attributes for sonicated (US) or control whey proteins with nose clips.

Attribute Taste definition

Treatment

Levels Scale value

Attribute

Control pH 3.5

Control pH 4.5

Control pH 7.5

US pH 3.5

US pH 4.5

US pH 7.5

P-value

Bitter

Caffeine

0.05% 0.08% 0.15% 0.20% 0.35% 0.50% 0.05% 0.08% 0.15% 2% 5% 10% 0.7% 1.4% 2.8%

Animal Astringent Bitter Brothy Cardboard Cereal Chalky Fecal Flour paste Fruity Malty Metallic Opacity Pasta Roasted Salty Soapy Sour Sweet Viscosity Yeasty

0.27 2.23a 0.36 0.73bc 0.64b 0.00b 0.00 0.00 0.32ab

0.27 0.5b 0.05 1.09abc 1.18ab 0.25b 0.00 0.00 0.50a

0.61 0.73 b 0.05 1.55 a 1.32 a 0.32ab 0.00 0.00 0.75a

0.43 2.77 0.27 0.52 0.68 0.09 0.00 0.00 0.00

0.27 1.14 0.09 1.30 0.68 0.14 0.00 0.00 0.41

0.41 0.45 0.09 1.34 0.64 0.68 0.00 0.00 0.70

0.2177 0.0001 0.0904 0.0171 0.0418 0.0151 0.0000 0.0000 0.0076

0.05 0.05 0.00 2.32 0.00 0.00 2.45 0.00 8.11 0.73 1.75 0.00

0.23 0.32ab 0.00 2.20 0.05 0.07 1.89 0.05 3.02b 0.80 1.66 0.05

0.00 0.48 0.00 2.23 0.09 0.00 1.70 0.05 1.48 0.55 1.68 0.09

0.00 0.05 0.00 1.89 0.09 0.00 2.43 0.00 8.75 0.80 1.48 0.00

Salty

Taste elicited by caffeine

Taste elicited by salts

Sodium chloride

Sour

Taste elicited by acids

Citric acid

Sweet

Taste elicited by sugar

Sucrose

Umami

Taste elicited by monosodium glutamate (MSG)

Monosodium glutamate

2 5 10 2.5 5 8.5 2 5 10 2 5 10 5 9 13

improved when US was used and that these effects were dependent on the acoustic frequency used: higher frequencies (40 kHz) were not as efficient as lower ones (20 kHz). Whey proteins are commonly used in sports beverages, which are generally formulated at acidic pH values (pH 3.5) because this results in a clear solution. Beverages produced at neutral pH are generally opaque or turbid, but require higher thermal processing temperatures than acidic beverages (Beecher, Drake, Luck, & Foegeding, 2008; LaClair & Etzel, 2009). Increasing the clarity of whey protein solutions at neutral pH values is of industrial importance. The sensory attributes of whey are also an important factor in beverage formulation. Previous research has shown that sonication of oils (sunflower, olive and soybean) can result in flavor changes. Specifically, sonication of sunflower oil leads to the generation of hydroperoxides and lipid oxidation (Chemat et al., 2004). Whey that is being processed to produce whey protein concentrate 80 can contain up to 5% fat, therefore, the use of US on whey has the potential to change the whey flavor. Most of the research on US and whey functionality described above was performed in model systems consisting of whey protein suspensions prepared from dry whey protein isolate and/or concentrate. The objective of this research was to evaluate the effects of US on whey protein's sensory and functionality using fresh whey. Whey was sonicated and the influence of sonication on the sensory, emulsification, particle size, thermal stability, and viscosity characteristics was investigated. 2. Materials and methods 2.1. Whey Liquid whey was obtained from a production line from Glanbia Foods Inc. (Twin Falls, ID, USA). The whey contained 28.2% solids of which 35.6% was protein. Samples were transported refrigerated to Utah State University and immediately frozen upon arrival. The protein content was confirmed using a Thermo Scientific Modified Lowry Protein Assay Kit (Waltham, MA, USA) with bovine serum albumin (BSA) as the standard. One percent solid matter suspensions were prepared using deionized water (pH 7.0) to determine protein content according to the manufacturer's protocol. 2.2. Ultrasound treatment Ultrasound treatment was according to Martini et al. (2010). Briefly, 50 ml of the whey was sonicated at 15 W for 15 min with a 3.2 mm titanium microtip using a Misonix Sonicator 3000 (Misonix Inc., NY, USA). The temperature was monitored during sonication

b

a

a

c

a

c b b

b

b

a

0.20 0.16 0.00 2.11 0.00 0.00 1.86 0.00 3.55 0.84 1.55 0.00

b

ab b b

ab

ab

b

0.05 0.20 0.09 2.30 0.23 0.11 1.86 0.02 1.20 0.73 1.61 0.00

b

ab b a

a

ab

c

0.1870 0.0445 0.4210 0.2629 0.3677 0.2303 0.2760 0.5797 0.0001 0.9345 0.3118 0.1254

Means with the same letter are not significantly different in each row.

and reached 60 °C after the sonication treatment. With this sonication treatment, previous research has shown that there was no denaturation of whey proteins leading to aggregation (Martini et al., 2010). After US, samples were poured into two 50 ml tubes and placed immediately into a − 20 °C freezer at a slight angle. Samples were kept at least 24 h in the freezer prior to freeze drying (Dura-Top, FTS Systems, NJ, USA). After freeze drying, the whey was pooled and ground using a pestle and mortar for a minimum of 5 min and stored at − 20 °C. 2.3. Descriptive sensory evaluation The whey powders were reconstituted at 10% solids (w/v). The samples were divided into three groups, and were reconstituted with 50 mM phosphate buffer pH 7.5, 50 mM acetate buffer pH 3.5 and 50 mM sodium acetate pH 4.5, in both the control samples and the samples that received US treatment. Samples were mixed by hand with a whisk, and mixed on stir plates in glass beakers at refrigeration temperatures (4 °C) for 24 h prior to sensory analysis. The samples were removed from refrigeration 1 h before the test, and approximately 30 ml of sample was dispensed into 59 ml plastic portion cups (Bakers & Chefs, Bentonville, AR, USA) with plastic lids. Samples were shaken gently prior to dispensing to avoid settling. A sensory descriptive panel (n = 11) was recruited from the local community to evaluate the whey samples. Potential participants were recruited using local newspapers and flyers. Participants were screened for their ability to differentiate between basic tastes in both identification and intensity ratings, according to established guidelines (American Society for Testing and Materials, 1991). Those who passed basic screening were recruited for the panel and monitored over time for ability to identify and quantify whey attributes in order to be included in the final evaluation. Panelists ranged in age from 18 to 60, with 8 males and 3 females, though demographics were not expected to influence the ratings in a trained descriptive panel. Panelists used in this test were also trained in descriptive methods and attributes on cheese for a minimum of 150 h, and were trained specifically on whey attributes for a minimum of 20 h. A 15-point category scale was used in the training of the panelists on the whey specific lexicon. This scale is commonly used among

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sucrose, citric acid, caffeine, sodium chloride, and monosodium glutamate were used, respectively. Concentrations used to train the panelists on specific taste intensities are shown in Table 1. Once the panelists were familiar in the use of the scale, they were introduced to the attributes that were specific to whey as described in Russell et al. (2006) and trained using the references described by Russell et al. (2006). The 21 attributes used included the basic tastes (sweet, sour, salty, bitter, and umami), astringency (a chemical feeling factor), viscosity, and opacity, among others. The panelists were familiarized with rating the attributes at different intensities during training, and were evaluated using analysis of variance (ANOVA) throughout training to ensure that they were able to provide consistent results. Panelists were asked to evaluate each sample in duplicate in a randomized and balanced manner, with 3-digit blinding codes on each sample cup. Six samples were presented at once. Panelists were asked to taste, expectorate, and identify and rate the intensity of 21 attributes using a 15-point category scale. This procedure was first performed using nose clips and repeated without nose clips. The attributes evaluated were: animal, astringent, bitter, brothy, cardboard, cereal, chalky, fecal, flour paste, fruity, malty, metallic, opacity, pasta, roasted, salty, soapy, sour, sweet, viscosity, and yeasty (Russell et al., 2006). The panelists were given distilled water and unsalted crackers to cleanse their palate between tasting the samples. After tasting all samples, panelists waited for 15 min prior to tasting a replicate of the samples in a different randomized order.

Table 3 Mean values of descriptive sensory attributes for sonicated (US) or control whey proteins without nose clips. Attribute

Control pH 3.5

Control pH 4.5

Control pH 7.5

US pH 3.5

US pH 4.5

US pH 7.5

P-value

Animal Astringent Bitter Brothy Cardboard Cereal Chalky Fecal Flour paste Fruity Malty Metallic Opacity Pasta Roasted Salty Soapy Sour Sweet Viscosity Yeasty

0.50 2.86 0.36 0.84 1.02 0.09 0.00 0.00 0.36

0.45 1.16 0.14 1.55 1.84 0.41 0.00 0.00 0.68

0.57 0.61 0.05 1.91 1.86 0.50 0.00 0.00 0.89

0.57 3.34 0.32 0.77 0.89 0.14 0.00 0.00 0.43

0.50 0.93 0.23 1.48 1.00 0.23 0.00 0.00 0.64

0.70 0.36 0.00 1.84 1.18 0.75 0.05 0.00 0.91

0.8103 0.0001 0.0473 0.0003 0.0082 0.0456 0.4210 0.0000 0.0434

0.20 0.14 0.00 2.23 0.07 0.00 3.39 0.00 9.27 0.95 1.61 0.09

a a b b b

b

c

a

a

0.14 0.32 0.05 2.20 0.05 0.00 1.95 0.00 3.82 1.16 1.66 0.00

b abc a a ab

ab

0.05 1.02 0.00 2.41 0.25 0.05 2.02 0.05 1.66 1.05 1.68 0.00

bc

b

b

bc bc a a ab

a

a

b

c

0.09 0.07 0.00 2.11 0.09 0.00 2.98 0.00 9.59 0.95 1.59 0.00

a ab b b b

b

c

a

a

0.30 0.52 0.05 2.07 0.14 0.09 1.98 0.05 4.14 1.14 1.50 0.05

bc abc a b b

ab

b

b

b

0.00 0.55 0.00 2.30 0.25 0.14 1.68 0.00 1.64 1.14 1.64 0.00

c c a ab a

a

b

b

c

0.1413 0.0001 0.5567 0.1324 0.6453 0.1821 0.0003 0.5567 0.0001 0.9684 0.3461 0.5279

Means with the same letter are not significantly different in each row.

descriptive panels across many products, including cheese and whey (Drake & Civille, 2003; Russell, Drake, & Gerard, 2006). The descriptive lexicon chosen for use by the panelists was based on previous research by Russell et al. (2006). Panelists were first trained on the identification and rating on the intensity scale of the five basic tastes: sweet, sour, bitter, salty, and umami. Solutions of

2.4. Thermal stability After freeze drying and grinding, suspensions of the treated samples were prepared at 5 or 10% solids using 50 mM phosphate buffer pH 7.5, 50 mM acetate buffer pH 3.5 or 50 mM sodium acetate

2

1.5 Control 7.5

1 Control 4.5

PC2 16.3%

0.5

Cardboard Malty

HIU 3.5

Sour Astringent Control 3.5

-2

-1.5

-1

0 -0.5

0

0.5

Brothy 1 1.5 Flour Paste

2

HIU 4.5

-0.5 Cereal

-1

-1.5 HIU 7.5

-2

PC1 76.8% Fig. 1. Principal component analysis of the sonicated (US) and control whey samples and significant attributes when nose clips were used.

S. Martini, M.K. Walsh / Food Research International 49 (2012) 694–701

697

2

1.5 Control 7.5

1

Control 4.5 Cardboard

0.5 PC2 7.4%

Control 3.5

-2

Malty

Salty Astringent Bitter Sour

-1.5

-1

Brothy

0 -0.5

0

0.5

1Flour paste1.5

2

Cereal

US 3.5

-0.5 US 4.5

-1

US 7.5

-1.5

-2 PC1 85.0% Fig. 2. Principal component analysis of the sonicated (US) and control whey samples and significant attributes when samples were smelled prior to tasting.

pH 4.5. The suspensions were kept under agitation for 2 h to maximize sample dissolution. Samples were heated in an oil bath set at 135 °C for 20 s (final sample temperature of 72 °C) or 35 s (final sample temperature of 85 °C). Samples were diluted to 0.2% solids and the turbidity was then measured with a Shimadzu Biospec1601 (Columbia, MD, USA) at 600 nm as percentage of transmittance (%T600 nm). An aliquot of each sample was also centrifuged at 9000 rpm (12,600 ×g) for 25 min. The supernatant and an aliquot of the sample before centrifugation were assayed for protein using the Thermo Scientific Modified Lowry Protein Assay Kit as described before. Data is reported as percentage of protein lost to precipitation. 2.5. Emulsion preparation and stability Oil-in-water emulsions, 80 ml water and 20 ml soybean oil, were prepared to contain 2% US or control whey. Samples were mixed with

a high-speed blender (polytron) (Ultra-turrax T25, Janke and Kunkel, Staufen, Germany) at 18,000 rpm for 5 min and then immediately passed through a microfluidizer (Microfluidics Corporation, Newton, MA, USA) at 17.4 ± 1.6 MPa (~ 25,000 psi) three times. All the emulsions were prepared in triplicate and stability and droplet size of the emulsions were measured from day zero (the day emulsion was prepared) to day ten. The stability of emulsions was determined using Turbiscan, a vertical scan macroscopic analyzer (TurbiScan MA2000, Toulouse, France). Approximately, 5 ml of each emulsion was dispensed into 11 cm long glass tubes to measure the percent change in backscattering as described by Garg, Martini, Britt, and Walsh (2010). A change in the backscattering of an emulsion at the bottom of the tube is related to the destabilization of emulsions via clarification. The absolute thickness of the clarification layer in mm over time was calculated to follow the extent of emulsion destabilization.

Table 4 Transmissions (600 nm) of heat-treated whey samples at 5 or 10% solids at different pH values and temperatures. pH

5%

10%

72 °C

7.5 4.5 3.5

85 °C

72 °C

85 °C

Control

US

Control

US

Control

US

Control

US

82.88 (0.56)AaAa 63.67 (0.57)BaAa 74.01 (0.23)CaAa

89.28 (0.41)AbAa 66.03 (0.51)BbAa 81.53 (0.16)CbAa

73.88 (1.21)AaAb 25.52 (0.43)BaAb 65.92 (0.93)CaAb

80.56 (1.07)AbAb 30.05 (1.43)BbAb 70.39 (1.51)CbAb

83.55 (o.42)AaBa 66.75 (1.37)BaBa 72.05 (1.73)CaBa

88.34 (0.49)AbBa 67.32 (1.32)BbBa 77.05 (0.51)CbBa

72.84 (0.32)AaBb 29.19 (1.63)BaBb 41.05 (0.63)CaBb

73.72 (0.97)AbBb 32.61 (3.47)BbBb 44.74 (1.24)CbBb

Standard deviations are given in parenthesis. First superscript denotes significant differences in values with respect to pH. Second superscript denotes significant differences in values with respect to sonication (US) or control. Third superscript denotes significant differences in values with respect to the concentration (5 or 10% solids). Fourth superscript denotes significant differences in values with respect to temperature (72 or 85 °C).

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Table 5 Percentage of protein lost in heat-treated whey samples at 5 or 10% solids at different pH values and temperatures. pH

5%

10%

72 °C

7.5 4.5 3.5

85 °C

72 °C

85 °C

Control

US

Control

US

Control

US

Control

US

0a 10.5 (0.50)a 3.2 (0.41)a

0a 9.8 (0.32)a 3.2 (0.33)a

6.2 (0.68)a 65.2 (0.88)a 11.3 (0.31)a

5.8 (0.52)a 66.2 (0.75)a 10.0 (0.41)b

0a 8.9 (1.20)a 5.6 (0.55)a

0a 9.8 (0.89)a 5.9 (0.32)a

7.9 (0.67)a 68.5 (0.44)a 35.6 (0.21)a

11.6 (0.39)b 68.9 (0.54)a 33.8 (0.77)b

Different superscripts denote significant differences between control and sonicated (US) whey samples. Standard deviations are given in parenthesis.

2.6. Droplet and particle size measurement The droplet size of the fat globules in the emulsions as well as a 10% solids solution of the US and control whey at three different pH values was measured using a LS Beckman Coulter droplet size analyzer (LS 230, Coulter Corporation, Miami, Florida, USA) with the polarization intensity differential scanning small fluid module. The oil droplet measurements were taken at angular dependence of the intensity of laser light (λ = 623.8 nm) scattered by emulsions, and then the mean oil droplet size was generated as the surface-volume mean particle diameter (as described by Garg et al., 2010). This data was reported as mean ± standard deviations of D(3, 2) for the whey samples and as volume % for the emulsions. 2.7. Viscosity measurements The apparent viscosity of whey suspensions (10% solids) was determined using an oscillatory rheometer (AR-G2, TA Instruments, New Castle, DE) equipped with a concentric cylinder geometry. Whey suspensions were prepared as previously described and viscosity was measured at 25 °C. A steady state flow procedure was used to measure viscosity as a function of shear rate (1 × 10 −4–100 s −1). The viscosity at zero rate (Pa.s) was reported in this study. 2.8. Statistical analysis Viscosity, particle size, and emulsion stability determinations were performed in triplicate. Data is reported as mean values and standard deviations. Significant differences were analyzed with an ANOVA using SAS 9.1.3 (SAS Institute Inc., Cary, N.C., USA). All significant differences were given at level of significance of α = 0.05. For the thermal stability data, determinations were performed in triplicate and SAS was used to analyze the data with treatments of three pH levels, two concentration levels, two temperature levels, and US. The SAS REGWQ grouping was used to determine significant

differences at an α = 0.05. For the sensory data statistical analysis was performed with the proc glm function for analysis of variance to identify statistically significant differences at the 95% confidence level in SAS 9.1.3. Comparison of the means was made based on P-values (α = 0.05) using the least significant difference adjustment to obtain differences of the least means squares. Principle component analysis (PCA) using proc corr was used to analyze the relationship of lexicon terms to the samples. Ratings obtained for each sample on the 21 attributes evaluated were used to develop the PCA plots. 3. Results and discussion 3.1. Descriptive sensory evaluation Results of the descriptive sensory analysis of the control and US whey are shown in Tables 2 and 3. ANOVA of the descriptive results showed differences between samples as their pH level changed (P b 0.05), as well as differences in detection by the panelists with and without nose clips. The mean panelist ratings with nose clips on are displayed in Table 2, while the mean panelist ratings without nose clips are displayed in Table 3. As seen in Table 2, pH dependent significant differences were found between samples in astringent, brothy, cardboard, cereal, flour paste, malty, and sour when nose clips were used. In both the control and US samples, astringent was highest in the sample with the lowest pH, and sour increased significantly as the pH decreased. This same trend was seen without the nose clips, and shows that lower pH levels in whey tend to give more of a sour and astringent perception, as would be expected. The relationship between astringency and low pH was previously reported by Beecher et al. (2008) where they reported astringency values as high as 8.8 for whey proteins at pH 3.4. These authors suggest that astringency is caused by compounds that bind salivary proteins. In the particular case of whey proteins, Beecher et al. suggest that low pH values result in positively charged whey proteins

7

9

6

8

5

6

Vol %

Thickness (mm)

7

5 4 3

4 3 2

2

1

1 0 0.0

2.5

5.0

7.5

10.0

12.5

Time (days) Fig. 3. Absolute thickness (at the bottom) of the clarification layer of emulsions formulated with 2% sonicated (▲) or control (■) whey.

0 0.01

0.1

1

10

100

1000

10000

D3,2 (µm) Fig. 4. Oil droplet distributions in emulsions formulated with 2% sonicated (thick black line) or control whey (fine black line) on day zero.

S. Martini, M.K. Walsh / Food Research International 49 (2012) 694–701

that can interact with the negatively charged salivary proteins causing a noticeable astringent sensation. Other attributes tended to be stronger in the higher pH samples when nose clips were used, such as brothy, flour paste, and malty. There were no significant differences between the control and US samples without nose clips when comparing the same pH levels, with the exception of cardboard which was found at a higher level in the pH 7.5 control sample than in the US pH 7.5 sample. As expected, when the whey was tasted without nose clips there were more differences found between samples since aromatics present in the samples are also detected by the panelists. The intensity of astringent, bitter, brothy, cardboard, cereal, flour paste, malty, salty, and sour at different pH values was significantly different (α = 0.05) when evaluated without nose clips. Similar to the ratings obtained with nose clips, astringent and sour were both significantly higher in the samples with the lowest pH. As the pH decreased, astringent, bitter, salty, and sour tended to increase in the samples, while brothy, cardboard, cereal, flour paste, and malty tended to decrease. As with the samples tasted with the nose clips, there were no significant differences between the control and US samples of the same pH, with the exception of cardboard being higher in the control pH 4.5 sample, and malty being higher in the control pH 7.5 sample compared to their respective US counterparts. The only significant difference between the evaluations with and without nose clips was for the flour paste attribute which was higher at pH 3.5 without nose clips with US. For both sets of sensory data, most attribute intensities were very low in the whey samples, similar to previous descriptive panels on dairy products where the majority of attributes fall between 0 and 4 (Croissant, Kang, Campbell, Bastian, & Drake, 2009; Jervis et al., 2012; Russell et al., 2006). Principal component analysis was also performed on the results obtained from the panelists. Fig. 1 shows the principal component analysis of the samples when nose clips were used. As shown in the figure, principal component 1 explains 76.8% of the variability, while principal component 2 explains 16.3% of the data variability. The control and US sample with a 3.5 pH were most strongly correlated to each other, and were the most strongly correlated with sour and astringent and were also the least correlated with brothy and flour paste attributes. The US sample with a 4.5 pH was not strongly correlated with any other sample or attribute. The control samples at 4.5 and 7.5 pH and the US sample at 7.5 pH were most closely related with cardboard, cereal, malty, brothy, and flour paste. Fig. 2 shows the principal component analysis of the samples when nose clips were not used and smelling was encouraged. Principal component 1 explained 85.0% of the data variability, while principal component 2 explained 7.4% of the variability. As in the previous figure (with nose clips), the control and US sample at pH 3.5 are strongly correlated with each other, as well as with sour, astringent, bitter, and sour while they were negatively correlated to cereal, flour paste, and brothy notes. The US at pH 4.5 was again not strongly correlated with any sample or attribute. The control at pH 4.5 was less strongly correlated with the control and US samples at pH 7.5 in this figure. The control at pH 4.5 and 7.5 and the US at pH 7.5 were most strongly correlated with cardboard, malty, cereal, brothy, and flour paste. 3.2. Thermal treatment of whey US and control whey samples were heated to 85 °C and 72 °C to simulate hot fill and pasteurization conditions at three different pH values and two different solids concentrations and the turbidity of the samples determined (Table 4). The 5% solids concentration contained 1.8% protein and the 10% solids concentration contained 3.6% protein. All sonicated samples had higher transmission measurements at each temperature, solids concentration, and pH value. A higher transmission correlates with less turbidity. There were higher

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transmissions on samples with the lower heating treatment of 72 °C compared to the samples heated to 85 °C and although there were significant differences between concentrations, there were no obvious trends, except that there was very low transmission in 10% solids samples heated to 85 °C, especially at pH 4.5. With respect to pH, the lowest transmissions were obtained at pH value 4.5 and the highest at pH 7.5. The transmission of the samples at pH 3.5 was lower than the samples at pH 7.5, yet higher than the samples at pH 4.5. The percentage of protein lost due to centrifugation in heattreated samples is shown in Table 5. Only the samples treated at pH 7.5 at 72 °C showed no protein loss. The amount of protein lost in US vs. not US samples was generally the same except for three samples (5 and 10% solids heated to 85 °C at pH 3.5 and 10% solids heated to 85 °C at pH 7.5). Therefore, the turbidity of the samples was generally not a function of the amount of protein in suspension. We are suggesting that a physical change, such as disruption of aggregates, occurred in the protein samples subjected to sonication that led to a decrease in turbidity. Whey protein solubility is influenced by pH, temperature, ionic strength, and concentration. In general, whey proteins are the least soluble at pH 4.5 with an increase in solubility at pH 3.5 and 7.8 (Pelegrine & Gasparetto, 2005). In addition, there is usually a decrease in whey protein solubility with an increase in temperature. Previous research has shown that at neutral pH, there is a decrease in solubility at 60 °C compared to 40 °C (Beecher et al., 2008; Pelegrine & Gasparetto, 2005). Whey proteins begin to denature at 50 °C and denaturation continues with an increase in temperature. Generally, the majority of whey proteins are denatured at 80 °C. Denatured whey proteins generally aggregate via intra- and intermolecular interactions forming large aggregates (Kazmierski & Corredig, 2003). Therefore, the combination of a higher protein concentration at 85 °C leads to the most turbid samples, yet the sonicated samples with this treatment were still less turbid than the controls, implying that less protein aggregates occurred in sonicated samples. Zisu et al. (2011) also showed a decrease in whey turbidity after sonication. 3.3. Emulsification activity of whey Emulsions (20:80 soybean oil:water) prepared with 2% protein were analyzed for stability and oil droplet size. The destabilization of the emulsions is shown in Fig. 3 as the thickness of the clarification layer at the bottom of the emulsion. Samples prepared with the control whey showed a clarification layer that reached 8 mm over 10 days (0.8 mm/day) while the US sample showed a much smaller clarification of approximately 3 mm (0.3 mm/day). These results suggest that emulsions made with the US sample were more stable than the control emulsions. Fig. 4 shows the initial oil droplet size distribution (the size distribution of oil droplets in percent volume) of the emulsions. Droplet sizes of emulsions formulated with the sonicated and control whey were not significantly different with values of D3,2 = 1.73 ± 0.01 and 1.82 ± 0.47 μm for emulsions formulated with the sonicated and control whey, respectively. Emulsions formulated with the control whey showed a very clear bimodal distribution. The bimodal distribution of droplets in the control may be due to a limited amount of flocculation or coalescence, which is observed in some whey stabilized emulsions (Demetriades, Coupland, & McClements, 1997). 3.4. Particle size and viscosity of whey suspensions The particle size of the US and control samples at the three different pH values is shown as the D3,2 data in Fig. 5. At each pH level, the control samples had significant (α = 0.05) larger particle sizes compared to the US samples. The controls at pH 4.5 and 7.5 were not different, as were the treatments at those pH values, but samples at pH

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0.06

2

a

a

a

b c

1

c d

d

0 pH 3.5

pH 4.5

pH 7.5

Fig. 5. Particle sizes of sonicated (black bars) and control (white bars) whey in a 10% suspension.

3.5 showed larger particle sizes than all other samples. It is unclear why the samples had larger particle sizes at pH 3.5 since whey protein is generally soluble at this pH value. It should not be due to the association of beta-lactoglobulin since this protein generally exists as a monomer (18 kDa) at pH values below 3.5 and associates into aggregates (144 kDa) between pH 3.7 and 6.5 (Hambling, McAlpine, & Sawyer, 1992). These results are similar to Zisu et al. (2011) who sonicated reconstituted whey at 12% protein. They showed a decrease in particle size with the application of sonication; specifically the elimination of large aggregates that ranged from 10 to 100 μm resulting in the majority of the whey less than 1 μm in size. Similar results in the particle size of sonicated whey samples were reported by Zisu et al. (2010), Gordon and Pilosof (2010) and Ashokkumar et al. (2009). All whey samples showed a shear thinning behavior. Viscosity values reported in this research correspond to zero-rate viscosity values. The zero-rate viscosity of samples at a 10% solids concentration is shown in Fig. 6. There were no significant differences between pH values, but there were significant differences between the US samples and the controls. In general, all the US samples had a higher viscosity than the controls. The samples at pH 3.5 showed the lowest viscosity. Kresic et al. (2008) also showed an increase in viscosity with sonicated whey and attributed this to the higher water binding capacity of sonicated whey. Yet, these results are in contrast to the data reported by Ashokkumar et al. (2009) and Zisu et al. (2010) who saw a significant decrease in viscosity of sonicated whey. It is interesting to note that even though significant differences were found in the viscosity measured using the dynamic rheometer, no significant difference was found in the viscosity of the samples when evaluated by the sensory panel. Oral perception of texture is affected by several factors such as temperature and shear (Dresselhuis, de Hoog, Cohen Stuart, & van Aken, 2008). Therefore, viscosity measurements performed in a rotational rheometer are usually not a good representation of oral perception of texture. As previously mentioned, whey solutions showed a shear-thinning behavior which means that the apparent viscosity decreases with shear rate. Shear rates experienced in the mouth range from 10 to 1000 s− 1 (Dresselhuis et al., 2008) while values obtained from the rheometer represent zero-rate viscosities. 4. Conclusions This study applied US at 15 W for 15 min to a whey sample obtained from a cheese production company that contained 10% protein and evaluated the whey with respect to descriptive sensory attributes, thermal stability, emulsion stability, particle size and viscosity. The use of US did not change the sensory attributes of the whey; in fact several detrimental attributes (cardboard and malty) were

Zero rate viscosity (Pa.s)

D3,2 (µm)

3

0.05 0.04

b

0.02

b

a

0.03

b

0.01 0.00 pH 3.5

pH 4.5

pH 7.5

Fig. 6. Zero rate viscosity of sonicated (black bars) or control (white bars) whey at a 10% solids concentration.

scored lower in US samples than the control using a trained panel. US increased the transmissions of whey samples across pH values, concentrations and temperatures studied, implying that US leads to a less turbid sample. Emulsions prepared with US whey were more stable than the control and US whey suspensions showed lower particle size and resulted in a suspension with higher viscosity. The improvement in the functional properties may be related to a decreased particle size (disruption in aggregates) in whey, which was shown here and in other studies. The use of US to improve the functional properties of whey may expand the uses of whey as an ingredient in food systems. Acknowledgment The authors would like to thank Mr. Ankur Jhanwar and Dennis Perry from Glanbia Foods Inc. for providing the samples and for the valuable discussions. This project was partially funded by the Dairy Research Institute and supported by the Utah Agricultural Experiment Station, Utah State University, and approved as journal paper number 8435. References American Society for Testing and Materials (1991). Guidelines for the selection and training of sensory panel members. ASTM Technical Publication 758ASTM Committee E-18 on Sensory Evaluation of Materials and Products. Pennsylvania: ASTM. Ashokkumar, M., Lee, J., Zisu, B., Bhaskarcharya, R., Palmer, M., & Kentish, S. (2009). Sonication increases the heat stability of whey proteins. Journal of Dairy Science, 92, 5353–5356. Beecher, J. W., Drake, M. A., Luck, P. J., & Foegeding, E. A. (2008). Factors regulating astringency of whey protein beverages. Journal of Dairy Science, 91, 2553–2560. Chandrapala, J. C., Oliver, S., Kentish, M., & Ashokkumar, M. (2012a). Ultrasonics in food processing. Ultrasonics Sonochemistry, http://dx.doi.org/10.1016/j.ultsonch.2012.01.010. Chandrapala, J. C., Oliver, S., Kentish, M., & Ashokkumar, M. (2012b). Ultrasonics in food processing—Food quality assurance and food safety. Trends in Food Science and Technology, http://dx.doi.org/10.1016/j.tifs.2012.01.010. Chemat, F., Grondin, I., Costes, P., Moutoussamy, L., Shum Cheong Sing, A., & Smadja, J. (2004). High power ultrasound effects on lipid oxidation of refined sunflower oil. Ultrasonics Sonochemistry, 11, 281–285. Chemat, F., & Zill-e-Huma, M. K. K. (2011). Applications of ultrasound in food technology: Processing, preservation and extraction. Ultrasonics Sonochemistry, 18, 813–835. Chow, R., Blindt, R., Chivers, R., & Povey, M. (2003). The sonocrystallization of ice in sucrose solutions: Primary and secondary nucleation. Ultrasonics, 41, 595–604. Chow, R., Blindt, R., Chivers, R., & Povey, M. (2005). A study on the primary and secondary nucleation of ice by power ultrasound. Ultrasonics, 43, 227–230. Croissant, A. E., Kang, E. J., Campbell, R. E., Bastian, E., & Drake, M. A. (2009). The effect of bleaching agent on the flavor of liquid whey and whey protein concentrate. Journal of Dairy Science, 92, 5917–5927. Demetriades, K., Coupland, J. N., & McClements, D. J. (1997). Physical properties of whey protein stabilized emulsions as related to pH and NaCl. Journal of Food Science, 62(2), 342–347. Drake, M. A., & Civille, G. V. (2003). Flavor lexicons. Comprehensive Reviews in Food Science and Food Safety, 2, 33–40. Dresselhuis, D. M., de Hoog, E. H. A., Cohen Stuart, M. A., & van Aken, G. A. (2008). Application of oral tissue in tribological measurements in an emulsion perception context. Food Hydrocolloids, 22, 323–335.

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