Proof-of-concept Study of a Whey Protein Isolate Based Carbon Dioxide Indicator to Measure the Shelf-life of Packaged Foods

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1 Food Sci. Biotechnol. 23(1): (2014) DOI /s RESEARCH ARTICLE Proof-of-concept Study of a Whey Protein Isolate Based Carbon Dioxide Indicator to Measure the Shelf-life of Packaged Foods Kyuho Lee and Sanghoon Ko Received: 12 July 2013 / Revised: 29 July 2013 / Accepted: 29 July 2013 / Published Online: 28 February 2014 KoSFoST and Springer 2014 Abstract A whey protein isolate (WPI) based carbon dioxide (CO 2 ) indicator was studied under different CO 2 conditions. WPI based CO 2 indicators were prepared and incubated in a CO 2 filled chamber in order to investigate the CO 2 dependent transition behavior in visual appearance. Transparency of the WPI based CO 2 indicator with 0.1% WPI changed from 91.8% to 24.6%. An indicator with 0.3% WPI changed from 73.7% to 7.7%. The change in visual appearance at the transition point was definite. The indicator showed an irreversible visual change due to the hysteresis behavior of the indicator. Desorption of CO 2 did not restore the transparency. The CO 2 dependent visual transition point was controlled by adding different concentrations of NaCl to the indicators. A WPI based CO 2 indicator has potential for application as a CO 2 dependent spoilage and over-ripeness indicator for a variety of foods. Keywords: carbon dioxide indicator, whey protein isolate, food freshness, intelligent food packaging Introduction Consumers are increasingly focusing on freshness and quality in food products and also safety of food from farm to table. Success in the farm-to-table approach for fresh and safe foods requires continuous monitoring of changes in food quality. Recently, various food quality indicators have been developed for consumer friendly food packages, for example, time-temperature indicators (TTIs) and oxygen indicators have be applied to agricultural products and Kyuho Lee, Sanghoon Ko ( ) Department of Food Science and Technology, Sejong University, Seoul , Korea Tel: ; Fax: sanghoonko@sejong.ac.kr packaged foods (1). The partial pressure of carbon dioxide (CO 2 ) has potential for use as an indication of food quality in packaged foods. Changes in food quality occur inside food packages due to fermentation, microbial spoilage, and/or respiration of agricultural products. A CO 2 indicator provides a way to easily check the food quality since a change in visual appearance is usually accompanied by an increased CO 2 concentration in the headspace of packaged foods. CO 2 can accumulate in the headspace of packaged foods due to spoilage or microbial fermentation. For example, the CO 2 concentration in the headspace of over-ripe kimchi was more than 1.5 mg/ml at which the ph and titratable acidity were below ph 4.0 and over 1.0%, respectively (2). A CO 2 indicator has been developed to measure the CO 2 concentration in the headspace of food packages (3) using chitosan as a reacting material to show an opaque apprarance at neutral ph and transparency at acidic ph values. The visual appearance of the chitosan based CO 2 indicator changes from initially opaque to transparency at the ripened or outdated stage. This is categorized as a binary, or OFF/ ON type of indicator (3). A simple OFF/ON type indicator is not prevalent with consumers, but an ON/OFF type indicator that shows a visual transition from transparency to opaque is favorable. Most consumers want a simple to recognize and intuitive indicator of food quality. A clearto-opaque type indicator is more acceptable for consumers since transparency is associated with freshness. Even though a CO 2 indicator is an ON/OFF type, it has potential for use in monitoring the food quality in packaged foods due to the quality cues of transparency and opaqueness. A whey protein isolate (WPI) is a collection of proteins in a drained liquid (whey) in the cheese making process. Whey contains β-lactoglobulin, α-lactalbumin, and small quantities of various water soluble proteins. A WPI solution is transparent at a neutral ph and becomes turbid at the

2 116 Lee and Ko Fig. 1. The WPI-based carbon dioxide indicator. isoelectric point (pi of approximately ph 5.5). In packaged food, a CO 2 dependent ph drop results from conversion of gaseous CO 2 in the headspace to carbonic acid (H 2 CO 3 ) in the aqueous medium of the CO 2 indicator (Fig. 1). The operating principle of the WPI based CO 2 indicator is that CO 2 in the headspace inside a food package dissolves in the WPI aqueous suspension and, subsequently, the visual appearance of the suspension changes from transparent (neutral ph) to opaque (slightly acidic ph) since the ph of the suspension decreases. Acidification of the WPI aqueous suspension by dissolution of CO 2 decreases the ph of the aqueous system toward the pi value of WPI, at which protein molecules lose aqueous solubility (4,5). A WPI aqueous suspension is transparent above ph 6.0, but becomes opaque below ph 6.0. This ph dependent property of visual appearance can be used as an indicator of the partial pressure of CO 2. In order to develop a consumer-friendly CO 2 indicator, it is important to determine optimum WPI preparation conditions for a CO 2 based indicator that show the most recognizable visual change for consumers. In addition, control of the visual transition point is important in order to expand the versatility of the CO 2 indicator for various packaged foods. Addition of electrolytes to the aqueous medium can delay the CO 2 absorption reaction with WPI molecules. NaCl was used for controlling the transition point of visual change by changing the responsiveness of the WPI based indicator for the CO 2 concentration. Controlling CO 2 absorption is useful for application of the WPI based CO 2 indicator to various packaged foods. A WPI based CO 2 indicator was prepared and the CO 2 dependent physicochemical properties were investigated based on ph and transparency. Responsiveness of the indicator was controlled by adjusting the concentrations of WPI and NaCl. Materials and Methods Materials WPI powder was obtained from Davisco Foods International, Inc. (Le Sueur, MN, USA). Hydrogen chloride and sodium hydroxide used were reagent grade (Daejung Chemicals & Metals, Shiheung, Korea). Preparation of a WPI based CO 2 indicator WPI powder (0.1, 0.3, 0.5, 1, 2, and 4 g) was added to 90 ml of distilled water and stirred for 30 min. The WPI aqueous suspension was adjusted to ph 7.0 using 1 M HCl and NaOH and stirred for 10 min. Then, distilled water was added to the WPI aqueous suspension to a final volume of 100 ml. The ph of the WPI aqueous suspensions was readjusted to 7.0 for the WPI based CO 2 indicator at the initial stage. Changes in ph and transparency of the indicator with different WPI concentrations were measured under 100% CO 2 conditions for 3 h. Transparency and ph measurement Five WPI based CO 2 indicator suspensions (5 ml) were collected in test tubes and was adjusted to ph 5.0, 5.5, 6.0, 6.5, and 7.0, respectively, using 0.1 or 1 M HCl. Changes in ph of the indicator were measured over time using a ph meter (SP Laboratory ph/orp Meter; Suntex Instruments Co., Ltd., New Taipei City, Taiwan) with an attached ph electrode and thermometer (Ioline Corp., Woodinville, WA, USA). Transparency was measured using UV visible spectrophotometry (DU 730; Beckman Coulter, Inc., Brea, CA, USA) at 325 nm. For a hysteresis study, WPI based CO 2 indicators were exposed to ambient air after incubation under 100% CO 2 for 1 day. Changes in the ph and transparency of the indicators were measured every day for 10 days.

3 Whey Protein Isolate Based CO 2 Indicator 117 Fig. 2. A comparison of turbidity at (A) ph 5.0 and (B) ph 7.0. Control of the visual transition point of the WPI based CO 2 indicator by addition of NaCl WPI powder (0.3 g) and NaCl (0.5, 1, and 2 g) were added to 90 ml of distilled water and stirred for 30 min. The aqueous mixture of WPI and NaCl was adjusted to ph 7.0 and stirred for 10 min. Then, distilled water was added to the aqueous mixture up to a 100 ml final volume and the ph was readjusted to ph 7.0. Changes in the ph and transparency were measured under 100% CO 2 conditions for 3 h. Preparation of a WPI based CO 2 indicator in a sachet The WPI based CO 2 indicator at an initial ph of 7.0 (0.3 g of WPI in 100 ml) was prepared using the method mentioned above. The indicator suspension (5 ml) was placed into a square (5 cm 5 cm) sachet of low density polyethylene (LDPE) film with a thickness of 50 µm. The sachet was stored under 100% CO 2 conditions and concomitant changes in ph and transparency were measured for 3 h. Results and Discussion Investigation of the CO 2 indicating capacity at different ph values Visual appearance of the CO 2 indicator changed from transparent to opaque depending on ph as shown in Fig. 2. Transparency changes in the CO 2 indicator with different WPI concentrations at different ph values are shown in Fig. 3. Transparency values for 0.1, 0.3, 0.5, 1, 2, and 4% WPI suspensions were 85.3, 68.9, 54.5, 35.1, 18.2 and 9.3, respectively, at ph 7.0. The transparency values of WPI suspensions were 46.6, 18.5, 13.4, 6.0, 5.6, and 6.6, respectively, at ph 5.5. The optical density of the CO 2 indicators changed from transparent to opaque near the pi value of WPI. At the transition point close to the pi value, the slope of the curve from transparent to opaque was steep (Fig. 3), indicating that the ph range at the transition point was narrow. All WPI based CO 2 indicators were transparent at ph 7.0, regardless of the WPI concentration. WPI molecules Fig. 3. Transparency change of the CO 2 indicators with different WPI concentrations at different ph values., 0.1% WPI suspension;, 0.3% WPI suspension;, 0.5% WPI suspension;, 1% WPI suspension;, 2% WPI suspension;, 4% WPI suspension exist in a native form with monomers or dimers at a neutral ph in an aqueous solution, but associate to form aggregates when the ph shifts towards the pi value. WPI molecules have a net negative charge at ph values above the pi value. Therefore, WPI molecules have an electrostatic repulsive force when the ph of an aqueous suspension is far from the pi value of WPI. This electrostatic repulsive force prevents aggregation of WPI molecules since it is stronger than the van der Waals force among WPI molecules. The size of WPI monomers and dimers suspended in an aqueous medium is too small to scatter light, which causes opaqueness. All WPI based CO 2 indicators were opaque at ph 5.5, regardless of the WPI concentration. WPI molecules aggregate when the ph of the WPI aqueous suspension is close to the pi value because molecules lose a net charge from the surface and have a negligible electrostatic repulsive force (6). Interactions between light photons and large WPI aggregates lead to scattering of light, resulting in an aqueous suspension with an opaque appearance. This scattered light joins diffusely reflected light, enhancing the opaque effect (7). At the transition point, aggregation causes WPI molecules to form large clusters of several hundred nanometers or micrometers in size. Thus, the WPI suspension at the transition point is opaque and clearly sensible to the human eye. CO 2 indicating capability in a 100% CO 2 chamber Changes in ph and transparency of CO 2 indicators prepared with 0.1, 0.3, 0.5, 1, 2, and 4% WPI concentrations during incubation in a 100% CO 2 chamber are shown in Fig. 4. Various concentrations of WPI suspensions were tested to determine optimum preparation conditions for development of a good CO 2 indicator. CO 2 indicators with high WPI

4 118 Lee and Ko Fig. 4. Changes in (A) ph and (B) transparency of CO 2 indicators during incubation under 100% CO 2 conditions., 0.1% WPI suspension;, 0.3% WPI suspension;, 0.5% WPI suspension;, 1% WPI suspension;, 2% WPI suspension;, 4% WPI suspension Fig. 5. Hysteresis of CO 2 (A) absorption and (B) desorption for 0.1, 0.3 and 0.5% WPI indicators incubated under 100% CO 2 conditions., 0.1% WPI suspension;, 0.3% WPI suspension;, 0.5% WPI suspension;, 1% WPI suspension;, 2% WPI suspension;, 4% WPI suspension concentrations (1, 2, and 4%) showed a narrow difference in visual appearance while indicators at 0.1, 0.3 and 0.5% WPI concentrations showed a clear change in visual appearance depending on the CO 2 concentration. The effect of the WPI concentration on the transparency was clearly shown in the response time of the CO 2 indicator. At WPI concentrations of 1, 2, and 4% the CO 2 indicators decreased to ph values of nearly 6 within 20 min, while indicators with 0.1, 0.3, and 0.5% WPI concentrations decreased to approximate ph values of 5.5 within the same period. Similarly, transparency values of indicators at 1, 2, and 4% CO 2 changed from 33.2 to 2.6, 16.4 to 3.2, and 6.5 to 3.7, respectively, within 3 h, while indicators with 0.1, 0.3, and 0.5% CO 2 changed from 89.5 to 25.2, 71.6 to 7.8, and 57.2 to 6.8, respectively, over the same period of time. The visual appearance of the WPI based CO 2 indicators was transparent at the initial stage (ph 7.0). An increase in the CO 2 concentration in the chamber decreased the ph of the WPI based CO 2 indicators due to formation of carbonic acid which dissociated to protons (H + ) and bicarbonate molecules (HCO 3 ) resulting in a decrease in the ph of the system (8). The lower ph induced aggregation among WPI molecules, forming large WPI clusters that caused an opaque appearance. Among various WPI concentrations, 0.1% showed the largest change in transparency while 0.3% showed the largest visual change in turbidity. When the WPI concentration increased, the amount of CO 2 absorption in the suspension decreased. The CO 2 indicator can be applied to packaged foods requiring a long shelf-life since CO 2 production in agricultural products and packaged foods is relatively slower than in the 100% CO 2 chamber used in this study. Irreversibility of the WPI based CO 2 indicator Figure 5 shows hysteresis of CO 2 absorption and desorption in 0.1, 0.3, and 0.5% WPI indicators incubated under 100% CO 2 conditions. The indicators showed a hysteresis tendency that CO 2 desorption was not identical to CO 2 absorption as the ph changed. In the absorption test, the ph values of the 0.1, 0.3, and 0.5% CO 2 indicators changed from 6.93 to

5 Whey Protein Isolate Based CO 2 Indicator 119 Fig. 6. (A) ph and (B) Transparency changes of CO 2 indicator with NaCl addition., 2% NaCl;, 1% NaCl;, 0.5% NaCl Fig. 7. (A) ph and (B) Transparency changes of the CO 2 indicator with 0.3% WPI in a sachet. 4.75, 7.05 to 5.16, and 7.03 to 5.23, respectively, during incubation under 100% CO 2 conditions for 24 h. However, in the desorption test, the ph values of CO 2 indicators with the same 3 WPI concentrations changed from 4.75 to 7.27, 5.16 to 7.01, and 5.23 to 6.97, respectively, when they were exposed to ambient air for 10 days. The change in transparency of the CO 2 indicators showed hysteresis behavior similar to the tendency in ph change. Moreover, the hysteresis in transparency changes was unique while the ph changes not. The opaque appearance of the indicator after incubation in the CO 2 chamber did not return to transparency. This hysteresis behavior may be caused by a partial denaturation of WPI molecules under lower ph conditions due to CO 2 absorption in the WPI aqueous suspension. Denatured WPI molecules (aggregated WPI clusters) were not divided into monomers and dimers even though the ph of the indicator was restored to 7.0 via exposure to ambient air for a long time. As a result, the indicator after the desorption test was still opaque at ph 7.0. Thus, the indicator showed a visual change in one direction from transparency at the initial stage to opaque at the final stage due to hysteresis in visual appearance. From the hysteresis tendencies in visual appearance, the WPI based CO 2 indicator has a property of irreversibility. Control of the visual transition point of the WPI based CO 2 indicator The effect of addition of NaCl on the WPI based CO 2 indicator is shown in Fig. 6. All CO 2 indicators with added NaCl changed ph values from approximately 7.0 to 5.2 while changes in the transparency behaviors were different. When 2% NaCl was added, indicator transparency values changed from 71% to 52.2% within 9 h. However, addition of 0.5 and 1% NaCl caused changes from 69.3% to 50.1% and from 69.9% to 65.7%, respectively, within 1 h. Subsequently, the transparency of CO 2 indicators with 1% and 2% added NaCl decreased consistently over time with final transparency values of 17.3% and 28.7% respectively. A higher NaCl concentration delayed the visual transition point, which depends on the absorption of CO 2. This method of controlling the visual transition point in the indicator can be used for various food packages for agricultural products and processed foods that develop different CO 2 levels due to respiration and fermentation.

6 120 Lee and Ko NaCl can be used to control the CO 2 dependent transition point since it lowers the degree of electrostatic repulsion in the WPI indicator. Salt (NaCl) can shield the surface charge of WPI protein molecules by altering the electrostatic properties of the molecules (9). When the surface charge of protein molecules is shielded, hydrophobic protein-protein interactions are promoted, resulting in an increase in aggregation among WPI molecules. Changes in the salt concentration induce different optical properties because the aggregation behavior of WPI molecules is changed. Performance of the WPI based CO 2 indicator in a sachet LDPE film sachets were used because of a high CO 2 permeability and a low water permeability. Transparency and ph changes of the CO 2 indicators with 0.3% WPI in sachet are shown in Fig. 7. The ph value of the indicators in the sachet changed from 7.05 to 5.50 while the transparency changed from 71.1 to 12.6 within 100 min. However, changes in ph and transparency were relatively small from 100 min to the end of the analysis. Results for the WPI based CO 2 indicator in a sachet were compared to results for a 0.3% WPI based CO 2 indicator without a sachet. A sample without the sachet was incubated until the ph and transparency values changed to 5.5 and The 20 min required to reach these values was relatively short compared to the 100 min required for the sachet. The WPI indicator reacted more slowly in the sachet, probably because the non-sachet indicator was in direct contact with CO 2 in the headspace. Thus, the CO 2 indicator has possibility to indicate CO 2 of various concentrations by changing films which are used at sachet as well as controlling concentration of WPI and NaCl. In addition, the WPI based CO 2 indicator shows a visual change in only one direction from transparency at the initial stage to opaque at the ripened or outdated stage due to an irreversible reaction. Thus, the WPI based CO 2 indicator can be used in various food packages to monitor food freshness since CO 2 is a by-product during spoilage and over-ripening of foods. Acknowledgments This research was supported by the Agriculture Research Center program of the Ministry for Food, Agriculture, Forestry and Fisheries, Korea. References 1. Ahvenainen R, Hurme E. Active and smart packaging for meeting consumer demands for quality and safety. Food Addit. Contam. 14: (1997) 2. Jung J, Lee K, Puligundla P, Ko S. Chitosan-based carbon dioxide indicator to communicate the onset of kimchi ripening. LWT-Food Sci. Technol. 54: (2013) 3. Puligundla P, Jung J, Ko S. Carbon dioxide sensors for intelligent food packaging applications. Food Control. 25: (2012) 4. Chanamai R, McClements DJ. Comparison of gum arabic, modified starch, and whey protein isolate as emulsifiers: influence of ph, CaCl 2 and temperature. J. Food Sci. 67: (2002) 5. Malamud D, Drysdale JW. Isoelectric points of proteins: A table. Anal. Biochem. 86: (1978) 6. Demetriades K, Coupland JN, McClements DJ. Physical properties of whey protein stabilized emulsions as related to ph and NaCl. J. Food Sci. 62: (1997) 7. Duran L, Calvo C. Optical properties of foods. Vol. I, pp In: Food Engineering. Barbosa-Cánovas GV (ed). Encyclopedia of Life Support Systems. Mississauga, Ontario, Canada (2009) 8. Jönsson B, Karlström G, Wennerström H, Roos B. Ab initio molecular orbital calculations on the water-carbon dioxide system: carbonic acid. Chem. Phys. Lett. 41: (1976) 9. Ko S, Gunasekaran S. In situ microstructure evaluation during gelation of â-lactoglobulin. J. Food Eng. 90: (2009)