University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Papers and Publications in Animal Science Animal Science Department 2016 Determination of yolk contamination in liquid egg white using Raman spectroscopy K. Cluff G. Konda Naganathan D. Jonnalagada I. Mortensen R. Wehling See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/animalscifacpub Part of the Genetics and Genomics Commons, and the Meat Science Commons This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Papers and Publications in Animal Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors K. Cluff, G. Konda Naganathan, D. Jonnalagada, I. Mortensen, R. Wehling, and Jeyamkondan Subbiah Determination of yolk contamination in liquid egg white using Raman spectroscopy K. Cluff,∗ G. Konda Naganathan,† D. Jonnalagada,† I. Mortensen,† R. Wehling,‡ and J. Subbiah†,‡,1 ∗Bioengineering, Wichita State University, Wichita, KS 67260-0035; †Biological Systems Engineering, University of Nebraska, Lincoln, NE 68583-0726; and ‡Food Science and Technology, University of Nebraska, Lincoln, NE 68588-0115 ABSTRACT Purified egg white is an important in- dard normal variate transformation. Samples were ran- gredient in a number of baked and confectionary foods domly divided into calibration (n = 77) and validation because of its foaming properties. However, yolk con- (n = 38) data sets. A partial least squares regression tamination in amounts as low as 0.01% can impede (PLSR) model was developed to predict yolk contami- the foaming ability of egg white. In this study, we nation levels, based on the Raman spectral fingerprint. used Raman spectroscopy to evaluate the hypothesis Raman spectral peaks, in the spectral region of 1,080 that yolk contamination in egg white could be detected and 1,666 cm−1, had the largest influence on detecting based on its molecular optical properties. Yolk contam- yolk contamination in egg white. The PLSR model was inated egg white samples (n = 115) with contamina- able to correctly predict yolk contamination levels with tion levels ranging from 0% to 0.25% (on weight ba- an R2 = 0.90 in the validation data set. These results sis) were prepared. The samples were excited with a demonstrate the capability of Raman spectroscopy for 785 nm laser and Raman spectra from 250 to 3,200 cm−1 detection of yolk contamination at very low levels in egg were recorded. The Raman spectra were baseline cor- white and present a strong case for development of an rected using an optimized piecewise cubic interpolation on-line system to be deployed in egg processing plants. on each spectrum and then normalized with a stan- Key words: foaming properties, functional properties, yolk contamination, Raman spectroscopy, partial least squares regression 2016 Poultry Science 95:1702–1708 http://dx.doi.org/10.3382/ps/pew095 INTRODUCTION and Wells, 1955;Smith,1959). Therefore, quality con- trol methods that are both rapid and accurate at low In the United States, 75 billion eggs are produced concentration levels are needed to ensure appropriate per year, which account for 10% of the world produc- egg white purity. tion (American Egg Board, 2015). Approximately 60% Earlier methods of measuring yolk contamination fo- of these eggs are used by consumers, 9% by food ser- cused on physico-chemical or gravimetric measurements vice sectors such as restaurants, and the rest are used (Murphy and Uhing, 1959; Cunningham and Cotterill, for manufacturing egg products (American Egg Board, 1964). More recent methods have used enzymatic deter- 2015). Purified egg white is an important egg product mination of phospholipids, or other yolk lipid compo- that is used extensively in the bakery and confectionary nents, or colorimetric determination of yolk cholesterol industries because of its foaming properties. Producing (Nielsen, 2000). The current industry standard relies pure egg white, without contamination of egg yolk, has on the physical dispersal of lipids in an aqueous solvent become a challenge because of the mechanization and (Bergquist and Wells, 1955); however, this method is speed of egg handling and breaking in egg processing fa- time consuming and requires an experienced technician cilities (Wang and Wang, 2009). Yolk contamination is to obtain reliable results. a critical issue because it affects the egg white’s ability Egg yolk is comprised of approximately 50% wa- to form stable and voluminous foams. Yolk contamina- ter, 16% protein, and 32% fat, whereas, egg white tion levels as low as 0.01% in egg white can significantly consists of nearly 90% water and only 10% protein affect foam volume (St. John and Flor, 1930; Bergquist (Li-Chan and Nakai, 1989). Due to the abundance of lipids in egg yolk, a recent study (Mortensen, et al., 2007) used near-infrared (NIR) spectroscopy to de- C 2016 Poultry Science Association Inc. tect yolk contamination in egg white by measuring Received August 1, 2015. Accepted February 4, 2016. the molecular vibrations of lipids. Mortensen et al. 1Corresponding author: [email protected] (2007) collected transmission spectra from 25 samples 1702 DETECTION OF YOLK CONTAMINATION IN EGG WHITE 1703 of egg white with yolk contamination levels ranging Poultry farm. After hand breaking the eggs, the egg from 0% to 0.25%. Partial least squares regression mod- white and egg yolk were separated using an egg sep- els were developed to predict yolk contamination with arator and pooled in separate beakers. Careful atten- an R2 value of 0.69 (Mortensen et al., 2007). While tion was given to ensure that no yolk was accidentally these results from the NIR technique are highly en- mixed in with the egg white. The egg white and yolk couraging, NIR is limited by the presence of water were then homogenized separately using a household molecules. The presence of water produces a broad ab- blender (Model: Hamilton Beach, Southern Pines, NC). sorption in NIR spectra, and thus can mask portions The blending speed was regulated using a voltage con- of the molecular vibrational spectral peaks of other troller such that there was minimal foaming of samples molecules. during blending operation to avoid denaturation of the Raman spectroscopy is a complementary technique egg white and yolk proteins. This process provided ho- to NIR spectroscopy, but is governed by different selec- mogenized pure egg white and yolk samples. tion rules. Raman spectroscopy responds to molecular Approximately 100 g of pure egg white was mea- vibrations that may not be observable by NIR absorp- sured out using an analytical balance with a precision tion. Raman and infrared (IR) spectroscopy both rely of ± 0.0001 g. To obtain different levels of yolk contam- on vibrational energies of molecules for detection, but ination, varying amounts of pure egg yolk were added each have different selection rules (Sasic and Ozaki, to the pure egg white using a disposable pipette. Each 2010). For example, vibrations that produce Raman egg white–yolk mixture was blended using a household spectral peaks frequently will not appear in IR spectra blender at low speeds with the help of a voltage reg- and vice versa. These selection rules are useful because ulator to obtain homogenized yolk contaminated egg they make the two spectroscopic methods complemen- white samples. The amount of egg yolk contaminant tary to each other. The selection rules are related to was recorded then divided by the amount of egg white the symmetry of the molecule. Molecules with a sym- and multiplied by 100% to obtain the percent contami- metric configuration are said to be Raman active be- nation. A total of 115 liquid egg white samples with yolk cause they will produce a peak in the Raman spectra contamination levels ranging from 0% to 0.25% were (Sasic and Ozaki, 2010). For example, a symmetric car- prepared. There were 31, 29, 19, 18, and 18 samples bon double-bond (C = C) will produce a Raman spec- with yolk contamination percentages of 0.00 to ≤0.05, tral peak around 1,657 cm−1 (Movasaghi, et al., 2007). 0.06 to ≤0.10, 0.11 to ≤0.15, 0.16 to ≤0.20, and 0.21 Conversely, molecules that have an asymmetrical con- to ≤0.25, respectively. figuration are said to be IR active, because they will produce peaks in IR spectra (Sasic and Ozaki, 2010). For example, a carbonyl (C = O), which has an asym- metrical configuration, will produce an IR spectral peak − at around 1,700 cm 1 (Sasic and Ozaki, 2010). Further- Instrumentation and Raman Spectra more, in contrast to NIR, Raman spectroscopy is not Collection affected by the presence of water molecules, which is critical for detecting yolk contamination in egg white. The instrumentation consisted of a Raman spec- In addition, the C = C stretching modes of unsatu- trometer (Model: EZRaman-M, Enwave Optronics Inc, rated fatty acids and CH2 scissoring modes of satu- Irvine, CA), with a fiber-optic probe, 785 nm excitation rated fatty acids, found in egg yolk, produce Raman laser, and sample holder. The fiber-optic probe had a peaks near 1,657 and 1,443 cm−1 respectively (Ozaki, 200 μm diameter collection fiber. The Raman spectrom- et al., 1992; Movasaghi, et al., 2007). Yolk contains eter had a spectral range of 250 cm−1 to 3,200 cm−1 and large amounts of lipids, while egg white contains no a spectral resolution of 6 cm−1. The Raman spectrome- lipids. Thus, Raman spectroscopy has a high potential ter was interfaced and controlled by a laptop computer for detecting yolk contamination in egg white and may using EZRaman software (Enwave Optronics). yield improved results compared to NIR. In this study, The Raman spectrometer was set up with a 90 second we evaluate the hypothesis that yolk contamination in integration time, three-point smoothing, and a three egg whites can be detected based on its optical proper- spectrum average.