RESEARCH NOTE. Sodium Bisulfate Analyses in Shrimp by Ion Selective Electrode, Sulfite Oxidase, and Modified Monier-Williams

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1 RESEARCH NOTE Sodium Bisulfate Analyses in Shrimp by Ion Selective Electrode, Sulfite Oxidase, and Modified Monier-Williams D. L. Gerdes R. D. Hirchak R. M, Grodner R. M. Martin ABSTRACT Sulfite determination in shrimp was accomplished using en ion-selective electrode, and enzymatic method and the Modified D.L. Gerdes is affiliated with University of Houston, Conrad N. Hilton College of Hotel and Restaurant Management, The Food & Beverage Research Institute, Houston. TX USA. R. D. Hirchak is associated with Allen Canning Company, PO. Box 189, Hessmer, LA USA. R. M. Grodner is affiliated with the Department of Food Science, Louisiana Agricultural Experiment Station, LSU Agricultural Center, Louisiana State University, Baton Rouge, LA USA. R. M. Martin is affiliated with National Fisheries Institute, 1901 North Fort Myer Drive, Suite 700, Arlington, VA USA. The authors wish to thank the National Fisheries institute for their financial support of this project. - Journal of Aquatic Food Product Technology, Vol. 8(1) by The Haworth Press, Inc. All rights reserved.

2 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY Monier-Williams procedure. A rapid electrode method using a double junction-sleeve type electrode with a hydrogen reference electrolyte was developed using sodium metabisulfite (0.01 M) as the reference electrolyte and saturated silver chloride as the internal electrolyte. Concentrations between 0 and 250 ppm were tested to produce a standard curve. The concentration range of Interest to the shrimp industry ties between 50 and 250pm. Response time was approximately 90 s with slight variations after- 120 s. This technique was completed in a fraction of the time required by the modified Monier-Williams and the enzyme method, which take 2.5 h and 45 min, respectively. (Article copies available for a fee from The Haworth Document Delivery Service: E-mall address: getinfo@haworthpressinc.com) KEYWORDS. Sulfite, ion-selective electrode, enzymatic method INTRODUCTION Recent interest due to possible health related problems over sulfite concentrations have prompted the development of a fast and accurate measurement of bisulfite for shrimp screening (DeWitt and Finne, 1985). Sulfites prevent melanosis (black spot) in shrimp but also have caused allergic reactions in "at risk" individuals (Taylor and Bush, 1983). Rapid, accurate sulfite analysis would allow regulatory officials to measure and enforce labeling regulations of the sulfite levels at the dock or point of entry, rather than waiting for time consuming laboratory analysis. Ion-selective electrodes have been used as indicators in determining sulfite levels in orange juice (Ingram, 1947). Ross et al. (1973) investigated the potentiometric gas-sensing electrodes using a hydrogen reference and gas permeable membranes. The major drawback to this method is that the membrane sites are quickly saturated, which prevents quantitation. The electrode method of Ross et al. (1973) has been modified for this study to decrease the analysis time for determination of sulfite in shrimp, while maintaining sensitivity and accuracy of the standard methods. Therefore, comparison was conducted between the Modified Monier-Williams procedure, the enzymatic methods and an electrode method. A working electrode was tested in a range of sulfite concentrations, which could be found in the shrimp industry with verification by the other available methods.

3 Gerdes et al. MATERIALS AND METHODS Shrimp Source Commercially frozen and peeled shrimp were thawed overnight at 4 C, washed for 1 min with deionized distilled water (ddh 2 O) and allowed to drain, Seven 50 g samples were weighed and placed into 150 ml beakers. Bisulfite Standards ' A stock solution of sodium bisulfite containing 1000 pasts per million (ppm) was prepared by dissolving 1.0 g sodium bisulfate (NaHS0 3 ) in a 1000 ml volumetric flask with ddh 2 0. This solution was prepared fresh daily. Working standard solutions were prepared from the stock solution (0, 50, 80,100,120,150 and 250 ppm). Shrimp samples (50 g) were each spiked with 50 ml of the bisulfite standard solutions and stirred. Ion-Selective Electrode (ISE) Preparation The sulfite-sensing electrode was a double junction-sleeve type electrode (Orion Model # , Orion Scientific Research, Cambridge, MA) with 0.01 M Na 2 S as the reference electrolyte and saturated silver chloride as the internal electrolyte. This electrode configuration was similar to that used by Ross et al. (1973). The reference electrode was a hydrogen electrode (Sargent-Welch S , Sargent-Welch Scientific Co., Skokie, IL). Calibration and Use of the ISE Shrimp spiked with 50 and 250 ppm bisulfate were used as standards to calibrate a Corning 135 ph/ion meter (Corning Glass Works, Medfield, MA). The electrodes were placed in the shrimp-bisulfate solutions and the millivolts were read after 90 s. The concentrations (ppm) were determined from a standard curve of millivolts versus standard concentration (ppm). Enzymatic Determination of Bisulfate A 0.10 ml sample was removed from the shrimp-bisulfate sample for testing by the enzyme test kit (Boehringer-Mannheim #725854,

4 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY Boehringer-Mannheim Biochemicals, Indianapolis, IN). Calculated results were expressed in ppm. Modified Monier-Williams Test for Bisulfite The remaining sample was analyzed using the Modified Monier-Williams procedure (AOAC, 1984). Results were also expressed in ppm. RESULTS AND DISCUSSION The calculated sulfite concentrations using the electrode ranged from 22 ppm (0 ppm standard solution) to 140 ppm (250 ppm standard solution), as shown in Table 1. These results indicated an overall recovery of 53% for the electrode procedure. Although this recovery was the lowest of the three procedures it should be rioted that the time required was less than two minutes. In addition to the short analysis time, the LSE gave highly reproducible results (coefficient of variation <2.0). There was a significant correlation between the sulfite concentration determined by the electrode procedure and the shrimp-sul fife standards (r = Ingram (194 reported that using an electrode to food systems estimates are within 2% of the known concentration with 5% error. Table 1: Average sulfite concentration (ppm) as determined by three methods. Standard Electrode Enzyme Modified (Shrimp + bisulfate) Methods ( ppm) Assay (ppm) Monier-Williams (ppm) a 21 a ab 49 b ab 37 abc 67 c ab 50 bc 85 d bc 71 cd 100 e c 94 d 112 e d 146 e 185 f Mean values are of triplicate samples of duplicate experiments for each feet Procedure. Values within columns followed by different subscripts are significantly different (p < 0.05) as tested by Duncan s New Multiple Range Test,

5 Gerdes et al. Sulfite concentration determined by the enzyme assay produced average values ranging from 11 ppm (0 ppm standard solution) to 146 ppm (250 ppm standard solution) (Table 1). These values represent a 55% recovery of added sulfite. The enzyme method required about 45 min for completion and had a coefficient of variation of >10.0. Correlation of these values with the shrimp-sulfite standard was significant (r = 0.987). Modified Monier-Williams recovered 83% of the sulfite using a standard curve ranging from 21 ppm (0 ppm standard solution) to 185 ppm (250 ppm standard solution) (Table 1). This method required in excess of 150 min to complete and produced variable results. The mean values when plotted versus standard ppm gave a correlation of r = The electrode procedure correlated well with both Modified Monier-Williams (r = 0.707) and enzyme method (r = 0.707). These results indicate that this electrode method decreased analysis time while maintaining comparable accuracy for sulfite determination as compared to the enzymatic assay. Grouping of the means using Duncan's New Multiple Range Test indicated no significant difference in grouping for the electrode and enzyme method in the range but significant difference at the 250 ppm level (Table 1). The mean values obtained using Modified Monier-Williams were significantly different in grouping at all levels except the ppm level. SIGNIFICANT FINDINGS The sulfite-sensing electrode produced a response in 90 s with only slight variations after 120 s in determining bisulfite concentration (0-250 ppm), This is a fraction of the analysis time required by the Modified Monier-Williams and the enzyme method, which take 2.5 h and 45 min, respectively. Since this study was conducted under controlled laboratory conditions, other considerations such as ph, salinity interference and temperature compensation should be investigated along with actual industry sampling to confirm the electrode procedure for regulatory usage. Further work is currently under way in the area of electrode miniaturization to allow field testing of this procedure. The Modified Monier-Williams in some cases lost more than 25%

6 JOURNAL OFAQUATIC FOOD PRODUCT TFCHNOLOCY of the sulfur dioxide, and the enzyme method produced several erratic sulfite values. The electrode readings produced a linear calibration curve that allowed extrapolation for readings in the range (0-250 ppm). The electrode readings represent free sulfite, while the other methods measure total sulfite. Reports by Ross et al. (1973) indicate that the ph of the sample may be altered to measure free or total sulfite. This would allow different uses for the electrode in regulated foods. It was noted in earlier work that the millivolts reading for levels below 50 ppm tended to drift upwards after 2 min. This may be due to leakage from the sleeve junction or the possibility that 50 ppm was tear the limit for accurate quantitation for a particular electrode. REFERENCES AOAC Official Methods o/ Analysis. Association of Official Analytical Chemists. Washington, DC. DeWitt, B. and G. Finne A comparison of methods for determining sulfur dioxide in shrimp. In Proceedings of the Tenth Annual Tropical and Subtropical Fisheries Conference of the Americas. New Orleans, LA, p.131. Ingram, M An Electrometric Indicator to Replace Starch in Iodine Titrations of Sulfurous Acid in Fruit Juices J. Soc. Chern. Ind. 66, Ross, J., J. Risemand and J. Krueger Potentiometric Gas-Sensing Electrodes. Pure and Applied Chemistry 36, Taylor, S. and R. Hush Sulfites: A Technical and Scientific Review. Prepared for: International Food Additive Council. Madison, WI.