JFS: Sensory and Nutritive Qualities of Food
Spectroscopic Differentiation and Quantification of Microorganisms in Apple Juice CHENXU YU, JOSEPH IRUDAYARAJ, CHITRITA DEBROY, ZE’EV SCHMILOVTICH, AMOS MIZRACH
ABSTRACT: A fast and easy-to-operate Fourier Transform Infrared (FTIR) spectrometry-based approach was developed for microbial differentiation and quantification in apple juice. Eight different microorganisms were evaluated: Enterobacter cloacae, Salmonella typhimurium, Enterobacter aerogenes, Salmonella choleraesuis, Serratia marcescens, Pseudomonas vulgaris, Vibrio cholerae, and Hafnia alvei. FTIR spectroscopy combined with chemometrics could differentiate the microorganisms studied at low concentration level of 103 colony-forming units (CFU)/mL in apple juice. The chemometric models developed to count microorganisms in apple juice were validated by an independent test set consisting of 18 samples and correlated against plate counts satisfactorily up to a detection limit of 103 CFU/mL. Keywords: FTIR spectroscopy, microorganism detection, chemometrics
Introduction
A
Sensory and Nutritive Qualities of Food
wide variety of fresh or minimally processed fruits and fruit juices have been linked to food-borne diseases (Beuchat 1996, 1998). Rapid tests and protocols for the detection and identification of food-borne pathogens present in food materials are of vital importance to public health. Traditional methods for identification and differentiation of microorganism are based on nutritional and biochemical, serological, fluorescent-based antibody and antigen interaction, and immunological techniques. Most of these methods are elaborate and require additional tests to identify bacteria, even at the genus level, and may take several days (Yang and Irudayaraj 2003). Molecular techniques are being used for the identification of microorganisms (Tang and others 1997, 1998; Nikkari and Relman 1999; Tenover and others 1994; Clark and others 1999). Although these techniques are potentially rapid, they are quite expensive and complex and require skilled personnel (Vaneechoutte and Van Eldere 1997; Noordhoek and others 1996; Ieven and Goossens 1997; Fredricks and Relman 1998). Quantification of microorganisms can generally be done by direct or indirect methods. Techniques for direct counting include phase contrast microscopy and fluorescence microscopy, which consists of counting the number of cells in a counting chamber such as the Thoma or Petroff-Hausser chamber. Such methods are suitable for water or liquid cultures (Perry and Staley 1997). In the case of food samples such as apple juice, the color of the background may make the counting of microbial cells more difficult. In flow cytometry, fluorescently labeled cells are detected and counted (Perry and Staley 1997). In microautoradiography, microbial cells are labeled with radioactive materials (3H labeled thymidine, 14C labeled leucine, 14C labeled glucose) and the number of the cells is determined according to the intensity of radioactivity (Perry and Staley 1997). Other methods include the electric particle count technique for counting of microbial cells. Direct methods for counting cells are
MS 20040143 Submitted 3/4/04, Revised 4/19/04, Accepted 5/27/04. Authors Yu and Irudayaraj are with Dept. of Agricultural and Biological Engineering, Pennsylvania State Univ., University Park, PA 16802. Author Debroy is with Gastroenteric Disease Center (GDC), Pennsylvania State Univ., University Park, Pa. Authors Schmilovtich and Mizrach are with Agricultural Research Organization, Volcani Center, Bet Dagan, Israel. Direct inquiries to author Irudayaraj (E-mail:
[email protected]).
S268 JOURNAL OF FOOD SCIENCE—Vol. 69, Nr. 7, 2004 Published on Web 8/17/2004
accurate, even down to a single cell. However, these methods are labor-intensive and require expensive instruments and sophisticated operating procedures and hence are not appropriate for fast analysis of a large number of samples. The major widely used indirect approach to microbial cell quantification is the colony count or plate count methods, which are based on the determination of the number of colony-forming units (CFU) in a sample. In most cases a dilution series needs to be produced to provide a feasible and statistically reliable result. At best, results from this method are accurate in magnitude only. Compared with direct count–based methods, colony count–based methods are less sophisticated and do not require expensive instrumentation. Although they are less accurate, labor intensive, and time consuming, for many applications an estimation of magnitude using this approach is an acceptable option (Bell and Kyriakides 1998). Turbidometry and photometry are also applied for microbial counting. In these approaches, measures of light refraction (turbidometry) and light absorbance (photometry) are correlated against plate count results to develop empirical curves and the concentration of unknown samples could then be estimated from these curves. Although they are fast and easy-to-operate, they usually are not sensitive enough to provide reliable estimations at low concentrations (
