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Changes in headspace volatile attributes of apple cider resulting from thermal processing and storage
Food Research International 36 (2003) 167–174 www.elsevier.com/locate/foodres
Changes in headspace volatile attributes of apple cider resulting from thermal processing and storage Geoffrey G. Ryea, Donald G. Mercerb,* a Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9
b
Received 17 February 2002; accepted 17 July 2002
Abstract One attribute that frequently reflects a product’s organoleptic impact is its aroma. In research presented here, a Fox-3000 Electronic Nose was used to determine and compare the aromatic profiles of samples of apple cider subjected to various thermal treatments. Initial results have indicated that exposure to temperatures of up to 90 C for approximately 28 s acts to stabilize the aromatic properties of apple cider over a 7-day period. In contrast, there are significant changes in the aromatic profiles of fresh apple cider having no such thermal treatment over the same time period. A comparison of samples thermally treated at 60, 70, and 80 C to non-processed cider showed minimal statistical difference on the basis of similarity indices obtained after 24-h of storage of the samples at 4 C. Treatment at 90 C showed significant differences from the unprocessed samples in the same test sequence. After storage for 7-days, samples that were thermally treated exhibited minimal differences from each other, however, were quite different from the non-processed sample on the basis of similarity index analysis. GC analysis was done to confirm potential differences between the samples measured using the electronic nose. Sensory testing is required to determine whether these differences affect the overall quality perception by consumers. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Apple cider; Electronic nose; Sensory; Thermal processing; HTST; Aroma; Thermal processing
1. Introduction Consumption of apple cider has been placed under a growing level of scrutiny over the past several years, as the number of occurrences of contamination with E. coli 0157:H7 has increased. Thermal processing is one acceptable method of reducing the microbial load present in cider, and consequently lowering the inherent risks associated with drinking this beverage. There is, however, reluctance by many consumers to accept any processing of fresh apple cider on the basis of its perceived effects on the flavour and other quality attributes of the product. Apple cider is the non-clarified juice product made from pressing fresh apples that is traditionally associated with the harvest, and the coming of fall and North American Thanksgiving. It has been consumed * Corresponding author. Tel.: +1-519-824-4120x6285; fax: +1519-824-6631. E-mail address: [email protected] (D.G. Mercer).
safely for many years. Apple cider typically has a high acid content with a pH of 3.6–4.0, giving it a CDC (Centers for Disease Control), CFIA (Canadian Food Inspection Agency) and FDA (Food and Drug Administration) label as a product that is generally regarded as safe due to its high acid content, therefore, not requiring pasteurization prior to distribution (CFIA, 1999a). The past 30 years have seen apple cider’s safety come under attack by consumer groups and government agencies pushing for mandatory pasteurization of apple cider due to outbreaks of Escherichia coli O157:H7 and Salmonellosis associated consumption (CFIA, 1999a). Mandatory pasteurization has sparked a debate among farming communities, traditional apple cider consumers, and food safety organizations. Manufacturers and some consumer factions supported by a USDA study claim that pasteurizing apple cider at temperatures in excess of 77 C imparts cooked non-traditional flavour to the product (CFIA, 1999b). Smith and Benson from the University of Nebraska-Lincoln claim that pasteurization at a temperature of 85 C for 20 s yields
0963-9969/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0963-9969(02)00133-3
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satisfactory cider that maintains fresh flavours if stored properly at refrigeration temperatures (Smith & Benson, 1998). The ultimate goal of processors is to produce a safe, high quality product without diminishing the rich flavours, aroma and texture attributable to apple cider. One method of quality control is through the utilization of human biological detection systems, such as the sense of smell and taste to determine differences and quality of food products. The human sense of smell uses olfactory sensors located in the nose to detect volatile components resulting in signals that are sent to the olfactory bulb where the signal is reduced for processing by the brain (Kress-Rogers, 1997). The higher olfactory centers in the brain then process the signals to generate an odour recognition response. Unfortunately, the human sense of smell is very subjective and differs among individuals and can be skewed by ailments such as colds, allergies or physical elements such as spices, temperature or sensory fatigue (Lawless & Heymann, 1998). These factors can cause human olfactory sensory evaluation to be unreliable as a method for quality control and quantification. Additionally digital sensors, such as electronic noses, also offer the potential for cost reduction, convenience and efficiency by reducing the reliance on sensory panels. Sensory inconsistency problems and cost efficiency have led to the development of various biosensor systems that can detect and quantify biological compounds accurately and reproducibly (Kress-Rogers, 1997; Persaud & Travers, 1997). The electronic nose was developed as a biosensor system that simulates the human olfactory system (refer to Fig. 1) by detecting volatile components producing an odour pattern that can be analyzed and quantified reproducibly (Kress-Rogers, 1997). The electronic nose, like the mammalian nose, detects gases using sensors that send the signals to a recognition organ; the computer (Schaller, Bosset, & Escher, 1998). Electronic nose technology is a unique and innovative technology that has incredible potential in the area of food analysis. The term electronic nose refers to an array of chemical sensors where each sensor has partial specificity to a wide range of aroma molecules with suitable analytical and/ or pattern recognition techniques (Shen et al., 2001). One basic configuration of an electronic nose system is the Fox 3000 which consists of a series of 12 semi-specific metal oxide detectors that are aligned in series in a resistor configuration in order to measure volatiles within an inert carrier gas. The resistance measurement from each of the sensors is sent to a computer where the signals are processed and reduced to provide a final odour spectrum (Kress-Rogers, 1997). Odour spectra can be utilized using computer software to determine differences among samples, to detect spoilage or as a quality control measure for product consistency. If differences between
samples can be determined, then the device will gain the potential for analytical use for particular food systems. Previous work by Young, Rossiter, Wang, and Miller (1999) and Oshita et al. (2000) demonstrated that differences between fruit ripeness could be detected using the electronic nose and in some cases compounds could be identified. These are important breakthroughs in the area of electronic nose odour recognition because in both of these cases the electronic nose identification took 3–4 times less time to complete then gas chromatography analysis. Works such as these demonstrate that the electronic nose has the potential to be an effective quality control tool due to its ease of use and efficiency. For this reason an expansion of reliable techniques used to detect product differences using the electronic nose is critical to its expanded capabilities. This study aims to determine whether differences are detectable between samples of apple cider processed using high temperature short time pasteurization as monitored by electronic nose technology.
2. Materials and methods 2.1. Sample preparation Fresh, unpasteurized apple cider, composed predominantly of McIntosh (greater than 50%) as well as other varieties of seasonal apples (Courtland, etc.), was obtained locally in 4-l plastic cider bottles and stored at 4 C for 24-h prior to sampling. Pre-processing involved emptying all bottles of apple cider into a clean food grade stainless steel vessel and thorough mixing. Aliquots of 3-l were taken using a clean plastic juice container and poured into the holding reservoir of an Armfield FT74 HTST Plate Heat Exchanger. The Armfield FT74 HTST Plate Heat Exchanger is a sub-pilot/ laboratory size (5-l holding reservoir) heat exchanger system with an internal pressurized heater and water chilling unit, that completely simulates a full size plate heat exchanging unit utilizing heating and cooling processes through the process of regeneration heat exchangers. 2.2. Sample processing The feed tank of the FT74 HTST Plate Heat Exchanger was filled with water prior to operation. The FT74 heat exchanger and the cold-water circulation from the FT61 cooling unit were activated at maximum pump frequency (approximately 5-l/min) and water was passed through the system for 5 min to remove possible substrates from plate surfaces and air from the system. As the water level in the feed tank diminished, the first 3-l sample of cider was poured into the holding tank. The machine was maintained at maximum pump frequency (speed) for 2 min to flush out remaining water
G.G. Rye, D.G. Mercer / Food Research International 36 (2003) 167–174
169
Fig. 1. Simplified schematic diagram comparing electronic nose sensory detection system to the human olfactory system.
and ensure minimal air entrapment in machine; apple cider was added as the holding reservoir approached emptiness. The pump speed was reduced to give a processing flow rate of 0.1 l/min providing an approximate holding time of 28 s. As the cider level decreased in the holding tank additional samples of apple cider were added to top up the tank and maintain a relatively constant hydrostatic head. The hot water circulation was then set to provide a holding temperature of 60 C. Cider was continuously processed through the machine until the temperature of 60 C was achieved and maintained for 5 min. Six 50-ml samples of processed cider were then collected at 30-s intervals, sealed in a snap closure style sample vial and stored immediately at 4 C. Samples were pre-chilled using the cooling plates and regeneration plates of the Armfield unit, yielding output samples at 10 C. Temperatures were then adjusted in turn for collection of samples, as described previously, at 70, 80, and 90 C, respectively. Six 50-ml control samples were collected by removing samples from the raw cider sample mixture at 15-min intervals during the course of the cider processing and were sealed and stored at 4 C. Samples remained in storage at 4 C until evaluation with the Fox 3000 electronic nose. Three samples of each temperature treatment and the control were stored for 24-h prior to analysis and the remaining three samples were stored for 7-days prior to analysis.
2.3. Sample analysis 2.3.1. Electronic nose Cider samples for Fox 3000 Electronic Nose analyses were thoroughly mixed by shaking the sample vials for 30 s. An aliquot of 10-ml of each sample was then pipetted into the electronic nose headspace vials. The sample vials were equilibrated at 40 C and held for 5 min in the electronic nose auto sampler prior to the injection of 2500-ml headspace into the electronic nose. The carrier gas was dry compressed instrument air that ran at a flow rate of 300-ml per minute. After sample analysis, the system was purged for 5 min with carrier gas prior to the next sample injection to allow for reestablishment of the instrument baseline. Each cider sample was evaluated in triplicate. 2.3.2. Gas chromatography A 35-ml aliquot of apple cider was placed into a 40-ml screw cap vial with a Teflon septum. Samples were stirred with an X-shaped magnetic stirring bar at 1000 rpm under ambient conditions. Headspaces were then sampled for 25 min with a SPME fibre coated with Divinylbenzene/Carboxen/Polydimethylsiloxane. The SPME fibre was placed into the injection port of a ThermoFinnigan GCQ GC-MS (Thermo-Finnigan, San Jose, CA) for 3 min where the injection port temperature was set at 250 C. The desorbed volatiles were swept onto a
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Supelco SPB-624 30 m0.25 mm capillary column with 1.4 um film by a 1-ml/min flow of helium. The column was heated at 50 C for 2 min then temperature programmed to 100 C at 5 C/min and held for 5-min after which the temperature was programmed to 190 C. Effluents were detected with an ion trap MS operating in the electron ionization mode at 70 eV.
3. Results and discussion The Fox 3000 electronic nose uses 12 metal oxide sensors to create a sensory profile or ‘‘fingerprint’’ that can be evaluated based on sample similarities. This is done by measuring the differences or distances between sensor values in a 12-dimensional array from sample to sample using the similarity index formula (Smith, 1998). The similarity index is a compilation of measurements of spaces between similar sensor points from two samples in 12-dimensional space known as the Euclidean distances (Smith, 1998). The similarity index is based on principle component analysis where several dimensions are compiled to create a single dimension parameter that will differentiate one sample from another. The equation compiles all 12 Euclidean distances between two samples and combines them to give a single number that represents the similarity index of the samples in 12 dimensional space (Smith, 1998). The equation for the similarity index is as follows in Eq. (1): N 0:5 P ðS1i S2i Þ2 Similarity Index ¼ i¼1 ð1Þ 0:5 100 N ðS S Þ2 P 1i 2i 2 i¼1 where: S1i=sensor value for sample 1, S2i=sensor value for sample 2, i=represents sensor number. Increasing similarity index numbers indicate samples with greater variation from each other (Smith, 1998). Previous research by Smith at Agriculture Canada revealed that a similarity index of 10 or greater is an indication of significant detectable human differences among food samples (Smith, 1998). By using Euclidean distances for analysis of the signals derived from the semi-specific metal oxide sensors we measure relative grouping factors allowing an individual sensor difference to affect the given results. This type of analysis gives a measurement of relative similarity among samples, but does not at this time give any indication of significant aromatic differences as perceived by the human nose. Tables 1 and 2 show the comparisons of the samples stored at 4 C for a period of 24-h and 7-days, respectively. In order to eliminate confusion the tables were designed to prevent data duplication and similarity indices were rounded to the nearest integer. Table 1 is a
Table 1 Similarity indexes for apple cider treated at various temperatures using the Armfield FT74 HTST plate heat exchanger with a holding time of 28 s followed by storage at 4 C for 24 h Treatment C
None
60 C
70 C
80 C
90 C
None 60 70 80 90
1 5 7 6 15
1 2 2 10
1 1 8
1 8
2
Table 2 Similarity indexes for apple cider treated at various temperatures using the Armfield FT74 HTST plate heat exchanger with a holding time of 28 s followed by storage at 4 C for 7 days Treatment C
None
60 C
70 C
80 C
90 C
None 60 70 80 90
4 34 35 36 40
2 2 2 6
1 1 5
1 4
2
comparison of the similarity indices for the replicate samples that were stored for 24-h prior to analysis. Numbers shown in bold are those considered to be significantly different from the others based on the similarity index principle. Table 2 is interpreted in a similar manner for the 7-day storage samples. Comparisons of the similarity indices for the five heat treatments analyzed 24-h after sampling is shown in Table 1. These comparisons indicate that processing the samples at 60, 70, and 80 C treatments yielded minimal differences when compared to the untreated sample after 24-h. The 90 C treatment yielded a similarity index of 15 indicating that this sample shows a more significant difference from the control than the lower temperature treated samples. It is noted that it is more significantly different from the untreated sample after 24-h storage. The 90 C sample does not show large differences from the 70 or 80 C, however yields a value of 10 when compared to the 60 C samples, indicating that a mild amount of volatile change is occurring in all of the heat treated samples and is magnified as the treatment temperature increases. The major peak values and mass percentage of all headspace volatiles analyzed using GC of the 24-h samples is shown in Fig. 2. Overall the GC peaks appear similar, however concentration on the major peak values at the times of 10.8, 11.27, 13.91, and 14.49 min as shown in Fig. 3 demonstrate some major differences that reinforce the similarity trends shown in Table 1. The major component peak at 10.8 min shows minimal differences among treatments, while the t=11.27, t=13.91 and t=14.49 indicate variations from sample to sample. Interestingly the peak at t=11.27 only appears when the sample is
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Fig. 2. Gas chromatogram peak values indicating volatile components found in headspace of samples of apple cider samples after storage for 24 h at 4 C.
Fig. 3. Major volatile headspace compounds in the headspace of the apple cider samples after storage for 24-h at 4 C.
exposed to a heat treatment and not in the control fresh apple cider sample. The compounding deviations when compared to the similarity indices indicate that the electronic nose is measuring differences in the volatile system and is also capable of identifying minute differences
between samples. Unfortunately the GC compound library was unavailable and the volatiles could not be individually identified. Comparison of the sample similarity indices after storage for 7 days yield the results shown in Table 2.
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This table indicates that all heat-treated samples show large similarity index differences relative to the control sample after 7 days of storage at 4 C. The heat-treated samples, however, do not show large differences from one another. Confirmation of these trends can be seen
in Fig. 4, which indicates the major peak values and mass percentage of all headspace volatiles analyzed using GC of the 7-day storage samples. The major component peaks highlighted from Fig. 4 in Fig. 5 at t=10.8 min, t=11.27, t=11.36, t=13.91 and t=14.49
Fig. 4. Gas chromatogram peak values indicating volatile components found in headspace of samples of apple cider samples after storage for 24 h at 4 C.
Fig. 5. Major volatile headspace compounds in the headspace of the apple cider samples after storage for 7 days at 4 C.
G.G. Rye, D.G. Mercer / Food Research International 36 (2003) 167–174 Table 3 Comparison of 24-h and 7-day storage similarity indexes for apple cider treated at various temperatures using the Armfield FT74 HTST Heat exchanger with a holding time of 28 s Treatment Samples stored for 7 days at 4 C C None 60 C 70 C 80 C 90 C None 60 Samples stored 70 for 24 h at 4 C 80 90
26 30 32 32 38
7 4 4 3 8
8 4 3 3 7
9 5 4 4 7
12 8 6 6 5
demonstrate the similarity index results shown in Table 2. The component values at t=10.8 in the heat treated samples show differences of less than 1%, while showing a difference as much as 7% compared the control treatment. The values of the components at t=11.27 and t=11.36 also show 2% differences in headspace composition while t=13.91 and t=14.49 show little differences indicating little change. Interestingly the volatile component at t=11.27 that was not detected in the control sample at 24-h, was in greater concentration in the control at 7-days storage than in the heat-treated samples. This evidence indicates that heat treated samples may maintain product similarities over time through preservation whereas the control sample may begin to degrade or spoil via natural mechanisms. Evidence of these criteria can be seen in Table 3. Table 3 shows the various treatments after 24-h of storage at 4 C to similar samples stored for 7-days at 4 C. The control samples showed a great deal of change between the storage intervals of 24-h and 7-days indicating that degradation of the product during storage is occurring. The heat-treated samples using treatments of 60, 70, and 80 C after storage for 7-days proved to be similar to the 24-h (fresh) control cider sample. This demonstrates that the heat treatment has helped to maintain aromatics that would otherwise degrade over a period of storage time in untreated apple cider. A comparison of each of the heat treatments at 24-h storage to the similar samples after 7-days demonstrates that minimal changes are occurring in these samples during storage.
4. Conclusions Heat treatments of 60, 70, and 80 C do not impose large differences in the aromatic profile of apple cider, measured using the electronic nose, when compared to untreated fresh apple cider after 24-h of storage at 4 C. The heat treatment of 90 C imposes larger differences in the aromatic profile of apple cider after 24-h storage when evaluated using the electronic nose.
173
Apple cider samples, with treatment applications of 60, 70, 80, and 90 C and storage at 4 C for 24-h do not demonstrate large similarity indices from fresh untreated cider. Untreated apple cider shows significant changes in the aromatic profile after storage for 7-days. Aromatic profiles and similarity indices for heat treated samples, with the exception of the 90 C sample, that were stored for 7-days are very similar, indicating minimal degradation of pasteurized apple cider during storage at 4 C. These data demonstrate that pasteurization does not appear to have drastic effects on the aromatic profile of apple cider when temperatures of 60, 70, or 80 C are used as treatment temperatures. There is also evidence that after heat treatment, apple cider becomes aromatically stable and does not degrade significantly at 4 C in 7-days. The electronic nose is effective in identifying differences among processed apple cider samples, however, further examination into identification of GC compounds and sensory evaluation are required to apply the electronic nose system to quality control and further organoleptic quality control in apple cider.
Acknowledgements The authors acknowledge the assistance and support of Agriculture and Agri-Food Canada for the use of the Armfield FT74 and FOX 3000 electronic nose; Brantview Apples & Cider; Andrea Johnston, Martin Chicoine, Kelley Knight, Dr. Robin McKellar and Dr. Chris Young all of Agriculture and Agri-Food Canada’s Food Research Program in Guelph, Ontario.
References Canadian Food Inspection Agency (CFIA). (1999a). Consumer fact sheet: unpasteurized juice/ciders. CFIA (1999b). Code of practice for the production and distribution of unpasteurized apple and other fruit juices/cider in Canada. Kress-Rogers, E. (1997). Biosensors and electronic noses for practical applications. In E. Kress-Rogers (Ed.), Handbook of biosensors and electronic noses: medicine, food and the environment. New York: CRC Press. Lawless, H. T., & Heymann, H. (1998). Sensory evaluation of food: principles and practices. New York: Chapman and Hall. Oshita, S., Shima, K., Haruta, T., Seo, Y., Kawagoe, S., Nakayama, S., & Takahara, H. (2000). Discrimination of odors emanating from ‘La France’ pear by semi-conducting polymer sensors. Computers and Electronics in Agriculture, 26, 209–216. Persaud, K., & Travers, P. (1997). Arrays of broad specificity films for sensing volatile chemicals. In E. Kress-Rogers (Ed.), Handbook of biosensors and electronic noses: medicine, food and the environment. New York: CRC Press. Schaller, E., Bosset, J. O., & Escher, F. (1998). ‘Electronic noses’ and their application to food. Lebensm-Wiss. U.-Technology, 31, 305– 316. Shen, N., et al. (2001). Relationship of electronic nose analyses and sensory evaluation of vegetable oils during storage. Journal of the American Oil Chemists Society, 78(9), 937–940.
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Smith, M. (1998). Pattern recognition and classification using the FOX 3000 electronic nose. Canada: Agriculture and Agri-Food. Smith, D., & Benson, A. K. (1998). Apple cider—reducing microbiological risks through good manufacturing practices. Lincoln: University of Nebraska.
Young, H., Rossiter, K., Wang, M., & Miller, M. (1999). Characterization of royal gala apple aroma using electronic nose technology—potential maturity indicator. Journal of Agriculture and Food Chemistry, 47, 5173–5177.
Changes in headspace volatile attributes of apple cider resulting from thermal processing and storage Geoffrey G. Ryea, Donald G. Mercerb,* a Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9
b
Received 17 February 2002; accepted 17 July 2002
Abstract One attribute that frequently reflects a product’s organoleptic impact is its aroma. In research presented here, a Fox-3000 Electronic Nose was used to determine and compare the aromatic profiles of samples of apple cider subjected to various thermal treatments. Initial results have indicated that exposure to temperatures of up to 90 C for approximately 28 s acts to stabilize the aromatic properties of apple cider over a 7-day period. In contrast, there are significant changes in the aromatic profiles of fresh apple cider having no such thermal treatment over the same time period. A comparison of samples thermally treated at 60, 70, and 80 C to non-processed cider showed minimal statistical difference on the basis of similarity indices obtained after 24-h of storage of the samples at 4 C. Treatment at 90 C showed significant differences from the unprocessed samples in the same test sequence. After storage for 7-days, samples that were thermally treated exhibited minimal differences from each other, however, were quite different from the non-processed sample on the basis of similarity index analysis. GC analysis was done to confirm potential differences between the samples measured using the electronic nose. Sensory testing is required to determine whether these differences affect the overall quality perception by consumers. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Apple cider; Electronic nose; Sensory; Thermal processing; HTST; Aroma; Thermal processing
1. Introduction Consumption of apple cider has been placed under a growing level of scrutiny over the past several years, as the number of occurrences of contamination with E. coli 0157:H7 has increased. Thermal processing is one acceptable method of reducing the microbial load present in cider, and consequently lowering the inherent risks associated with drinking this beverage. There is, however, reluctance by many consumers to accept any processing of fresh apple cider on the basis of its perceived effects on the flavour and other quality attributes of the product. Apple cider is the non-clarified juice product made from pressing fresh apples that is traditionally associated with the harvest, and the coming of fall and North American Thanksgiving. It has been consumed * Corresponding author. Tel.: +1-519-824-4120x6285; fax: +1519-824-6631. E-mail address: [email protected] (D.G. Mercer).
safely for many years. Apple cider typically has a high acid content with a pH of 3.6–4.0, giving it a CDC (Centers for Disease Control), CFIA (Canadian Food Inspection Agency) and FDA (Food and Drug Administration) label as a product that is generally regarded as safe due to its high acid content, therefore, not requiring pasteurization prior to distribution (CFIA, 1999a). The past 30 years have seen apple cider’s safety come under attack by consumer groups and government agencies pushing for mandatory pasteurization of apple cider due to outbreaks of Escherichia coli O157:H7 and Salmonellosis associated consumption (CFIA, 1999a). Mandatory pasteurization has sparked a debate among farming communities, traditional apple cider consumers, and food safety organizations. Manufacturers and some consumer factions supported by a USDA study claim that pasteurizing apple cider at temperatures in excess of 77 C imparts cooked non-traditional flavour to the product (CFIA, 1999b). Smith and Benson from the University of Nebraska-Lincoln claim that pasteurization at a temperature of 85 C for 20 s yields
0963-9969/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0963-9969(02)00133-3
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satisfactory cider that maintains fresh flavours if stored properly at refrigeration temperatures (Smith & Benson, 1998). The ultimate goal of processors is to produce a safe, high quality product without diminishing the rich flavours, aroma and texture attributable to apple cider. One method of quality control is through the utilization of human biological detection systems, such as the sense of smell and taste to determine differences and quality of food products. The human sense of smell uses olfactory sensors located in the nose to detect volatile components resulting in signals that are sent to the olfactory bulb where the signal is reduced for processing by the brain (Kress-Rogers, 1997). The higher olfactory centers in the brain then process the signals to generate an odour recognition response. Unfortunately, the human sense of smell is very subjective and differs among individuals and can be skewed by ailments such as colds, allergies or physical elements such as spices, temperature or sensory fatigue (Lawless & Heymann, 1998). These factors can cause human olfactory sensory evaluation to be unreliable as a method for quality control and quantification. Additionally digital sensors, such as electronic noses, also offer the potential for cost reduction, convenience and efficiency by reducing the reliance on sensory panels. Sensory inconsistency problems and cost efficiency have led to the development of various biosensor systems that can detect and quantify biological compounds accurately and reproducibly (Kress-Rogers, 1997; Persaud & Travers, 1997). The electronic nose was developed as a biosensor system that simulates the human olfactory system (refer to Fig. 1) by detecting volatile components producing an odour pattern that can be analyzed and quantified reproducibly (Kress-Rogers, 1997). The electronic nose, like the mammalian nose, detects gases using sensors that send the signals to a recognition organ; the computer (Schaller, Bosset, & Escher, 1998). Electronic nose technology is a unique and innovative technology that has incredible potential in the area of food analysis. The term electronic nose refers to an array of chemical sensors where each sensor has partial specificity to a wide range of aroma molecules with suitable analytical and/ or pattern recognition techniques (Shen et al., 2001). One basic configuration of an electronic nose system is the Fox 3000 which consists of a series of 12 semi-specific metal oxide detectors that are aligned in series in a resistor configuration in order to measure volatiles within an inert carrier gas. The resistance measurement from each of the sensors is sent to a computer where the signals are processed and reduced to provide a final odour spectrum (Kress-Rogers, 1997). Odour spectra can be utilized using computer software to determine differences among samples, to detect spoilage or as a quality control measure for product consistency. If differences between
samples can be determined, then the device will gain the potential for analytical use for particular food systems. Previous work by Young, Rossiter, Wang, and Miller (1999) and Oshita et al. (2000) demonstrated that differences between fruit ripeness could be detected using the electronic nose and in some cases compounds could be identified. These are important breakthroughs in the area of electronic nose odour recognition because in both of these cases the electronic nose identification took 3–4 times less time to complete then gas chromatography analysis. Works such as these demonstrate that the electronic nose has the potential to be an effective quality control tool due to its ease of use and efficiency. For this reason an expansion of reliable techniques used to detect product differences using the electronic nose is critical to its expanded capabilities. This study aims to determine whether differences are detectable between samples of apple cider processed using high temperature short time pasteurization as monitored by electronic nose technology.
2. Materials and methods 2.1. Sample preparation Fresh, unpasteurized apple cider, composed predominantly of McIntosh (greater than 50%) as well as other varieties of seasonal apples (Courtland, etc.), was obtained locally in 4-l plastic cider bottles and stored at 4 C for 24-h prior to sampling. Pre-processing involved emptying all bottles of apple cider into a clean food grade stainless steel vessel and thorough mixing. Aliquots of 3-l were taken using a clean plastic juice container and poured into the holding reservoir of an Armfield FT74 HTST Plate Heat Exchanger. The Armfield FT74 HTST Plate Heat Exchanger is a sub-pilot/ laboratory size (5-l holding reservoir) heat exchanger system with an internal pressurized heater and water chilling unit, that completely simulates a full size plate heat exchanging unit utilizing heating and cooling processes through the process of regeneration heat exchangers. 2.2. Sample processing The feed tank of the FT74 HTST Plate Heat Exchanger was filled with water prior to operation. The FT74 heat exchanger and the cold-water circulation from the FT61 cooling unit were activated at maximum pump frequency (approximately 5-l/min) and water was passed through the system for 5 min to remove possible substrates from plate surfaces and air from the system. As the water level in the feed tank diminished, the first 3-l sample of cider was poured into the holding tank. The machine was maintained at maximum pump frequency (speed) for 2 min to flush out remaining water
G.G. Rye, D.G. Mercer / Food Research International 36 (2003) 167–174
169
Fig. 1. Simplified schematic diagram comparing electronic nose sensory detection system to the human olfactory system.
and ensure minimal air entrapment in machine; apple cider was added as the holding reservoir approached emptiness. The pump speed was reduced to give a processing flow rate of 0.1 l/min providing an approximate holding time of 28 s. As the cider level decreased in the holding tank additional samples of apple cider were added to top up the tank and maintain a relatively constant hydrostatic head. The hot water circulation was then set to provide a holding temperature of 60 C. Cider was continuously processed through the machine until the temperature of 60 C was achieved and maintained for 5 min. Six 50-ml samples of processed cider were then collected at 30-s intervals, sealed in a snap closure style sample vial and stored immediately at 4 C. Samples were pre-chilled using the cooling plates and regeneration plates of the Armfield unit, yielding output samples at 10 C. Temperatures were then adjusted in turn for collection of samples, as described previously, at 70, 80, and 90 C, respectively. Six 50-ml control samples were collected by removing samples from the raw cider sample mixture at 15-min intervals during the course of the cider processing and were sealed and stored at 4 C. Samples remained in storage at 4 C until evaluation with the Fox 3000 electronic nose. Three samples of each temperature treatment and the control were stored for 24-h prior to analysis and the remaining three samples were stored for 7-days prior to analysis.
2.3. Sample analysis 2.3.1. Electronic nose Cider samples for Fox 3000 Electronic Nose analyses were thoroughly mixed by shaking the sample vials for 30 s. An aliquot of 10-ml of each sample was then pipetted into the electronic nose headspace vials. The sample vials were equilibrated at 40 C and held for 5 min in the electronic nose auto sampler prior to the injection of 2500-ml headspace into the electronic nose. The carrier gas was dry compressed instrument air that ran at a flow rate of 300-ml per minute. After sample analysis, the system was purged for 5 min with carrier gas prior to the next sample injection to allow for reestablishment of the instrument baseline. Each cider sample was evaluated in triplicate. 2.3.2. Gas chromatography A 35-ml aliquot of apple cider was placed into a 40-ml screw cap vial with a Teflon septum. Samples were stirred with an X-shaped magnetic stirring bar at 1000 rpm under ambient conditions. Headspaces were then sampled for 25 min with a SPME fibre coated with Divinylbenzene/Carboxen/Polydimethylsiloxane. The SPME fibre was placed into the injection port of a ThermoFinnigan GCQ GC-MS (Thermo-Finnigan, San Jose, CA) for 3 min where the injection port temperature was set at 250 C. The desorbed volatiles were swept onto a
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Supelco SPB-624 30 m0.25 mm capillary column with 1.4 um film by a 1-ml/min flow of helium. The column was heated at 50 C for 2 min then temperature programmed to 100 C at 5 C/min and held for 5-min after which the temperature was programmed to 190 C. Effluents were detected with an ion trap MS operating in the electron ionization mode at 70 eV.
3. Results and discussion The Fox 3000 electronic nose uses 12 metal oxide sensors to create a sensory profile or ‘‘fingerprint’’ that can be evaluated based on sample similarities. This is done by measuring the differences or distances between sensor values in a 12-dimensional array from sample to sample using the similarity index formula (Smith, 1998). The similarity index is a compilation of measurements of spaces between similar sensor points from two samples in 12-dimensional space known as the Euclidean distances (Smith, 1998). The similarity index is based on principle component analysis where several dimensions are compiled to create a single dimension parameter that will differentiate one sample from another. The equation compiles all 12 Euclidean distances between two samples and combines them to give a single number that represents the similarity index of the samples in 12 dimensional space (Smith, 1998). The equation for the similarity index is as follows in Eq. (1): N 0:5 P ðS1i S2i Þ2 Similarity Index ¼ i¼1 ð1Þ 0:5 100 N ðS S Þ2 P 1i 2i 2 i¼1 where: S1i=sensor value for sample 1, S2i=sensor value for sample 2, i=represents sensor number. Increasing similarity index numbers indicate samples with greater variation from each other (Smith, 1998). Previous research by Smith at Agriculture Canada revealed that a similarity index of 10 or greater is an indication of significant detectable human differences among food samples (Smith, 1998). By using Euclidean distances for analysis of the signals derived from the semi-specific metal oxide sensors we measure relative grouping factors allowing an individual sensor difference to affect the given results. This type of analysis gives a measurement of relative similarity among samples, but does not at this time give any indication of significant aromatic differences as perceived by the human nose. Tables 1 and 2 show the comparisons of the samples stored at 4 C for a period of 24-h and 7-days, respectively. In order to eliminate confusion the tables were designed to prevent data duplication and similarity indices were rounded to the nearest integer. Table 1 is a
Table 1 Similarity indexes for apple cider treated at various temperatures using the Armfield FT74 HTST plate heat exchanger with a holding time of 28 s followed by storage at 4 C for 24 h Treatment C
None
60 C
70 C
80 C
90 C
None 60 70 80 90
1 5 7 6 15
1 2 2 10
1 1 8
1 8
2
Table 2 Similarity indexes for apple cider treated at various temperatures using the Armfield FT74 HTST plate heat exchanger with a holding time of 28 s followed by storage at 4 C for 7 days Treatment C
None
60 C
70 C
80 C
90 C
None 60 70 80 90
4 34 35 36 40
2 2 2 6
1 1 5
1 4
2
comparison of the similarity indices for the replicate samples that were stored for 24-h prior to analysis. Numbers shown in bold are those considered to be significantly different from the others based on the similarity index principle. Table 2 is interpreted in a similar manner for the 7-day storage samples. Comparisons of the similarity indices for the five heat treatments analyzed 24-h after sampling is shown in Table 1. These comparisons indicate that processing the samples at 60, 70, and 80 C treatments yielded minimal differences when compared to the untreated sample after 24-h. The 90 C treatment yielded a similarity index of 15 indicating that this sample shows a more significant difference from the control than the lower temperature treated samples. It is noted that it is more significantly different from the untreated sample after 24-h storage. The 90 C sample does not show large differences from the 70 or 80 C, however yields a value of 10 when compared to the 60 C samples, indicating that a mild amount of volatile change is occurring in all of the heat treated samples and is magnified as the treatment temperature increases. The major peak values and mass percentage of all headspace volatiles analyzed using GC of the 24-h samples is shown in Fig. 2. Overall the GC peaks appear similar, however concentration on the major peak values at the times of 10.8, 11.27, 13.91, and 14.49 min as shown in Fig. 3 demonstrate some major differences that reinforce the similarity trends shown in Table 1. The major component peak at 10.8 min shows minimal differences among treatments, while the t=11.27, t=13.91 and t=14.49 indicate variations from sample to sample. Interestingly the peak at t=11.27 only appears when the sample is
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Fig. 2. Gas chromatogram peak values indicating volatile components found in headspace of samples of apple cider samples after storage for 24 h at 4 C.
Fig. 3. Major volatile headspace compounds in the headspace of the apple cider samples after storage for 24-h at 4 C.
exposed to a heat treatment and not in the control fresh apple cider sample. The compounding deviations when compared to the similarity indices indicate that the electronic nose is measuring differences in the volatile system and is also capable of identifying minute differences
between samples. Unfortunately the GC compound library was unavailable and the volatiles could not be individually identified. Comparison of the sample similarity indices after storage for 7 days yield the results shown in Table 2.
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This table indicates that all heat-treated samples show large similarity index differences relative to the control sample after 7 days of storage at 4 C. The heat-treated samples, however, do not show large differences from one another. Confirmation of these trends can be seen
in Fig. 4, which indicates the major peak values and mass percentage of all headspace volatiles analyzed using GC of the 7-day storage samples. The major component peaks highlighted from Fig. 4 in Fig. 5 at t=10.8 min, t=11.27, t=11.36, t=13.91 and t=14.49
Fig. 4. Gas chromatogram peak values indicating volatile components found in headspace of samples of apple cider samples after storage for 24 h at 4 C.
Fig. 5. Major volatile headspace compounds in the headspace of the apple cider samples after storage for 7 days at 4 C.
G.G. Rye, D.G. Mercer / Food Research International 36 (2003) 167–174 Table 3 Comparison of 24-h and 7-day storage similarity indexes for apple cider treated at various temperatures using the Armfield FT74 HTST Heat exchanger with a holding time of 28 s Treatment Samples stored for 7 days at 4 C C None 60 C 70 C 80 C 90 C None 60 Samples stored 70 for 24 h at 4 C 80 90
26 30 32 32 38
7 4 4 3 8
8 4 3 3 7
9 5 4 4 7
12 8 6 6 5
demonstrate the similarity index results shown in Table 2. The component values at t=10.8 in the heat treated samples show differences of less than 1%, while showing a difference as much as 7% compared the control treatment. The values of the components at t=11.27 and t=11.36 also show 2% differences in headspace composition while t=13.91 and t=14.49 show little differences indicating little change. Interestingly the volatile component at t=11.27 that was not detected in the control sample at 24-h, was in greater concentration in the control at 7-days storage than in the heat-treated samples. This evidence indicates that heat treated samples may maintain product similarities over time through preservation whereas the control sample may begin to degrade or spoil via natural mechanisms. Evidence of these criteria can be seen in Table 3. Table 3 shows the various treatments after 24-h of storage at 4 C to similar samples stored for 7-days at 4 C. The control samples showed a great deal of change between the storage intervals of 24-h and 7-days indicating that degradation of the product during storage is occurring. The heat-treated samples using treatments of 60, 70, and 80 C after storage for 7-days proved to be similar to the 24-h (fresh) control cider sample. This demonstrates that the heat treatment has helped to maintain aromatics that would otherwise degrade over a period of storage time in untreated apple cider. A comparison of each of the heat treatments at 24-h storage to the similar samples after 7-days demonstrates that minimal changes are occurring in these samples during storage.
4. Conclusions Heat treatments of 60, 70, and 80 C do not impose large differences in the aromatic profile of apple cider, measured using the electronic nose, when compared to untreated fresh apple cider after 24-h of storage at 4 C. The heat treatment of 90 C imposes larger differences in the aromatic profile of apple cider after 24-h storage when evaluated using the electronic nose.
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Apple cider samples, with treatment applications of 60, 70, 80, and 90 C and storage at 4 C for 24-h do not demonstrate large similarity indices from fresh untreated cider. Untreated apple cider shows significant changes in the aromatic profile after storage for 7-days. Aromatic profiles and similarity indices for heat treated samples, with the exception of the 90 C sample, that were stored for 7-days are very similar, indicating minimal degradation of pasteurized apple cider during storage at 4 C. These data demonstrate that pasteurization does not appear to have drastic effects on the aromatic profile of apple cider when temperatures of 60, 70, or 80 C are used as treatment temperatures. There is also evidence that after heat treatment, apple cider becomes aromatically stable and does not degrade significantly at 4 C in 7-days. The electronic nose is effective in identifying differences among processed apple cider samples, however, further examination into identification of GC compounds and sensory evaluation are required to apply the electronic nose system to quality control and further organoleptic quality control in apple cider.
Acknowledgements The authors acknowledge the assistance and support of Agriculture and Agri-Food Canada for the use of the Armfield FT74 and FOX 3000 electronic nose; Brantview Apples & Cider; Andrea Johnston, Martin Chicoine, Kelley Knight, Dr. Robin McKellar and Dr. Chris Young all of Agriculture and Agri-Food Canada’s Food Research Program in Guelph, Ontario.
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