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Southeast Dairy Foods Research Center, Department of Food Science, North Carolina State University, Raleigh 27695-7624
Corresponding author: M. A. Drake; e-mail: mdrake{at}unity.ncsu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: ultra-high temperature • microwave • sulf-hydryl oxidase • sensory
Abbreviation key: DTNB = 5, 5'-dithio-bis(2-nitro-benzoic acid), SHOX = sulfhydryl oxidase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Conventional pasteurization methods have long been in place and with the advent of UHT technology, the sterilization of fluid milk was achieved using higher temperature treatments for shorter periods. However, shelf-stable milk has met with limited acceptability by the consumer, especially in the United States, due in part to a high cooked flavor. Several attempts to improve the quality of UHT-treated milk products proved successful to varying degrees. Previously, Swaisgood and coworkers used immobilized sulfhydryl oxidase to reduce the thiol content of UHT-heated skim milk and described an improved flavor after enzymatic oxidation to form protein disulfide bonds (Swaisgood et al., 1987). Other studies have showed that altering UHT processing parameters, such as indirect vs. direct steam injection systems, cooling rates, and long-term storage conditions have a significant impact on sensory attributes (Browning et al., 2001). Most recently, epicatechin, a flavonoid compound, was added to UHT milk prior to heating, and the results revealed partial inhibition of thermally generated cooked aroma (Colahan-Sederstrom and Peterson, 2005).
Alternative technologies, such as microwave processing, have also evolved. Microwaves, an element of the electromagnetic spectrum, range in frequency between 300 MHz and 3 GHz, and their routine application in heating foods is already prevalent, especially in developed countries. Since the early 1960s, microwave energy has been used for cooking, baking, and thawing. Hamid et al. (1969) were the first group to use the technology for milk pasteurization. Unlike other energy delivery systems, microwave heating involves a rapid and direct heating process that reduces the time required to achieve a desired temperature. Hence, the total cumulative thermal treatment is much reduced, better preserving the thermolabile constituents of foods, such as aromas, vitamins, and pigments (Mudgett, 1986) while maintaining the sterility of the final product by minimizing bacterial growth (Merin and Rosenthal, 1984).
Based on our review of the literature, there are relatively few reports that describe the effects of microwave processing on sensory, microbiological, rheological, and biochemical parameters of heated milk during long-term storage. In fact, we found only one article that discussed chemical and sensorial changes that occurred during cold storage of microwave-treated milk over a 15-d period (Valero et al., 2001). Therefore, our study was designed to evaluate the effects of microwave vs. indirect UHT heating regimens on these attributes using fluid skim milk products, both white and chocolate varieties, stored at room temperature (21° C) over a much longer period (12 mo). Specifically, the objectives were focused on determining the differences between these processes in 1) bacterial counts, 2) plasmin activity, 3) DTNB reactive protein sulfhydryl groups, 4) sulfhydryl oxidase activity, 5) viscosity, 6) color parameters, and 7) descriptive sensory attributes. These results suggested that microwave processing regimens might deliver an equivalent quality fluid milk product that retains a long-term, stable, shelf life.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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UHT Processing Methodology
Skim milk was subsequently heated using a traditional UHT processing unit that delivers thermal energy via an indirect steam injection system (No-Bac Unitherm model VIII Cherry Burrell, Corp., Louisville, KY). In this paper, the abbreviation UHT is specifically defined as indirect UHT milk, based on this particular method of preparation (indirect steam injection). White milk was heat-treated at 137.8° C for 10 s fastest particle (heating rate: 3.26° C/s, 20-s average hold time, Fo = 8), whereas chocolate milk was thermally processed at 140.6° C for 10 s fastest particle (heating rate: 3.33° C/s, 20-s average hold time, Fo = 15). Fo is a process temperature equivalent unit, whereby an Fo = 1 indicates that a product received a heat treatment equivalent to 121° C for 1 min. The flow rate was approximately 2.0 L/min. It is important to note that with steam heating, there is a gradation of thermal energy because heat is delivered from the outside area of the processing chamber to the interior (indirect heating). As a result, the outermost surfaces of the milk product were likely superheated.
Microwave Processing Methodology
For microwave processing, a 60-kW continuous flow microwave-heating unit (IMS 60 kW microwave system, Industrial Microwave Systems, Morrisville, NC), operating at 915 MHz, was used to process skim milks. Microwaves were focused to a cylindrical applicator, and the power relayed from the generator was monitored using a control panel supplied by the manufacturer. Microwaves were then delivered to the product by a waveguide of rectangular cross-section, which was split into 2 sections and geared towards 2 specially designed applicators, with a directional coupler in each. A polytetrafluoroethylene tube (0.038 m i.d.) was placed at the center of each applicator and the exposure region was 0.2 m long in each applicator (Coronel et al., 2003). A positive displacement pump (model A7000, Marlen Research Corp., Overland Park, KS) was used to move the milk through the system at a flow rate of 3.8 L/min. Temperatures were measured at the inlet of the system, the inlet and exit of each applicator, and at the holding tube exit with thermocouples arranged as previously described (Coronel et al., 2003). Thermal readings were recorded at 4-s intervals using a data logging system (HP 3497A, Hewlett Packard, Palo Alto, CA). Heat processing conditions were essentially equivalent to those previously described in the UHT methodology section (i.e., white milk, heating rate 3.42° C/s, Fo = 8, and chocolate milk, heating rate 3.51° C/s, Fo = 15). In contrast to UHT processing methods, microwave energy, or cold wall heat, was transferred directly into the food product via the microwave itself.
Aseptic Packaging
Milk was aseptically filled (250-mL aseptic filler, model SA-50, International Paper Co., Memphis, TN) and packaged into 250-mL brick-style aseptic cartons formed from standard aseptic roll-stock board composed of laminated layers of polyethylene, aluminum foil, and paperboard (International Paper Co.). The aluminum foil roll-stock provides protection against light and gas penetration. Twenty boxes, collected from the beginning and end of each run, were excluded from the study to obtain uniformly heat-exposed, representative products from each trial. Cartons were stored at 21° C in the dark over a 12-mo period, and sampling was conducted every 3 mo. Time-zero analyses were performed within 96 h after processing. For each testing period (sampling), 20 boxes of each milk type (white and chocolate) were pulled, and 5 boxes aseptically combined for micro-biological plate counts, enzymatic assays, instrumental analyses, and descriptive sensory evaluations. Pooled milk samples were stored at 5° C, and microbiological assays were conducted on all experimental milks before sensory analyses.
Bacteriological Tests
Initially, total plate and coliform counts were conducted on raw milks. The microbiological quality of shelf-stable milks was evaluated for total numbers of bacteria including coliforms, spore-forming, and psychrotrophic microorganisms according to standard methods (American Public Health Association, 2004). Briefly, total plate counts were evaluated using standard methods agar (Difco, Sparks, MD) followed by incubation at 32° C for 48 h. Coliforms were enumerated on violet red bile agar (Difco) followed by incubation at 32 ° C for 48 h. To quantify the numbers of spore-forming bacteria, 200 mL of milk was heated to 80° C in a water bath and held for 12 min. Afterwards, milk samples were immediately cooled in an ice water bath, and pour-plated in duplicate onto standard methods agar containing 0.1% (wt/wt) soluble starch (Sigma, St. Louis, MO) followed by incubation at 32° C for 48 h. Psychrotrophic bacteria were counted using crystal violet tetrazolium agar. Appropriate dilutions were made using sterile 0.1% peptone water, and plates were incubated at 21° C for 48 h before enumeration of visible colonies.
Plasmin
Plasmin activity was monitored at 25° C using a chromozym PL kit purchased from Roche Molecular Biochemicals (Mannheim, Germany). Hydrolysis of the substrate, tosyl-glycyl-prolyl-lysine-4-nitroanilide-acetate, was followed at A405 nm. One unit was defined as that activity liberating 1 micromole of 4-nitroaniline per minute at 25° C under the defined experimental conditions. Two samples from each trial run were measured in duplicate and the numbers averaged.
Reactive Sulfhydryl Groups
The reagent, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), was purchased from Sigma Chemical Company. The level of reactive protein sulfhydryl group was measured according to a modified assay technique in which DTNB reacted with free SH groups producing a yellow color with a maximum absorbance at 412 nm (Ellman, 1959). For these experiments, milks were diluted 1:50 with 0.1 M phosphate buffer, pH 8.0, and 1.0 mL of a 1 mM DTNB solution, prepared with 50 mM sodium phosphate, pH 7, was added. The solution was maintained at room temperature (21° C) for 30 min before reading the absorbance at 412 nm. A blank sample was prepared in an analogous manner with the addition of 1 mL of 50 mM phosphate buffer, pH 7, rather than the DTNB reagent, serving to correct for milk turbidity. A DTNB control (minus milk sample) was also prepared to adjust for the slight increase in the absorbancy of the DTNB solution itself, that occurred during the incubation period. This value was subtracted from the experimental sample reading before calculation of the reactive sulfhydryl concentration. Two samples from each trial run were measured in duplicate and the numbers averaged.
Sulfhydryl Oxidase
Sulfhydryl oxidase (SHOX) activity was determined as the rate of oxidation of 0.8 mM glutathione, prepared in 50 mM sodium phosphate buffer, pH 7, at 37° C, essentially according to the method of Janolino and Swaisgood (1975). Typically, the reaction mixture contained 0.1 mL of test milk added to 1.0 mL of glutathione substrate. At timed intervals, 0.1-mL aliquots were removed and mixed with 5.0 mL of 100 µM DTNB prepared with 0.1 M sodium phosphate buffer, pH 8. The concentration of remaining substrate (reduced glutathione) was calculated from the absorbance at 412 nm (
412nm = 13,600 m–1cm–1). Sulfhydryl oxidase activity was expressed as micromoles of thiol oxidized per minute (units) under the experimental conditions as defined. Two samples from each trial run were measured in duplicate and the numbers averaged.
Protein Assays
A bicinchoninic acid protein assay kit was purchased from Pierce Chemical Company (Rockford, IL) with BSA (2 mg/mL) as the standard. Protein measurements of skim milks were made to calculate the specific activity of sulfhydryl oxidase.
Instrumental Color Analysis
Reflectance measurements of all milks were recorded using a Gardner Spectrogard Color system (Pacific Scientific Instrument Division, Silver Spring, MD) with daylight illumination. The tristimulus parameters were measured in the L*a*b* mode, in which L* represents the lightness value, and a* and b* represent chromaticity coordinates. The colorimeter was calibrated against white and black tile standards, and all samples were tempered to 25° C before performing duplicate readings of each skim milk product.
Viscosity Analysis
Apparent viscosity readings for all milk samples were achieved using a stress-controlled rheometer (Stress-Tech, Rheologica Instruments AB, Lund, Sweden) equipped with a CC 25 concentric cylinder geometry. Two samples from each trial run were measured in duplicate, the numbers averaged, and the apparent viscosity reported as the mean shear rate of the measurement taken at 50 s–1.
Descriptive Sensory Analysis
Milk products, at each sampling, were evaluated in triplicate by each member of a trained descriptive panel (n = 9, 7 females, 2 males, ages 22 to 45). Sensory testing was conducted in compliance with North Carolina State University’s Institutional Review Board for Human Subjects. Each panelist had at least 60 h of previous training on sensory analysis of dairy products using the Spectrum descriptive analysis technique (Meilgaard et al., 1999). Twelve 30-min training sessions were conducted to focus on sensory properties of shelf-stable milks. Flavor, mouthfeel, and color terms identified and selected by the panelists are listed in Table 1
. Throughout all training sessions, panelists evaluated and discussed milks to clarify descriptor concepts and to consistently scale product attributes. The statistical analysis of data collected from these training sessions confirmed that panel results were consistent and that terms were not redundant (Drake et al., 2003). Descriptive analysis was conducted by each panelist in triplicate on each milk using a randomized block design. Samples (20 mL) were served in 59-mL plastic cups fitted with plastic lids (Sweetheart Cup Co., Owings Mills, MD) and labeled with 3-digit codes. Milks were served at 10 ± 2° C, and panelists evaluated 4 milks per session. Chocolate milks were evaluated in separate sessions. Visual attributes were determined at different times to prevent the influence of flavor and mouthfeel attributes on appearance. For tasting purposes, panelists were first presented with a warm-up sample of commercial shelf-stable milk, previously discussed during training sessions. Panelists then proceeded to evaluate each sample. Ambient-temperature spring water was available for palate cleansing.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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2 log cfu/mL) after treatment under several temperature and time combinations. Ultra-high temperature technology has also been developed for food safety applications. Browning et al. (2001) demonstrated the impact of UHT heating parameters on sterility and presented a spreadsheet for predicting safety parameters, such as lethality and microbial inactivation, using UHT milk products subjected to different heating and cooling profiles and varying holding times in a continuous heat exchanger. The raw milk used for these studies showed 1600 ± 350 cfu/mL when plated on standard methods agar, and 25 ± 20 cfu/mL when grown on violet red bile medium. Both UHT and microwave processing regimens virtually eliminated all bacterial growth in the milk as evidenced by the lack of colony formation using various microbiological media. Furthermore, sterility was maintained throughout a 1-yr storage period, indicative of effective aseptic packaging methodologies.
Plasmin Enzyme Assays
Plasmin, an enzyme naturally occurring in milk, cleaves casein and often causes coagulation and sometimes gelation in milk products over time. Plasmin activity is often associated with the development of off-flavors, especially bitterness, during storage (Baker, 1983). Previously, Alichanidis et al. (1986) noted that plasmin retained as much as 30 to 40% of the total activity even after exposure to UHT processing conditions; however, in our own work, plasmin was totally inactivated after both UHT and microwave thermal treatments. Presumably, the absence of plasmin activity may improve the long-term shelf stability of the final fluid milk product.
Reactive Protein Sulfhydryl Groups and SHOX Activity
Off-flavors in heated milk often result from creation of organic sulfur compounds that arise during the decomposition of reactive protein sulfhydryl groups associated with the amino acids, methionine and cysteine. Often, their concentration has been correlated with the sensory detection of undesirable "cooked" flavors in milk samples exposed to high temperatures (Swaisgood et al., 1987). Several examples of such compounds produced upon heating include hydrogen sulfide (rotten egg odor), dimethyl sulfide (rotten vegetables), dimethyl disulfide (putrid), and dimethyl trisulfide (onion, garlic), all of which have extremely low thresholds for detection (DMI, 2003).
Protein sulfhydryl groups also contribute directly to undesirable effects such as these. Whey protein sulfhydryl groups, typically buried within the core of the protein structure, are exposed to the surface because of heating. Furthermore, protein sulfhydryl groups may be formed because of hydrolysis or ß-elimination of disulfide bonds during thermal treatment (Patrick and Swaisgood, 1976). ß-Lactoglobulin, a major protein component of the whey fraction (accounting for over 50% of the total protein) is often involved in these processes (Mulvihell and Kinsella, 1987). The dimeric structure of ß-lactoglobulin comprises 2 free [-SH-] groups and 4 disulfide [-S-S-] bonds with a molecular weight of 36,000 (McKenzie et al., 1972). Thus, during heat treatments, the protein is rapidly denatured and aggregated through disulfide interchange.
Using Ellman’s (1959) procedure, we measured the concentration of reactive protein sulfhydryl groups, generated because of each heat treatment, by performing DTNB assays. The results showed that there were no significant differences between the microwave-treated skim milk products (both white and chocolate varieties) compared with the indirect UHT-processed milks (Figure 1). However, both heating methods resulted in an increased thiol content as compared with conventionally pasteurized milk samples due to the higher thermal temperatures attained during processing. Within 3 mo of storage at room temperature, the (-SH-) content for all heated test milks decreased to essentially equivalent low level values (~0.20 to 0.30 µmol/g of protein), and remained at minimal concentrations throughout the remainder of the study (data not shown). Interestingly, Patrick and Swaisgood (1976) noted similar data patterns using UHT-treated skim milk stored at 25° C.
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Previously, it was noted that SHOX retained enzymatic activity after pasteurization; however, UHT treatment resulted in a significant loss of catalytic capacity (H. E. Swaisgood, Raleigh, NC; personal communication). Therefore, we measured SHOX activity in both heated milk products to investigate the retention of enzymatic catalytic capacity after each type of thermal exposure (microwave vs. indirect UHT). The results established that residual SHOX activity was detected in microwave-processed skim milks (white and chocolate) compared with UHT-sterilized samples that were essentially devoid of active enzyme (Figure 2). Seemingly, microwave conditions favored retention of SHOX activity because of shorter ramping times required to reach the target temperature and uniform thermal distribution throughout the milk. Both of these parameters may significantly affect denaturation of the enzyme. Previously, others noted a low degree of whey protein denaturation, specifically ß-lactoglobulin, after application of continuous microwave treatment using both cow and goat milk (Villamiel et al., 1997).
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Viscosity and Color Measurements
Casein micelles play a major role with respect to the viscosity parameters of skim milk (Walstra and Jenness, 1984); therefore, any factor that affects the aggregational state of the micelle such as ionic strength, pH, or heat will influence resistance to flow. Furthermore, when milk is heated above 70° C, there is a notable effect on the viscosity attributed to the denaturation of whey proteins. As discussed before, denatured whey proteins often undergo sulfhydryl-disulfide interchange reactions, an effect that additionally promotes increased viscosity.
As depicted in Figure 3, both UHT- and microwave-treated chocolate skim milks exhibited the highest viscosity throughout the entire experimental trial. This result would likely be anticipated because chocolate milk formulations were prepared by adding UHT SIPT 8, a UHT chocolate ingredient mixture prepared with cocoa processed with alkali, starch, salt, carrageenan, vanillin, and other artificial flavorings. In addition, the Fo heating parameters were higher for chocolate milks, a factor that could elevate these readings. Statistical analysis verified that the viscosity of the microwave-treated chocolate skim milk was comparable to that of the UHT-heated samples at each sampling time during the storage study.
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). The viscosity of all skim milks increased during storage at room temperature although this observation was most apparent at 6 mo. A slight increase in these values occurred at 9 mo, after which time these measurements remained similar for the duration of the study (12 mo). Viscosity changes may be at least partially attributed to age gelation effects, an aggregation phenomenon that typically affects shelf-stable, sterilized dairy products, although the actual mechanism has not yet been fully defined.
With regard to color, there were no major variations between chocolate milks processed by either heat treatment (P > 0.05); however, significant differences were observed between white UHT- and microwave-treated samples (P < 0.05; Figure 4). Ultra-high temperature—treated white milks exhibited less green (a*) and more yellow (b*) hues compared with microwave-treated milks (Figure 4). Over time, the lightness (L*) of both white milks decreased (P < 0.05), whereas redness decreased in both chocolate milks (P < 0.05).
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Villamiel et al. (1996b) were among the first to show that microwave processing by continuous flow could deliver a sterile fluid milk product with satisfactory sensory quality. In later work, Valero et al. (2001) established that trained sensory panelists were unable to distinguish between microwave treated vs. conventionally pasteurized milk samples based on taste and odor differences. In fact, the 2 products remained indistinguishable based on their sensory qualities, even after storage at refrigeration temperatures over a 15-d period.
For the purposes of this study, the most notable sensory-perceived differences were observed between white milks. Statistical analysis of the data showed that the major dissimilarities between the 2 heated milk products could be attributed to the type of thermal treatment used, i.e., UHT vs. microwave processing (P < 0.05), independent of storage time (P > 0.05). At all time points, microwave milk products were characterized by lower caramelized flavor, decreased astringency, less fatty/stale flavors, and less brownish hues (Figure 5). Such sensory traits are considered negative qualities of shelf-stable milks generally associated with high heat treatment or extended storage time. As these attributes were consistently lower in microwave-heated milks, the results suggested that microwave technology may deliver milk products that exhibit superior sensory characteristics at given time intervals. Future work should be designed to address consumer acceptance and perception of such products. Other sensory attributes did not differ between the 2 heat treatments (P > 0.05; data not shown).
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. It should be noted that there is relatively little information with respect to Maillard reactivity in microwave milk products. In one report, Villamiel et al. (1996a) detected elevated levels of furosine (an indicator of the Maillard reaction) in microwave milks compared with conventionally treated milk, a finding attributed to uneven heating in the microwave oven. In contrast, the unique microwave system used for this study offers an infinitely variable, continuous power source with improved temperature control. Such a system should circumvent potential problems associated with nonuniform temperature distribution and deliver a product with low potential for excessive Maillard browning.
The length of the storage time influenced the sensory profiles of UHT- and microwave-sterilized milk products in a similar manner (Figure 6). In each case, sweet aromatic flavors and sweet taste decreased with time, whereas fatty/stale flavor, astringency, and color intensity were increased (P < 0.05). These attributes were influenced by storage time regardless of type of heat treatment (no statistical interaction, P > 0.05). Other sensory attributes did not change with storage time (P > 0.05; data not shown). Sensory differences were not detected between UHT- vs. microwave-treated chocolate skim milks (P > 0.05; data not shown); chocolate flavor alone may have masked any subtle differences. However, after 12 mo of storage at room temperature, visible separation occurred in microwaved chocolate milks, as observed by all sensory panelists, indicating a failure of the stabilizer system over this time. Brief swirling of the container eliminated the phase separation. Similar effects were not observed in the indirect UHT-treated chocolate milk samples, which undergo more shearing throughout processing, a factor that could contribute to greater constancy of the stabilizer during storage.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication May 13, 2005. Accepted for publication August 2, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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