Note 33: Changes in Volatile Organic Composition in Milk Over Time

Introduction

Volatile and semi-volatile organic compounds present both in the matrix and the headspace aroma are largely responsible for the flavor qualities of the foods we eat. Flavor is always considered the most important factor of any of the quality categories when comparing various dairy products. Dairy products provide a great sense of eating pleasure for their characteristic flavor and smooth taste as well as have an important role in a well- balanced diet. Because milk possesses a bland and soft flavor, the appearance of an objectionable off-flavor or off-odor is readily noticeable. There is a delicate balance between many flavor compounds that produce a desirable milk flavor and if this balance is disturbed, off-flavors may occur. In assessing its overall flavor quality, it would be extremely advantageous to have a reliable and efficient method for the detection, identification and quantification of the volatile organic compounds responsible for the degree of freshness in milk.

Analytical techniques are needed to profile a wider range of volatile and semi-volatile organics in milk and to identify the flavors, off-flavors, off-odors and potential contaminants that may be present. The purpose of this investigation is to develop an analytical technique that could detect and identify a wide range of volatile and semi-volatile compounds in milk over time and relate the differences in volatile organic composition to the quality of milk. For this study, volatile organic compounds were purged from samples of milk followed by trapping onto Tenax® TA adsorbent resin using a dynamic purge and trap technique (P&T). The adsorbent traps were subsequently analyzed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). The dynamic P&T technique together with TD-GC-MS permits the analysis of a wider range of both volatile and semi-volatile organic compounds and is more sensitive than the routinely used static headspace techniques, cryofocusing techniques and high resolution GC.

Instrumentation

All experiments were conducted using a Scientific Instrument Services model TD3 Short Path Thermal Desorption System accessory connected to the injection port of an HP 5890 Series II interfaced to an HP 5971 Mass Selective Detector. The mass spectrometer was operated in the electron impact mode (EI) at 70 eV and scanned from 35 to 450 daltons during the GC run for the total ion chromatogram.

A short 0.5 meter by 0.53 mm diameter fused-silica precolumn was attached to the injection port end of a 60 meter x 0.25 mm i.d. DB-5MS capillary column containing a 0.25 µm film thickness. The GC injection port was set to 260 degrees C with no split. The head of the column was maintained at -70 degrees C using a GC Cryo-Trap model 951 (Scientific Instrument Services, Ringoes, NJ) during the desorption and extraction process and then ballistically heated to 220 degrees C after which the GC oven was temperature programmed from 35 degrees C (hold for 5 minutes) to 80 degrees C at 10 deg/min, then to 200 degrees C at 4 deg/min and finally to 260 degrees C at a rate of 10 deg/min.

Experimental

Several brands of milk that were freshly opened and allowed to sit at room temperature for two days were analyzed by a Purge and Trap technique to relate the off-flavor/off-odor to changes in the volatile organic composition of milk. In addition, several milk cartons were analyzed by Direct Thermal Extraction to determine if any volatile organics from the cartons contributed to the flavor profiles of milk.

Figure 1 - Purge and Trap System For the Extraction Of Volatiles From Milk Samples

Sample sizes of 0.5 ml of the various milks were pipetted into a 10 ml test tube and heated to 60 degrees C for 90 minutes. The surface of the samples was purged with high purity helium at 20 ml/min with an additional 25 ml/min dry purge using the S.I.S. Purge and Trap System (Fig. 1). Volatile analytes were purged from the liquid matrix and carried to a preconditioned 4.0 mm i.d. glass-lined stainless steel desorption tube packed with 200 mg of Tenax TA. Approximately 25 mg of each of the milk carton samples with different colored inks were inserted into a 4.0 mm i.d. glass-lined stainless steel desorption tube and purged for 2 minutes at 80 ml/min to remove any moisture from the sample.

The desorption tube with sample was then attached to the Short Path Thermal Desorption System and a syringe needle attached. The desorption tube was injected into the GC injection port at desorption block temperatures of 250 degrees C for 10 minutes and a flow rate of 1 ml/min for samples collected via purge and trap. For samples to be analyzed by Direct Thermal Extraction, the desorption block temperatures were set at 220 degrees C.

Figure 2 - Grade A fresh milk, 0.5 ml. Collected By P&T Onto Tenax TA For 90 minutes At 60 deg. C At 20 ml/min With Additional 25 ml/min Dry Purge. Desorb At 250 deg. C For 10 min.

Figure 3 - Grade A 2 days Old Milk, 0.5 ml. Collected By P&T Onto Tenax TA For 90 minutes At 60 deg. C At 20 ml/min With Additional 25 ml/min Dry Purge. Desorb At 250 deg. C For 10 min.

Results and Discussion

Three different manufacturers of milk (Grade A, Half & half and a glass bottled milk) as well as samples from milk cartons with different colored inks were analyzed by Purge and Trap techniques and Direct Thermal Extraction to identify and compare the volatile organics present, and from the data, relate off-flavor/off-odor to the differences in composition of the volatile organic compounds. Over 100 volatile organics were identified in the fresh and two day old milk. Most of the milk studied produced 50 or more volatile organics which were identified in addition to many more that were either too weak to identify or in which a good NBS library match was not achievable (Figs. 2-9). The milk samples were found to contain numerous straight and branched chain hydrocarbons, aldehydes, alcohols, ketones, fatty acids, esters, phenolic compounds, lactones and other miscellaneous compounds. Significant differences in the volatiles present occurred in the fresh milk samples versus the 2 day old samples. Although many of the same volatiles are present in all of the milk samples, there is a distinct variation in the quantities as well as the variety of volatile organics present. This variation could be due to the cow's diet, environment, heat treatment, bacterial spoilage, storage and the oxidation of lipids. In addition, the milk packaged in carton containers contained several volatile organic compounds that appeared to be derived from the ink used in the manufacturing of the milk cartons.

Figure 4 - Hald & Half - fresh, 0.5 ml. Collected By P&T Onto Tenax TA For 90 minutes At 60 deg. C At 20 ml/min With Additional 25 ml/min Dry Purge. Desorb at 250 deg. C For 10 min.

Figure 5 - Half & Half - 2 Days Old, 0.5 ml. Collected By P&T Onto Tenax TA For 90 minutes At 60 deg. C At 20 ml/min With Additional 25 ml/min Dry Purge. Desorb At 250 deg. C For 10 min.

Figure 6 - Glass Bottled Milk, Fresh, 0.5 ml. Collected By P&T Onto Tenax TA For 90 minutes At 60 deg. C At 20 ml/min With Additional 25 ml/min Dry Purge. Desorb At 250 deg. C For 10 min.

Figure 7 - Glass Bottled Milk, 2 Days Old, 0.5 ml. Collected By P&T Onto Tenax TA For 90 minutes At 60 deg. C At 20 ml/min With Additional 25 ml/min Dry Purge. Desorb At 250 deg. C For 10 min.

Among the hydrocarbons identified, tetradecane, pentadecane, hexadecane and heptadecane were the most abundant (Figs. 2-7). They are derived either from the autooxidation of free fatty acids or from plants used in cow feed. Limonene was the predominant terpene hydrocarbon identified in the milk samples. Because only plants and some microorganisms synthesize terpenes, they must be transferred to the milk through the rumen. The aliphatic compounds octanal, nonanal and decanal which are typical products of lipid oxidation were identified in each of the milk samples (Figs. 2-7). When these compounds pass a certain threshold, they are considered significant contributors to the development of rancid flavor. Among the ketones, 4-methyl-3-penten-2-one, 4-hydroxy-4-methyl-2-pentanone, 3-methyl-2-cyclohexen-1-one, 3,5,5-trimethyl-2-cyclohexen-1-one and 2-nonanone were identified. However, 1-phenyl-ethanone was only detected in the 2 day old milk samples packaged in carton containers (Figs. 3&5). In addition, 1-butanol, isoamyl alcohol and acetic acid, propanoic acid and butanoic acid were identified in the 2-day-old milk samples. The differences in composition of the alcohols between the fresh and 2 day old samples may contribute to the difference of odor characteristics between the samples. Most of the esters were found in the 2 day old milk samples (Figs. 3&5). 2,6-Di-tert-butyl-p-cresol (BHT) was the predominant phenolic compound detected in each of the milk samples and may have been added as an antioxidant (Figs. 2-7).

Figure 8 - Milk Carton With Red Ink, 22.3 mg. Analyzed By Direct Thermal Extraction For 10 minutes At 220 deg. C.

Figure 9 - Milk Carton With Purple Ink, 25.6mg. Analyzed By Direct Thermal Extraction For 10 minutes At 220 deg. C.

The compounds 2-(2-methoxyethoxy)-ethanol and 2-(2-butoxyethoxy)-ethanol were only found in the milk samples packaged in the carton containers (Figs. 8 & 9) and not in the glass bottled sample. These compounds are believed to be derived from the ink used in the manufacturing process of the cartons. Both of these compounds were also detected in carton samples containing different colored inks using the Direct Thermal Extraction technique. In addition, the milk cartons contained numerous straight and branched chain hydrocarbons, aldehydes, alcohols and terpene compounds.

Conclusion

The dynamic P&T technique together with TD-GC-MS has been utilized for the detection and identification of volatile organics, aromas and flavor components in milk over time and how these differences in volatile organic composition relate to the quality of milk. This analytical technique can be utilized for the quality control during the production of milk as well as a technique for the detection of potential contamination of milk. The dynamic P&T technique offers several unique advantages over static headspace including greater sensitivity and the detection of a wider range of volatile organics including higher molecular weight compounds and is more sensitive by a factor of at least 100 as compared to the static headspace technique. This technique can easily be incorporated into troubleshooting techniques to detect problems in a wide variety of commercial food products, to compare various competing manufacturers products as well as the implementation of a quality control program for the food industry.