Changes of Headspace Volatile Compounds Due to Oxidation of Milk

Chapter 7

Changes of Headspace Volatile Compounds Due to Oxidation of Milk Fat During Storage of Dried Dairy Products

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Y. B. Lee and C. V. Morr Department of Food Science and Technology, Ohio State University, Columbus, OH 43210

Flavor quality is one of the most important factors for determining consumer acceptance of dairy and other food products. Lipid oxidation and Maillard browning reactions that occur during storage of spray dried dairy products result in the formation of volatile off -flavors that greatly impair theirflavorquality, thereby limiting their value as ingredients in formulated food products. Analytical methodologies previously available for isolation, fractionation and identification of volatile compounds formed during storage of dried dairy products are time consuming, tedious and are sometimes subject to error due to artifact formation and incomplete compound recovery. The recently developed dynamic headspace analysis (DHA) technique is suitable for identifying and monitoring changes in concentrations of volatile compounds produced in dried dairy products during storage. The major objective of this research was to study the changes of headspace volatile compounds produced by oxidation of milkfat in selected, commercial, spray dried dairy products by DHA. A secondary objective was to design a miniaturized water activity (aw) control chamber for conducting accelerated storage studies on dried dairy products, and that can be sampled for headspace volatile compounds.

Although considerable research has been done on the development of off-flavors in milk and fermented dairy products over the past 10-15 years, relatively little attention has been given to research on off-flavors in dried milk and dairy products (7-4). Two major mechanisms are usually involved in flavor deterioration of dried milk and dairy products, i.e., lipid oxidation and Maillard browning reaction (3,5,6). Ferretti and Flanagan (2) identified 24 volatile compounds in the steam distillate from

0097-6156/94/0558-0098$08.00/0 © 1994 American Chemical Society Ho and Hartman; Lipids in Food Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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Headspace Volatile Compounds Due to Oxidation

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commercial spray dried whey powder that included alkylpyrazines, furans, pyrroles, ct-methyl-y-butyrolactone, isobutyramide, N-methyl-2-pyrrolidinone, 3-hydroxy-2butanone, benzaldehyde, phenol, benzyl alcohol, maltol, dimethylsulfone, and several organic acids. No attempt was made to distinguish those compounds produced by lipid oxidation from those produced by Maillard browning reaction. Although storage temperature is very important for preventing or inhibiting these chemical reactions (7), moisture and lipid concentration of the dried products is also extremely important in this regard (8). Atomization of milk or whey concentrates into > 150°C air during spray drying would likely promote initiation of the lipid oxidation mechanism. Once initiated, reaction of unsaturated lipids with peroxides, hydroperoxides and free radicals to form the undesireable volatile compounds responsible for the "oxidized" off-flavor would be accelerated. Oxidized milkfat is commonly described as having an "oxidized" or "cardboard" flavor (9), which differs from the "stale" flavor formed in dried whey and whey protein concentrate (WPC) products. Oxidation of milkfat results in the formation of undesireable volatile compounds in dried milk and whey products (10) and is one of the most important factors limiting the manufacture and utilization of these products. Linoleic, linolenic and several unsaturated fatty acids in milk are especially susceptible to oxidation (9). Aldehydes and other lipid oxidation products from these unsaturated fatty acids are chemically reactive and are believed to promote the Maillard browning reaction (10). Phospholipids are more readily oxidized than the neutral glyceride lipid fraction and is therefore considered as one of the off-flavor precursors in milk (77). Experimental Dried milk products. Commercial dried dairy products obtained for this study included: whole milk powder (WMP) that contained 26% milkfat from Maple Island, Inc., Stillwater, MN; nonfat milk powder (NFMP) that contained 0.8 % milkfat, potassium caseinate (PC) that contained ^ 1% milkfat, and whey protein concentrate (WPC) that contained 7% milkfat from DMV Ridgeview, La Crosse, WI; whey protein isolate (WPI) that contained < 1% milkfat from Le Sueur Isolates, Le Sueur, MN; and sweet whey powder (WP) that contained 1% milkfat from Holmes Cheese Company, Millersburg,OH. Miniaturized a control chamber. The control chamber design is shown in Figure 1. A saturated slurry of sodium nitrite (NaN0 ) crystals in water was layered on the bottom of a 125 ml serum bottle (Wheaton, Millville, NJ). This arrangement provided a theoretical a^ of 0.59 (72). Six 0.5g aliquots of each dried dairy product were weighed into separate 10 χ 75 mm, borosilicate glass, disposable culture tubes (VWR Scientific, Cleveland, OH) and sealed with a teflon-coated septum (Supelco, Bellefonte, PA) and an aluminum cap (Supelco). w

2

Accelerated storage of dried dairy products. The serum bottle was held 2 days in the dark at room temperature to equilibrate a^ of the samples to 0.59. It was

Ho and Hartman; Lipids in Food Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

LIPIDS IN FOOD FLAVORS


100

Figure 1. Design of humidity (aj control device consisting of a saturated aqueous NaN0 slurry in a sealed 125 ml serum bottle. 3

subsequently exposed to accelerated storage for 6 days at 60°C in the dark and stored in a -20°C freezer until analyzed by DHA. Dynamic headspace analysis (DHA) of volatile compounds. Serum bottles were held at room temperature, i.e., 20-24°C, for 30 min and attached to the dynamic headspace analyzer (DHA) as shown in Figure 2. Headspace volatile compounds were purged 10 min with ultrapure helium carrier gas at a flow rate of 40 ml/min and trapped onto a Tenax TA chemical column (Tekmar, Cincinnati, OH). Trapped volatiles were desorbed by heating the trap 4 minutes at 160°C and cryogenically focussed at a 150°C capillary interface that was cooled with liquid nitrogen. Focussed compounds were thermally injected onto the capillary gas chromatographic column by heating to 180°C within 1 minute. Compounds were separated with a 30 m χ 0.25 mm χ 0.25 pm film thickness DB-WAX column (J&W Scientific, Folsom, CA) in a gas chromatograph (model 5890 II, Hewlett Packard). The column was heated from an initial temperatures of 32°C to 160°C at a rate of 2°C/min. A total ion chromatograph (TIC) of eluted compounds was obtained with a mass-selective detector (model 5971 A, Hewlett Packard) and compounds were identified by comparison of their retention index values with those of reference compounds (13) and by computer-matching their mass spectra with that in reference NBS49K.L (Hewlett Packard).


7. LEE AND MORR



t

Sample

Purge

101

Λ

Carrier

Figure 2. Diagram of the dynamic headspace analysis (DHA) equipment with the 125 ml sealed serum bottle used as control and external sample device.

Results and Discussion Volatile compounds in control, dried dairy products. The six spray dried dairy products prior to accelerated storage exhibited a highly acceptable bland and clean odor. The mass spectrum of the unidentified volatile compound exhibiting the largest TIC peak fractionated from all dried milk products except WPC prior to accelerated storage, with a retention time of approximately 6 min, has a base peak at m/z 41. The largest TIC peak eluted from the headspace of WPC with a retention time of 6.8 minutes, was identified as trichloromethane. None of these compounds are believed to be responsible for the off-flavors observed in stored, dried dairy products. Volatile compounds in stored, dried dairy products. Identification of volatile compounds from the spray dried dairy products that have been stored 6 days at 60°C and a 0.59 are provided in Table I. Uniquely different TIC profiles were obtained for each spray dried dairy product after accelerated storage. Contrary to expectations, NFMP exhibited a TIC profile corresponding to higher concentrations of most volatile compounds than in the TIC from WMP. Since NFMP contains a much lower concentration of milkfat than WMP, this phenomenon may likely be due to differences in lipid composition of the two products. For example, NFMP lipids might be expected to contain predominantly smaller sized milkfat globules with higher proportions of phospholipids and lipoprotein-containing milkfat globule membranes and proportionately less neutral triglycerides than WMP w



d

d

d

Aldehydes 2-methyl propanal Butanal 2-methy butanal 3-methyl butanal Hexanal Heptanal Octanal Nonanal Ketones 2-Butanone 2-Heptanone 6-methyl-2-Heptanone 2-Octanone 2-Nonanone Alcohols 2-Butanol 1-Pentanol 2-Furanmethanol Esters Ethyl acetate S-methyl ethanethioate Methyl thiocyanate Hydrocarbons Pentane Hexane Heptane

Compounds

18 28 62 ND 29 47 291 61 38

12 ND 28 7 24 12 39 12 49

17 21 223 14 61 14 75 29 ND

7 ND 142 4 17 4 38 11 ND

1032 1259 1644 890 1048 1262 500 600 700

C

Continued on next page.

647 108 13 5 4 460 13 3 2 ND

WPC

587 16 13 ND 3

C

563 93 ND ND ND

WP

900 1177 1233 1281 1386

C

517 21 84 63 346 ND 34 67

NFMP 644 21 436 386 121 30 ND 33

C

533 27 267 468 99 50 24 60

WMP 476 22 131 192 59 ND ND ND

b

6

Peak area (x 10 )'

816 873 914 918 1086 1182 1286 1390

RI

w

Table I. Total Ion Chromatogram Peak Areas for Headspace Volatile Compounds Isolated from Spray Dried Dairy Products Stored 6 Days at 60°C and 0.59 a



d

d

d

1

d

d

d

ND 25 118 91 ND ND 39 18 ND

689 730 751 1067 1363 1964 927 1023 1324

WMP 106 61 12 ND 41 ND 9 ND

b

870 898 954 1225 1457 938 1037 1250

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C

20 ND 31

29 42 237 158 11 25

259 ND 25 5 74 ND 52 ND

NFMP

0

6

Peak area (x 10 )

a

ND 6 15 1792 31 129 21 803 31

47 46 244 70 ND 129 20 32 41

C

129 39 44 ND 16 651 51 5

WPC

152 70 34 8 30 18 61 2

pc

W

d

c

b

• Average values of three replicate determinations. ND means less than 1 χ 10*. Experimentally determined retention index. Product identification: WMP = whole milk powder, NFMP = nonfat milk powder, WP = whey powder, and WPC « whey protein concentrate. Tentatively identified by mass spectrometry.

d

Aromatic hydrocarbons 2-methyl furan 3-methyl furan 2-ethyl furan 2-pentyl furan 2-Furancarboxaldehyde Benzene Toluene Styrene Sulfur-containing compounds Carbon disulfide* Thiobis methane Ethanethiol Dimethyl disulfide Dimethyl trisulfide Benzothiazole Miscellaneous compounds Dichloro methane Trichloro methane Ν,Ν-dimethyl formamide

Compounds

Table I Continued



104


lipids. Concentrations of 18:1, 18:2; and 18:3 fatty acids in bovine milk phospholipids are 46.7, 12.4, and 3.4 mole %, respectively (14). Differences in phospholipid concentrations in milk and milk products are given by Kurtz (15). Milkfat in whole milk and skim milk contains 0.87 and 17.29% phospholipid, respectively. These lipid compositional differences could well explain the the greater production of volatile compounds in NFMP than in WMP. In addition, differences in the design and operation of the spray dryer as well as in compositional and processing-related factors, i.e., antioxidants, prooxidants, heat treatment, pH, and homogenization, affect the oxidation reaction in milk and dairy products (16). Major differences were observed in the TIC profiles for WP, WPI and WPC, which were more complicated than for WMP or NFMP. The reasons for these differences are not known, but may be due to the major variations in processing technologies used to manufacture them. For example, WP is made from Swiss cheese whey, a by-product of the cheese manufacturing industry. Milk is subjected to biochemical action of casein coagulating enzymes and lactic acid bacteria during cheese curd formation which may alter the susceptibility of the residual whey lipids to oxidation after spray drying. Membrane fractionation and other processing steps in the manufacture of WPC and WPI may also alter the susceptibility of residual whey lipids after drying. Differences in protein-milkfat ratios in the various whey and milk powders may also result in an altered microstructure of their particles such that the milkfat is more or less protected against oxidation. Differences in polar lipids, lipoproteins and other milkfat globule membrane components may also have a bearing on the formation of volatile compounds in dried dairy products during accelerated storage. The highly unsaturated phospholipid fraction associated with the milkfat globule membrane material is believed to be the site of oxidative deterioration in fluid milk an cream (17). PC and WPI, the two dried dairy products with the lowest milkfat concentrations, exhibited considerably less production of headspace volatile compounds than the other products during accelerated storage. The best spray dried dairy products from a flavor stability standpoint should be PC and WPI, which contain lowest lipid concentrations. However, additional and more costly processing steps, i.e., ion exchange adsorption for making WPI, are required for manufacturing these latter products (18). Mechanisms for volatile compound formation in dried dairy products. The two main mechanisms for volatile compound formation in dried dairy products during storage are Maillard browning reaction and lipid oxidation (3,5,6). The substantial concentration of protein and lactose in all of these dried dairy products makes it virtually impossible to rule out the Maillard browning reaction in these studies. In fact, it is possible that these two reactions may be synergistic in nature in dried dairy products. Intermediate and final products from one reaction may actually catalyze the other reaction. Thus, many of the volatile compounds identified in TIC from stored, dried dairy products undoubtedly result from both of these chemical reactions. Volatile compounds expected to arise from milkfat oxidation, were recovered from the headspace of dried dairy products by DHA after being subjected to



7. LEE AND MORR


105

accelerated storage for 6 days at 60°C by DHA (Table I). These included: aldehydes i.e., 2-methyl propanal, butanal, 2-methyl butanal, 3-methyl butanal, hexanal, heptanal, octanal and nonanal; and ketones, i.e., 2-butanone, 2-heptanone, 6-methyl2-heptanone, 2-octanone, and 2-nonanone. Esters that were identified included: acetic acid, ethyl ester; ethanethioic acid, S-methyl ester; and thiocyanic acid, methyl ester. Acids and lactones were not detected in the headspace of stored, dried dairy products, due likely to their low volatility at the temperature employed for purging. Milkfat contains roughly 30% oleic acid and approximately 3% linoleic acid (19). Oleic acid is oxidized to form octanal, nonanal, 2-undecenal and 2-decenal, and oxidation of linoleic acid results in the formation of hexanal, 2-octenal and 2,4decadienal via the hydroperoxide mechanism (20). Aldehydes, which possess a common ion fragment with a mass/charge (m/z) ratio of 44 (27), were among the most abundant compounds formed by lipid oxidation of dried dairy products in the present study. Chromatograms of all volatile compounds isolated from NFMP, WP, and WPC that exhibited a m/z ratio of 44 are shown in Figure 3. Comparison of results for these three spray dried dairy products indicates that the three most abundant aldehydes were in decreasing order hexanal (RT 8.3 min), heptanal (RT 12.8 min), nonanal (RT 25.2 min), and octanal (RT 18.7 min). These results would not be expected on the basis of the percentages of oleic and linoleic acids WPC stored at 60°C over saturated NaN0 solution to maintain an a of 0.59 for 6 days exhibited the highest concentrations of the four aldehydes, hexanal, nonanal, heptanal, and octanal, of all dried dairy products studied. WPC also showed the poorest flavor stability among the dried dairy products studied. The reasons for the high susceptibility of WPC to lipid oxidation is not known. WPC contained 7% milkfat and the powder was quite fluffy, thus allowing a more intimate contact of substrate with the residual oxygen from the headspace atmosphere in the serum bottle. It is also likely that WPC had a smaller particle size distribution than the other dried products. Information on the fatty acid composition of the residual lipids in WPC is lacking but it is possible that these lipids may contain a higher percentage of fatty acids that are more suscptible to oxidation than the other dried dairy products. It is for this reason that researchers are attempting to remove the residual lipids from cheese whey prior to manufacturing WPC (18). Water activity is an important factor that affects the off-flavor formation of spray dried dairy products during storage. The minimum rate of lipid oxidation in milk-based infant foods is at or near an a,^ of 0.24 (22). However, Maillard reaction products, which are simultaneously formed during storage of dried dairy products, functioned as antioxidants (23), thus making it difficult to evaluate the relationship between a^, and lipid oxidation rate. It was shown previously that the use of NaN0 for controlling a,, during accelerated storage resulted in a greater degree of lipid oxidation in dried dairy products than was obtained with other a -controlling chemicals (24). The release of N 0 by NaN0 into the headspace is believed to catalyze the lipid oxidation reaction (24,25). 2

w

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Conclusions Although all of the dried dairy products, i.e., whole milk powder, nonfat dry milk, potassium caseinate, whey protein concentrate, whey protein isolate and whey


106


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Figure 3. Forty four ion chromatogram (44 IC) of volatile compounds from whole milk powder (WMP), whey powder (WP), and whey protein concentrate (WPC) after accelerated storage. powder, exhibited a clean and bland flavor before accelerated storage, DHA results revealed different susceptibilities to lipid oxidation during 6 days of accelerated storage at 60°C and a,, of 0.59. DHA 44 ion chromatograms were used as a reliable indicator of the degree oxidation experienced by these dried dairy products during storage. The humidity (a )-controlling device effectively controlled a^ during accelerated storage and also allowed direct sampling of headspace volatiles for DHA of the stored products. The order of decreasing lipid oxidation rates for dried dairy products was: WPI and PC < WMP < WP, NFMP and WPC. w

Literature cited 1. 2. 3.

Ferretti, Α.; Flanagan, V.P. J. Dairy Sci. 1971a, 54, 1764. Ferretti, Α.; Flanagan, V.P. J. Dairy Sci. 1971b, 54, 1769. Ferretti, Α.; Flanagan, V.P. J. Agric. Food Chem. 1972, 20, 695.


7. LEE AND MORR 4. 5. 6.


7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.


107

McGugan, W.A., Larmond, E.; Emmons, D.B. J. Inst. Can. Sci. Technol. 1979, 12, 32. Ramshaw, E.H.; Dunstone, E.A. J. Dairy Res. 1969, 36, 215. Min, D.B.; Tickner, D.B.; Lee, S.H.; Reineccius, G.A. J. Food Sci. 1990, 55, 401. Driscoll, N.R.; Brennand, C.P.; Handricks, D.G. J. Dairy Sci. 1985, 68, 1931. Morr, C.V.; Ha, E.Y.W. Int. Dairy J. 1991, 1, 1. Hammond, E.G. In Flavor Chemistry of Lipid Foods; Min, D.B.; Smouse, T.H., Eds.; American Oil Chemist's Society: Champaign, IL, 1989; pp 222236. Hill, L.M.; Hammond, E.G.; Carlin, A.F.; Seals, R.G. J. Dairy Sci. 1969, 52, 1917. Sattar, Α.; deMan, J.M. CRC Crit. Rev. Food Sci. Nutr. 1975, 7, 13. Dean, J. Α.; Lange's Handbook of Chemistry; McGraw-Hill: New York, NY, 1985; pp 10-84. Jennings, W.; Shibamoto, T. Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography; Academic Press: New York, NY, 1980; pp 30-113. Morrison, W.I. In Topics in Lipid Chemistry; Gunstone, F.D., Ed.; Logos Press Ltd.: London, England, 1970, Vol. 1; pp 51-106. Kurtz, F.E., In Fundamentals of Dairy Chemistry; Webb, B.H.; Johnson, A.H.; Alford, J.A., Eds.; AVI Publishing Co.: Westport, CT, 1974 2nd Edition; pp 125-219. Weihrauch, J.L. In Fundamentals of Dairy Chemistry; Wong, N.P.; Jenness, R.; Keeney, M.; Marth, E.H., Eds.; Van Nostrand Reinhold Co.: New York, NY, 1988, 3rd Edition; pp 215-278. Badings, H.T. Cold-storage Defects in Butter and their Relation to the Autoxidation of Unsaturated Fatty Acids; Ph.D. Thesis, Agricultural University, Vageningen, The Netherlands, 1970. Morr, C.V. Food Technol. 1992, 46, 110. Dairy Handbook; Alfa-Laval: Lund, Sweden, pp 13. Kinsella, J. E. Chem. Ind. 1969, 36-42. Silverstein, R. M . ; Basserler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds; John Wiley & Sons, Inc.: New York, NY, 1991. Roozen, J. P.; Linssen, J. P. H. In Lipid Oxidation in Food; St. Angelo, A. J., Ed.; ACS Symp. Ser. 500; American Chemical Society: Washington, D. C., 1992, pp 302-309. Waller, G. R.; Beckel, R. W.; Adeleye, B. O. In The Maillard Reaction in Foods and Nutrition; Waller, G. R.; Feather, M. S., Eds.; ACS Symp. Ser. 215; American Chemical Society: Washington, D. C., 1983, pp 125-140. Lee, Y. B. Ph.D. Dissertation, Ohio State University, Columbus, OH, 1993. Labuza, T. P. In Moisture Sorption:Practical Aspects of Isotherm Measurement and Use; American Association of Cereal Chemists: St. Paul, MN, 1984;p65.

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Changes of Headspace Volatile Compounds Due to Oxidation of Milk

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