Antioxidant Measurement and Applications - American Chemical Society

Chapter 25

Formation of Off-Odorants during Light Exposure of Milk and Its Inhibition by Antioxidants 1

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Toshio Ueno , Yuto Suzuki , Chi-Tang H o , and Hideki Masuda Downloaded by IOWA STATE UNIV on March 1, 2017 | http://pubs.acs.org Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch025

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Material R&D Laboratories, Ogawa and Company, Ltd., 15-7 Chidori, Urayasushi, Chiba 279-0032, Japan Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901-8520 2

Potent off-odorants formed during light exposure of milk were identified and the effects of added antioxidants on their formation investigated. The ultra-high temperature (UHT) milk purchased from a local market was stored under fluorescent light (15,000 lux) or in the dark for 16 h at 10 °C. The flavor differences between the light-exposed and nonexposed milk were monitored by aroma extract dilution analysis using the aroma extracts prepared from the milk. Offflavor formation in the milk upon light exposure can be attributed to the formation of nine potent odorants, namely hexanal, (Z)-4-heptenal, 1-octen-3-one, methional, (E)- and (Z)-2-nonenal, (E,Z)-2,6-nonadienal, (E,E)-2,4-nonadienal, and (E,E)-2,4-decadienal. The contribution of these odorants to the light-induced off-flavor was further confirmed by flavor reconstitution experiments using the reference substances. Among the tested antioxidants, (-)-epicatechin and chlorogenic acid were the most effective inhibitors of the formation of the identified off-odorants during light exposure of milk and also most effective for reducing off-flavor intensities of light-exposed milk.

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© 2007 American Chemical Society

Shahidi and Ho; Antioxidant Measurement and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

Downloaded by IOWA STATE UNIV on March 1, 2017 | http://pubs.acs.org Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch025

391 Off-flavor formation in milk due to light exposure has already been extensively studied. Sulfur-containing volatiles such as methional, methanethiol, dimethyl sulfide, and dimethyl disulfide have been identified in milk exposed to light (1,2). These compounds probably result from the degradation of the sulfurcontaining amino acids of the serum (whey) proteins (3), and are considered to be responsible for the off-flavor components described as cabbage, burnt protein, or "activated" (4). On the other hand, carbonyl compounds such as 2-alkenals, 2-alkanones, acetaldehyde, and alkanals are formed by the light-induced lipid oxidation in milk (5,6), and are considered to be responsible for the off-flavor components described as metallic and tallowy (4). Recently, Cadwallader and Howard employed GC-sniffing techniques to detect potent odorants formed in milk exposed to light (7). They revealed that methional, 2-acetyl-l-pyrroline, pentanal, hexanal, l-hexen-3-one, l-octen-3-one, and (Z)-l,5-octadien-3-one are the major contributors of light-induced off-flavor. Thus, many odorants are suggested to be responsible for the off-flavor formation in milk due to light exposure. However, it has not been clarified whether the mixture of these odorants actually represents the flavor character of the light-exposed milk. Only a few studies have reported the effects of added antioxidants on the formation of off-flavor in milk during light exposure. Jung et al. reported that the addition of L-ascorbic acid reduced the formation of dimethyl disulfide and improved the sensory quality of light-exposed skim milk (8). Muranishi et al. reported that phenolic antioxidants, such as catechins and rosmarinic acid inhibited the formation of methional, dimethyl disulfide, and dimethyl trisulfide in an aqueous solution of methionine under riboflavin photo-sensitized conditions (9). The aims of the present study were (1) to identify the potent off-odorants formed during light exposure of milk, (2) to verify the analytical results by flavor reconstitution using reference substances, and (3) to clarify the effect of added antioxidants on the formation of off-odorants during light exposure of milk.

Materials and Methods Materials Ultra-high-temperature (UHT) treated whole milk was purchased from a local market. A l l chemicals were of the highest grade commercially available and were used without any further purification.

Light Exposure of M i l k One hundred grams of milk were sealed in a 180-mL glass bottle with a plastic-lined aluminum cap. The samples were stored under fluorescent light


392 (15,000 lux) at 10 °C for 16 h in an Eyela LST-300 light box (Tokyo Rikakikai Co. Ltd., Tokyo, Japan). When using an antioxidant, 1.0 g of a solution containing 10 mg of the antioxidant and 100 mg of Tween 20 in water were added to 99 g of the milk. The milk containing an antioxidant was exposed to light under the same conditions as described earlier.


Preparation of Aroma Extracts The volatile fraction was isolated from 100 g of the milk by high-vacuum distillation using a solvent-assisted flavor evaporation (SAFE) apparatus as previously described by Engel et al. (10). The obtained distillate was spiked with 100 pL of an internal standard solution containing 2-octanol in dichloromethane (50 mg/kg), and then extracted with dichloromethane (50 mL χ 2). The extract was dried over sodium sulfate, concentrated in vacuo to ~5 mL, and further concentrated under a stream of nitrogen to -50 pL. Gas Chromatography-Olfactometry (GC-O) A n Agilent 6850 series gas chromatograph equipped with a thermal conductivity detector (TCD) and a D B - W A X fused silica capillary column (30 m χ 0.25 mm i.d.; film thickness of 0.25 pm; J & W Scientific was used. The outlet from the detector was connected to a sniffing port flushed with humidified air at 100 mL/min. The operating conditions were as follows: detector temperature, 250 °C; nitrogen carrier gas flow rate, 1 mL/min; oven temperature program, 40 °C, raised at 5 °C/min to 210 °C (60 min); 1 p L of sample was injected in the splitless mode. A n aroma extract dilution analysis (11) was conducted using serial 1:3 dilutions of the original aroma extract with dichloromethane.

Gas Chromatography-Mass Spectrometry ( G C - M S ) An Agilent 6890 Ν gas chromatograph equipped with an Agilent Model 5973 Ν series mass spectrometer and a D B - W A X fused silica capillary column (60 m χ 0.25 mm i.d.; film thickness of 0.25 pm; J & W Scientific was used. The operating conditions were as follows: injector temperature, 250 °C; helium carrier gas flow rate, 1 mL/min; oven temperature program, 40 °C, raised at 3 °C/min to 210 °C (60 min); 1 pL of sample was injected in the splitless mode; ionization voltage, 70 eV; ion source temperature, 140 °C.

Sensory Evaluation The flavor profiles of the milk were evaluated by fourteen trained assessors consisting of three females and eleven males. They were asked to rate the


393 intensities of the five odor attributes ("freshness," "fatty," "metallic," "dusty," and overall off-odor) using a linear scale ranging from 1 (= absent) to 7 (= very strong). Ranking tests were conducted according to a method described by Meilgaard et al. (12). Twenty untrained panelists consisting of eight females and twelve males were asked to rank a series of six samples from first to sixth in the increasing order of off-flavor intensities. The statistical significance was evaluated by Friedman's test.


Results and Discussion Identification and Quantification of Off-Odorants Flavor changes in milk due to light exposure were monitored by a comparative aroma extract dilution analysis using the aroma extracts prepared from the milk. Figure 1 shows the flavor dilution (FD) chromatograms obtained from the milk stored under florescent light (Figure la) and stored in the dark (Figure lb). At an F D factor of sixteen and above, a total of fifteen odorants were detected. Among them, the F D factors of nine odorants, i.e., peak numbers 2, 3,4, 7, 8, 9, 10, 12, and 14 increased with light exposure, whereas those of the other odorants did not change or decreased with light exposure. These results indicate that the off-flavor formation in the light-exposed milk can be attributed to the increase in these nine odorants. Based on G C - M S and GC-Olfactometry analyses using reference substances, the nine odorants whose F D factors increased with light exposure were identified as listed in Table I. With the exception of (2T)-2-nonenal, whose M S spectrum was too small to be quantified, the amounts of the identified odorants in the milk were determined using calibration curves obtained by the addition of the reference compounds to the milk. In agreement with the values of the FD-factor, the concentration of the nine odorants significantly increased with light exposure (Table I).

Reconstitution of the Light-Induced Off-Flavor In order to verify the analytical results, flavor reconstitution experiments using the reference substances were attempted. Differences in the quantified amounts of off-odorants between the light-exposed and non-exposed milk (Table I) were added to the non-exposed milk. Sensory evaluation was then performed to compare the flavor profiles. Figure 2 shows the results of the flavor profiling analysis among the light-exposed milk, non-exposed milk, and non-exposed milk with the added off-odorants. The difference between the lightexposed milk and non-exposed milk (Figure 2a, b) indicates that the "freshness" of the milk almost totally deteriorated and "fatty," "metallic," and "dusty" offodors developed with light exposure. With the addition of the identified odorants


394 Table I. Concentrations and Flavor Dilution Factors of Potent Off-Odorants in Light-Exposed and Non-Exposed Milk


peak α no. 2 3 4 7 8 9 10 12 14

til 1089 1237 1296 1447 1516 1525 1575 1689 1805

compound

odor description

concn in milk"

FD-factor d

LP hexanal green 16 (Z)-4-heptenal tomato-like 16 l-octen-3-one 256 metallic methional potato-like 64 (Z)-2-nonenal earthy 16 (£)-2-nonenal fatty 256 (£,Z)-2,6-nonadienal green 16 fatty 64 (£, E )-2,4-nonadienal 64 fatty (E, E )-2,4-decadienal

e

NP < 16 < 16 16 < 16 < 16 < 16 < 16 < 16 16

e

LP" 37.0 0.3 0.9 7.6

NP 5.7 0.0 0.0 3.6

nd' 8.3 1.0 1.6 2.4

n/ 0.4 0.2 0.1 0.2 c

" Numbers correspond to those in Figure 1. * Retention index on DB-WAX (60 m). Each value is the mean of three experiments. Light exposed milk: milk was stored under fluorescent light (15,000 lux) at 10 °C for 16 hr. Non-exposed milk: milk was stored in the dark at 10 °C for 16 hr/Not determined. d

e

Figure 1. Flavor dilution chromatograms obtained by an aroma extract dilution analysis of milk (a) stored under florescent light and (b) stored in the dark.



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to the non-exposed milk, the light-induced off-odors were well reproduced except for the "dusty" off-odor (Figure 2c). No significant difference was observed in the intensities of the "fatty," "metallic," and "overall off-odor" between the light-exposed milk and non-exposed milk with added off-odorants, whereas a significant difference was observed in the intensity of the "dusty" offodor between them (Figure 2a, c). These results indicate that unknown compounds can contribute to the "dusty" off-odor of the light-exposed milk. Effects of Antioxidants In order to inhibit light-induced off-flavor formation in milk, we tested the addition of five antioxidants, (-)-epicatechin, chlorogenic acid, α-tocopherol, Lascorbic acid, and Trolox C. Figure 3 shows the effects of these antioxidants on the formation of the off-odorants whose FD-factors were greater than 64 in the light-exposed milk (Table I). Among the tested antioxidants, (-)-epicatechin and chlorogenic acid showed the strongest inhibitory effects against most of the offodorants. These two antioxidants almost completely inhibited the formation of (£,£)-2,4-nonadienal, (£)-2-nonenal, and (£,£)-2,4-decadienal (Figure 3a-c). On the other hand, the inhibitory effects of (-)-epicatechin and chlorogenic acid on



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Figure 3. Effects of added antioxidants (100 mg/Kg) on the formation of (a) (E,E)-2,4nonadienal, (b) (E)-2-nonenal, (c) (E,E)-2,4-decadienal, (d) 1-octen-3-one, (e) methional during light exposure of milk. The error bar represents standard deviation (n = 3)



398 the formation of l-octen-3-one and methional were moderate, but were still equal to or stronger than the effects of the other antioxidants (Figure 3d, e). Both (-)-epicatechin and chlorogenic acid were reported to inhibit the formation of methional from methionine in an aqueous solution under riboflavin photo sensitized conditions (9). The results in this study demonstrate that (-)epicatechin and chlorogenic acid are also effective for inhibiting the formation of lipid-derived carbonyl compounds during the light-exposure of milk. The results of the sensory ranking tests according to the off-flavor intensities of the milk are shown in Figure 4, in which the higher ranking sums indicate the stronger off-flavor intensities. The addition of (-)-epicatechin followed by chlorogenic acid show the lowest ranking sum, indicating the weakest off-flavor intensity. Thus, the effectiveness of (-)-epicatechin and chlorogenic acid on the inhibition of the light-induced off-flavor was demonstrated in both the instrumental and sensory analyses.

Mechanisms of Antioxidant Actions A l l the tested antioxidants are well-known radical scavengers, which can inhibit lipid oxidation proceeding via a free-radical chain mechanism. This might explain the inhibitory effects of the tested antioxidants on the formation of the lipid-derived odorants such as (£,£)-2,4-nonadienal, (£)-2-nonenal, and (£,£)-2,4-decadienal (Figure 3a-c). However, all the tested antioxidants including (-)-epicatechin and chlorogenic acid could not fully inhibit the formation of l-octen-3-one and methional (Figure 3 d, e). It has been proposed that l-octen-3-one in oxidized lipids comes from l-octen-3-ol (/3), which is a unique product whose formation is clearly from the oxidation of linoleic acid with singlet oxygen (14). Regarding the formation of methional, a mechanism like Strecker degradation in which methionine as a free amino acid reacts with the excited triplet riboflavin has been proposed (75). Therefore, the moderate inhibitory effects of (-)-epicatechin and chlorogenic acid on the formation of 1octen-3-one and methional might be due to their lack of ability to quench both singlet oxygen and the excited triplet riboflavin.

Conclusions This study provides new information regarding light-induced off-flavor formation in milk and its prevention using antioxidants. The results from this study indicated that nine off-odorants, hexanal, (Z)-4-heptenal, l-octen-3-one, methional, (£)- and (2!)-2-nonenals, (£,Z)-2,6-nonadienal, (£,£)-2,4-nonadienal, and (£,£)-2,4-decadienal are the major contributors of light-induced off-flavor in milk. In addition, the use of antioxidants, especially (-)-epicatechin and chlorogenic acid, were found to effectively inhibit the formation of these offodorants. The added antioxidants might inhibit the riboflavin-sensitized photo-



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Figure 4. Sum of sensory ranking among milk exposed to light with and without added antioxidants. Samples were ranked in the increasing order of off-flavor intensities.

oxidation of lipids and proteins in three possible ways: (1) by scavenging free radicals, (2) by quenching singlet oxygen, and (3) by quenching the excited triplet riboflavin. Further studies are needed to clarify the antioxidant actions of the tested compounds.

References 1. 2. 3. 4. 5. 6. 7.

8.

Allen, C; Parks, O. W. J. Dairy Sci. 1975, 58, 1609-1611. Forss, D. A . J. Dairy Res. 1979, 46, 691-706. Marsili, R. In Techniques for Analyzing Food Aroma; Marsily, R., Ed.; Dekker: New York, 1997; pp. 237-264. Badings, H . T. In Volatile Compounds in Foods and Beverages; Maarse, H . , Ed.; Dekker: New York, 1991; pp 91-106. Wishner, L . Α.; Keeney, M. J. Dairy Sci. 1963, 46, 785-788. Bassette, R. J. Milk Food Technol. 1976, 39, 10-12. Cadwallader, K . R.; Howard, C. L . In Flavor Analysis-Developments in Isolation and Characterization; Mussinan, C. J., Morello, M. J., Eds.; A C S Symposium Series 705; American Chemical Society: Washington, D C , 1998; pp 343-358. Jung, M. Y.; Yoon, S. H . ; Lee, H . O.; M i n , D. B . J. Food Sci. 1998, 63, 408-412.


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Muranishi, S., Masuda, H . , Kiyohara, S., Ueno, T., Ho, C.-T. In Food Factors in Health Promotion and Disease Prevention; A C S Symposium Series 851; American Chemical Society: Washington, D C , 2003; pp. 400 409. Engel, W.; Bahr, W.; Schieberle P. Eur. Food Res. Technol. 1999, 209, 237-241. Grosch, W. Trends Food Sci. Technol. 1993, 4, 68-73. Meilgaard, M.; Civille, G.; Carr, Β. T. In Sensory Evaluation Techniques, 2nd ed.; C R C Press: Boca Raton, FL, 1991. Michalski, S. T; Hammond, E. G . J. Am. Oil Chem. Soc. 1972, 49, 563-566. Frankel, E. N.; Neff, W. E.; Selke, E. Lipids 1981, 16, 279-285. Dimick, P. S. Can. Inst. Food Sci. Technol. J. 1982, 15, 247-256.


Antioxidant Measurement and Applications - American Chemical Society

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