Chemical and Sensory Evaluation of Flavor in Untreated and

Chapter 9

Chemical and Sensory Evaluation of Flavor in Untreated and Antioxidant-Treated Meat Allen J. St. Angelo, Arthur M. Spanier, and Karen L. Bett Agricultural Research Service, U.S. Department of Agriculture, Southern Regional Research Center, 1100 Robert E. Lee Boulevard, New Orleans, LA 70124

Meat flavor deterioration primarily develops from the oxidation of polyunsaturated fatty acids catalyzed by metal ions and other free radical generating systems. As off-flavors intensify with storage, the desirable meaty flavor notes decrease. Experiments were designed to examine mechanisms that prevent theseflavorchanges. Ground beef patties, freshly cooked and cooked/stored for up to 5 days at 4°C, were examined by direct gas chromatography and chemical and sensory methods of analysis. Results demonstrated that combined vacuum packaging and use of chelators and antioxidants act synergistically to prevent lipid oxidation and to preserve desirable meat flavor.

In reports on the mechanism responsible for warmed-over flavor ( W O F ) i n cooked meat (also referred to as meat flavor deterioration, or M F D ) , the generally accepted theory is that M F D is caused by the oxidation of membrane phospholipids, catalyzed by some ionic form of iron (1, 2). When lipids oxidize, as i n the case of M F D , a mixture of aldehydes, ketones and alcohols are produced. Their concentrations increase with storage. Appreciable amounts of hexanal, one of the primary oxidation products originating from linoleic acid (3), can be observed by G C within hours after storage (4). Many other oxidation products, such as propanal, butanal, pentanal, heptanal, 2,3-octanedione, and nonanal, were readily measured upon analyses of M F D roasts (4, 5). In addition to the increase in lipid oxidation products, which generate offflavor notes such as painty ( P T Y ) and cardboard (CBD), a loss of desirable beefy flavor notes, such as cooked beef/brothy (CBB) occurs (6-8). Sulfurcontaining compounds that also contribute to the desirable meaty flavor will also

This chapter not subject to U.S. copyright Published 1992 American Chemical Society

9. ST. ANGELO ET AL.

Flavor in Untreated and Antioxidant' Treated Meat

141

change (9). Thus, M F D appears to result from a combined chemical process whereby lipid oxidation products are evolved and desirable meaty flavor compounds not of lipid origin are diminished. According to L i u et al. (10), lipid oxidation products are found in concentrations of ppm, whereas the desirable meaty compounds are present at ppb levels. Volatile heteroatomic meat flavor principles were also confirmed at ppb levels by Vercellotti et al. (11). Several mechanisms cause food flavors to deteriorate. Owing to the abundance of lipid oxidation products formed during the development of M F D , lipid oxidation is the most frequently cited mechanism. Over the past three decades, chelators have been used successfully to retard or inhibit iron-catalyzed lipid oxidation. However, several of these chelators do not prevent the loss of the desirable beefy flavor, C B B . O n the other hand, antioxidants that function as free radical scavengers were not only successful in inhibiting W O F formation, but also appeared to retain C B B at a higher level than the chelators (12). Obviously, free radical mechanisms not only effect lipid oxidation, but are also involved in the oxidation of compounds other than lipids, such as proteins (13). Other factors that contribute to M F D are final cooking temperature, length of refrigerated storage, and the availability of oxygen (7,14,15). Using combined instrumental, chemical and descriptive sensory methodologies, the M F D process was shown to be inhibited or retarded by the use of antioxidants (9) or the exclusion of oxygen (75). More recently, results from studies on combined use of chelators and free radical scavengers as antioxidants have shown a significant amount of flavor loss when the antioxidant-treated cooked meat was maintained under vacuum (16). Based on the results from the doseresponse combinations obtained in those initial studies, the objectives of this paper were to determine the correlation between chemical and flavor attributes and to investigate the synergism of antioxidants and chelators in beef stored both in the presence and absence of oxygen. Experimental Procedures Sample Preparation. Samples consisted of USDA-Choice, top round steaks (semimembranosus muscle). The meat was trimmed of separable fat and then ground by two passes through each of two grinding discs with holes of 1.0 and 0.75cm diameter. Fat content ranged from 4-5%. Glass petri dishes (100mm x 20mm) were used as molds to form 85 grams of beef into patties of uniform shape. A 425g portion of the ground meat was used to make five 85 gram patties as standards. These reference standards, placed in covered petri dishes, were stored in a freezer immediately after preparation. The remaining ground meat was divided into 850g portions to make ten 85 gram patties per replicate per treatment. Each portion was mixed with either 10 ml of the additive in water or water only since water served as the additive carrier. The additives used were propyl gallate (PG) and the tetrasodium salt of ethylenediaminetetraacetic acid ( E D T A ) . The additives were presented at 50 ppm for P G and 100 ppm for E D T A , based on the wet weight of raw ground meat. Experimental samples were allowed to stand (marinate) overnight, ca. 18 hrs, at 4 ° C. A l l patties were cooked on a Farberware grill (Model 455ND) for 7

142

LIPID OXIDATION IN FOOD

min/side. The internal temperature of the patties was 65 ± 1°C. After the "marinated" experimental samples were cooked, they were placed in covered petri dishes, which were then placed in vacuum desiccator jars and stored in a refrigerator for a period of 3 days. Simulated vacuum packaging was accomplished by purging the desiccator jars twice with nitrogen. Final vacuum was less than 4 mm Hg. Samples stored without vacuum were placed in vacuum jars that were left open to the atmosphere. O n the morning of the experiment, the cooked/refrigerated samples were rewarmed for 25 minutes at 121 °C (250 °F). Frozen samples were thawed and then cooked on the grill. A l l samples were placed in warming trays (Hobart, Model CF021) at 52 °C until evaluated by the panel. Samples to be analyzed by chemical and instrumental methods were placed in the freezer until assayed. The experiment was statistically designed as a balanced incomplete block. The blind standard was given the notation "BNO" to represent the "standard" (B) with "no vacuum" (N) and "no chelator or antioxidant" (O). The experimental groups or "treatments" were given the following notation: (1) "ENO" represented the samples in which the flavor had been allowed to deteriorate for 3 days with "no vacuum"; this sample represented the M F D sample. (2) " E V O " represented the M F D sample maintained under "vacuum" (V). (3) " E N A , E N B , or E N X " represented the M F D samples containing the "additives" (where A = Propyl Gallate, B = E D T A , and X = both P G & E D T A ) . (4) " E V A , E V B , or E V X " represented the M F D samples maintained with both "vacuum and additives". Samples that were used in the 5-day correlation (chemical and flavor attributes) study, were prepared as described above, except that cooked patties were stored in covered petri dishes at 4 ° C until assayed. They contained no additive other than water and they were not stored under vacuum. Sensory Analysis. Sensory profiling of the patties was performed by the method described by Meilgaard et al. (17). Evaluations were accomplished by a trained panel of 12 using the spectrum universal intensity scale, 0-15, as described by Meilgaard et al. (17). Sensory attributes included the following descriptors as originally described by Johnsen and Civille (6) and modified by Love (8): "salty", "cooked beef/brothy", "painty", "serum/raw," "browned/caramel", "cooked liver", "cardboard", "sour", "sweet", and "bitter". Chemical Analysis. Chemical attributes such as thiobarbituric acid reactive substances (TBARS) were measured by the distillation method of Tarladgis (18), and were reported as mg malonaldehyde per kg sample. A standard curve was prepared with 1,1,3,3-tetraethoxypropane. Instrumental Methodology. Instrumental attributes were determined by obtaining the volatile profile of meat samples utilizing the direct gas chromatographic (GC) method described by Dupuy et al. (4). This method separates the volatiles by G C after applying the sample into an external closed inlet device interfaced with a packed Tenax G C / P M P E column.


Flavor in Untreated and Antioxidant-Treated Meat

143

Statistical Analysis. Results were statistically analyzed using SAS (19) for both analysis of variance ( A N O V A ) and principal components analysis (PCA). P C A is a multivariate statistical method that is used when an empirical summary of patterns of inter-correlations among variables is desired. The purpose of P C A is to reduce a large number of variables to as small a number of factors as possible. In the present work, the variables were the experimental treatments and the sensory, chemical and instrumental attributes. Results Correlation of Chemical and Flavor Attributes. Cooked ground meat that was stored for 5 days was examined for hexanal content by direct G C and for desirable and off-flavor attributes by a trained sensory panel. Hexanal production and the off-flavor aromatic descriptor called painty (PTY) increased with storage (Figure 1). As noted, there was a direct correlation between the two. Other off-flavors that increased with M F D were cardboard (CBD), bitter (BTR) and sour (SOU) (data not shown). As M F D progressed during storage, the desirable flavor notes, such as cooked beef/brothy (CBB), decreased. To date, no compound has been directly correlated to off-flavors other than P T Y or to the desirable flavors as hexanal has been to P T Y . Synergism of Antioxidants and Chelators. Concomitantly with the production of lipid oxidation products, proteins and peptides were shown to decompose via a free-radical mechanism (20). Consequently, the two compounds chosen to serve as antioxidants in these experiments were E D T A , which functions primarily as a strong chelator, and P G , which can chelate iron, but functions primarily as a free radical scavenger. Hopefully, E D T A , being the stronger chelator, would bind the metal and thereby allow the P G to react with the free radicals. The action of these two compounds coupled with oxygen exclusion (via vacuum) would create an atmosphere that would inhibit or retard M F D . Chemical and Instrumental Attributes. The effect of vacuum and additives on chemical (TBARS) and instrumental (hexanal, 2,3-octanedione and nonanal) attributes of beef is shown in Figures 2 and 3. A l l bars were presented as the mean ( ± s.e.m.) result of data obtained from A N O V A with appropriate linear contrasts tested. Each graph was organized into 3 clusters with each bar representing a different treatment. The first cluster represents the blindstandard and the 3 day M F D beef pattie. The second cluster represents the effect of the different additives, i.e., A = propyl gallate, B = E D T A , X = the two additives combined. The third cluster was similar to the second except that all samples were maintained under vacuum so that the bars from left to right represented "vacuum only", "vacuum + PG", "vacuum + E D T A " and "vacuum + both P G and E D T A " . In Figure 2, the upper graph shows the relative change in the level of T B A R S whereas the bottom graph and those in Figure 3 describe the G C area count of three selected products of lipid oxidation, i . e., hexanal, 2,3octanedione, and nonanal. The GC-volatiles and the T B A R S data show

144


Refrigerated Storage

(Days; 4 ° C )

Figure 1. Cooked beef flavor changes during storage.


TBARS


(ppm) TBA (ppm) ^

s.e.m.

HH

Mean

•

I BNO

I ENO

I

I ENA

I ENB

•

I ENX

I

1 111

1 EVO

I EVA

I EVB

I EVX

HEXANAL (Area Counts) 300 -A

I BNO

I ENO

1

I ENA

I ENB

I ENX

1

I EVO

I

I

I

EVA

EVB

EVX

Figure 2. Effect of vacuum and additives on beef as measured by chemical (TBARS) and instrumental (hexanal) methods. Treatment codes are BNO, blind standard, no storage, no vacuum (N), and no additives (O); E N O , experimental M F D sample with no vacuum and no additives; E N A , ENB, and E N X , experimental samples with no vacuum and either 50 ppm P G (A), 100 ppm E D T A (B), or both (X) added; E V O , experimental samples with vacuum and no additives; E V A , E V B , and E V X , experimental samples with vacuum and either A , B, or X added.

145

146


2,3 Octanedione (Area Counts)

BNO

ENO

ENA

ENB

ENX

EVO

EVA

EVB

EVX

EVO

EVA

EVB

EVX

NONANAL (Area Counts)

BNO

ENO

ENA

ENB

ENX

Figure 3. Effect of vacuum and additives on beef as measured by instrumental methods. Treatment codes are same as described in Figure 2.



response to each treatment that is similar to one another. For example, in each instance the 3-day M F D sample ( E N O ) showed a significant increase in the intensity of the attribute measured when compared to the blind standard (BNO). Vacuum alone ( E V O ) showed a lowering effect of the intensity of each attribute when compared to the M F D samples (ENO). In general, each additive had its own individual response, with the antioxidant, P G , being more efficient in decreasing the intensity of the chemical and instrumental attributes (compare treatments E N A to E N B ) . When vacuum was not used, combining P G with E D T A ( E N X ) reduced hexanal and 2,3-octanedione production, but not as much as P G alone ( E N A ) . The decrease in T B A R S and nonanal production were comparable. When E D T A was combined with vacuum ( E V B ) , there was a significant reduction of lipid oxidation when compared to that without vacuum (ENB). When P G was combined with vacuum ( E V A ) or with E D T A plus vacuum ( E V X ) , lipid oxidation was reduced (lower than the blind standard that had been stored frozen, BNO), and the difference was statistically significant when compared to E N A . Combining additives with vacuum, resulted in a synergistic response, which suggested that more than one type of free radical chemistry may be occurring during M F D . Furthermore, these data showed that M F D definitely involves more complex chemistry than mere lipid oxidation. Off-Flavor Attributes. The effect of vacuum and additives on off-flavor attributes is shown in Figures 4 and 5. A l l four graphs illustrate the intensities of off-flavor descriptors, i.e., painty, cardboard, bitter and sour, representing two aromas, Figure 4, and two tastes, Figure 5, respectively. A l l bars are presented as the mean results of data from treatments as described in Figures 2 and 3. The 3-day M F D sample (ENO) showed a significant increase in each of the four off-flavor attributes when compared to the blind standard (BNO). The response of all four descriptors to the different additives is seen in the second cluster. These data indicate that all four descriptors responded positively to the 50 ppm concentration of the antioxidant, P G (samples E N A ) . O n the other hand, only two of the descriptors, i.e. painty and sour, showed a response to the 100 ppm of the chelator, E D T A (ENB). However, similarly shown by the chemical data in Figures 2 and 3, combining E D T A with vacuum ( E V B ) caused a synergistic effect for all sensory notes (compare E V B to E N B and E V O ) . There also was a synergistic sensory response to a mixture of P G with the chelator ( E N X ) . In this case, the perception of the flavor intensities approached the intensity levels of the standard patties (BNO). Again, these data showed the presence of more than one mechanism involved in M F D . Samples treated with vacuum alone ( E V O ) had the effect of reducing the intensity of all four offflavor descriptors of the cooked patties at levels lower than without vacuum (ENO), but higher than the standard patties (BNO). Combining the vacuum treatment with the additives indicated a synergistic response to the additives as seen by data for all four descriptors in the second cluster. Desirable Flavor Attributes. The effect of vacuum on the desirable onflavor attributes of beef patties is shown in Figure 6. This figure is similar to Figures 4 and 5 with the exception that it pertains to the desirable flavor

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147

148


CARDBOARD

BNO

ENO

ENA

ENB

ENX

EVO

EVA

EVB

EVX

EVO

EVA

EVB

EVX

TREATMENT

PAINTY

BNO

ENO

ENA

ENB

ENX

TREATMENT

Figure 4. Effect of vacuum and additives on beef as measured by off-flavor aromas. Treatment codes are same as described in Figure 2.



SOUR 0.9 0.75 0.6 0.45 0.3 0.15 o

| BNO

|" ENO

ENA

ENB

ENX

EVO

EVA

EVB

EVX

TREATMENT

BITTER 0.8 -,

0.6 -

0.4

0.2

BNO

ENO

ENA

ENB

ENX

EVO

EVA

EVB

EVX

TREATMENT

Figure 5. Effect of vacuum and additives on beef as measured by off-flavor tastes. Treatment codes are same as described in Figure 2.

149

150


COOKED B E E F / B R O T H Y

i

i

BNO

ENO

—

i

—

i

ENA

i ENB

i —

i

ENX

—i EVO

i

i

i

EVA

EVB

EVX

TREATMENT

BROWNED/CARAMEL

BNO

ENO

ENA

ENB

ENX

EVO

EVA

EVB

EVX

TREATMENT

Figure 6. Effect of vacuum and additives on beef as measured by on-flavor aromas. Treatment codes are same as described in Figure 2.



151

attributes, cooked beef/brothy and browned/caramel (BRC). As shown by the 3-day M F D sample (ENO), the two desirable attributes decreased compared to those of the blind standard. The sample treated with no vacuum but with P G ( E N A ) retained the high intensity of C B B during storage for 3 days. E D T A alone did not have an effect during storage, as seen from sample E N B . Again, samples treated with E D T A plus vacuum ( E V B ) showed a synergistic effect. Based on the retention of high intensity values of the desirable notes, C B B and B R C , and coupled with the rentention of low intensity values for the off-flavor notes ( C B D , P T Y ) , the most successful treatment was observed in patties treated with vacuum plus both compounds, P G and E D T A , as seen in sample E V X . This point was also confirmed in Table I, which expressed significant differences between means of E V X and E N O for all flavor descriptors. C B D , P T Y , S O U and B T R were significantly lower in E V X than in E N O . C B B and B R C were significantly higher in E V X than E N O . E N X and E V B treated samples had significantly different means from E N O for all 6 discriptors also. Although C B B and B R C were not as intense in the E N X and E V B samples as in the E V X samples, P T Y and C B D were higher in intensity for the E N X and E V B samples than for the E V X samples. Statistical Evaluation by Pearson Correlation Coefficients Sensory Data. Statistical analysis of the raw sample sensory data, including 290 - 323 separate observations, revealed several significant correlations among the sensory flavor descriptors examined (Table II). Values that were greater than 0.5 were considered "very" highly correlated, while values greater than 0.3 but less than 0.5 were considered "highly" correlated. Values greater than 0.25 but less than 0.3 were correlated to a lesser degree, but were still highly significant. In all cases, the level of significance is 0.0001, which indicated statistically that these correlations were highly significant. For example, in the column listed as C B B , painty, cardboard, sour and bitter were negatively correlated with cooked beef/brothy, whereas browned/caramel and sweet had a strong positive correlation with cooked beef/brothy. In all cases, those descriptors that were negatively correlated to one descriptor, were positively correlated with each other. For example, the off-flavor descriptors, cardboard and painty, were negatively correlated to the desirable-flavor descriptor, cooked beef/brothy. Yet, these descriptors yielded the highest positive statistical correlation between themselves with a correlation of 0.80. Chemical and Instrumental Data. Statistical analysis of chemical and instrumental attributes, including 56 separate observations, also revealed several significant correlations among the attributes examined (Table III). The intensity of these attributes increased with storage and the onset of M F D . Therefore, as the analysis indicated, they were positively correlated to one another, and the correlations were highly significant, i . e., a significance level of less than 0.0001. The correlation between T B A R S and 2,3-octanedione had the highest positive correlation, 0.90. The second highest was between T B A R S and hexanal, 0.79. The lowest was between hexanal and nonanal, 0.59.

D D

b

b

b

b

6

b

b

b

b

b

b

PTY

b

D

6

b

b

b

b

0.0130 0.0156 0.0557 0.0035 0.1106 0.0071 0.0056 0.0194

SOU D

b

b

b

b

0.0029 0.0927 0.2985 0.0118 0.1835 0.0177 0.0091 0.0165

BTR

0

D

b

b

b

b

0.0001 0.0645 0.8619 0.0015 0.0590 0.0070 0.0029 0.0006

CBB

(P -values) o f Sensory D e s c r i p t o r s

0.0001 0.0001 0.0437 0.0001 0.0013 0.0001 0.0001 0.0001

8

b

b

b

b

0.0001 0.0909 0.4638 0.0307 0.0698 0.1025 0.0062 0.0021

BRC

d

C

Degrees o f freedom = 1. ''Means were s i g n i f i c a n t l y d i f f e r e n t a t a p r o b a b i l i t y o f 0.05. CBD, cardboard; PTY, p a i n t y ; SOU, sour; BTR, b i t t e r ; CBB, cooked beef/brothy; BRC, browned/caramel. ENO, experimental, no vacuum, no a d d i t i v e s ; BNO, b l i n d standard, no vacuum, no a d d i t i v e s ; ENA, experimental, no vacuum, p l u s PG; ENB, experimental, no vacuum, p l u s EDTA; ENX, experimental, no vacuum, p l u s PG and EDTA; EVO, experimental, p l u s vacuum, no a d d i t i v e s ; EVA, experimental, p l u s vacuum and PG; EVB, experimental, p l u s vacuum and EDTA; EVX, experimental, p l u s vacuum, PG and EDTA.

a

0.0001 0.0019 0.2198 0.0001 0.0053 0.0001 0.0002 0.0001

BNO ENA ENB ENX EVO EVA EVB EVX

6

CBD

Treatment C o n t r a s t s

Treatment (ENO vs)

Table I .

O

3

2

1

0

ST. ANGELO ET AL.


in

H 0 1

co

in VO VO CN co VO

•

o

•

H o o

CO VO in

o

rsj CN co eg CO

Q U

•

•

•

H o o o

VO vo 00 CN

00 o\ • co vo co in co in CN in co H o co in o in in co co ^

o o o o o o II

II

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154

LIPID OXIDATION IN FOOD 3

5

Table III. Pearson Correlation Coefficients of Chemical Attributes

TBARS HEXANAL 2,3-OCT NONANAL a

b

TBARS

HEXANAL

1 0.7854 0.8977 0.6848

1 0.7438 0.5873

2,3-OCT

1 0.7604

NONANAL

1

Values >0.5 are very highly significant, >0.3 but 0.25 but

Chemical and Sensory Evaluation of Flavor in Untreated and

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