STABILIZATION OF MEAT LIPIDS WITH FLAVONOIDS AND FLAVONOID-RELATED COMPOUNDS

FEREIDOON SHAHIDI, ZHENG YANG and ZAN 0. SALEEMI

Department of Biochemistly Memorial University of Newfoundland

St. John’s, NF, Canada, A1B 3x9

Received for Publication December 16, 1992 Accepted for Publication February 10, 1993

ABSTRACT

The antioxidant activity of a number of jlavonoid and flavonoid-related phenolic acids was evaluated in meat model systems over a three-week storage period. The activity of compounds investigated depended on their chemical structure. Among the jlavonods tested in meat systems, morin, myricetin, kaempferol and quercetin at a level of 200ppm were most active leading to TBA (2-thiobarbituric acid) values of S 0.55 pgmalondialdehyde equivalentslg sample after a 21 -day storage at 4C. Among the phenolic compounds tested, ellagic acid followed by sesamol, tannic acid, eugenol and isoeugenol were most effective antioxidants. The activity of phenolic acids belonging to the hydroxy- benzoic and caffeic acid families was moderate with a 2449% inhibition of formation of TBA reactive substances.

INTRODUCTION

Oxidation of lipids is a common cause of quality deterioration and production of off-flavors and off-odors in meats (Dziezak 1986). Malondial- dehyde (MDA), a breakdown product of polyunsaturated fatty acids (PUFA), may be formed by either enzymatic or nonenzymatic pathways. The 2-thiobarbituric acid (TBA) test measures the concentration of MDA and other TBA reactive substances (TBARS) and is commonly used as an indicator of lipid oxidation in muscle foods (Shahidi and Hong 1991).

Journal of Food Lipids 1 (1993) 69-78. All Rights Reserved OCopyrighr I993 by Food & Numtion Press, Inc., Tnrrnbull, Connecticur. 69

70 F. SHAHIDI, Z YANG and ZO. SALEEMI

Numerous lipid oxidation products have been implicated as having mutagenic and perhaps carcinogenic, toxic and teratogenic effects in animals (Shamburger et al. 1974; Kubow 1990). Hence, lipid oxidation not only lowers the acceptability of muscle foods but it may also affect their nutritional value and wholesomeness.

Flavonoids and flavonoid-related compounds are found naturally in plants such as oilseed meals, which are often used as protein extenders in emulsified meat products. The human dietary consumption of these natural phenolics generally exceeds 1 g per day (Kuhnau 1976). The major portion of the antioxidant activity of oilseeds, oilseed flours and concentrates is attributable to flavonoids and hydroxylated cinnamic and benzoic acids. The combined influence of these phenolic compounds was found to account for nearly all of the antioxidant activity of soybean meal, soy flour and concen- trate (Arai ef al. 1966, Naim et al. 1973; Pratt and Birac 1979). Similar results were reported for cottonseed, cottonseed meal and peanuts (Rhee et al. 1979; Ziprin et al. 1981; Pratt and Miller 1984; Whittern et al. 1984).

Flavonoids, and phenolic compounds, in general, exhibit antioxidant effects by acting as free radical scavengers and possibly as chelators of metal ion catalysts (Pratt and Hudson 1990). Thus, they may affect the production of TBARS in meat systems and preserve the flavor and sensory quality of muscle foods.

Due to the lack of any detailed reports on antioxidant properties of individual flavonoids and natural phenolic compoundsof plant origin in meat systems, the present study was carried out. Inhibition of " B A R S production in cooked meats due to natural phenolics, including llavonoids, as well as phenolic acids belonging to p-hydroxybenzoicand coumaric acid families was also investigated.

MATERIALS AND METHODS

Materials

Ail flavonoids and phenolic compounds as well as other antioxidants used in this study were purchased from either Sigma (St. Louis, MO) or Aldrich (Milwaukee, WI) Chemical Companies. Lean pork loins were obtained from the Newfoundland Farm Products Corporation (St. John's, NF) and were then ground twice using an Oster meat grinder, Model 99068 (4.8 mm plate).

Sample Preparation and Methods

Phenolic compoundswere added to ground pork slurries containing20%

STABILIZATION OF MEAT LIPIDS WITH FLAVONOIDS 71

FLAVONOLS FLAVANONES

Kaempfcrol : 3,5,7,4'-taraOH Naringenin : 5.7.4'iriOH

Morin : 3.5.7,2',4'pentaOH Naringin : 7-rhamoglueose,

Quercetin : 3,5,1,3',4'-pntaOH 5,4'diOH

Myricetin : 3,5,7.3'.4'.5'-hurOH

Rutin : 3Qmtiwsc, 5.7.3 '.4'-tecnOH

3

HO,@ 1 0

BENZOlC ACIDS

Gallic acid : 3.4.5biOH

Vlnillic acid : 44H. 3-OMe

Syringic acid : 4 4 H . 3.5diOMc

Ellagic acid

Tanoic acid

: Dimer of gdlic acid

: Ester of glumW with 3 ta 5 gnllic acids

Eugmol

HO

0

CINNAMIC ACIDS

Coumuic acid : 4-OH

Caffeic acid : 3,SdiOH

Faulic acid : W H , 3-OMe

S i i c acid

chlorogenic acid : 3.cdiOH. ester

: 4 4 H , 3, SdiOME

with C, of quinic acid

m - 1 Servllol

0 0

CH3O 0 \

OH I

HO ' curatmia

FIG. 1. CHEMICAL. STRUCTURES OF SOME PHENOLIC COMPOUNDS

72 F. SHAHIDI, Z YANG and 20. SALEEMI

added distilled water at 30 or 200 ppm (200 ppm is equivalent to approxi- mately 0.6-0.7 mmoVkg flavonols and 0.33 mmoVkg flavanones) levels in an aqueous or 50% ethanolic solution prior to heat processing. Ascorbic acid was added at 500 ppm. Meat systems were cooked in plastic bags in a thermostated water bath to reach an internal temperature of 75 f 1C. The cooked meat samples were kept in Nasco Whirl-Pak bags at 4C until used.

The content of TBARS in meat samples was quantified after their distillation in acidified water (97.5 ml water and 2.5 m14 N HCl) using the procedure of Tarladgis et aZ. (1960) as modified by Shahidiet al. (1987). The TBARS content, reported as pg MDA equivalents& sample, was calculated by multiplying the absorbance readings of the TBARS-TBA chromogen at 532 nm by a factor of 8.1. The latter factor was deduced using 1,1,3,3-tetra- methoxypropane as a standard (Shahidi et al. 1987). The percent inhibition of TBARS formation due to the additives tested was calculated as lOO[l-(TBARS content of meat with additive/TBARS content of meat without additive)].

RESULTS AND DISCUSSION

Table 1 summarizes the content of TBARS of treated cooked meat systems as affected by selected flavonoids (flavonols and flavanones) over a 3-week storage period at 4C. In all cases, the antioxidant efficacy of flavonoids tested was superior at the higher concentration. Furthermore, flavonols were more powerful antioxidants in meat systems than flavanones at equal concentrations. Prolonging the storage period generally resulted in meat flavor deterioration (MFD) as higher concentrations of TBARS were produced.

A close scrutiny of the results in Table 1 leads to the deduction of the following trends with respect to structure-activity of flavonoids examined in this study.

(1) Presence of a G-C, olefinic bond and a free hydroxyl group at the C, position of ring C is essential for the antioxidant activicy of flavonoids. Therefore, naringin and naringenin, both flavonones devoid of a Q-C, olefinic bond and a hydroxyl group at the C3 position had no antioxidant effect.

(2) Presence of a 5,7dihydroxy grouping on the A ring had little effect on the activity of flavonoids as antioxidants. Thus, naringin with obscured 7-hydroxy group on ring A due to its etherification with a rhamnoglucose had similar activity to naringenin which possesses a free OH group in position 7.

STABILIZATION OF MEAT LIPIDS WITH FLAVONOIDS 73

TABLE 1. TBARS CONTENT (pg MDA EQUIVALENTS/$ SAMPLE) OF FLAVONOID- TREATED COOKED GROUND PORK DURING A 3-WEEK STORAGE AT 4 C

Flavonoid @pm)

Control

Kaempferol (30)

Kaempferol (200)

Morin (30)

Morin (200)

Myricetin (200)

Naringenin (30)

Naringenin (200)

Naringin (30)

Naringin (200)

Quercetin (30)

Quercetin (200)

Rutin (30)

Rutin (200)

Storaae Period. Davs

1

5.95k0.09

3.62k0.12

0.10~0.02

3.77k0.09

0.11 k0.02

0.12*0.02

6.00k0.25

5.4250.18

4.85kO. 10

4.37k0.18

0.22k0.02

0.10k0.M

4.10k0.42

3.8050.22

7 I 14

9.64k0.12 11.59k0.10

4.20k0.10 8.02k0.21

0.20+0.05 0.76+0.10

7.42k0.10 9.99k0.12

0.26k0.02 0.47k0.05

0.11*0.02 0.12*0.01

8.85k0.20 11.68kO. 11

8.2950.20 11.50+0.18

9.99k0.30 11.40_+0.12

9.32k0.10 11.08kO. 12

0.38k0.02 0.42+0.05

0.16&0.01 0.17k0.02

6.30k0.42 7.92k0.38

5.82k0.21 7.00+0.20

21

11.79k0.20

7.72k0.30

0.55f0.12

6.80k0.20

0.42+0.02

0.15 k0.02

11.30k0.14

11.40+0.20

11.52k0.20

12.34 k0.20

0.48k0.05

0.13k0.05

10.20~0.11

7.90k0.32

(3) Presence of ortho-dihydroxylation at the 3 ’ ,4 ‘ -configurationon ring B enhances the antioxidant activity of flavonoids as compared with those possessing a metadihydroxylation arrangement. Therefore, quercetin was found to possess a stronger antioxidant activity than morin.

(4) The antioxidant activity of flavonoids was somewhat influenced by the number of hydroxyl groups present on ring B. Thus, myricetin with 3 ‘ ,4 ’ ,5 ’ -trihydroxylation had a higher antioxidant activity than quercetin and morin which were still more active than kaempferol.

(5) Etherification of the 3-hydroxyl group in ring C drastically reduced the antioxidant effect of flavonoids. Therefore, rutin with obscured -OH group by etherification with a rutinose moiety had a much lower antioxidant activity than quercetin.

74 F. SHAHIDI, Z YANG and ZO. SALEEMI

While flavonoids act as primary antioxidants by a free-radical scavenging mechanism (Shahidi and Wanasundara 1992), presence of ortho hydroxyl groups in the B ring leads to facile formation of a stable orthoquinone structure. Furthermore, ortho hydroxylation on the B ring as well as presence of a ketol group on the C ring potentially allows chelation of prooxidants such as metal ions in meats. Extensive hydroxylation accentuates the latter effect. The relationship between flavone structures and their antioxidant activity has been welldocumented (Dziedzic and Hudson 1983; Das and Pereira 1990, Shahidiand Wanasundara 1992). The present results, for meat systems, lend further support to those findings.

Of the other phenolic compounds tested (Table 2), ellagic acid, a dimer of gallic acid, was the most effective phenolic substance tested. At both 30 and 200 ppm levels of addition to meat, it inhibited lipid oxidation as the TBARS concentrations over a 3-week storage period remained constant at about 0.1 pg MDA equivalents/g sample. In this respect, ellagic acid was a superior antioxidant compared to most synthetic antioxidants commonly used in food processing. In view of its desirable pharmacological effects and its antimutagenic and anticarcinogenic properties (Castonguay et al. 1990), use of ellagic acid in formulated muscle foods and for preserving meat flavor quality may have applied significance. Antioxygenic action of tannic acid, eugenol, isoeugenol and sesamol (Tables 2 and 3) was very strong at a 200 ppm level of addition and was equivalent or superior to that of BHT. These phenolic antioxidants are naturally present in plant materials, some of which, like soy products, may be used as protein extenders in emulsified meat products. The effectiveness of these latter compounds at a 30 ppm level of addition was not sufficient to control MFD.

The effectiveness of other phenolic acids belonging to the family of p-hydroxybenzoic and cinnamic acids, generally found in oilseed meals and flours, varied between 24 and 69% with respect to inhibition of TBARS formation (Table 3). The efficiency of the phenolic acids in retarding lipid oxidation followed the trend given below.

Tannic > Gallic L- Caffeic > Coumaric L- Ferulic z Sinapic Syringic > Chlorogenic = Vanillic

Thus, the activity of phenolic acids in retarding lipid oxidation is primarily due to the number of free hydroxyl groups in their molecules. Gallic acid (also present in tannic acid) with three hydroxyl groups and caffeic acid with two OH groups were most effective. Effectiveness of coumaric, ferulic, sinapic and syringic acids with one free hydroxyl group was similar and somewhat less than those with two or three hydroxyl groups. Vanillic and chlorogenic acids were least effective. The lower activity of chlorogenic acid

STABILIZATION OF MEAT LIPIDS WITH FLAVONOIDS 75

TBARS CONTENT 01 PORK '

Antioxidant @pm)

Control

a-Tocopherol (30)

a-Tocopherol (200)

L-Ascorbic Acid (500)

BHT (30)

BHT (200)

Ellagic acid (30)

Ellagic acid (200)

Eugenol (30)

Eugenol (200)

Isoeugenol (30)

Isoeugenol (200)

Sesamol (30)

Sesamol (200)

Curcumin (30)

Curcumin (200)

TABLE 2. MDA EQUIVALENTS/g SAMPLE) OF COOKED GROUND EATED WITH SELECTED ANTIOXIDANTS

1

2.69 f 0.04

0.78 f 0.22

0.36 f 0.01

0.63 f 0.05

1.33 f 0.05

0.45 f 0.05

0.10 f 0.02

0.08 f 0.02

1.43 f 0.05

0.26 f 0.05

0.72 f 0.08

0.06 f 0.01

0.09 f 0.02

0.07 f 0.01

4.50 f 0.12

2.03 f 0.23

Storage I

7

7.41 f 0.24

4.58 f 0.11

3.55 f 0.10

2.95 f 0.12

4.24 f 0.14

0.92 f 0.12

0.10 f 0.01

0.09 & 0.01

1.51 f 0.04

0.38 f 0.02

0.65 & 0.10

0.08 f 0.01

2.97 & 0.05

0.07 f 0.01

8.31 f 0.18

4.54 f 0.12

nod, Days

14

9.85 f 0.03

6.46 f 0.05

4.18 5 0.12

5.39 f 0.25

7.88 5 0.32

1.33 f 0.38

0.11 f 0.01

0.09 f 0.03

3.04 f 0.08

0.34 5 0.02

2.20 5 0.05

0.11 f 0.02

3.64 & 0.08

0.09 i- 0.02

9.09 f 0.15

4.84 f 0.10

21

9.68 f 0.56

8.31 5 0.08

5.01 + 0.18

5.81 0.10

7.80 & 0.54

0.99 f 0.15

0.12 & 0.02

0.10 f 0.02

4.39 * 0.10

0.37 0.03

2.69 f 0.04

0.12 + 0.01

3.47 f 0.05

0.09 f 0.02

9.31 0.32

6.08 + 0.15

is due to the fact that its sugar moiety is neutral and thus its effective concentration, as caffeic acid equivalents, is only about 50% of that of other phenolics tested. Activity of phenolic acids extracted from canola meal has recently been reported in a B-carotene-linoleate model system (Zadernowski et al. 1991).

It has been reported that cooked beef possessing a TBA value of larger than 1 may not be sensorially fit for consumption. Therefore, natural antioxidants should give TBARS content of less than 1 pg MDA equiva- lents/g sample over their intended period of storage prior to consumption.

76 F. SHAHIDI, Z YANG and ZO. S U E M I

TABLE 3. TEiARS CONTENT (pg MDA EQWALENTSlg SAMPLE) OF COOKED

ROUND PORK TREATED WITH NATURAL PHENOLIC ACIDS 7

Phenolic acid I @Pm)

Control

Gallic (30)

Gallic (200)

Caffeic (30)

Caffeic (200)

Coumaric (30)

Coumaric (200)

Ferulic (30)

Ferulic (200)

Syringic (30)

Syringic (200)

Vanillic (30)

Vanillic (200)

Sinapic (30)

Sinapic (200)

Chlorogenic (30)

Chlorogenic (200) Tannic (30)

Tannic (200)

Storage Period. Days I

1 I 7

3.12 f 0.28

1.58 f 0.08

0.83 f 0.05

0.92 f 0.10

0.90 f 0.09

2.50 f 0.06

2.38 f 0.05

1.60 f 0.12

1.17 f 0.09

1.71 f 0.05

1.50 f 0.03

2.62 f 0.14

1.97 f 0.15

2.64 f 0.32

1.65 f 0.18 3.02 f 0.10 - 1.12 f 0.16

1.07 f 0.05

0.07 f 0.01

5.35 f 0.20

2.52 f 0.12

1.21 f 0.101

2.84 f 0.11

1.61 f 0.21

3.23 f 0.05

2.90 f 0.12

4.88 f 0.23

2.97 f 0.12

2.62 f 0.02

2.09 f 0.09

3.93 f 0.21

4.01 f 0.09

3.35 f 0.12

2.46 f 0.15

1.73 f 0.21

3.39 f 0.08

0.08 f 0.01

. -

14

6.58 f 0.18

5.03 f 0.05

2.12 f 0.08

3.61 f 0.08

2.12 f 0.18

4.70 f 0.10

4.47 f 0.07

4.50 f 0.18

4.18 f 0.12

4.86 f 0.05 3.28 f 0.22

5.26 f 0.10

4.78 f 0.18

4.54 f 0.11

3.83 f 0.10 -

3.64 f 0.10

3.73 f 0.05

0.15 f 0.02 -

21

9.11 f 0.33

4.78 f 0.41

2.41 f 0.05

3.25 f 0.12

2.80 f 0.10

4.24 f 0.20

4.16 f 0.18

4.88 f 0.05

3.95 f 0.18

5.90 f 0.10

4.07 f 0.16

7.08 f 0.32

6.96 f 0.25

6.84 f 0.28

5.60 f 0.14

6.60 f 0.32

5.92 f 0.28

5.27 f 0.12

0.10 f 0.03

Nonetheless, in view of the sensitivity of consumers to synthetic additives, utilization of novel antioxidants from natural sources is desirable. Thus, the strong an tioxidant activity of flavonoids, flavonoid-related compounds and phenolic acids, and their natural occurrence in many plant protein extenders may offer practical application for their use in processed meats. Addition of plant protein extenders, spices, herbs and vegetables in formulated muscle foods, in addition to conveying their intended effects, might prove beneficial in flavor retention and prevention of flavor quality deterioration of meats.

STABILIZATION OF MEAT LIPIDS WITH FLAVONOIDS 17

ACKNOWLEDGMENT

We are grateful to the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support.

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