The pre- and post-grinding application of rosemary and its effects on lipid oxidation and color during storage of ground beef

Meat Science 73 (2006) 413–421 www.elsevier.com/locate/meatsci

The pre- and post-grinding application of rosemary and its eVects on lipid oxidation and color during storage of ground beef C.W. Balentine a, P.G. Crandall a b

a,¤

, C.A. O’Bryan a, D.Q. Duong a, F.W. Pohlman

b

Department of Food Science, University of Arkansas, Fayetteville, AR 72701, United States Department of Meat Science, University of Arkansas, Fayetteville, AR 72701, United States

Received 21 February 2005; received in revised form 8 December 2005; accepted 8 December 2005

Abstract The timing of the application of rosemary extract was evaluated as one-way of minimizing myoglobin and lipid oxidation in ground beef. In experiment 1, rosemary extract was added to beef at four diVerent stages namely trim, cube, coarse, and Wne ground beef. The beef was evaluated for color and TBARS values during 144 h of storage (4 °C). Results showed that when rosemary was added to the pregrinding treatments of trim and cube, ground beef had the highest a¤ values (redness), oxymyoglobin content, and lowest TBARS values at 144 h. In experiment 2, the eVect of rosemary extract was evaluated on the color quality of case ready ground beef inoculated with 107 CFU/g Escherichia coli. Microbial counts, color, and TBARS values were measured during 144 h of simulated storage. The results showed that both the rosemary treated samples that were inoculated and uninoculated remained redder longer and had lower TBARS values than the untreated inoculated and uninoculated controls. There was no signiWcant inhibition of E. coli by the rosemary extract. © 2006 Elsevier Ltd. All rights reserved. Keywords: Ground beef; Antioxidants; Meat color; Rosemary

1. Introduction Ground beef comprises over 40% of all of the cuts of beef consumed in the US (AMI, 2002). However, ground beef is the most susceptible form of meat to microbial contamination during processing and handling (Nam & Ahn, 2003), and it is also the most susceptible to discoloration. Color has been found to be the most important factor aVecting the consumer’s buying decisions concerning ground beef (Faustman & Cassens, 1990). The loss of value due to discoloration in beef at the retail level in the US costs American consumers about three quarters of a billion dollars per year as estimated by Liu, Lanari, and Schaefer (1995). The main cause of discoloration and oxidative rancidity or other oV-odor or oV-Xavor compounds in red meat is the *

Corresponding author. Tel.: +1 479 575 7686; fax: +1 479 575 6936. E-mail address: [email protected] (P.G. Crandall).

0309-1740/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2005.12.003

oxidation of myoglobin and the autoxidation of fat (Montgomery, Parrish, Olson, Dickson, & Niebuhr, 2003). Chan, Faustman, and Decker (1997) showed that lipid autoxidation products increased the rate of oxidation of oxymyoglobin to metmyoglobin. Also, heme and non-heme iron contained in the muscle tissue of animals has been reported as catalyzing lipid oxidation in meat. The iron atom itself promotes autoxidation of fats and the heme iron molecule has been claimed to participate in the oxidation reaction (Liu & Watts, 1970). By minimizing the rate of the lipid oxidation reaction, there should be reduced color deterioration of red meat because these reactions are linked (Chan et al., 1997). For more than 50 years it has been known that aerobic bacteria in their logarithmic phase of growth can increase the rate of beef discoloration (Butler, Bratzler, & Mallman, 1953). This is believed to be due to the high oxygen demand of aerobic bacteria in their logarithmic growth phase, which results in the oxidation of myoglobin to metmyoglobin (Seideman, Cross, Smith, & Durland, 1984).

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Robach and Costilow (1962) concluded that aerobic bacteria in high enough numbers could cause metmyoglobin formation by reducing the oxygen tension at the meat surface. With low oxygen levels, brown metmyoglobin occurs as the heme Fe becomes oxidized to Fe+++. However, Robach and Costilow (1962) also stated that bacterial counts in high enough numbers can actually prevent oxygen from reaching the meat surface, thus permitting rapid enzymatic reduction of brown metmyoglobin to purple myoglobin after an extended period of time. With no oxygen present, purple myoglobin occurs as the heme Fe becomes reduced to Fe++. Other discoloration of meat by bacteria includes the production of byproducts that oxidize the Fe molecule and become attached to the free binding site of the heme group. Natural antioxidants and antimicrobials such as rosemary extract have been shown to help prevent oxidation and the resulting color loss as well as lowering microbial contamination in red meat packaged with modiWed atmosphere (Sanchez-Escalante, Djenane, Torrescano, Beltran, & Roncales, 2001; Djenane, Sanchez-Escalante, Beltran, & Roncales, 2002). In one study, rosemary powder alone (1000 ppm) and rosemary with ascorbic acid (500 ppm) when combined with ground beef patties showed increased inhibition of metmyoglobin formation and lipid oxidation superior to that of ascorbic acid (500 ppm), taurine (50 mM), carnosine (50 mM), and their combinations (Sanchez-Escalante et al., 2001). Djenane et al. (2002) showed that when used in combination with vitamin C (500 ppm), rosemary (1000 ppm) was the most eVective in delaying myoglobin oxidation and lipid oxidation when sprayed on the surface of beef steaks as compared to combinations of taurine (50 mM) and vitamin E (100 ppm) along with a water control. Numerous phenolic compounds have been isolated from rosemary extract including carnosic acid, carnosol, epirosmanol, isorosmanol, rosmaridiphenol, rosmanol and rosmarinic acid. The antioxidant properties of rosemary are believed to come from these phenolic compounds (Houlihan, Ho, & Chang, 1984; Zheng & Wang, 2001). These phenolic compounds often have antimicrobial properties as well. The mechanism by which these phenolic antioxidants inhibit bacteria is believed to aVect the function and composition of the bacterial cellular membrane, the synthesis of DNA, RNA, proteins and lipids, and the function of the mitochondrion (Raccach, 1984). However, Farbood, MacNeil, and Ostovar (1976) showed that a 1.0% concentration of rosemary extract had little eVect on Escherichia coli, E. aerogenes, and P. Xuorescens but reduced S. typhimurium and S. aureus growth by 43.2% and 99.9%, respectively, in meats. Shelef, Naglik, and Bogen (1980) also found that rosemary was more eVective in inhibiting microbial growth in gram-positive organisms than in gram-negative. Grampositive bacteria may be more sensitive to external environmental changes such as temperature, pH, and natural extracts due to the absence of an outer membrane in their cell membrane (Shelef et al., 1980). In contrast, results by Ahn, Grun, and Mustapha (2004), showed approximately

1 log reduction of E. coli by oleoresin rosemary (1%) over nine days. To optimize the beneWcial eVects of natural antioxidants like rosemary, research needs to be done to determine the optimum stage for the addition of the rosemary extract to act as an antioxidant/antimicrobial during the production of ground beef. Also, studies on the antioxidant/antimicrobial eVects against the microbes that are detrimental to meat color need to be performed to determine if these compounds have an eVect on preserving the color of meat that contains a relatively high level of microorganisms. This study was divided into two experiments. The Wrst investigated the color eVects on ground beef from adding a commercial rosemary extract with antioxidant/antimicrobial properties at four diVerent stages during the processing of ground beef. The second experiment built on the Wrst experiment and evaluated the eVect of a commercial rosemary extract antioxidant/antimicrobial on the color quality of case ready ground beef inoculated with a bacteria load versus an uninoculated control. By adding rosemary extract to red meat, it is proposed that discoloration will be minimized, and bacterial growth will be slowed. 2. Materials and methods 2.1. Experiment 1 Experiment 1 was designed to assess the eVects of adding rosemary extract during diVerent stages of ground beef processing. Instrumental color and lipid oxidation measures were taken every 12 h for the Wrst 72 h then every 24 h for the next 72 h in order to determine if rosemary extract would prevent discoloration and lipid oxidation. Experiment 1 was replicated with two runs on diVerent lots of beef trim purchased from a local meat processor who routinely supplies ground beef retailers. Average time post-mortem was 7–10 days. 2.1.1. Sample preparation For each of the two replicates, inside round (M. semitendinosus), about 1 week post-mortem, was used that was identical to that used in the normal retail chains consisting of 85% lean. The meat was received in vacuum packaged bags and refrigerated at 4 °C until the next day. It was then cut into trim pieces approximately 12 cm £ 12 cm in size and the trim pieces were carefully mixed to achieve uniformity before dividing into Wve diVerent treatments. All of the samples except the control received 3000 ppm of a commercial rosemary extract, as recommended by the manufacturer (Fortium R10, Kemin Americas, Des Moines, IA). Each treatment received the antioxidant at a diVerent stage during processing. The treatment stages were as follows: (1) no antioxidant, (2) treatment of beef trim, (3) treatment of cubed beef, (4) treatment of coarse ground beef, and (5) treatment of Wne ground beef with rosemary extract. The antioxidant was topically applied by a spray mister to insure uniform coverage and allowed to set 1 h on the sam-

C.W. Balentine et al. / Meat Science 73 (2006) 413–421

ples. The trim was cut into approximately 3 cm cubes. Then the cubes were immediately coarse ground through a 0.9525 cm plate with a Hobart grinder (Model 4822, Hobart Inc., Troy, OH). After coarse grinding, the samples were then immediately Wne ground with a 0.635 cm plate. 2.1.2. Packaging and storage The Wne ground samples (454 g) were placed onto trays and sealed with a PVC Wlm overwrap with an oxygen transmission rate of 1400/cc/m2/24 h/1 atm (Borden Inc., Dallas, TX). Twenty trays of each of the Wve treatments tested were collected for a total of 100 trays. After sealing the packages, they were placed in a 4 °C refrigerated display case 45 cm from white Xuorescent lighting (1630 1x, Phillips Inc., Somerset, NJ) to simulate commercial display and to evaluate for color at 0, 12, 24, 36, 48, 60, 72, 96, 120, and 144 h of display. All of the samples were placed in the refrigerated display case at zero time. 2.1.3. Color analysis At each sampling interval, a random number generator was used to select two numbered trays from each treatment that were analyzed for CIE L¤ (lightness), a¤ (redness), and b¤ (yellowness) values. Color analysis was performed with a Hunter Lab ColorXex colorimeter (Model 45/0, Hunter Associates Laboratories, Inc., Reston, VA). Illuminate A, 10° standard observer, and 3.18 cm view and port areas were used. The averages of three measurements from random locations on two diVerent samples (3 £ 2 D 6) from each treatment were averaged used for statistical analysis to give a total of two readings for each treatment per sample time. The colorimeter was calibrated with a layer of packaging Wlm over the white and black reference tiles before use. Ratios of a¤/b¤, hue angle (h¤ D tan¡1b¤/a¤) and saturation index (C¤ D (a¤2 + b¤2)1/2) were also determined using CIE values. In addition to CIE values, reXectance measurements were taken in the visible spectrum of 400–700 nm. In particular, the 630/580 nm reXectance ratio was calculated to determine oxymyoglobin proportion in the ground beef sample according to Hunt et al. (1991). 2.1.4. TBARS analysis Lipid oxidation was investigated using the thiobarbituric acid reactive substances (TBARS) method. The method was modiWed from that given by Tarladgis, Watts, Younathan, and Dugan (1960) and Rhee (1978). In sampling meat from each treatment, 2 g was homogenized for 20 s with 8 ml of cold 50% mM phosphate buVer mix standardized to a pH of 7 and also containing 0.1% EDTA and 0.1% n-propyl gallate. Next, 2 ml of trichloroacetic acid was added to the tubes then centrifuged at 2300 rpm for 5 min and the supernatants were Wltered. Two milliliters of the Wltrate was mixed with 2 ml of 20 mM TBA and put in a boiling water bath for 20 min. Then the tubes were cooled in an ice bath for 5 min. After cooling, the 533 nm absorbance was measured. Results were expressed as TBARS in mg malonaldehyde/kg sample.

415

2.1.5. Statistical analysis The data in the statistical analysis resulted from averaging the three measurements from two trays within each run for each response variable. An analysis of variance was conducted for each response variable in which the Wxed sources of variation were treatment (four stages of rosemary addition and the control), hours of display, and their interaction. Run and its interaction with each of the Wxed sources were the random sources of variation. Means were separated using multiple t tests, each at the 0.05 signiWcance level. Statistical computing was done using the MIXED procedure of SAS (SAS Institute Inc., 2001). 2.2. Experiment 2 Based on the results of experiment 1, this second experiment was designed to evaluate the eVectiveness of rosemary extract on minimizing discoloration, lipid oxidation, and microbial growth of inoculated ground beef when treated at the trim stage of ground beef processing. Instrumental color and lipid oxidation measures were taken every 12 h for the Wrst three days then every 24 h for the next three days in order to determine if rosemary extract would prevent discoloration and lipid oxidation. In addition, microbial analysis and pH was performed every 24 h. In order for further analysis, the rosemary treatments were repeated on a separate run for color and TBARS. 2.2.1. Sample preparation Inside round (M. semitendinosus) trim consisting of 85% lean was purchased from a local meat company for use in the study. The meat was 7–10 days post-mortem. The meat was kept in vacuum packaged bags and refrigerated at 4 °C until use the next day. A total of 36.3 kg of meat was cut into trim pieces approximately 12 cm £ 12 cm in size and mixed together to achieve uniformity then divided into four diVerent treatments. Treatments consisted of a (1) control, (2) inoculated control, (3) rosemary treated, and (4) inoculated rosemary treated beef. The inoculated treatments received a bacteria load consisting of 107 CFU/g of a nonpathogenic Rifampicin antibiotic resistant E. coli ATCC 25922. Rifampicin resistance was induced in this E. coli strain using the method of Dickson and Olson (2001). The E. coli was selected for Rifampicin resistance in order to minimize background growth on the media since Rifampicin has been shown to inhibit coliform growth (Dickson & Olson, 2001). Seven liters of inoculum was added to 36 kg of meat and allowed contact for 1 h, then drained. The inoculated meat was stored overnight (12 h) under refrigeration to allow time for microbial attachment. After 12 h, a commercial rosemary extract (Fortium R10, Product # 15435, Kemin Americas, Des Moines, IA) was topically added by a spray mister to the rosemary treatments at 3000 ppm at the trim stage. At 1 h, the trim was cut into approximately 3 cm cubes. The cubes were then immediately coarse ground through a 0.9525 cm plate with a Hobart grinder (Model 4822, Hobart Inc., Troy, OH). After coarse grinding, the

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samples were then immediately Wne ground with a 0.635 cm plate. 2.2.2. Packaging and storage The Wne ground samples were put into 454 g trays and sealed with atmospheric overwrap Wlm packaging with an oxygen transmission rate of 1400/cc/m2/24 h/1 atm (Borden Inc., Dallas, TX). A total of 20 trays of each of the four treatments were collected. After sealing, all of the packages were placed in a display case at 4 °C 45 cm under white Xuorescent lighting (1630 1x, Phillips Inc., Somerset, NJ) to simulate commercial display and evaluated for oxidation at 0, 12, 24, 36, 48, 60, 72, 96, 120, and 144 h of display. 2.2.3. Microbiological analysis Inoculated samples were plated at zero time on TPC agar containing Rifampicin to determine the actual level of E. coli each treatment received. At each sampling period, a composite sample from each of two randomly selected trays of each group was taken. The count of microorganisms was determined by aseptically removing a representative 25 g sample from the trays into a sterile stomacher bag. The sample was diluted by hand kneading with 225 ml of ButterWeld’s solution (Difco) for a 1–10 dilution. Each sample was homogenized by hand massage in stomacher bags for 2 min and serially diluted with ButterWeld’s to the desired dilution. The diluted samples were plated on Hektoen Enteric Agar (Difco) with 200 ppm Rifampicin added and incubated at 37 °C for 24–48 h. The plates were then counted and results recorded as colony forming units per gram (CFU/g) of E. coli. 2.2.4. Color analysis During each of the sampling intervals, CIE L¤, a¤, and b¤ and reXectance measures were taken as previously described. 2.2.5. TBARS analysis During each of the sampling intervals, TBARS measures were taken as previously described. In addition, the pH of the samples was taken at hour 0, 24, 48, 72, 96, 120, and 144 using a Corning pH Meter 440 by taking 3 g of sample and homogenizing with 27 ml water.

2.2.6. Statistical analysis The data in the statistical analysis resulted from averaging the three measurements from two trays for each response variable involving color and TBARS. An analysis of variance was conducted for each response variable in which the Wxed sources of variation were treatment (control, inoculated control, rosemary, inoculated rosemary), hours of display, and their interaction. Run and its interactions with the Wxed sources were the random sources of variation. Means were separated using multiple t tests, each at the 0.05 signiWcance level. Statistical computing was done using the MIXED procedure of SAS (SAS Institute Inc., 2001). Data from the log CFU/g E. coli and pH measured on each tray in a single run were analyzed by two-way analysis of variance with treatment and hour as the factors in the analysis. 3. Results and discussion 3.1. Experiment 1 3.1.1. EVect of stage of addition of rosemary extract on instrumental color and TBARS characteristics The addition of the rosemary extract at all stages exerted an eVect on the myoglobin and lipid oxidation of the ground beef. The eVects of the diVerent stage addition of the rosemary extract on color and lipid oxidation are shown in Table 1. The L¤ value (lightness) of the control, cube, and coarse addition stages were similar as were the control, cube, and Wne stages. The addition at the trim stage was signiWcantly darker (P < 0.05) than all others with the lowest L¤ value. The a¤ values (redness) of ground beef treated with rosemary at the trim and cube stage were similar, and they were signiWcantly redder than all the other treatments (P < 0.05). The b¤ values (yellowness) of ground beef from the trim and cube stages were signiWcantly more yellow (P < 0.05) than all the other treatments. Both the a¤ and b¤ values seemed to be higher on the pre-grinding treatment applications. The 630/580 nm values showed that the oxymyoglobin content of the trim stage had the highest ratio (greatest oxymyoglobin content), and it was signiWcantly diVerent from the other treatments (P < 0.05) except the cube treatment. These highest oxymyoglobin values

Table 1 EVect of rosemary extract treatments applied to diVerent stages of ground beef on mean § standard error of CIE L¤A, a¤A and b¤A values, 630/580 nm reXectance values, saturation index, hue angle, and TBARS through 144 h of simulated retail storage Attribute ¤

CIE L CIE a¤ CIE b¤ 630/580 nm Sat. index Hue angle TBARS A B C

Treatment control 48.84ab 23.17b 21.92b 3.28bc 32.19b 45.88ab 3.75a

B

Trim

Cube

Coarse

Fine

SEC

46.68c 25.68a 23.74a 3.86a 35.16a 44.22c 0.78c

48.67ab 24.72a 23.48a 3.54ab 34.33a 45.22bc 0.83c

49.54a 22.82b 22.37b 3.10c 32.23b 46.49a 1.15c

47.96b 22.15b 22.02b 3.01c 31.47b 46.85a 2.77b

0.70 1.76 1.23 0.29 2.03 1.17 0.38

L¤ values: 0 D black and 100 D white; a¤ values: ¡60 D green and 60 D red; b¤ values: ¡60 D blue and 60 D yellow. Means within row not having a common letter are signiWcantly diVerent (p < 0.05). SE, standard error.

C.W. Balentine et al. / Meat Science 73 (2006) 413–421

The most commonly used naturally occurring exogenous antioxidant for meat is rosemary (Mielnik, Aaby, & Skrede, 2003). It is antioxidative properties are strongly related to the presence of numerous phenolic compounds which are speculated to act in the same manner as synthetic phenolic antioxidants such as BHA, BHT and TBHQ (Cuvelier, Richard, & Berset, 1996). 3.1.2. EVect of hours of display on instrumental color and lipid oxidation characteristics Figs. 1–3 summarize the eVect of duration of display on instrumental color and lipid oxidation of ground beef treated with rosemary extract at diVerent stages. The L¤ values shown in Fig. 1a seemed to vary over time with no deWnite pattern, though the trim stage had the lowest L¤ L* Value 54

coarse

control

cube

fine

trim

L* value

52 50 48 46 44 42 0

12

24

36

48

60

a

72 84 time (hr)

96 108

120 132 144

96 108

120 132 144

a* Value 35

a* value

30 25 20 15 10

coarse

control

cube

fine

trim

5 0

12

24

36

48

60

72

84

time (hr)

b

b* Value 31

coars e cube trim

29 27 b* value

agreed with the a¤ values in which the trim and cube stage were the reddest. Saturation index (vividness) showed that the trim and cube stages are similar, and they were also signiWcantly more vivid (P < 0.05) than the other treatments. The hue angle values shows that the control, coarse, and Wne stages were similar in orange hue to each other, and the control was signiWcantly more orange than the trim. When comparing the control to samples with the addition of the rosemary extract at the trim stage, there was signiWcant diVerence in L¤, a¤, b¤, 630/580 nm, hue angle, saturation index, and TBARS values (P < 0.05). Most importantly, the addition of rosemary to the trim before grinding had higher a¤ values, 630/580 nm ratio, and saturation index and a lower hue angle value when compared with the control. These values correlate to a redder ground beef product. These results are in agreement with the results of Sanchez-Escalante et al. (2001) and Sanchez-Escalante, Djenane, Torrescano, Beltran, and Roncales (2003) who used modiWed atmosphere packaging in addition to the rosemary. The rosemary treatments were highly eVective in inhibiting both metmyoglobin formation for 4 days and lipid oxidation for more than 6 days. For lipid oxidation, all of the stage treatments were signiWcantly diVerent than the control (P < 0.05). The trim stage had the lowest TBARS value (0.78 § 0.38 mg/kg malonaldehyde) followed by the cubed (0.83 § 0.38 mg/kg), coarse (1.15 § 0.38 mg/kg), Wne (2.77 § 0.38 mg/kg), and control (3.75 § 0.38 mg/kg), respectively. The TBARS values when rosemary was added to the trim, cube, and coarse stages were signiWcantly diVerent (P < 0.05) and dramatically lower than the control just as it had been in the color values. These results are similar to Sanchez-Escalante et al. (2003) and Djenane et al. (2002), who found rosemary extract was able to retard lipid oxidation for more than six days. These lipid oxidation results of TBARS coincide with the myoglobin oxidation results of a¤ value and 630/580 nm. This is important since Chan et al. (1997) showed that lipid autoxidation products can increase the oxidation of oxymyoglobin to metmyoglobin. So in essence, controlling the autoxidation with antioxidants such as rosemary extract should, in turn, reduce the rate of myoglobin color degradation. Overall, the trim and cubed treatments had the reddest color and were the least oxidized samples. The addition of the rosemary extract at the pre-grinding stages of trim and cube was shown to be the best application time. The rosemary treatment should be applied to meat before it is ground to ensure the maximum beneWt. These stages are also the most practical in the commercial processing of ground beef. By adding this antioxidant early in processing, the rosemary extract has more contact time and contact area as it is thoroughly mixed into the ground beef through grinding. It is hypothesized that the increased eVect of inhibition of oxidation observed in the pre-grinding stages of the trim and cube treated meat has to do with the ability of the rosemary to come in contact with more of the meat as the cell membranes of the meat muscle are destroyed.

417

control fine

25 23 21 19 17 15 0

c

12

24

36

48

60

72 84 time (hr)

96 108

120 132 144

Fig. 1. EVect of simulated retail display time on least square mean (a) L¤ values SE § 1.03 LSD 1.95, (b) a¤ values SE § 2.08 LSD 2.02, and (c) b¤ values SE § 1.58 LSD 1.38 of ground beef treated with rosemary extract at 4 °C.

418

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Oxymyoglobin coarse cube trim

630/580 nm

6 5

TBARS

6

control fine

4 3 2

coarse cube trim

5 mal (mg/kg)

7

control fine

4 3 2

1

1

0 0

12

24

36

48

60

a

72 84 time (hr)

96 108 120 132 144

0

Saturation Index 50

sat. index

45

coarse cube trim

40

control fine

35 30 25 20 15 0

12

24

36

48

b

60

72 84 time (hr)

96

108 120 132 144

96

108 120 132 144

Hue Angle 65

coars cube trim

hue angle

60 55

control fine

50 45 40 35 0

c

12

24

36

48

60

72 84 time (hr)

0

Fig. 2. EVect of simulated retail display time on least square mean (a) 630/ 580 nm ratios § 0.37 LSD 0.44, (b) saturation index SE § 2.40 LSD 2.07, (c) hue angle SE § 2.59 LSD 3.29 of ground beef treated with rosemary extract at 4 °C.

value throughout the study. Fig. 1b shows the a¤ values of the diVerent stages of treatment. The values tend to decline for all treatments after 60 h of display but with the trim being signiWcantly redder (P < 0.05) from the control at hours 48 and 72–144. This would provide a longer shelf life in the current atmospheric packaging retail environment. Values for the Wrst 96 h of all treatments in this experiment were higher than the a¤ values of modiWed atmosphere packaged ground beef with rosemary extract of SanchezEscalante et al. (2003) at 96 h. As seen in Table 1, the trim and cube had the highest a¤ values over 144 h at 25.68 § 0.70 and 24.72 § 0.70. Ground beef b¤ values in Fig. 1c showed that the trim and cube treatments were signiWcantly more yellow (P < 0.05) than the control on hour 12, 24, 48, and 72–144. Fig. 2a shows the oxymyoglobin

12

24

36

48

60

72 84 time (hr)

96 108 120 132 144

Fig. 3. EVect of simulated retail display time on least square mean TBARS values SE § 0.46 LSD 0.96 of ground beef treated with rosemary extract at 4 °C.

proportion of each stage over 144 h. Again, the trim treatment had a signiWcantly higher (P < 0.05) oxymyoglobin ratio than the control at hours 48–96. This conWrms the results obtained for the a¤ values. All stages decreased in redness (a¤) as the proportion of oxymyoglobin decreased over time. A decline in 630/580 nm values after 60 h also occurred, and the control had the lowest proportion from hour 48–96. Saturation index and hue angles are shown in Fig. 2b and c. The trim and cube treatments had higher saturation index values than the control from hours 48 to 144. This relates to a more vivid color. The trim and control were similar in hue angle through display except the trim was signiWcantly lower (P < 0.05) at hours 96 and 120. Fig. 3 shows the TBARS values over 144 h of display. There was a dramatic diVerence in values at hour 0. This is believed to be the result of immediate antioxidant activity of the rosemary extract as the beef was ground. This Wnding tends to support our hypothesis of how rosemary is reacting. The addition of rosemary at the trim, cube, and coarse stages resulted in signiWcantly lower TBARS values. The TBARS values remained relatively constant within each treatment over the 144 h. The control values were the highest (P < 0.05) through display. Based on the results of lowering lipid oxidation and reducing myoglobin oxidation, adding rosemary extract should result in longer shelf life for retail applications. 3.2. Experiment 2 3.2.1. EVect on duration of display on instrumental color and lipid oxidation characteristics of ground beef inoculated with E. coli and treated with rosemary extract Figs. 4–6 summarize the eVect of duration of display on instrumental color and lipid oxidation. Fig. 4a shows L¤ values with the values varied by day. Both rosemary treatments had the highest numerical values (lightest color) throughout the display life with the rosemary treatment signiWcantly lighter (P < 0.05) than the control from hour 36 to 144. In Fig. 4b, the rosemary treatment had higher a¤ values

C.W. Balentine et al. / Meat Science 73 (2006) 413–421

L* Values 54

L* value

Oxymyoglobin

630/580 nm

10

0

12

24

36

48

60

a

50

Control Inoc. Con.

45 35

0

Rosemary Inoc. Ros.

12

24

36

48

60

72 84 96 108 120 132 144 time (hr)

a* Values

hue angle

38

40 Control Inoc. Con.

35

Rosemary Inoc. Ros.

30

12

24

36

48

60

20 15 10 0

12

24

36

48

b

60 72 84 time (hr)

96

108 120 132 144

72

84

96 108 120 132 144

time (hr)

Hue Angle

58 56 54 52 50 48 46 44 42 40 38 36

Control Inoc. Con.

0

c

25

Rosemary Inoc. Ros.

40

b

0

96 108 120 132 144

Saturation Index

20

a

84

55

25

Control Inoc. Con.

72

time (hr)

48

40

Inoc. Ros.

0

30

44

Rosemary

Inoc. Con.

4

50 46

Control

6

52

42

a* value

8

2

sat. index

(P < 0.05) for each hour after 24 h than the inoculated control, and signiWcantly higher (P < 0.05) than the uninoculated control from hours 48 to 144. There was a noticeable decline after 72 h for all treatments except the rosemary treatment. The values in Fig. 4c show b¤ values (yellowness) of the rosemary treatment were the highest at hours 48 and 96. The values in Fig. 5a show that neither rosemary nor E. coli had much eVect on oxymyoglobin proportion over the Wrst 72 h, but the rosemary treatment had the highest oxymyoglobin content (P < 0.05) at 96 and 120 h. The a¤ values are related to the oxymyoglobin proportion in that they were signiWcantly higher (P < 0.05) at 96 and 120 h. The saturation index (Fig. 5b) shows that the rosemary

419

12

24

36

48

Rosemary Inoc. Ros.

60

72

84

96 108 120 132 144

time (hr)

Fig. 5. EVect of simulated retail display time on least square mean (a) 630/ 580 nm reXectance values SE § 0.56 for rosemary treatments and 0.60 for control treatments, (b) saturation index SE § 2.00 for rosemary treatments and 2.13 for control treatments, (c) hue angle SE § 1.21 for rosemary treatments and 1.29 for control treatments of ground beef with or without E. coli inoculation and/or rosemary extract at 4 °C.

b* value

b* Values 34 32 30 28 26 24 22 20 18 16

Control Inoc. Con.

0

c

12

24

36

48

60 72 84 time (hr)

Rosemary Inoc. Ros.

96 108 120 132 144

Fig. 4. EVect of simulated retail display time on least square mean (a) L¤ values SE § 2.43 for rosemary treatments and 2.60 for control treatments (b) a¤ values SE § 1.72 for rosemary treatments and 1.80 for control treatments (c) b¤ values SE § 1.19 for rosemary treatments and 1.32 for control treatments of ground beef with or without E. coli inoculation and/or rosemary extract at 4 °C.

treatment and inoculated rosemary treatment had the most vivid color (P < 0.05) from hour 48 through 144. Hue angle values in Fig. 5c show similar values for the treatments for the Wrst 72 h. At 96 h, the hue angles for all the treatments increase with the rosemary treatment remaining the lowest from hour 96 to 144. Fig. 6a shows the TBARS values over 144 h. The rosemary treated samples had the lowest TBARS values (P < 0.05) throughout. The TBARS values rose slightly and Xuctuated little within each treatment over the 144 h of display. The staggered start at hour 0 for the treatments is believed to be due to the immediate eVect of the antioxidant addition before the grinding of the meat and its earlier contact time in the ground beef processing. Fig. 6b shows the pH of the treatments through display. The pH of the

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96 108 120 132 144

inhibition of E. coli growth seen when comparing the counts for rosemary extract treated beef and the control. These results were similar to Farbood et al. (1976) in which rosemary extract had no inhibitory eVect on E. coli. The log CFU of E. coli in the inoculated samples of both the rosemary and control treatments remained constant at approximately the 5 log CFU/g range over the 144 h of storage as seen in Fig. 6c. Del Campo, Amiot, and Nguyen-The (2000) also found that rosemary extract was not eVective in inhibiting gram-negative bacteria such as E. coli. In comparing the two treatments, the inoculated rosemary treated beef was signiWcantly diVerent than the inoculated control beef for the L¤, a¤, b¤, 630/580 nm, saturation index, hue angle, and TBARS values (P < 0.05). Visually, minimal diVerence in redness was noted for the Wrst 48 h, but thereafter, the rosemary treated ground beef samples appeared redder. Our data indicated that the addition of E. coli seemed to have less of an eVect on the rosemary treated beef than the control beef when looking at myoglobin and lipid oxidation; however, there were no inhibitory eVect of rosemary extract on growth or survival of inoculated E. coli.

96 108 120 132 144

4. Conclusion

mal (mg/kg)

TBARS 9 8 7 6 5 4 3 2 1 0

Control Inoc. Con.

0

12

24

36

Rosemary Inoc. Ros.

48

60

a

72

84

time (hr)

pH Values 5.8 5.7

pH

5.6 5.5 5.4 5.3

Control Rosemary

5.2

Inoc. Con. Inoc. Ros.

5.1 0

12

24

36

48

60

b

72

84

time (hr)

Log CFU/g E.coli log CFU/g of E.coli

5.5

Inoc. Con.

Inoc. Ros.

5.25 5 4.75 4.5 0

c

24

48

72

96

120

144

time (hr)

Fig. 6. EVect of simulated retail display time on least square mean (a) TBARS values SE § 0.49 for rosemary treatments and 0.50 for control treatments, (b) pH values SE § 0.03, and (c) log CFU/g E. coli SE § 0.07 of ground beef with or without E. coli inoculation and/or rosemary extract at 4 °C.

inoculated rosemary and inoculated control were both higher than their corresponding uninoculated treatments except at hours 24 and 48. We have speculated that this may be due to competitive inhibition of the spoilage bacteria by the E. coli present keeping the pH less acidic. Another explanation for the diVerence could be the result of the added buVer eVect from inoculation, or a natural variation in the Wnal product. 3.2.2. EVect of rosemary extract treated ground beef on microbial inhibition of E. coli The inoculated treatments had a constant level of E. coli throughout the experiment (Fig. 6c) to contend with the rosemary extract. Unlike previous results of Ahn et al. (2004) in which oleoresin rosemary inhibited E. coli approximately 1 log CFU/g after nine days, there was no

In experiment 1, the rosemary extract best minimized myoglobin oxidation in the ground beef during the pregrinding stages when the rosemary was added to the trim or cube. These stages of addition also had the greatest eVect on delaying lipid oxidation. Therefore, experiment 1 showed that stage of rosemary extract antioxidant addition impacts color and lipid stability. It is likely that the more area that the antioxidant comes in contact with, the better the red color retention. It is also our hypothesis that having the antioxidant present at the instant that the cells are disrupted better protects the oxymyoglobin from further oxidation. This is due to the more complete mixing of the rosemary extract within the meat as it is processed. Experiment 2 showed that the antioxidant potential of the rosemary extract at the level used is still suYcient to protect color and lipid oxidation in ground beef despite a large E. coli bacteria load. The inoculated rosemary extract treated ground beef held a higher a¤ value than the inoculated control from hours 24 to 144. The TBARS values remained lowest in both rosemary treatments after 144 h. This experiment showed that the microbial contamination eVects on myoglobin and lipid oxidation in ground beef could be minimized by rosemary extract though no microbial inhibition was seen. Rosemary extract is a safe and eVective ground beef additive with antioxidant properties. It appears that controlling the autoxidation with antioxidants such as rosemary extract should reduce the rate of myoglobin color degradation. Further studies with rosemary are needed in order to apply Wndings to commercial applications, which could result in a higher quality ground beef product that is safe with a longer shelf life for consumers. Future experiments should look at the inhibition of other bacteria and

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