CHARACTERISTICS OF MINCED BEEF STORED AT CHILLED AND ABUSE TEMPERATURES

H.A. ABD EL-RHMANI, N.G. MARRIOTT~, H.WANG~, M.M.A. YASSEI" and A.M. AHMED'

'Suez Canal University, Ismailia, Egypt 'Virginia Polytechnic Institute and State University

Blacksburg, Virginia 24061-0418

Submitted for Publication on December 16, 1996 Accepted for Publication on June 20, 1997

ABSTRACT

Fresh minced beef samples were collectedfiom retail food stores in the Eastern United States and stored for various perioak at 4 and 25C. Evaluations included psychrotophic counts, microbial taxonomy, pH, total volatile nitrogen, malonaldehyde (TBAm)), color and o f odor. Minimal discoloration was observed through 5 days of storage at 4C; whereas, minced beef stored at 25C sustained major discoloration prior to storage for 12 h. Ajier fwe days of storage at 4C, I 1.6% of the samples exhibited an off odor compared to 29.8% ajier storage for I2 h at 25C. Psychrotrophic counts of less than I a'/g were attained during storage at 4C for 5 days; whereas, samples stored at 25C exceeded this level ajier 12 h. Chemical indices suggested similar product deterioration. These observations suggested that fiesh minced and aerobically packaged in a 4C environment will attain acceptable color without oflodor development through 5 days of storage.

INTRODUCTION

The livestock and meat industry has been challenged to seek ways of producing meat products that will enable the consumer to receive maximum safety and palatability benefits. Such appeal depends on factors including meat color, odor, and flavor. At the point of purchase, meat quality is judged by the consumer mainly by its appearance. Consumers relate the color of lean to freshness (Adams and Huffman 1972). Urbain (1952) reported that consumers have learned through experience that the color of fresh beef is bright red and any deviation is unacceptable. Inger (1977) found that sensory methods are the most common techniques used for judging meat freshness.

During carcass chilling, degradation is initiated because of cold tolerant aerobic bacteria (psychrotrophs) that may discolor fresh meat through oxidation of the

Journal of Muscle Foods 9 (1998). 139-152. All Rights Resewed. Topyright 1998 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 139

140 H.A. ABD EL-RHMAN ETAL.

muscle pigment myoglobin. Aerobic bacteria have been found to cause the formation of metmyoglobin by reducing the oxygen tension on the meat surface (Robach and Costilow 1962). Greenish discoloration is produced by catalase negative bacteria which cause an accumulation of hydrogen peroxide in the tissue and subsequent reaction with myoglobin to produce the green pigment hydroperoxymetmyoglobin (Fox et al. 1974). Furthermore, sulfhydryl producing bacteria can cause a green discoloration of meat (Nicol et al. 1970) due to the formation of sulfmyoglobin. Discoloration may also be due to the elaboration of the foreign pigments of Pseudomonas spp. and pink pigment of various Micrococcus spp. (Haines 1937).

Many psychrotrophic bacteria, when present in large numbers, can cause a variety of off odors as well as physical defects in foods. Their growth rate is highly temperature dependent, and becomes increasingly slower as the temperature is reduced. Therefore, shelf-life or the rate of quality loss, and subsequent spoilage of a refrigerated food is also highly temperature dependent (Tompkin 1973).

Many foods contain a significant amount of lipids which are susceptible to hydrolysis and oxidation with a resultant change in flavor. Although many of the problems of fat breakdown are not microbial in origin, numerous bacteria are believed to be capable of causing hydrolytic deterioration (APHA 1992). Fat may undergo changes during storage that result in the production of an unpleasant taste and odor, which is commonly referred to as rancidity. Rancidity is brought about by the action of oxygen (oxidative rancidity) and believed to be affected by lipolytic microorganisms (ketonic rancidity) (Pearson 1977). Alford and Pierce (1 97 I), Papan and Talon (1 988), and APHA (1 992) confirmed that the storage temperature of meat affects the growth of lipolytic bacteria, but their lipase enzymes remain active to -3OC and may be associated with quality loss or rancidity.

During the past, aerobic spoilage of meat has been attributed to the proteolytic and lipolytic activities of aerobic spoilage flora. An alternative view (Gill 1997) is that microorganisms associated with the putrid spoilage of meat, such as pseudomonads and psychrotropic Enterobacter, display a preference for glucose while it is available as a substrate. When glucose is exhausted, enzyme systems are induced for the catabolism of free amino acids, which are abundantly available in meat. The breakdown of amino acids, with the release of organoleptically offensive by-products, such as ammonia and small quantities of amines and sulfides, is the principal cause of the changes which are perceived as putrid spoilage.

As previously indicated, beef is vulnerable to microbial degradation which can cause discoloration and off odor and flavor. Thus, this study was conducted to identify the psychrotropic bacteria in comminuted beef. Since the minced beef in this study was aerobically packaged, enumeration of lactic acid bacteria was not conducted.

TEMPERATURE EFFECTS ON MINCED BEEF 141

MATERIALS AND METHODS

A total of 100 samples (unknown age of raw materials) of minced beef packaged less than 12 h (1 kg each, packaged in polyvinyl chloride film, ~30% fat stored at ca 2C) were collected from different retail markets in the Eastern United States. The samples, which were ground and packaged at the meat market, were transferred within 10 min after collection to an ice box container and sent to the Virginia Polytechnic Institute and State University Muscle Foods Laboratory.

Seventy samples were divided into seven groups (1 0 samples per group) and stored at 4C. Each group was examined periodically after 0,2.5, 5,7.5, 10, 12.5 and 15 days, respectively. Thirty samples were divided into three groups and maintained at room temperature 25C. Each group was examined after 0,12 and 24 h, respectively. All samples were evaluated for physical, microbial, and chemical indices.

Physical Indices

The samples were evaluated for discoloration (l=very abundant; 8=none) and odor (l=extreme off odor; 5=no off odor) during storage. This method involved seven trained panelists that examined the samples for the different physical characters and the results were recorded on score sheets (Marriott 1995).

Microbial Indices

Collected samples were prepared according to the technique recommended by Thatcher and Clark (1975) as follows: Twenty five grams of the sample under investigation were transferred into a sterile blender jar to which 225 mL of 0.1% sterile peptone water were added and blended for 2.5 min to provide a dilution of 10 - I . The mixture was then allowed to stand for 2 min at room temperature. One mL of the original homogenate was transferred to a test tube containing 9 mL of 0.1% sterile peptone water to prepare a dilution of lo2. After thorough mixing, further decimal dilutions of up to lo’were prepared.

Determination of Psychrotrophic Counts (APHA 1992). One mL from the previously prepared dilutions was plated in duplicate, using Standard Plate Count Medium (Difco Laboratories, Detroit). The inoculated plates were incubated at 20C for two days. The average number of colonies per countable plates was determined and the total number of colonies per gram was then calculated and recorded. Approximately five colonies from each plate were selected and streaked onto slant nutrient agar tubes for fbrther purification and identification.

142 H.A. ABD EL-RHMAN ETAL.

Determination of Psychrotrophic Proteolytic Counts (Martley et aL 1970). The same procedure as for the previous determination was incorporated except that the Standard Methods Caseinate Agar (Difco Laboratories, Detroit) and the inoculated plates were incubated at 20C for 3 days.

Determination of Psychrotrophic Lipolytic Counts (Jonsson and Snygg 1974). The same procedure as for the previous determination was involved except that Tributyrin Agar Medium was used and the inoculated plates were incubated at 20C for 5-7 days.

Identification of Isolated Bacteria. API Strips (BioMerieux Sa. 1992) and (MacFaddin 1980) were incorporated for the identification technique. Microorganisms that were not determined to be psychrotrophs were identified as other bacteria.

Chemical Indices

pH Determination (Dodge and Stadelmin 1960). Twenty five grams from the sample were mixed thoroughly with 50 mL of neutral distilled water and left to stand for 10 min. pH was determined by using an electric pH meter (Corning Model 340, Scientific Products, McGaw Park, Ill.).

Determination of Total Volatile Nitrogen (Lee 1975). In a distillation flask of a Kjeldahl unit, 10 g from the sample, 2 g of magnesium oxide, 3 drops of silicone antifoam liquid, 350 mL of distilled water and a pinch of anti-bumping granules were added. While in the collection flask, 25 mL of 2% boric acid solution with the addition of a few drops of screened indicator (0.02% methyl red and 0.01% bromocresol green ethanol) were added. The liquid was heated so that boiling occurred within 10 min and distillation was continued at a constant rate of heating for 25 min. 'Ihe condenser tip was washed for collecting any liquor in the lower flask. Distilled liquor and acid was titrated with 0.05 M sulfuric acid (titer a). Then, 25 mL of 2% boric acid solution was added to a few drops of screened indicator and titrated against the 0.05 M sulfuric acid (titre b). The total volatile base was calculated as:

Total volatile base = (a - b) 14 mg nitroged100g.

Determination of Thiobarbituric Acid Reactive Substances (Vyncke 1970). Twenty g of each sample was homogenized with 100 mL of 7.5% trichloroacetic acid solution for 1 min in a blender and the homogenate was filtrated through the filter paper. Five mL of TBA reagent ( 0.2883 M TBA reagent g/100 mL of 90% glacial acetic acid) was added to 5 mL of the filtrate in test tubes with screw caps. The blank and standard sample were prepared. Then, the tubes were placed for 40

TEMPERATURE EFFECTS ON MINCED BEEF 143

min in a boiling water bath. The tubes were cooled in water for 10 min and the absorbance (D) was measured against the blank at 538 nm using 1 cm cells.

Concentration of Malonaldehyde In Sample Distillate. Concentration was determined by:

- Yi-A Where - Xi

Ks

Xi - - Malonaldehyde concentration of sample distillate, mole/mL

Absorbance of the sample distillate - Yi -

Ks,A = Slope and constant from standard curve

TBARS Value of Samples. TBARS was calculated as:

TBARS(mg/Kg) = XixVixMWx 106 W t x R

Vi - - Volume of sample distillate, mL

Mw - - Molecular weight of malonaldehyde, 72.03 g/mole

Wt - - Sample weight, g

Recovery rate, no unit - - R

Statistical Analysis

The data obtained were analyzed to determine the effect of treatment (time) on the physical, microbial and chemical indices of minced beef samples. Analysis of Variance (ANOVA) and the General Linear Model (GLM) procedures of the Statistical Analysis System (SAS 1987) were used to determined whether there was a significant effect of treatment (time) on the mean values of physical, microbial and chemical indices of examined minced beef samples if the model contained significant differences (a = 0.05).

I44 H.A. ABD E L - W A N ETAL.

RESULTS AND DISCUSSION

Meat spoilage should not be considered an individual condition, but the result of many changes in a food that render the product unacceptable to the consumer. This process includes physical, microbial and chemical changes (Gill 1982) .

The mean discoloration values (Table 1) for minced beef samples stored at 4C were not different (P > 0.05) between 0 and 2.5 days or 2.5 and 5 days. But, there were differences (P < 0.05) between 0 and 5 days and after storage for 5,7.5 and 10 days. The mean values for discoloration were different (P < 0.05) among samples stored at 0, 5, 10, 15 days. Differences (P < 0.05) in mean values of discoloration for minced beef samples stored at 25C existed between 0, 12,24 h, respectively (Table 3). Our results revealed that the discoloration of minced beef samples stored at 4C and 25C ranged from none (bright red color) to very abundant discoloration (greenish grey or dark brown). Prolonged storage time increased color deterioration of minced meat stored at 4C. Samples stored at 4C sustained minimal discoloration through 5 days of storage. Beyond this period, significant (P < 0.05) color deterioration occurred between each storage time. Our samples maintained color stability longer than what has been reported by other researchers. The increased color stability of these samples was attributable to the very constant temperature that was attained during storage. It is unlikely that these results could be duplicated if the samples were stored in a retail meat case with normal temperature fluctuation.

Odor

Amount of off odor of minced beef samples stored at 4C did not differ (P > 0.05) between 0, 2.5 and 5 days (Table 1). Other samples differed (P < 0.05) between 5,7.5, 10 and 15 days (Table 2). The odor values in minced beef samples stored at 25C were different (P < 0.05) between 0, 12 and 24 h (Table 3 and 4), due to the high storage temperature which prompted increased microbial growth. The off odor of minced beef may be attributable to bacteria that develop proteolytic or putrid odor from protein decomposition and rancid odor from fat oxidation or hydrolysis. These results agree with those obtained by Ingram and Dainty (1 97 1).

Microbial Indices

As beef carcasses are moved into the chill room at near OC, a selective environment is developed gradually that favors growth of psychrotrophic bacteria. Pseudomonas identified by API Strips appeared to grow most rapidly and will ultimately predominate if a chilled, moist environment is maintained long enough (Price and Schweigert 1987).

TEMPERATURE EFFECTS ON MINCED BEEF 145

TABLE 1. PHYSICAL, MICROBIAL AND CHEMICAL INDICES OF MINCED

BEEF STORED AT 4C

' I=very abundant; 8=none I=extreme off odor; 5=no odor total psychrotrophic counVg of sample (log CFUlg) total psychrotrophic proteolytic count/g of sample (log CFU/g) total psychrotrophic lipolytic count/g of sample (log CFU/g)

', total volatile base ' m a g of malonaldehyde Ykdmeans in the same column with unlike superscripts are different (P<O.OS)

146 H.A. ABD EL-RHMAN ETAL.

TABLE 2. PERCENTAGE OF DISCOLORATION AND ODOR OF MINCED

BEEF STORED AT 4C

Comminution of meat creates additional surface contamination. In addition, some comminuted meats are prepared fiom meat trimmings at the retail level under less than desirable hygienic conditions. Most chilled comminuted meats are packed in oxygen permeable films to retain bloom. If the meat initially has a low bacterial population, spoilage on the surface will result from the growth of pscychrotrophic bacteria.

These data suggest that the psychrotrophic bacteria in samples stored at 4C require 15 or more days to reach a maximum microbial population. Samples stored at 25C attained a higher microbial load after storage for 24 h than counterparts held for 15 days.

Psychrotropbic Counts (T.Ps.C.)

The mean log values of T.Ps.C. of minced beef samples stored at 4C increased (P < 0.05) between 0,5, 10 and 15 days (Table I), while at 25C (Table 3) the mean log values of T.Ps.C. were higher (P < 0.05) between 0, 12 and 24 h. than counter parts held for 15 days.

Pivnick e? ul. (1 975) proposed lo’ as a maximum limit of bacteria per gram for ground beef in Canada, while Chambers e? ul. (1 976) indicated that acceptable minced meat in Ohio should have a count of less than 1.5 x 10’. Geopfert (1 976) proposed bacterial standards for minced beef in some states (USA) with a maximum range of 1 . 0 ~ 1090 5 . 0 ~ 1 0 ~ bacteria /g. The minced beef samples in this study that were stored at 4C had aerobic plate counts below these values. However, no correlation has been established between amount of spoilage and food safety.

TEMPERATURE EFFECTS ON MINCED BEEF 147

' # Time Discoloration YO Odor% I

1 O h 0 0

2 12 h 50.25 29.8

3 24 h 82.5 45.8

Our results revealed that Pseudomonm spp predominated others during refrigerated storage of minced beef. These results agree with those obtained by Holzapfel and Holy ( 1 984).

TABLE 3. PHYSICAL, MICROBIAL AND CHEMICAL INDICES OF MINCED

BEEF STORED AT 25C

I=extreme off odor; 5-0 odor total psychrotrophic count/g of sample (log CFU/g)

'total psychrotrophic proteolytic counVg of sample (log CFU/g) 'total psychrotrophic lipolytic count/g of sample (log CFU/g) total volatile nitrogen m@g of malonaldehyde

'"means in the same column with unlike superscripts are different (P<0.05)

148 H.A. ABD E L - M A N ETAL.

Psychrotrophic Proteolytic Count (T.Ps.P.C.). Results presented in Table 1 indicate that the samples examined for TPsPC had low counts through 5 days of storage at 4C and 12 h at 25C. We hypothesized that the other spoilage bacteria first consume the soluble substrate (extractive) in the minced beef, then the proteolytic bacteria become active and produce the protease enzyme which enhances decomposition of protein and creates the necessary substrate for microbial growth.

The frequency percentage of psychrotrophic Pseudomonas spp from the minced beef samples after storage for 0 and 24 h at 4C were 10% and 19%, respectively (Table 5), and other psychrotrophic proteolytic species at the same temperature and time of storage were 90% and 81%, respectively (Table 6). Limited research has addressed the proteolytic psychrotrophic bacteria in minced beef (Abd El-Rhman and Ahmed 1988; Robin 1989), but similar results were obtained by Abd El- Rhman and Ahmed (1988).

Psychrotrophic Lipolytic Count (T.Ps.L.C.). Results from Table 3 reveal that the log mean values of T.Ps.C. reached a maximum level in samples stored at 4C for 15 days (log 4.64 cfdg). In other samples stored at 25C, the log mean values were (log 5.5 cfu/g) after storage for 24 h.

The log mean value of T.Ps.L.C. of minced beef samples stored at 4C differed (P < 0.05) between storage periods of 0, 5 , 10 and 15 days (Table 1). At 25C (Table 3), the log mean value of T.Ps.C. of minced beef samples increased (P < 0.05) with storage time (0, 12, and 24 h).

The frequency percentage of psychrotrophic Pseudomonas spp from the minced beef samples after 0,5, 10 and 15 days of storage at 4C were 9%, 42%, 57.5% and 71.5%, respectively, and those of other psychrotrophic lipolytic species at the same temperature and time of storage were 91%, 58%, 42.5% and 28.5%, respectively (Table 5) . Table 6 indicates that the frequency percentages of psychrotrophic Pseudomom spp from the minced beef samples after 0 and 24 h of storage at 4C were 16% and 22.5%, respectively, and other psychrotrophic lipolytic microbes at the same temperature and time of storage were 84% and 77.5%, respectively. Fewer data have been published on bacterial lipases in meat and meat products (Robin 1989; Papon ef al. 1990).

A spoilage microbial flora is dominated by microorganisms that grow most rapidly in the environment provided by the meat and the storage conditions with temperature, pH, and atmospheric gas compositions dominating. At storage temperatures above 20C, psychrotrophic Enterobacter can compete effectively with lactic acid bacteria (Gill 1997). Thus, at these abusive temperatures, psychrotrophic Enterobacter can dominate the spoilage microbial flora. The ready detection of Enterobacter (but not lactic acid bacteria in the flora) because of methods used, could account for small maximum numbers recovered from the samples stored at 4C as opposed to those stored at 25C.

TEMPERATURE EFFECTS ON MINCED BEEF I49

TABLE 5. FREQUENCY PERCENTAGE OF PSEUDOMONAS SPP. IN RELATIONSHIP TO OTHER

IDENTIFIED BACTERIA

total psychrotrophic count total psychrotrophic proteolytic count

'total psychrotrophic lipolytic count

TABLE 6. FREQUENCY PERCENTAGE OF PSEUDOMONAS SPP. IN RELATIONSHIP TO OTHER

IDENTIFIED BACTERIA

' total psychrotrophic count

' total psychrotrophic lipolytic count total psychrotrophic proteolytic count

Chemical Indices

Hydrogen Ion Concentration (pH). pH increased (P < 0.05) among minced beef samples stored at 4C for 0,5, 10 and 15 days and for those samples stored at 25C for 0, 12 and 24 h suggesting that increased temperature and time of storage resulted in an increase of pH and change in the quality of such products. pH of minced beef is considered to be acceptable until 6.4. Higher values alter the

150 H.A. ABD EL-RHMAN ETAL.

physical appearance of the product and enhance microbial growth. Our results revealed that the mean pH values of samples stored at 4C were acceptable until 12.5 days. Other samples stored at 25C were acceptable up to 12 h. Our results revealed that an increase in storage time of minced beef stored at 4C for more than 5 to 7.5 days enhances microbial growth and increases the pH to 6.2-6.4 and with a resultant product that is unfit for human consumption.

Total Volatile Nitrogen (TVN). Our data indicate that TVN elevates with increased time and storage temperature accompanied by an increase in psychrotrophic bacteria (Table 1 and 3). Total volatile nitrogen (TVN) consists almost entirely of ammonia. The changes in TVN are considered a relatively good index of the fluctuation in protein solubility (Awad et ul. 1968), and an indicator of microbial spoilage (Gehart and Quang 1979) and aging of meat (Brooks and Ammennan 1978). Most beef is acceptable if TVN does not exceed 16.5 mg/l OOg. Sengupta et ul. ( 1975) reported that unspoiled beef should contain less than 17 mg of TvN/lOOg.

According to these suggestions, minced beef samples stored at 4C were acceptable for up to 10 days. Acceptance of the description of the aerobic spoilage of meat in which spoilage is precipitated by exhaustion of glucose available to the microbial flora implies that chemical indicators of spoilage are of limited value, because spoilage is an abrupt phenomenon which will be well advanced before any substantial chemical changes are detected. Nevertheless, these results suggested a relationship between psychrotropic counts and TVN.

Thiobarbituric Acid Reactive Substances ("BARS). Our samples yielded acceptable TBARS values for up to 15 days of storage at 4C; whereas, the values were higher after 12 h of storage at 25C than any observed for the 4C environment. This observation may occur because most rancidity problems encountered in muscle foods are not of microbial origin (Price and Schweigert 1987). In addition, bacteria do not depend on fat as energy sources until other substrates in meat such as protein are spent.

CONCLUSIONS

Results suggest that fresh minced and aerobically packaged meat samples stored at 4C will not sustain major discoloration or off odor development through 5 days of storage. A relationship existed between the psychrotrophic bacteria and total volatile nitrogen and TBARS values since increased psychrotrophic counts resulted in increased TVN and TBARS values, respectively. Pseudomonas was the major contributor to the spoilage of refrigerated meat products. These results suggest need for the production of minced beef under hygienic conditions to decrease the initial microbial load.

TEMPERATURE EFFECTS ON MINCED BEEF I51

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