Microbiological and Sensory Changes in Minced Beef Treated with Potassium Lactate and Sodium Diacetate during Refrigerated Storage

Abstract

The effect of potassium lactate and sodium diacetate on the microbiological changes and sensory properties of vacuum-packaged minced beef was investigated. The meat samples both with a preservative (in the amounts 0.65% and 1.3%) and without were stored at temperatures of 0–1°C and 5–6°C. The influence of storage time on changes in total bacteria count (TBC), lactic acid bacteria (LAB), Brochothrix thermosphacta, and the microbes of the Enterobacteriaceae family was investigated, as well as changes in pH and sensory quality. It was found that the addition of the preservative to the minced meat caused a significant extension (p < 0.05) of the lag phase and an inhibition of microbial growth rate, depending on temperature, storage time, and its concentration. The antibacterial effect was significantly higher (p < 0.05) at a temperature of 0–1°C than at 5–6°C and most susceptible to it were the bacteria belonging to the Enterobacteriaceae. The study results showed that the minced beef containing the preservative which had been vacuum stored at 0–1°C, presented a better sensory quality and had a shelf-life of about 6 days longer, in relation to the quality and shelf-life of the control samples. For each of the refrigeration storage temperatures however, there was no statistically significant change (p < 0.05) in the pH for the various storage periods and preservative quantities present.

Keywords:

• Beef mince
• Microbiological quality
• Potassium lactate
• Refrigeration storage
• Sensory properties
• Sodium diacetate
• Vacuum package

INTRODUCTION

In the meat industry, like in other food industry branches, safety, and storage life of the products and half-finished products is of importance, and it depends on the development of microorganisms. This mainly concerns minced meat, which, as a consequence of mincing as well as of the bacterial microflora and oxygen from the air entering it in the process, constitutes a favourable environment for microbial growth. Their development may cause not only undesired organoleptic changes but sometimes even present danger for the consumers' health.

Minced meat usually has a far shorter refrigerated storage life when compared to meat that had not been minced, and is particularly affected by microbiological changes. Generally, bacterial spoilage of meat is most commonly caused by microbes showing the highest growth rate during storage. Hence, it is essential for the stability of fresh meat to ensure that it has a low pH and the lowest possible number of contaminant microorganisms.[1] Also, the packaging methods can be of significance for the speed of microbiological spoilage. In aerobic refrigerated storage, the most common factor causing spoilage is the bacteria of the Pseudomonas species. These microbes, while growing, produce proteases and lipases which catalyze protein and lipid degradation reactions. The consequence of such changes is the release of peptides, fatty acids, and other decomposition products which cause unfavourable changes in meat colour, taste, and odour.[2] On the other hand, in the refrigerated meat stored in vacuum packages or a modified atmosphere, typical spoilage is generally related to the considerable growth of psychrotolerant lactic acid bacteria.[1,3,4] These bacteria may also produce various components antagonistic to the development of other microorganisms present in this product, and inhibit their growth through lowering the environment pH.

Increasing the storage life of minced meat requires either the use of approved preservatives, capable of inhibiting excessively fast microbial growth, or freezing, and frozen storage. At present, of large interest for the meat industry are mostly those natural substances, which are safe for consumers, and, in particular, lactic acid and its salts.[5,6,7,8] The principal role of these substances in meat and meat products is to inhibit the microflora growth rate through lengthening the lag phase. In the hydrous environment and in the buffer environment of meat and its products, lactates dissociate into metal cations and lactic anions. The latter, as a result of the reversal in dissociation of a weak acid, are largely transferred into an undissociated and therefore, more active form of lactic acid. This acid, in its undissociated form, can penetrate through the cell membrane to the microbial cytoplasm and as a result of its growing concentration, inhibit their development.[7,9] Apart from the bacteriostatic activity, lactates can also affect the stabilization of products, lower the water activity, and show antioxidant properties.[10,11,12] The level of inhibitory effect on microbial growth depends not only on their influence in lowering the environment pH but also on the types of the microbes and environmental reactions, including the specific effect of the lactic ion.[7,8] Of all the lactates, the most commonly used to preserve meat products is the sodium lactate. It inhibits bacterial growth and extends the refrigerated and frozen storage life of ground pork,[13] and the vacuum packaged meat products.[9] Sallam and Samejima[14] showed that the 3% addition of this lactate to vacuum packaged minced beef causes significant inhibition of its microflora, and an extension by 7 days of the shelf life of the product when stored at 2°C.

Nowadays, many readily-available preservatives are used, but at the same time, there is often little or nothing known about their antibacterial efficiency. This concerns, among others, a preservative of potassium lactate and sodium diacetate. Therefore, the objective of the study was to assess its effect on the microbial and sensory quality of vacuum-packaged minced beef stored at two different refrigeration temperatures.

MATERIALS AND METHODS

The Preservative Used

Used for the preservation of meat was Protec K-DI preparation (manufactured by Dutch Protein Services) which is a 59.2–63.5% solution of potassium lactate and sodium diacetate (the content of sodium diacetate in the solution is 4–5%) of pH 6.0–7.5 and density 1.3 g ml⁻¹.

Material and Its Preparation

The raw material was the meat of class 0–2—meat taken from the legs of 6 beef sides of 6-year-old cattle—which after standard slaughtering and cooling was stored for 5 days in cold storage at 0–2°C. Samples for the tests were prepared in industrial conditions on the company's processing line. The beef was ground through a 3 mm grinder plate. The ground meat, weighing 45 kg, was carefully mixed and divided into 3 equal parts of 15 kg, one of which was the control sample (with no preservative added), while the remaining two were used to make two variants of test samples, by mixing them for ten minutes in a Spomasz (Poland) M-450P mixer with 0.65% and 1.3% of the preservative, respectively. The meat samples were then vacuum-packaged on a Variovac Primus (Germany) packaging machine, in 200 g portions, with a polyamide/polyethylene (PA/PE) laminate allowed to come in contact with the food products by the EC 90/128/EEC. Immediately after packaging, the material was brought in plastic containers filled with ice to our laboratory in Krakow and stored for tests at two different refrigeration temperatures, i.e., at 0–1° and 5–6°C.

Determination of pH

Initially and after 2, 4, 6, 8, 10, 13, 15, 17, 20, and 22 days of storage representative ground beef samples from each treatment were homogenized with distilled water using a blender RW 11 basic (IKA-Werke GMBH & Co. KG, Germany) for 30 sec (high speed). The pH of prepared homogenates was determined with Hanna Instruments (Model HI 9025) pH meter equipped with a combinet electrode.

Microbiological Analyses

Microbial evaluation of the minced beef was performed initially and thereafter at regular intervals up to 22 and 13 days of storage at 0–1°C and 5–6°C, respectively. For all microbiological counts, 10 g samples from each pack were aseptically weighed into sterile lab bags with 90 ml 0.1% peptone water (Merck) and then mixed for 2 min in a laboratory stomacher (Seward, UK). From the 10⁻¹ dilution, other decimal dilutions were prepared in peptone water and its aliquots (1.0 ml) were plated out into appropriate media using the pour plate technique.

The total bacteria count (TBC) was cultivated on a Plate Count Agar (PCA, Difco Laboratories) and enumerated after incubation at 30°C for 48 h. The lactic acid bacteria count (LAB) was determined using a pour plate method with de Man, Rogosa and Sharpe (MRS) agar (Merck, Germany) as the medium.[15] Plates were incubated at 30°C for 48 h. Bacteria belonging to the Enterobacteriaceae were enumerated in Violet Red Bile Dextrose (VRBD) agar (Merck) after incubation at 37°C for 48 h.[16] The count of Brochothrix thermosphacta was evaluated using Streptomycin Thallous Acetate Actidione (STAA) agar (Merck) after the plates incubation at 30°C for 48 h.[17] All samples were analysed in duplicate and the average was taken for statistical analysis. Analyses were carried out on the materials from each pack separately. All bacterial populations were determined as the log colony forming units (cfu g⁻¹).

Sensory Evaluation

Sensory properties of raw vacuum-packaged minced beef during refrigerated storage were evaluated by a six-member panel appropriately trained and tested in sensory sensitivity. Training sessions were concluded when individual scores did not vary by more than 1 unit from the mean score and the panelists were familiar with the evaluation system. Representative samples from stored meat were randomly selected and served on porcelain plates in the laboratory (open area). Panel members were asked to evaluate the freshness grade using a 5-point scale—each attribute being scored from 1 to 5 points depending on specifications of sensory quality. The following properties were evaluated: colour, flavour, appearance and consistence. The overall sensory quality scores 5, 4, 3, 2, and 1 corresponded to the beef mince qualities evaluated as very good, good, acceptable, unacceptable, and bad, respectively.

Statistical Analysis

Data were analysed statistically with the CSS Statistica Package (Stat Soft, Tulsa, OK, USA). The significance of differences between means was determined by variance analysis (ANOVA) at the 5% confidence level. The evaluation of the relationships between temperature (0–1° or 5–6°C), storage times (0, 2, 4, 6, 8, 10, 13, 15, 17, 20, or 22 days), level of preservative in samples (0, 0.65, or 1.3%), microbial growth and sensory quality was carried out by computing the relevant correlation coefficients at p < 0.05.

RESULTS AND DISCUSSION

Chemical Composition and pH

The preservative added to the minced beef at the amounts of 0.65 and 1.3%, had no statistically significant effect (p < 0.05) on its basic chemical composition. Therefore, all the three tested groups, control sample included, contained the mean percentages of protein—20.76, fat—2.59, and water—75.03. Some differences were found only in the content of ash which increased slightly with the growing concentration of the preservative, reaching 1.35% for samples with its 1.3% addition. The preservative's effect on the initial pH values of minced beef before storage, as well as changes occurring during storage at both refrigeration temperatures (Table 1), was also statistically insignificant (p < 0.05). The pH values were 5.67–5.70 at the start of storage (day 0), 5.70–5.80 after 22 days of storage at 0–1°C, and 5.54–5.62 after 17 days at 5–6°C. The pH values at the beginning of storage were within the normal range for fresh beef.[3] The slight decrease in the pH of samples at temp. 5–6°C may be attributed to the predominance of lactic acid bacteria.

Table 1 Changes in the pH of vacuum-packaged minced beef during refrigerated storage

Storage duration(days)	Storage temperature
	0–1°C			5–6°C
	Level of preservative in beef mince (%)
	0	0.65	1.3	0	0.65	1.3
0	5.68 ± 0.06	5.68 ± 0.03	5.70 ± 0.07	5.67 ± 0.05	5.68 ± 0.03	5.70 ± 0.07
2	5.73 ± 0.05	5.76 ± 0.01	5.74 ± 0.00	5.77 ± 0.01	5.75 ± 0.05	5.75 ± 0.01
4	5.69 ± 0.01	5.77 ± 0.01	5.77 ± 0.01	5.94 ± 0.06	5.76 ± 0.05	5.74 ± 0.00
6	5.77 ± 0.01	5.80 ± 0.02	5.78 ± 0.05	5.87 ± 0.06	5.80 ± 0.06	5.77 ± 0.01
8	5.80 ± 0.00	5.79 ± 0.02	5.82 ± 0.06	5.66 ± 0.05	5.80 ± 0.03	5.78 ± 0.04
10	5.83 ± 0.05	5.78 ± 0.04	5.77 ± 0.01	5.65 ± 0.01	5.71 ± 0.06	5.73 ± 0.06
13	5.73 ± 0.05	5.79 ± 0.05	5.77 ± 0.01	5.43 ± 0.07	5.63 ± 0.05	5.68 ± 0.01
15	5.69 ± 0.03	5.78 ± 0.02	5.76 ± 0.01	5.32 ± 0.05	5.59 ± 0.05	5.67 ± 0.05
17	5.82 ± 0.01	5.82 ± 0.05	5.80 ± 0.05	5.62 ± 0.00	5.54 ± 0.00	5.54 ± 0.04
20	5.82 ± 0.02	5.80 ± 0.06	5.80 ± 0.00	nd	nd	nd
22	5.70 ± 0.00	5.80 ± 0.01	5.80 ± 0.01	nd	nd	nd
The values are given as mean ±SD for three measurements; nd: not determined.

Microbial Growth

In general, the counts for the two replicate beef mince samples taken at each sampling time were in reasonable agreement with one another. Figures 1 to 4—based on the mean of the replicate counts—show the growth curves measured after the storage of the ground beef for: total bacteria count, lactic acid bacteria, Brochothrix thermosphacta and Enterobacteriaceae, respectively. Before storage, minced beef presented the following microbial contamination: total bacteria count 4.7 log cfu g ⁻¹, lactic acid bacteria 3.9 log cfu g⁻¹, Brochothrix thermosphacta 3.2 log cfu g⁻¹, microorganisms of the Enterobacteriaceaefamily, below 2.5 log cfu g⁻¹. The process of adding preservatives and mixing meat, caused the number of the Enterobacteriaceae family bacteria to go up to 2.9 log cfu g⁻¹. Escherichia coli was absent in the initial material.

Figure 1 Changes in the total bacteria count in vacuum-packaged minced beef containing potassium lactate and sodium diacetate preservative during storage at 0–1°C (a) and 5–6°C (b).

During the vacuum storage of all the minced beef samples, the growth in the number of all the mentioned microorganisms was observed, which was significantly (p < 0.05) dependent on the temperature, storage time and the addition of the preservative used. The numbers of the total and lactic acid bacteria increased exponentially at the same rate while Brochothrix thermosphacta growth was significantly slower. In samples containing a 1.3% addition of the preservative the bacteria remained in the lag phase of growth at different periods of time depending on the temperature (Figure 1), i.e., for 6 days at 0–1°C (Figure 1) and for 4 days at 5–6°C. In the case of the control samples, however, these periods were 4 and 2 days respectively. After longer storage, the preservative presence in the meat significantly inhibited (p < 0.05) the bacterial growth. In the control samples, total bacteria counts reached 7 log cfu g⁻¹ (the level generally initiating meat spoilage) after 4 days of storage at 5–6°C, and after 8 days at 0–1°C (Figure 1). The 0.65% concentration of the preservative extended these periods, at the same temperatures, to 6 and 13 days, respectively, whereas a 1.3% addition resulted in further extension of these periods to 7 and 17 days, respectively. It should be stressed that this antimicrobial effect was stronger at the lower storage temperature (0–1°C): to reach a total bacteria count of 7 log cfu g⁻¹, the meat with a 1.3% preservative concentration took twice as long as the meat to which no preservative was added. Also, according to Egbert et al.,[18] the bacterial growth in low-fat carrageenan-based patties was reduced through the use of a 2 or 3% potassium lactate concentration with no deleterious effect on the sensory properties of the low-fat minced beef. Gill and Newton[19] indicated that the inhibitory effect of lactic acid on microbial growth in meat was the result of a decreased pH. However, the addition of such undissociated acids as potassium or sodium lactate has little effect on the pH value.

The bacteriostatic effect of the preservative in beef mince has also been found in reference to other microbial groups studied. Most susceptible to it were the Enterobacteriaceae family bacteria which, in tests with a 1.3% content of the preservative, maintained their number at the same level for a 3-week storage period at 0–1°C, and for 10 days at 5–6°C (Figure 2). The effect of this preservative at a 0.65% concentration in meat on the growth repression of these microorganisms, compared with their growth in control samples, was significantly lower (p < 0.05), particularly at the higher storage temperature (5–6°C).

Figure 2 Changes in the Enterobacteriaceae count in vacuum-packaged minced beef with potassium lactate and sodium diacetate preservative during storage at 0–1°C (a) and 5–6°C (b).

The bacteria which may grow under the conditions of refrigerated meat storage and adversely affect sensory value, is Brochothrix thermosphacta.[16,20] The studies show that the growth of this bacteria in vacuum-packaged beef mince is very slow and significantly inhibited (p < 0.05) by the addition of the preservative (Figure 3). The bacteria reached a maximum number of around 5 log cfu g⁻¹. At the temperature 0–1°C it occurred after around 10 days in the control samples, while in their counterparts with the addition of the preservative, after 22 days. At the temperature 5–6°C, these changes in the stored meat were faster, but the maximum number of Brochothrix thermosphacta did not exceed 5 log cfu g⁻¹. Also, Sakala et al.,[21] when analysing the microbial changes which occurred during the refrigerated storage of the vacuum-packaged beef, showed that these bacteria proliferated in it only up to 5 log cfu g⁻¹ in number. This may mean that they are incapable of competing with lactic acid bacteria in refrigerated, vacuum-packaged beef. Since Brochothrix thermosphacta does not grow anaerobically in meat below pH 5.8,[22] the lowering of the pH by lactobacilli is likely to have some effect on the repression of the B. thermosphacta growth. There may be additional factors, such as oxygen permeability of the film, substrate availability, and the production of antimicrobial agents, which also attributed to the limited growth of Brochothrix spp.

Figure 3 Changes in the Brochothrix thermosphacta count in vacuum-packaged minced beef containing potassium lactate and sodium diacetate preservative during storage at 0–1°C (a) and 5–6°C (b).

The dominating microflora in the vacuum-stored beef mince were the lactic acid bacteria (LAB). Such an effect was expected and stayed in agreement with the former data given by many authors for vacuum stored meat.[1,16,20,21,23] These microflora reached 8–9 log cfu g⁻¹ in all conditions of the test, after a period of time dependent on temperature as well as on the addition of the preservative (Figure 4). As expected, LAB growth was accelerated by storage time at 5–6°C and thus was greater than during storage at 0–1°C. In the control samples, significant growth (p < 0.05) was observed after 4 and 6 days of storage at the respective temperatures of 5–6°C and 0–1°C, and in the case of samples with a preservative added, after 4–6 and 8–10 days at the same temperatures. The addition of 0.65% and 1.3% of preservative to the meat significantly inhibited (p < 0.05) the LAB growth during storage at both refrigeration temperatures. Although development of lactic microflora markedly extends the storage life of meats packaged under vacuum or in a modified atmosphere with increased CO₂,[23,24] LAB can cause spoilage of beef stored anaerobically due to the development of objectionable odours.[25,26]

Figure 4 Changes in the lactic acid bacteria count in vacuum-packaged minced beef with potassium lactate and sodium diacetate preservative during storage at 0–1°C (a) and 5–6°C (b).

Generally, very high correlation coefficients (r₁ at temp. 0–1°C and r₂ at 5–6°C, respectively) at the level p < 0.05 were found between the storage time of the minced beef and the counts of total bacteria (r₁ = 0.96 and r₂ = 0.96), LAB (r₁ = 0.96 and r₂ = 0.88), Brochothrix thermosphacta (r₁ = 0.91 and r₂ = 0.95), and Enterobacteriaceae (r₁ = 0.65 and r₂ = 0.81).

Sensory Properties

Through the results of the sensory evaluation it has been found that the desired quality of vacuum-packaged beef mince is made significantly longer (p < 0.05) when stored at 0–1°C than at 5–6°C (Table 2). Irrespective of the addition of the preservative, at the lower temperature (0–1°C) it obtained scores nearing “very good” as late as 8 days into storage, and at the higher temperature after around 4 days. During further storage, samples with the addition of the preservative received at both temperatures higher scores than the control samples, and their storage life according to the sensory panelists was 13 days at temperatures of 0–1°C and 8 days at 5–6°C. The stored meat effectively preserved its desired colour, while the addition of the preservative, especially 1.3%, by the sixth day had caused a slight change of the natural raw meat flavour toward the characteristic sour odour. This odour, however, totally disappeared after a short thermal processing of the samples and the flavour typical for fresh meat was restored. Very high negative correlation coefficients (r₁ at temperature 0–1°C and r₂ at 5–6°C) at the level p < 0.05 were found between the sensory properties of stored minced beef and storage time (r₁ = −0.95 and r₂ = −0.96), TBC (r₁ = −0.91 and r₂ = −0.96), LAB (r₁ = −0.87 and r₂ = −0.95), Brochothrix thermosphacta (r₁ = −0.81 and r₂ = −0.89), and Enterobacteriaceae (r₁ = −0.78 and r₂ = −0.93).

Table 2 Changes in overall sensory quality of vacuum-packaged minced beef during refrigerated storage

Storage duration(days)	Storage temperature
	0–1°C			5–6°C
	Level of preservative in beef mince (%)
	0	0.65	1.3	0	0.65	1.3
0	5.0 ± 0.0	5.0 ± 0.0	5.0 ± 0.0	5.0 ± 0.0	5.0 ± 0.0	5.0 ± 0.0
2	5.0 ± 0.0	5.0 ± 0.0	5.0 ± 0.0	5.0 ± 0.0	5.0 ± 0.0	5.0 ± 0.0
4	5.0 ± 0.0	5.0 ± 0.0	5.0 ± 0.0	4.5 ± 0.0	4.9 ± 0.2	4.7 ± 0.4
6	4.8 ± 0.3	4.8 ± 0.2	4.9 ± 0.2	3.8 ± 0.4	4.0 ± 0.2	3.9 ± 0.3
8	4.0 ± 0.3	4.6 ± 0.3	4.6 ± 0.4	3.4 ± 0.3	4.0 ± 0.3	3.9 ± 0.4
10	3.5 ± 0.5	4.4 ± 0.5	4.3 ± 0.3	2.9 ± 1.3	3.6 ± 0.3	3.6 ± 0.3
13	2.9 ± 0.6	4.0 ± 0.4	3.9 ± 0.2	2.6 ± 0.2	2.9 ± 0.4	2.8 ± 0.5
15	2.8 ± 0.2	3.4 ± 0.3	3.5 ± 0.3	1.6 ± 0.4	2.3 ± 0.3	2.6 ± 0.5
17	2.6 ± 0.4	3.1 ± 0.5	3.0 ± 0.4	nd	nd	nd
20	2.3 ± 0.5	2.2 ± 0.6	2.2 ± 0.6	nd	nd	nd
22	1.6 ± 0.4	1.7 ± 0.3	1.6 ± 0.4	nd	nd	nd
The values are given as mean ±SD for six evaluations; nd: not determined.

CONCLUSIONS

The addition of a potassium lactate and sodium diacetate preservative in the amounts of 0.65% and 1.3% to minced beef had a significant inhibitory effect (p < 0.05) on the bacterial growth rate during refrigerated storage under vacuum and improved microbial stability of stored meat. The bacteria inhibitory effect depended on the preservative's concentration, storage temperature and storage time. However, there was a significant difference in microbial growth rate between the control and samples with preservative addition, although these differences were less evident when evaluated by sensory analyses. Antibacterial action of the preservative was significantly higher (p < 0.05) at temperatures 0–1°C than at 5–6°C. The use of potassium lactate and sodium diacetate enables the extension of vacuum-packaged minced beef shelf-life by about 13 days (as apposed to 7 days for the control), provided it is of good initial microbiological quality and has a storage temperature of 0–1°C. Therefore, the study carried out has proved that the potassium lactate and sodium diacetate preservative can be useful in industrial minced meat production as an effective inhibitor of microbial growth.