Effect of the addition of starter cultures to ground meat for hamburger preparation

The aim of this work was to study the effect of a selected lactic acid bacteria formulation on the microbiological characteristics and colour of beef hamburgers stored at different temperatures. All hamburgers were evaluated on day-0, day-1, day-3, and day-5 for the following microbiological parameters (Staphylococcus spp., enterococci, Lactococcus spp., Lactobacillus spp., total mesophilic aerobes, Pseudomonas spp., total coliforms) according to standard methods and for colorimetric measurements performed with Colorimeter Digital Color Picker for iOS 10, under a 6500K light, with the CIELab system. All data (geometric mean for microbiological data) were elaborated with GraphPad InStat, 3.0b and GraphPad Prism 6.0d for Mac OS X. Twoway analysis of variance (ANOVA) followed by the Tukey’s multiple comparisons test was performed. The analysis of the colour proved that the addition of Lactic Acid Bacteria (LAB) does not affect the natural colour of ground meat, avoiding the risk of hiding the spoilage or fastening it. The addition of the starter has preserved the colour stability throughout the preservation period, with the same behaviour both in the hamburgers stored at 4°C and in those at 10°C after thermal abuse or not. In conclusion, the application of the proposed LAB formulation maintains hamburgers quality standards and can be a potential tool to increase their shelf-life.


Introduction
Meat products and meat preparations are very sensitive to external factors, such as temperature, and have a very sensitive substrate for the biochemical processes that occur during storage. These products are characterized by a short shelf-life and are good substrates for microbial growth. 1,2 In food industry starter cultures are currently used in a wide variety of products in order to transform raw prime materials and to safeguard their microbial and sensory quality. 3,4 Most of the commercially available starter cultures contains different strains of the so-called Lactic Acid Bacteria (LAB). LAB can be divided in two groups: heterofermentative LAB of the Carnobacterium, Leuconostoc and Weissella genera which are usually more involved in meat spoilage and homofermentative LAB of the Lactobacillus, Lactococcus and Pediococcus genera. Therefore, commercially available meat starter cultures for meat products and preparations exclusively belong to the latter two. 5 Heterofermentative LAB species produce significant amounts of non-desirable fermentation end products, such as ropy slime, CO 2 gas, ethanol, acetic acid, butanoic acid and acetoin while homofermentative LAB produce almost exclusively lactic acid from fermentable carbohydrates present in meats, which is relatively mild and palatable. 6 However, it has been reported that Lactobacillus spp., under particular conditions, may also produce significant amounts of acetic acid, ropy slime and discolouration (greening) of meats. 7,8 Lactic acid bacteria have been shown to inhibit the growth and decrease the load of foodborne pathogens such as Escherichia coli, Salmonella spp. and Listeria monocytogenes. 9,10 They can create a competitive environment through several different mechanisms, including the production of bacteriocins, hydrogen peroxide and weak acids such as acetic and lactic acid. 11 The antagonistic action of LAB takes place when they compete with other non-desirable bacteria for a common niche, when an extracellular agent is produced, or when the environment is modified to inhibit the growth of the other microorganism. 12 To be acceptable for use by the meat industry, the addition of LAB cultures must not change the sensory properties of ground beef or cause spoilage masking within the package of the product. 13 The aim of this study was to analyse the effect of the addition of LAB on the normal microflora present in ground beef used for the production of hamburgers and on the colour of the beef hamburgers stored at different temperatures including temperature abuse.

Material and Methods
Two batches of hamburgers (average weight of about 100g each) were prepared in the experimental processing plant of the laboratory of inspection of foods of animal origin of the department of veterinary medicine, University of Perugia. Hamburgers were prepared according to the classical method of production: minced beef was mixed with NaCl (20g/kg), prepackaged egg mixture (300ml/kg), skim milk (250ml/kg), bread crumbs and hard cheese (2g/kg), without the addition of food additives. One batch was prepared with the addition of LAB (Lactococcus lactis ssp. lactis, strain 340; L. lactis ssp. lactis, strain 16; Lactobacillus casei ssp. casei, strain 208 and Enterococcus faecium strain 614 in a ratio lactococci:lactobacilli:enterococci of 2:1:1; level of inoculum 10 7 cfu g -1 ) and one without, in three replicates done in three different days. The morphological, biochemical and physiological characterisation, the growth curves at several temperatures, including refrigeration conditions, the acidifying activity of these four bacterial strains and their ability to improve palatability of dry salami along with safety considerations for the commercial probiotic E. faecium UBEF-41 have been reported by the authors in previous papers. 14  37°C in air for 24 hrs) at 24 hours was approximately 1 x 10 9 cfu ml -1 .
For each batch, a subset of samples was stored under proper conditions (4°C) and the other at 40°C (temperature abuse conditions, to mimic inadequate storage conditions after purchase by consumer) both for 5 hours. After 5 hours, each subset was further divided into two subgroups: one maintained at 4°C and the other at 10°C, in order to simulate both a good functioning and a bad functioning refrigerator. All hamburgers were evaluated on day-0, day-1, day-3, and day-5 for the following microbiological parameters (Staphylococcus spp., enterococci, Lactococcus spp., Lactobacillus spp., total mesophilic aerobes, Pseudomonas spp., total coliforms) according to standard methods; in brief, at each analyses time, 25 g of meat were homogenised in a sterile bag with 225 ml of pepton water (PW, Oxoid, Basingstoke, Hampshire, UK) by using a stomacher. 10-fold dilutions were prepared by using sterile tubes with 9 ml of Maximum Recovery Diluent (MRD, Oxoid Moreover, colorimetric measurements were performed with Colorimeter -Digital Color Picker for iOS 10, under a 6500K light, with the CIELAB system, by taking three readings for each sample. The arithmetic means within each sampling was computed, subsequently all data (geometric mean for microbiological data) were elaborated with GraphPad InStat, 3.0b and GraphPad Prism 6.0d for Mac OS X. Twoway analysis of variance (ANOVA) followed by the Tukey's multiple comparisons test was performed.

Results
All the microbiological analysis results are reported in Table 1 (hamburgers stored at 4°C) and in Table 2 (hamburgers stored at 10°C).
The initial total aerobic mesophilic count was 6.86 cfu g -1 in the batch with LAB and 4.19 cfu g -1 in the batch without LAB. The initial enterococci count was 6.64 cfu g -1 and 2.10 cfu g -1 respectively. The initial Lactococcus spp. count was 6.93 cfu g -1 and 4.11 cfu g -1 . The initial Lactobacillus spp. count was 7.08 cfu g -1 and 4.15 cfu g -1 .
After 5 days, in the batch stored immediately at 4°C the total aerobic mesophilic count was 6.56 cfu g -1 in the batch with LAB and 5.88 cfu g -1 in the batch without LAB. The enterococci count was 6.29 cfu g -1 and 2.36 cfu g -1 respectively. The Lactococcus spp. count was 6.23 cfu g -1 and 3.80 cfu g -1 . The Lactobacillus spp. count was 6.91 cfu g -1 and 6.23 cfu g -1 . The total coliforms count was 2.00 cfu g -1 for both. The Pseudomonas spp. count was 6.11 cfu g -1 and 6.46 cfu g -1 . The Staphylococcus spp. count was 3.55 cfu g -1 and 3.51 cfu g -1 .
After 5 days, in the batch stored at 4°C after 5 hours of temperature abuse (40°C) the total aerobic mesophilic count was 6.50 cfu g -1 in the batch with LAB and 5.99 cfu g -1 in the batch without LAB. The enterococci count was 6.32 cfu g -1 and 2.70 cfu g -1 respectively. The Lactococcus spp. count was 6.37 cfu g -1 and 4.28 cfu g -1 . The Lactobacillus spp. count was 7.06 cfu g -1 and 6.24 cfu g -1 . The total coliforms count was 2.26 cfu g -1 and 1.33 cfu g -1 . The Pseudomonas spp. count was 6.39 cfu g -1 and 6.51 cfu g -1 . The Staphylococcus spp. count was 3.68 cfu g -1 and 3.80 cfu g -1 .
After 5 days, in the batch stored immediately at 10°C the total aerobic mesophilic count was 6.63 cfu g -1 in the batch with LAB and 6.01 cfu g -1 in the batch without LAB. The enterococci count was 6.35 cfu g -1 and 2.16 cfu g -1 respectively. The Lactococcus spp. count was 6.42 cfu g -1 and 4.04 cfu g -1 . The Lactobacillus spp. count was 7.46 cfu g -1 and 6.23 cfu g -1 . The total coliforms count was 2.30 cfu g -1 and 2.20 cfu g -1 . The Pseudomonas spp. count was 6.27 cfu g -1 and 6.37 cfu g -1 . The Staphylococcus spp. count was 3.43 cfu g -1 and 3.65 cfu g -1 .
After 5 days, in the batch stored at 10°C after 5 hours of temperature abuse (40°C) the total aerobic mesophilic count was 6.88 cfu g -1 in the batch with LAB and 6.01 cfu g -1 in the batch without LAB. The enterococci count was 6.39 cfu g -1 and 2.10 cfu g -1 respectively. The Lactococcus spp. count was 6.50 cfu g -1 and 4.31 cfu g -1 . The Lactobacillus spp. count was 7.92 cfu g -1 and 6.27 cfu g -1 . The total coliforms count was 1.33 cfu g -1 and 0.67 cfu g -1 . The Pseudomonas spp. count was 6.12 cfu g -1 and 6.43 cfu g -1 . The Staphylococcus spp. count was 3.34 cfu g -1 and 3.53 cfu g -1 .
Staphylococcus aureus was never isolated in any sample.
The results of the colorimetric analysis are shown in

Discussion
The application of starter cultures and bio protective microbial cultures is a much discussed innovative and sustainable technology for improving the microbiological safety and overall quality of meat products, in particular pre-packaged cold cuts. 5 It could represent an alternative to chemical preservatives or to a second pasteurisation step after packaging which both have a negative sensory impact. 18 Starter cultures allow a standardization of the product quality and considerably reduce the risk of product defects. However, it should be kept in mind that starter cultures cannot replace good manufacturing practice which besides the selection of the appropriate raw materials with acceptable hygienic parameters also includes the implementation and control of appropriate processing conditions. 19,20 The official authorities of food control and the big retail chains often look with suspicion at high microbial counts in meat preparations, regardless of the responsible microflora. The German Society for Hygiene and Microbiology, e.g., recommends a maximum of 5x10 6 cfu g -1 . 21 However, many of the pre-packaged sliced cold cuts display 10-100 times higher counts at the end of their indicated shelf lives without being recognized as spoiled by sensory panels. On the other hand, unpleasant tastes and smells (not fresh, sour) are often associated with high LAB counts. 22 In minced meat, LAB can be added during the grinding process or during the trim-ming prior to grinding. 11 Our study confirms that the addition of LAB in hamburgers produced with high hygienic parameters helps to maintain the microbiology quality of the products. In addition, it is important to note that the populations of LAB in the inoculated samples did not increase over time, avoiding the risk of a bacterial over population. On the other hand, common spoilage microorganisms such as Pseudomonas spp. did not increase as usual in temperature abused foods. 23,24 Article  Data on the sensory acceptance by the consumer of non-fermented meat products with addition of LAB are scarce. It is important that proposed food additives not mask factors that consumers would typically associate with a spoiled product, so that they do not consume a product that would normally be discarded. 10 The analysis of the hamburgers' colour performed in our study confirms that the addition of LAB does not affect the natural colour of ground meat, avoiding the risk of hiding the spoilage or fastening it. The addition of the starter has preserved the colour stability throughout the preservation period, with the same behaviour both in the hamburgers stored at 4°C and in those at 10°C after thermal abuse.

Conclusions
Meat and meat products provide a concentrated source of protein of high biological value and can make a valuable contribution to human diets. However, they are also highly perishable foods which rapidly spoil and may even allow the growth of food-  borne pathogenic microorganisms if no suitable preservative actions are taken. The addition of LAB beside good manufacturing practices can help to maintain the safety of these products without altering their sensory quality and the consumer perception.