Evaluation of proteolytic and antagonistic activity of dairy lactic acid bacteria
using skim milk as a model system
Andrea Caridi, Felice Panebianco
Department of AGRARIA, Mediterranea University of Reggio Calabria, Feo di Vito, 89122 Reggio Calabria (Italy)
1. INTRODUCTION
In food microbiology, the study of microbial interactions may be useful to select both starter and adjunct cultures, allowing the prior evaluation of their technological effects. In dairy production, the interaction between lactic acid bacteria (LAB) and undesirable microorganisms may be evaluated both in vitro and in vivo. However, not always the performance observed in vitro is exactly corresponding to that observed during cheese-making. Our aim was to evaluate the reliability of skim milk as a model system to study the proteolytic activity of LAB and to predict their antagonistic activity against Pseudomonas spp. and Escherichia coli in mozzarella cheese.
2. MATERIALS AND METHODS
2.1 Evaluation of proteolytic activity
The proteolytic activity of over 200 strains of LAB - previously isolated from different food matrices (cheese, olive, sourdough) - was evaluated in Skim Milk Agar (El-Gaish et al., 2010) and in MRS-Milk Agar (Pereira et al., 2001). Plates were incubated at
37C for 24 hours (Skim Milk Agar) and at 30C for 16 hours in
anaerobic conditions (MRS-Milk Agar). Proteolytic activity of LAB was assessed by the presence of clear/white halos around the inoculum spot (Figure 1). Regarding mozzarella cheese, the strains that exhibited medium or high proteolytic activity were excluded to avoid the loss of elasticity.
2.2 Antagonistic activity in skim milk
The remaining strains were tested in skim milk for their antagonistic activity against P. putida, P. fluorescens and E. coli. Overnight cultures of LAB were used for the inoculation (1% v/v) of 15 ml of skim milk, previously inoculated (0.2% v/v) with 24 h precultures of the target bacteria. Co-cultures were
incubated at 38C for 4 hours followed by a storage at 10C for
5 days, to simulate the cheese-making and the storage conditions of mozzarella cheese. LAB were determined on MRS Agar and M17 Agar; Pseudomonas spp. were determined on Pseudomonas Agar Base with CFC selective supplement; E. coli viable cells were determined on Violet Red Bile Agar.
2.3 Antagonistic activity in mozzarella cheese
The strain that exhibited the best global performance was used as adjunct culture in mozzarella cheese production. It was inoculated in milk (1% v/v) together with a commercial starter culture used for mozzarella cheese production. Samples of
mozzarella cheese were stored at 10C for 7 days.
3. RESULTS
3.1 Evaluation of proteolytic activity
11 strains of LAB exhibited low proteolytic activity in both
media.
3.2 Antagonistic activity in skim milk
All the 11 LAB exhibited good antagonistic activity against of P. putida, P. fluorescens and E. coli (Table 1).
Table 1. Antagonistic activity of LAB in skim milk.
* Control sample
ATCC = American Type Culture Collection
3.3 Antagonistic activity in mozzarella cheese
The strain B155, identified as Lactococcus lactis, exhibited the lowest proteolytic activity and good inhibition activity against all the target bacteria. Consequently, it was used as adjunct
culture in mozzarella cheese. After 7 days at 10C, the reduction
of the number of E. coli and Pseudomonas spp. in cheese inoculated with strain B155 was 1.3 log CFU/g and 0.3 log CFU/g, respectively (Table 2); the reduction of undesirable bacteria was similar also in the governing liquid (1.13 log CFU/g for E. coli; 0.11 log CFU/g for Pseudomonas spp.).
Strain ID Origin Identification Antagonistic activity in skim milk (log CFU/ml)
P. fluorescens P. putida E. coli
LAB B155 Cheese Lactococcus lactis 1.70 2.58 2.83
LAB B284 Olive Lactobacillus spp. 1.30 1.60 2.34
LAB B288 Olive Lactobacillus spp. 1.48 1.90 >5.48
LAB B289 Olive Lactobacillus spp. 1.85 1.95 1.00
LAB B321 Olive Lactobacillus spp. 1.60 2.08 <1.00
LAB B322 Olive Lactobacillus spp. <1.00 2.20 1.00
LAB B339 Olive Lactobacillus spp. >3.48 >3.48 >5.48
LAB B351 Olive Lactobacillus spp. <1.00 3.00 2.54
LAB B393 Olive Lactobacillus delbrueckii subsp. delbrueckii 1.70 2.30 2.30 LAB B499 Sourdough Lactobacillus sanfranciscensis <1.00 2.51 2.78 LAB B500 Sourdough Lactobacillus sanfranciscensis 1.48 1.90 1.78
Ps B128* Cheese Pseudomonas fluorescens 6.05 -
-Ps B141* Cheese Pseudomonas putida - 6.78
-E. coli B65* ATCC Escherichia coli - - >5.48
References
• El-Ghaish, S., Dalgalarrondo, M., Choiset, Y., Sitohy, M., Ivanova, I., Haertlé, T., & Chobert, J. M. (2010). Screening of strains of lactococci isolated from Egyptian dairy products for their proteolytic activity. Food Chemistry, 120(3), 758-764.
• Pereira, C. I., Crespo, M. B., & San Romao, M. V. (2001). Evidence for proteolytic activity and biogenic amines production in Lactobacillus curvatus and L. homohiochii.
International Journal of Food Microbiology, 68(3), 211-216.
Figure 1. A proteolytic LAB in Skim Milk Agar (on the left) and in MRS-Milk Agar (on the right).
Count (log CFU/g)
Standard cheese-making Cheese-making with adjunct culture
Cheese Day 1 Day 7 Day 1 Day 7
Pseudomonas spp. 1.28 6.50 <1.00 6.17
E. coli <1.00 3.47 <1.00 2.19
Governing liquid Day 1 Day 7 Day 1 Day 7
Pseudomonas spp. 1.85 7.71 1.30 7.60
E. coli 1.04 3.98 <1.00 2.85
Table 2. Microbiological counts of mozzarella cheese and governing liquid.
4. CONCLUSIONS
The reduction of Pseudomonas and E. coli in mozzarella cheese was slightly lower than that detected in skim milk. However, the use of skim milk as a model system should be emphasized, considering the advantages for dairy industry and the consequences for starter and adjunct culture selection.
