NEW APPROACH TO ASSESS LACTIC ACID BACTERIA
COMPATIBILITY DURING CHEESE-MAKING
Andrea CARIDI
Department of AGRARIA , Mediterranea University of Reggio Calabria,
Loc. Feo di Vito, 89124 Reggio Calabria, Italy
Compatibility tests constitute an effective way to assess interactions among lactic acid bacteria (LAB). It is obviously necessary to avoid strain combinations displaying mutually inhibitory properties and, on the other hand, it may be beneficial to combine strains based on their ability to coexist [1]. Working on antimicrobial activity of 755 wild LAB isolated from Egyptian dairy products [2], to accelerate the screening procedures to obtain starter, adjunct, and protective culture strains LAB of identical genus were examined by internal interaction: so each strain was applied as an inhibitor microorganism while another was taken as indicator microorganism. Compatibility among adjunct dairy LAB identified as Lactobacillus paracasei subsp.paracasei and Lactobacillus brevis and non-starter dairy microflora of the species Enterococcus durans, Enterococcus faecium, and Pediococcus spp. was
evaluated using the agar-spot method [3]. To screen LAB for associative growth in mixed cultivation, a tube containing 4 ml of 10% skim milk was inoculated with 1% of 2 different strains in pure and mixed culture: the size of the inoculum in the mixed culture was therefore at the 0.5% level for each strain. The cultures were incubated at 30 °C or 37 °C for 24 h, at which time the pH value of the culture was determined. Any interaction occurring in mixed cultures is reflected by acid production. The acidification of growth medium in batch culture is a good reflection of bacterial growth, which is why pH measurement is sometimes used to track growth [4]. Another screening assay to evaluate the compatibility between different LAB can be performed by using the plate diffusion technique [5]. Aliquots (35 μl) of cell-free supernatant obtained from the early stationary phase of the third subculture of the microorganisms grown in MRS broth were placed into holes (4 mm diameter) of MRS 1% agar plates with 109 CFU ml–1, 107 CFU ml–1,
and 105 CFU ml–1 with the indicator lactobacilli strains. The plates were incubated for 2 h at room temperature and then 48 h at 37 °C, when the inhibition
was assessed.
Mozzarella cheese (Fig. 1) has a shelf life of 5-7 days due to the fast growth of proteolytic Pseudomonas strains. Pseudomonas can grow in Mozzarella cheese modifying texture and reducing its shelf life; several strains of Pseudomonas are able to hydrolyse casein on skim milk (Fig. 2). Another factor that reduces its shelf life is the coliform occurrence; effectively, Escherichia coli can grow in Mozzarella cheese reducing its safe life and shelf life. Obviously, proteolysis and lipolysis control is of high importance in its preservation. Investigation on shelf life extension of Mozzarella cheese was recently focused on active packaging systems and antimicrobial peptides. However, in spite of the research carried out, at present no completely efficacious method is available to inhibit the fast microbial spoilage of Mozzarella cheese. In our opinion, utilization of antimicrobial microbes can be an innovative strategy to gain this result; so, our aim was to select adjunct cultures of Lactobacillus spp. possessing antagonistic activity against both Pseudomonas and E. coli [6]. Considering the ability to inhibit Pseudomonas growth and using standard culture media, we identified LAB strains possessing high, low, or absent activity (Fig. 3 a, b, c). However, after the identification of an antagonistic strain of LAB, it is necessary to control if this strain (adjunct culture) can coexist with the LAB used to control cheese-making (starter culture). Sometimes it is sufficient to perform a simple compatibility test in plate (Fig. 4); however, before to employ the two LAB - starter and adjunct culture - to produce Mozzarella cheese more frequently it is necessary to perform other compatibility tests.
To study the compatibility of starter cultures and adjunct cultures mixed to control the production of mozzarella cheese, a tube containing 10 ml of UHT milk was inoculated in triplicate with three different strains in pure and mixed culture: the size of the inoculum in the mixed culture was at the 5% level for the starter strain and at the 10% level for the adjunct strain. The cultures were incubated at 37°C and after 90, 180, 270, and 360 min minutes were analysed for pH and antioxidant activity, measured by analysing the radical scavenging activity using a spectrophotometric decolourization assay (ABTS) [7]. The evolution of the pH in pure and mixed cultures is reported in Tab. 1; it is evident that the interaction between the starter culture (B199) and each adjunct culture (B74 and B151) is positive, seeing as the pH decreases more quickly in the mixed cultures than in the single one. The evolution of the antioxidant activity in pure and mixed cultures is reported in Tab. 2; in this case, the interaction between the starter culture (B199) and each adjunct culture (B74 and B151) is not similarly evident, at least in the earliest hours of monitoring. Considering the advantages for dairy industry and the consequences for starter and adjunct culture selection deriving from the possibility to simply predict interaction among LAB during cheese-making, in our opinion the proposed methodology can be really useful.
[1] Geria M. and Caridi A. (2014). Methods to assess lactic acid bacteria diversity and compatibility in food. Acta Alimentaria, 43(1), 96-104.
[2] Ayad E.H.E. et al. (2004). Selection of wild lactic acid bacteria isolated from traditional Egyptian dairy products according to production and technological criteria. Food Microbiology, 21, 715-725.
[3] Spelhaug S.R. and Harlander S.K. (1989). Inhibition of food-borne bacterial pathogens by bacteriocins from
Lactococcus lactis and Pediococcus pentosaceus. International Journal of Food Protection, 52, 856-862.
[4] Kimoto-Nira H. et al. (2012). Interaction between Lactococcus lactis and Lactococcus raffinolactis during growth in milk: development of a new starter culture. Journal of Dairy Science, 95, 2176-2185.
[5] Maldonado N.C. et al. (2012). Lactic acid bacteria isolated from young calves. Characterization and potential as probiotics. Research in Veterinary Science, 92, 342-349.
[6] Caridi A. et al. (2015). New approach to extend shelf life of Mozzarella cheese using antimicrobial microbes. In: Multidisciplinary approaches for studying and combating microbial pathogens, Editor: A. Méndez-Vilas, 102-106, Publisher: BrownWalker Press.
[7] Virtanen T. et al. (2007). Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria. Journal of Applied Microbiology 102, 106-115.
pH
90 min
180 min
270 min
360 min
24 h
Lactococcus spp. strain B199
1
(starter culture)
6.22±0.03
6.14±0.01
6.07±0.00
5.94±0.01
4.24±0.00
Lactococcus lactis subsp. lactis strain B74
2
(adjunct culture)
6.21±0.02
5.95±0.01
5.74±0.02
5.60±0.01
4.81±0.01
Lactobacillus delbrueckii strain B151
2
(adjunct culture)
6.29±0.01
6.17±0.01
6.06±0.00
5.95±0.00
4.94±0.00
Mixed culture: B199
1
and B74
2
5.65±0.03
4.96±0.00
4.68±0.01
4.42±0.01
nd
3
Mixed culture: B199
1
and B151
2
5.88±0.04
5.80±0.01
5.25±0.01
4.92±0.01
nd
Table 1 - Evolution of the pH in pure and mixed cultures
1
5% of inoculum;
2
10% of inoculum;
3
not determined
ABTS
90 min
180 min
270 min
360 min
24 h
Lactococcus spp. strain B199 (starter culture
1
)
13.00±0.20
8.37±0.10
11.72±0.11
16.34±0.11
33.14±0.20
Lactococcus lactis subsp. lactis strain B74 (adjunct culture
2
)
15.14±0.20
10.00±0.10
14.78±0.21
14.70±0.11
32.93±0.10
Lactobacillus delbrueckii strain B151 (adjunct culture)
24.43±0.20
9.41±0.31
13.36±0.11
13.96±0.11
35.21±0.10
Mixed culture
3
: B199 and B74
18.14±0.20
25.78±0.21
30.07±0.11
29.93±0.11
nd
3
Mixed culture: B199 and B151
16.36±0.10
20.07±0.10
26.19±0.11
30.22±0.32
nd
Table 2 - Evolution of the antioxidant activity in pure and mixed cultures
1
5% of inoculum;
2
10% of inoculum;
3
not determined
Figure 1 Figure 2 Figure 3 Figure 4
