The aim of the present study was to describe the microbial groups of the traditional Slovak cheese Parenica during rippening. The microbial group included the total bacterial count, coliform bacteria, enterococci, lactic acid bacteria, and microscopic filamentous fungi, which may affect the organoleptic characteristics of this product. A total of 42 cheese samples were collected from four different farms during three months. The total bacterial counts were cultivated on Plate count agar at 30 °C, lactic acid bacteria (LAB) on MRS, APT and MSE at 37 °C, coliform bacteria on VRBL at 37 °C. Gram-positive and Gram-negative isolates were identified by MALDI-TOF MS profiling.
Cheese is one of the oldest fermented foods (
Diversity of sensory characteristics as flavor, smell, and texture of cheese are linked to microbiological activity in the product. Microorganisms show large metabolic capacities, and contribute the sensory parameters through the production of digestive enzymes and small molecules. Different cheese production technologies can lead to the growth of different microbial groups or microorganisms. The source and raw milk treatment (raw or pasteurized) used for cheesemaking can result in different microbiota of cheese. Changes in product characteristics during aging can significantly influence the cheese-associated microbiota with pH, salt, moisture, and temperature of cheese reveal the biggest impact on microorganisms (
The microbiota of cheese rinds vary from simple to complex with
From a technological point of view, the contamination of processed cheese by Gram-positive spore-forming rodshaped bacteria of the genera
The aim of our study was to detect the microbiota of the traditional Slovak cheese „Parenica“ and to identify the microorganisms by mass spectrometry.
Are there different bacteria and yeast species present in smoked and non-smoked traditional Slovak cheese?
Are microscopic filamentous fungi presented in traditional Slovak cheese?
In our study, 42 samples of Slovak traditional cows cheese “Parenica“ were examined during three moths. The cheese samples included non-smoked cheese (n = 21) and smoked cheese (n = 21). Additionally, 42 cow milk cheese samples from the western and middle Slovak producers were collected (Bánovce nad Bebravou, Liptovský Mikuláš, Červený Kameň, Važec). Samples were collected in sterilized sample containers and brought to laboratory in icebox for microbiological investigations. Samples were kept in a refrigerator (4 ±1 °C) until the testing began.
The primary dilution of cheese were made by adding of 5 g of sample to 45 mL of 0.87% sterile saline. Then, the serial dilutions (10-2 to 10-4) were done and 100 μL of each dilution was plated out onto agars.
Plate count agar (PCA, (Sigma-Aldrich®, St. Louis, USA) for total count bacteria enumeration was used. Inoculated plates were incubated for 48 – 72 h at 30 °C and then examined for the presence of bacterial colonies.
Violet red bile lactose agar (VRBGA, Sigma-Aldrich®, St. Louis, USA) for enumeration of coliforms bacteria was used. Inoculated plates were incubated at 37 °C for 24 – 48 h and examined for the presence of typical colonies.
MRS (Main Rogose agar), MSE (Mayeux, Sandine and Elliker) and APT (All Purpose TWEEN® agar, Sigma-Aldrich®, St. Louis, USA) agars were used for cultivation of lactic acid bacteria. Inoculated plates were incubated at 37 °C for 72 h anaerobically and then the bacterial growth was evaluated.
Malt extract agar (Sigma-Aldrich®, St. Louis, USA) and acid base indicator bromocresol green (Sigma-Aldrich®, St. Louis, USA) (0.020 g.l-1) were used for microscopic fungi and yeasts identification. Inoculated plates were incubated at 25 °C for 5 days aerobically and then the growth was evaluated.
The colonies from total bacterial counts,
One colony of each bacterial isolate was transferred into an Eppendorf tubes and mixed with 300 μL of sterile water. After addition of ethanol (900 μL), the suspension was mixed and centrifuged (13,000 g, 2 min). After removal of supernatant, the pellets were dried at room temperature at least for 5 min. The bacterial pellets were resuspended in 20 – 50 μL of formic acid (70%) and the same amount of acetonitrile. After centrifugation (2 min at 13,000 g), a 1 μL of supernatant was spotted onto a sample position of a polished steel MALDI target plate and dried at room temperature. A 1 μL of MALDI matrix (solution of α-cyano-4-hydroxycinnamic acid (HCCA) in 50% acetonitrile/2.5% trifluoro-acetic acid) was added to the spot and dried.
The MALDI target plate was introduced into the MALDI-TOF mass spectrometer for automated measurement and data interpretation. MALDI-TOF profile mass spectra were imported into the MALDI Biotyper 3.0 software and processed automatically after measurement. The logarithm of the score (log[score]) was displayed as the matching result. The MALDI Biotyper output was a log(score) between 0 and 3.0, which was calculated from a comparison of the peak list from an unknown isolate with the reference MSP in the database. A log(score) ≥1.7 indicated identification at the genus level, log(score) ≥2.0 was set as the threshold for a match at the species level. Isolates with ≥2.0 were accepted as a correct identification.
All experiments were carried out in triplicate. Standard deviations were calculated for replications. The experimental data were subjected to analysis of variance (Duncan's test) at the 95% confidence level of 0.05 (software XL STAT, 2019).
The results on non-smoked and smoked cheese bacterial counts, including coliform bacteria, enterococci, total bacterial count, lactic acid bacteria and microscopic filamentous fungi are shown in Table
Microbial counts in traditional Slovak cheese.
| CB | E | TCB | LAB | MFY | |
|---|---|---|---|---|---|
|
|
|||||
| 0.00 ±0.00 | 2.46 ±0.05 |
3.67 ±0.06 |
3.77 ±0.16 |
2.72 ±0.14 |
|
| 0.00 ±0.00 | 2.29 ±0.07 |
3.54 ±0.09 |
3.47 ±0.20 |
2.30 ±0.13 |
|
| 0.00 ±0.00 | 2.60 ±0.07 |
3.62 ±0.09 |
3.87 ±0.09 |
2.79 ±0.05 |
|
| 0.00 ±0.00 | 2.23 ±0.02 |
3.17 ±0.05 |
3.52 ±0.06 |
2.46 ±0.14 |
|
| 0.00 ±0.00 | 2.41 ±0.15 |
3.74 ±0.15 |
3.64 ±0.10 |
2.52 ±0.07 |
|
| 0.00 ±0.00 | 2.17 ±0.02 |
3.24 ±0.04 |
3.46 ±0.19 |
2.26 ±0.05 |
|
| 0.00 ±0.00 | 2.55 ±0.02 |
3.70 ±0.14 |
3.70 ±0.17 |
2.75 ±0.10 |
|
| 0.00 ±0.00 | 2.32 ±0.11 |
3.34 ±0.08 |
3.40 ±0.17cdef | 2.43 ±0.09 |
|
| 0.00 ±0.00 | 2.44 ±0.11 |
3.88 ±0.09 |
3.51 ±0.19 |
2.72 ±0.17 |
|
| 0.00 ±0.00 | 2.09 ±0.04 |
3.41 ±0.13fg | 3.23 ±0.09 |
2.38 ±0.15 |
|
| 3.17 ±0.03 |
4.32 ±0.23 |
4.36 ±0.14 |
3.37 ±0.09 |
3.38 ±0.12 |
|
| 3.43 ±0.10 |
3.53 ±0.06 |
4.08 ±0.03 |
3.19 ±0.07 |
3.28 ±0.06 |
|
| 0.00 ±0.00 | 2.84 ±0.09 |
3.60 ±0.07 |
3.68 ±0.10 |
2.75 ±0.09 |
|
| 0.00 ±0.00 | 2.27 ±0.06 |
3.27 ±0.03 |
3.50 ±0.14 |
2.37 ±0.05 |
|
Note: mean ± standard deviation; different letters in column mean that the differences were significant.
From the genus Acinetobacter 4 different strains were isolated:
Microorganisms isolated from non-smoked and smoked cheese “Parenica”.
| Sample | Isolated microorganisms |
|---|---|
|
|
|
Secondary microflora such as yeasts could be frequently isolated from different types of cheeses with counts from 104 – 106 cfu.g-1 to 107 – 108 cfu.g-1. The presence of yeasts in cheese could be attributed to the favourable conditions for their growth and the wide distribution in dairy environment (
Among lactic acid bacteria the following species were isolated:
Lactic acid bacteria are the most studied microorganisms in milk fermentation (
Among staphylococci
Three
This study was designed to evaluate coliform bacteria, enterococci, yeasts and LAB population’s in Slovak traditional non-smoked and smoked cheese. Our results show that the traditional Slovak cheese contains very diverse microbiota. In our study, the mass spectrometry method allowed the accurate identification of microorganisms and this method was reliable and easy done for identification in comparison with molecular methods.
This work has been supported by the grant APVV-16-0244 "Qualitative factors affecting the production and consumption of milk and cheese".