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Article

Impact of Commercial Protective Culture on Manouri PDO Cheese

by
Thomas Bintsis
1,* and
Maria A. Kyritsi
2
1
Laboratory of Safety and Quality of Milk and Dairy Products, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
2
Laboratory of Hygiene & Epidemiology, University of Thessaly, 41222 Larissa, Greece
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(1), 35; https://doi.org/10.3390/fermentation11010035
Submission received: 24 December 2024 / Revised: 10 January 2025 / Accepted: 14 January 2025 / Published: 16 January 2025
(This article belongs to the Special Issue Dairy Fermentation, 3rd Edition)
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Abstract

Manouri is a Greek whey cheese, with a Protected Denomination of Origin recognition, produced by heating the cheese whey and added milk and/or cream at high temperatures (88–90 °C) to form a coagulum. High-heat treatment results in the inactivation of any indigenous microorganisms from the raw materials. However, the high moisture, fat and pH of the cheese make it a favorable medium for the growth of any microbial contamination. The objective of this study was to investigate the application of a commercial protective culture (CPC) on the microbial profile of Manouri cheese during storage. Three treatments were prepared: S1 was the control cheese with sterile water sprayed on the surface, S2 was sprayed with a dense CPC and S3 with a diluted CPC. The experimental cheeses were covered with greaseproof paper and stored at 5 °C for 21 days. For all three treatments, the fat content and total solids showed a significant increase during storage, while protein and carbohydrates showed a significant decrease at 14 days. The application of the CPC resulted in cheeses with higher pH than the control cheese, probably due to the growth of acidifying lactic acid bacteria in the microbiota of the S2 and S3 cheeses. Manouri cheese sprayed with the CPC showed a reduction of 1.60–1.69 log CFU/g in the population of yeasts; no effect was observed on Enterobacteriaceae and Staphylococcus spp. The dominant yeast microbiota was identified as Candida zeylanoides (63.5%), Candida parapsilosis (21.1%) and Candida famata (15.4%). Although the application of the CPC was not able to control the spoilage bacteria, it showed an effective way to control the growth of yeasts in Manouri cheese. However, the presence of certain Candida spp. reveals the significance of applying good hygiene practices throughout the cheesemaking process.

1. Introduction

Whey cheeses are heat- and/or acid-coagulated cheeses produced by heating the cheese whey, with or without the addition of milk and/or cream, at high temperatures (88–90 °C) to form a coagulum; the coagulum is formed by the denaturation of whey proteins, mainly α-lactalbumin and β-lactoglobulin, and the subsequent entrapment of the milk fat globules into the protein network [1,2,3,4]. Ricotta is the most studied whey cheese [4], and Manouri, Myzithra, Anthotyros, Xynomyzithra and Urda are the most popular Greek whey cheeses [2]. Manouri has been registered as a Protected Designation of Origin (PDO) cheese [1], and the cheesemaking technology is presented in Figure 1. Manouri has a high fat content, with a minimum fat-in-dry matter (FDM) of 70% and a creamy body, delicate texture and is consumed fresh. Fresh cheeses have high levels of water activity (aw), fat content and pH and, consequently, a short shelf-life [2,4,5].
Several innovative strategies have been proposed to increase the shelf-life of whey cheeses, especially for Ricotta, mainly focusing on the application of modified atmosphere packaging (MAP) [2,4]. The use of natural bio-active preservatives and protective cultures has been proposed to reduce the risk of survival and growth of both pathogenic and spoilage-causing microbes in different types of cheese [6]. Most studies on biopreservation of whey cheeses have focused on Listeria monocytogenes [7,8], whereas Spanu et al. studied the combined effect of protective cultures and MAP on the spoilage microorganisms in Ricotta fresca [9,10]. However, there is a limited number of studies on the impact of antifungal protective cultures to prevent spoilage from yeasts and molds in whey cheeses [6]. The application of protective cultures, consisting of lactic acid bacteria (LAB) with antagonistic actions against pathogens and spoilage microbes, can be an effective and consumer-friendly strategy to extend the shelf-life of certain cheeses, thereby reducing food waste, without using chemical preservatives [11].
Commercially available protective cultures (CPCs), composed of selected LAB, are considered safe and easy to use in food [12]. Various CPCs have been developed and applied in cheesemaking to control the growth of spoilage yeasts and molds; FreshQ (Chr. Hansen) and Holdbac YM (DuPont) have been developed to target yeasts and molds in cheese [13]. The effectiveness and their mode of action is dependent on the cheese matrix and the interactions with the dominant cheese microbiota. According to the manufacturer’s instructions, the protective culture is added in combination with the normal starter culture in the milk. However, due to the high heat treatment, the addition of a protective culture is not recommended for Manouri cheese. Thus, a possible solution was investigated in this study, that is, to spray the culture on the surface of the fresh cheese after the heat treatment step and monitor the microbiological quality of unpacked Manouri cheese during storage.
To the best of our knowledge, no study has been published on the investigation of the application of protective cultures in Manouri and/or similar Greek whey cheeses. Lioliou et al. studied the presence of yeasts in artisanal Manouri cheese [14]; understanding the role of yeasts in the spoilage of cheese and their behavior against bioprotective cultures can provide insights into the quality of Manouri and similar cheeses.
The objective of this study was to apply a CPC by spraying it on the surface of Manouri cheese and to study its effects on the microbial profile during cheese storage. Since the selected protective culture is recommended for the inhibition of yeasts and molds, the additional aim was to identify the dominant and resistant yeasts.

2. Materials and Methods

2.1. Experimental Design

Experimental Manouri PDO was manufactured, according to the registered cheesemaking process, in a local dairy plant in Thessaloniki. Three consecutive batches were manufactured; two cheese samples from each batch were randomly selected and transferred to the laboratory in insulated boxes for treatment and analysis. After thorough mixing, 4.1 g of the CPC (FreshQ9—Lacticaseibacillus rhamnosus, Chr. Hansen, Hørsholm, Denmark) was dissolved in 40 mL of sterile distilled water and an aliquot of 5 mL of the suspension was evenly sprayed on the surface of experimental Manouri cheese; each cylindrical cheese had a weight of ca. 300 g, with a surface of ca. 340–350 cm2. Control cheese (S1) was sprayed with 5 mL of sterile water, S2 was sprayed with 5 mL of the dense CPC and S3 was sprayed with 5 mL of a mixture 0.5 mL of the dense CPC plus 4.5 mL of water (diluted CPC). The cheeses were sprayed in the morning, stored in the fridge and analyzed after 4 h. Cheeses were covered with greaseproof paper, to mimic the handling from a consumer in the household fridge, and stored at 5 °C. Duplicate samples of each of the three treatments (S1, S2 and S3) of Manouri were analyzed at the day of inoculation (d0), and after 7 (d7), 14 (d14) and 21 (d21) days of storage at 5 °C. At each time-point, S1, S2 and S3 were analyzed for the determination of the microbiological profile, chemical composition and intrinsic properties.

2.2. Physicochemical and Microbiological Analyses

Total fat, protein, lactose and total solids content of Manouri cheese were measured using the Milko-ScanMinorTM analyser (Foss Electric, Hillerod, Denmark), as previously described by Bintsis et al. [2]. The pH was measured directly with a pH meter (XS Instruments, Carpi, Italy), and water activity (aw) with an AquaLab instrument (Decagon Devices, Pullman, WA, USA). Physicochemical analyses were carried out in quadruplicate.
For the microbiological analyses, 10 g of cheese was aseptically diluted in 90 mL of sterile quarter-strength Ringer’s solution in a stomacher bag and homogenized in a stomacher. Decimal dilutions were prepared in 9 mL of sterile Ringer’ s solution, and the following microbiological analyses were carried out: total mesophilic count in Plate Count Agar (Biolife, Monza, Italy), incubated at 30 °C for 72 h, aerobically; Enterobacteriaceae in Violet Red Bile Glucose Agar (Biolife, Italy), incubated at 37 °C for 24 h, under microaerophilic conditions; LAB in Man, Rogosa and Sharpe (MRS) Agar (Biolife, Italy), incubated at 30 °C for 72 h and in M17 Agar (Biolife, Italy), incubated at 30 °C for 48 h, under microaerophilic conditions; and yeasts and molds in Rose Bengal Chloramphenicol Agar (Biolife, Italy), incubated at 25 °C for 5 days, aerobically. Microbiological analyses were carried out in duplicate.
The microbial counts of the CPC were confirmed by the plate count method after decimal dilutions in MRS and M17. Counts on MRS were equal to counts on M17, being 11.08 log CFU/mL in both media.

2.3. Identification of Yeasts

Approximately 8–10 colonies from the Rose Bengal agar plates were randomly selected from the three batches of Manouri cheese on day 21. The colonies were inoculated in Tryptic Soy Yeast Extract broth (Biolife, Italy) and incubated at 25 °C for 48 h. The grown cultures were stored at −80 °C in the broth with 20% glycerol. A loopful of each culture was streaked on Tryptic Glucose Yeast Extract Agar (Biolife, Italy). For the isolated yeasts, proteolytic activity was tested in Tryptic Glucose Yeast Agar enriched with 1% (w/v) skim milk powder (Neogen, Ayrshire, UK) and lipolytic on Tributyrin Agar (Sigma-Aldrich, St. Louis, MO, USA) [15].
A total of 52 yeast isolates were identified at the species level by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) using the MALDI Microflex LT (Bruker Daltonic GmbH, Bremen, Germany). The on-target-plate protein extraction protocol was used to increase the spectrum quality, as recommended by the manufacturer (Bruker Daltonic). Protein profiles were acquired using linear positive mode analysis with laser frequency at 60 Hz, through AutoXecute software (Flex control 3.4; Bruker Daltonics, Bremen, Germany). The method was externally calibrated using the Bruker Bacterial Test Standard (BTS) as a reference protein extract. The obtained raw spectra were compared in the mass spectral library (6093 MSPs), and finally the isolated strains were identified using MALDI Biotyper Software (version 4.0). The results were categorized according to the manufacturer’s recommended score values. The score value given by MALDI Biotyper RTC represents the probability that the unknown microorganism is a species in the reference MALDI Biotyper database. An identification score value between 0.000 and 1.699 represents a not reliable identification, between 1.700 and 1.999 a probable genus identification, between 2.000 and 2.299 secure genus identification and probable species identification, and finally score values between 2.300 and 3.000 a highly probable species identification.

2.4. Statistical Analysis

Microbiological counts were converted to log CFU/g and together with physicochemical data were subjected to a General Linear Model with a repeated measures ANOVA, by using the SPSS Statistics program 29.0.0.0 (IBM, Armonk, NY, USA). Pairwise multiple comparisons were carried out by using Tukey’s test (p < 0.05).

3. Results and Discussion

Physicochemical and microbiological analyses were carried out to evaluate the effects of the dense and diluted CPC on the characteristics of Manouri cheese throughout the storage period. The results obtained from the physicochemical analyses of the experimental Manouri cheeses treated with the CPC are shown in Table 1.
Total solids were constantly increased during storage for all three treatments, and this was due to the slight drying that occurred. The fat content showed a significant increase during storage (p < 0.05), and this was concomitant with the moisture loss. Protein showed some fluctuations, a decrease at 7 days and increase thereafter; the decrease was probably the result of proteolysis and release of amino acids, whereas, the increase thereafter was, as for fat, from the loss of moisture. Carbohydrates showed a significant decrease at 14 days (p < 0.05), probably due to the fermentation of lactose by LAB.
No difference was observed between the three treatments for fat, protein and carbohydrates. In general, the application of the CPC in Manouri cheese had no effect on the chemical composition of the experimental cheeses. More carbohydrates were utilized in S3 and S2 than S1; however, the pH of S1 was lower than that of S2 and S3 at 14 and 21 days. It seems that the native LAB grown in the control cheese (Table 2) was more acidifying than the strains of L. rhamnosus present in the CPC. A constant decrease was observed for aw values, and this was in agreement with the decrease in the moisture content.
Typical Manouri has 52% total solids (37% fat, 11% protein) and 1% salt (on fresh weight basis) [3]; Govari and Vartzis found 57% total solids, 48% fat, 6% protein and pH values of 5.5 in commercial Manouri cheese [16], while lower values for pH were previously reported [17]. Bintsis et al. reported high variability in the chemical composition of whey cheeses [2], reflecting differences in the origin of cheese milk and the technology used for the primary cheese product, the composition of the whey, the use of sweet or acid whey as well as differences in the cheesemaking process of the whey cheese (artisanal or industrial method, the animal origin of milk and/or cream added, temperature of heating etc.) [3,4].
The results obtained from the microbiological analyses of the experimental Manouri cheeses treated with the CPC are shown in Table 2.
The total mesophilic counts in the experimental Manouri cheese samples were of the order of 6.96 (S1), 7.78 (S2) and 7.88 log CFU/g (S3) on day 0. Total mesophilic counts were significantly increased at 7 days (p < 0.05), and remained constant during the storage, but no significant differences were observed between the treatments. Mesophilic lactobacilli showed significant differences on day 0 (p < 0.05), and this was the result of the added protective culture; all samples showed a gradual increase during storage and S1 reached 5.69 log CFU/g, whereas S2 and S3 had 9.06 and 8.89 log CFU/g at 21 days, respectively. A similar trend was observed for presumptive lactcoccoci and pediococci at 21 days, with counts of 8.29, 9.15 and 8.90 log CFU/g, for S1, S2 and S3, respectively.
Enterobacteriaceae were at low numbers in all three batches at day 0 and increased during storage. Staphylococcus spp. was found at high numbers on day 0 in all three batches and increased at day 14 and remained constant at day 21. The addition of the protective culture had no effect on the inhibition of either Enterobacteriaceae or staphylococci. The counts of Enterobacteriaceae at 21 days were much higher than that of other commercial Manouri cheese [2]; however, Lioliou et al. [14] reported that artisanal Manouri had higher counts of Enterobacteriaceae (7.26 log CFU/g) on the surface of Manouri cheese, and these numbers suggested post-heat treatment contamination and subsequent growth during draining and storage. Similar counts to the present study were reported for Anthotyros cheese [18]. Greek whey cheeses analyzed for their microbiological profile were found to have total microbial counts at a level of 8.19 log CFU/g, LAB of 8.13 log CFU/g and non-LAB of 6.55 log CFU/g [19]. Pappa et al. reported lower populations of LAB and dairy cocci for Urda cheese, another Greek artisanal whey cheese; Urda has a different composition than Manouri (higher total solids and protein, lower fat and salt can reach values of 4.1%) [20]. Kalogridou-Vassiliadou et al. reported high total mesophilic counts (7.84 log CFU/g), coliforms (6.74log CFU/g) and LAB (7.18 CFU/g) in Anthotyros cheese [21].
Yeasts and molds were found in the present study at less than 2 log CFU/g on day 0, but the counts significantly increased (p < 0.05) in all three batches at day 7. Similar counts for yeasts were found in industrial Manouri cheese [2], Anthotyros [22], Lor cheese [23] and buffalo Ricotta cheese [24], whereas higher counts for yeasts were reported for artisanal Manouri (4.19–6.46 log CFU/g) [2,14] and Anthotyros (5.48 log CFU/g) [21]. The counts of yeasts in experimental Manouri were increased by 2 log units in the control cheese (S1) on day 14 and reached 6.69 log CFU/g in 21 days. For S2, the counts showed a much smaller increase on day 14 and remained constant on day 21. Similarly to S2, the yeast counts in S3 reached 5.00 log CFU/g on day 21. Thus, the addition of the protective culture had a significant effect on the growth of yeasts (p < 0.05), resulting in a reduction of 1.60 for the dense culture and 1.69 log CFU/g for the diluted culture. Interestingly, although the diluted culture was slightly more effective, no significant difference (p > 0.05) was observed between the dense and the diluted protective culture. Since the application of the CPC was made by spraying on the surface of the cheese, a 10-fold reduction in S3 could be as effective as the dense culture; the presence of the protective culture at a level of 1.4 × 108 CFU/cm2 in S3 was able to control the yeast counts. Pires et al. investigated the effect of a CPC in combination with probiotic culture to produce a whey cheese using ultra-filtrated bovine whey, and reported an increase in the shelf-life of the cheese by reducing yeast and mold counts by one log cycle [25]. The antifungal efficacy of CPC in Queso fresco showed widely variable results [26], whereas it was ineffective at controlling the growth of yeast in Cottage cheese [27].
The results from the microbiological profile of the experimental Manouri cheese confirm the results from previous studies that this type of cheese is a favorable medium for the growth of different microbial groups [3,4]. Post-heating contamination with bacteria and yeasts could grow and cause spoilage, even at refrigeration temperatures. It is crucial, therefore, to strictly apply good hygiene practices during manufacture and storage. Yeast strains were identified with the MALDI-TOF MS; this method generates spectral fingerprints from specific peptides released from the cell surface by special acidic treatment [28,29,30] and has been successfully used for the identification of milk microbiota [31] and yeasts in different cheeses [13,32,33,34,35]. The results from the identification of yeasts from experimental Manouri cheese are presented in Table 3.
A limited yeast species diversity was found in the experimental Manouri cheese; Candida zeylanoides (63.5%) was the most dominant yeast, followed by Candida famata (21.1%) and Candida parapsilosis (15.4%). No species showed proteolytic activity, and five strains of C. zeylanoides, three strains of C. famata and one strain of C. parapsilosis showed very weak lipolytic activity. Lioliou et al. found a more diverse yeast microbiota in artisanal Manouri cheese with eight species identified (D. hansenii, Pichia membranaefasciens, Pichia farinose, C. mogii, Torulopsis delbrueckii, C. intermedia, Zygossacharomyces rouxii and Sacchromyces cerevisiae) [14].
C. zeylanoides is a species found in raw milk [36] and fermented milk [37,38]. It is frequently isolated from cheese where it may play an important role in both the flavour development and possibly in the cheese spoilage. C. zeylanoides can be part of the complex cheese surface microbiota in surface-ripened varieties [39], and has been isolated from Fiore Sardo, Pecorino di Farindola, semi-hard ovine cheese, Gorgonzola-type [40], artisanal Minas cheese [35] and Brazilian artisanal cheeses [33].
C. parapsilosis has been isolated from raw milk [37,41] and mastitic bovine milk [42]. The species has been isolated from Pecorino di Farindola, semi-hard ovine and caprine cheese and Halloumi [40], raw milk Salers cheese [43] and Brazilian artisanal cheeses [32]. Garnier et al. reported that C. parapsilosis, isolated from fresh cheese and yogurt, was one of the most resistant yeasts against chemical preservatives [44]. C. parapsilosis has been detected in the air from the draining room in a dairy plant producing white brined cheese [45]. C. parapsilosis is a yeast species often associated with human infections in immunocompromised patients [46,47]. Although dairy yeasts that caused foodborne infections have not been reported, the species C. parapsilosis was identified as the causative agent of a fatal case of endocarditis in a drug user [48]. Its involvement in endo-nosocomial infections is facilitated by its ability to adhere to surfaces and form biofilms [49]. To the best of our knowledge, this is the first time it is reported in whey cheese. The presence of C. parapsilosis in Manouri may be attributed to this ability to form biofilms, although contamination from infected personnel can not be excluded.
C. famata (teleomorph D. hansenii) is a heterogeneous species with a remarkable genetic and phenotypic diversity [39]; its presence has been reported in Feta cheese, Feta cheese brine, Camembert and Brie, Danish surface-ripened cheese, Rokpol and blue cheeses, e.g., Stilton, Roquefort, Mycella and Gorgonzola-type [40]. D. hansenii (anamorph C. famata) is probably the most abundant cheese yeast and has been isolated from a great number of cheeses: hard cheeses, e.g., Fiore Sardo, Pecorino Romano and Serro Minas; semi-hard cheeses, e.g., bovine, ovine and caprine milk cheeses, Canastra, Fontina and Tomme d’Orchies; and soft cheeses such as Bryndza and Manouri (whey cheese) [40].
The role of yeasts in the spoilage and/or development of flavour and other organoleptic characteristics has been studied for cheeses that undergo ripening [39,40,50]. However, studies on the role of yeasts in whey cheeses are rare. Since contamination with airborne or biofilm-forming yeasts on surfaces of cheesemaking equipment is possible, further studies are required on the role of yeasts in fresh cheeses.

4. Conclusions

The application of the CPC to a high-heat-treated cheese by spraying on the surface after the heat treatment had no effect on the chemical composition; higher pH values were found compared with the control cheese. No inhibition in Enterobacteriaceae and Staphylococcus spp. was observed. These groups of bacteria may be found in the Manouri cheese as post-heating contaminants. The increase in the numbers of Enterobacteriaceae, Staphylococcus spp. and yeasts during the storage of Manouri confirms that it is a favorable medium for microbial growth and is susceptible to spoilage. The treatment with the protective culture was not able to fully control the spoilage microorganisms; however, it enhanced the microbiological quality by decreasing the yeast counts 1.6–1.7 log units relative to the control cheese. The production of a more stable cheese with no increased yeast counts on day 14 may extend the shelf-life of the cheese and reduce food waste with possible economic benefits for the consumers.
Further research focused on the effect of other protective cultures and combinations of them on the microbiological quality of Manouri and related cheeses would provide more insights into the potential of extending the shelf-life and improving the safety of such types of fresh cheeses.
Overall, the use of protective cultures against spoilage yeasts in whey cheeses should be seen as an additional preventative measure, in parallel with the correct application of good hygienic practices during manufacturing and the post-processing stages.

Author Contributions

Conceptualization, T.B.; methodology, T.B. and M.A.K.; validation, T.B.; formal analysis, T.B. and M.A.K.; resources, T.B.; data curation, T.B.; writing—original draft preparation, T.B.; writing—review and editing, T.B.; supervision, T.B. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Special Account for Research Funds of the Aristotle University of Thessaloniki (Grant No. 75143).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

The authors acknowledge the support of the Provogal Dairy, Thessaloniki, for the manufacture of the Manouri cheese samples.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 11 00035 g001
Figure 1. Flow diagram of the Manouri PDO cheesemaking process.
Figure 1. Flow diagram of the Manouri PDO cheesemaking process.
Fermentation 11 00035 g001
Table 1. Physicochemical characteristics (mean ± standard deviation) of experimental Manouri cheese samples.
Table 1. Physicochemical characteristics (mean ± standard deviation) of experimental Manouri cheese samples.
ParametersCheese Samples 1,2Storage Time
0 Day7 Days14 Days21 Days
Fat (% w/w)S132.49 ± 0.64 A,a42.71 ± 2.38 B,a47.10 ± 5.60 C,a50.6 ± 3.73 C,a
S233.42 ± 0.42 A,a43.00 ± 2.29 B,a42.86 ± 3.76 B,b47.50 ± 3.06 C,b
S335.16 ± 2.38 A,a44.84 ± 3.33 B,a41.52 ± 4.42 B,b44.9 ± 3.21 B,c
Protein (% w/w)S113.14 ± 0.07 A,a12.11 ± 0.51 B,a13.72 ± 1.28 A,a15.78 ± 1.86 D,a
S214.01 ± 0.30 A,a12.21 ± 0.68 B,a14.09 ± 1.58 A,a15.07 ± 1.86 C,a
S313.14 ± 0.59 A,a11.83 ± 0.58 B,a13.07 ± 0.53 A,a14.65± 0.22 C,a
Carbohydrates (% w/w)S13.36 ± 0.08 A,a3.58 ± 0.27 A,a2.55 ± 0.84 B,a2.51 ± 0.45 B,a
S23.30 ± 0.08 A,a3.40 ± 0.11 A,a2.22 ± 0.98 B,a2.18 ± 0.56 B,a
S33.45 ± 0.04 A,a3.44 ± 0.06 A,a1.65 ± 0.13 B,b1.90 ± 0.18 B,b
Total solids (% w/w)S155.80 ± 0.42 A,a59.92 ± 3.10 B,a64.86 ± 3.47 C,a74.28 ± 5.28 D,a
S257.09 ± 0.55 A,a60.48 ± 1.82 B,a62.20 ± 3.73 B,b70.04 ± 2.52 C,b
S357.84 ± 2.80 A,a59.99 ± 1.77 B,a60.06 ± 1.75 B,c69.26 ± 1.56 C,b
pHS16.25 ± 0.02 A,a5.93 ± 0.10 B,a5.75 ± 0.12 C,a5.59 ± 0.08 D,a
S26.30 ± 0.03 A,a5.91 ± 0.08 B,a5.86 ± 0.07 C,b5.73 ± 0.07 D,b
S36.22 ± 0.04 A,a5.89 ± 0.08 B,a5.80 ± 0.06 C,c5.70 ± 0.06 D,c
Water activity (aw)S10.969 ± 0.001 A,a0.942 ± 0.006 B,a0.922 ± 0.008 C,a0.912 ± 0.007 D,a
S20.969 ± 0.002 A,a0.948 ± 0.008 B,a0.935 ± 0.014 C,a0.917 ± 0.012 D,a
S30.967 ± 0.001 A,a0.954 ± 0.007 B,a0.933 ± 0.010 C,a0.916 ± 0.008 D,a
S1: Control cheese; S2: cheese sprayed with 5 mL of protective culture; S3: cheese sprayed with 0.5 mL of protective culture plus 4.5 mL of water. 1 Values are means of four replicates from duplicate samples from three batches. 2 Means of each parameter in the same row (effect of time) with different capital letter superscripts and in the same column (effect of treatment) with different lowercase letter superscripts were significantly different at a level of significance of 5%.
Table 2. Microbial populations (log CFU/g; mean ± standard deviation) in the experimental Manouri cheese samples during storage at 4 °C.
Table 2. Microbial populations (log CFU/g; mean ± standard deviation) in the experimental Manouri cheese samples during storage at 4 °C.
Microbial GroupCheese Samples 1,2Storage Time
0 Day7 Days14 Days21 Days
Total mesophilic countS16.96 ± 0.01 A,a8.64 ± 0.29 B,a8.57 ± 0.22 B,a8.75 ± 0.19 B,a
S27.78 ± 0.07 A,b9.15 ± 0.32 B,b9.05 ± 0.20 B,b9.20 ± 0.19 B,b
S37.88 ± 0.08 A,b9.05 ± 0.28 B,b9.12 ± 0.55 B,b9.26 ± 0.58 B,b
Mesophilic lactobacilliS13.46 ± 0.17 A,a4.65 ± 0.14 B,a5.43 ± 0.19 C,a5.69 ± 0.08 D,a
S27.24 ± 0.10 A,b9.00 ± 0.33 B,b8.94 ± 0.12 B,b9.06 ± 0.14 B,b
S37.31 ± 0.22 A,b8.34 ± 0.20 B,b8.76 ± 0.51 C,b8.89 ± 0.45 B,b
LAB on M17S15.97 ± 0.09 A,a7.07 ± 0.77 B,a8.11 ± 0.36 C,a8.29 ± 0.33 D,a
S26.95 ± 0.19 A,b8.98 ± 0.33 B,b8.92 ± 0.20 B,b9.15 ± 0.14 C,b
S37.16 ± 0.38 A,b8.45 ± 0.15 B,b8.66 ± 0.07 C,b8.90 ± 0.04 D,b
EnterobacteriaceaeS13.32 ± 0.17 A,a4.63 ± 0.15 B,a4.99 ± 0.31 C,a5.22 ± 0.29 D,a
S23.03 ± 0.15 A,a4.45 ± 0.03 B,a5.03 ± 0.30 C,a5.18 ± 0.34 D,a
S33.21 ± 4.35 A,a4.35 ± 0.09 B,a4.91 ± 0.25 C,a5.06 ± 0.23 D,a
StaphylococciS15.90 ± 0.15 A,a6.05 ± 0.41 A,a6.38 ± 0.39 B,a6.55 ± 0.37 B,a
S25.86 ± 0.02 A,a5.84 ± 0.07 A,a6.35 ± 0.20 B,a6.54 ± 0.20 B,a
S35.73 ± 0.09 A,a5.93 ± 0.01 A,a6.45 ± 0.31 B,a6.66 ± 0.33 B,a
Yeasts and moldsS1<24.82 ± 0.13 A,a6.46 ± 0.13 B,a6.69 ± 0.15 B,a
S2<24.25 ± 0.16 A,b5.03 ± 0.24 B,b5.06 ± 0.14 B,b
S3<24.14 ± 0.05 A,b5.05 ± 0.22 B,b5.00 ± 0.12 B,b
S1: Control cheese; S2: cheese sprayed with 5 mL of protective culture; S3: cheese sprayed with 0.5 mL of protective culture plus 4.5 mL of water. 1 Counts were presented as mean log CFU/g from two replicates from duplicate samples from three batches. 2 Means of counts of each microbial group in the same row (effect of time) with different capital letter superscripts and in the same column (effect of treatment) with different lowercase letter superscripts were significantly different at a level of significance of 5%.
Table 3. Yeast strains identified using MALDI-TOF MS and their score ranges and relative abundances in Manouri cheese.
Table 3. Yeast strains identified using MALDI-TOF MS and their score ranges and relative abundances in Manouri cheese.
Score RangeRelative Abundance (%)
Candida zeylanoides2.022–2.33263.5
Candida famata2.031–2.43521.1
Candida parapsilosis1.997–2.19915.4
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Bintsis, T.; Kyritsi, M.A. Impact of Commercial Protective Culture on Manouri PDO Cheese. Fermentation 2025, 11, 35. https://doi.org/10.3390/fermentation11010035

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Bintsis T, Kyritsi MA. Impact of Commercial Protective Culture on Manouri PDO Cheese. Fermentation. 2025; 11(1):35. https://doi.org/10.3390/fermentation11010035

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Bintsis, Thomas, and Maria A. Kyritsi. 2025. "Impact of Commercial Protective Culture on Manouri PDO Cheese" Fermentation 11, no. 1: 35. https://doi.org/10.3390/fermentation11010035

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Bintsis, T., & Kyritsi, M. A. (2025). Impact of Commercial Protective Culture on Manouri PDO Cheese. Fermentation, 11(1), 35. https://doi.org/10.3390/fermentation11010035

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