Microbes in Cheese: Isolation, Molecular Detection and Characterization

A special issue of Foods (ISSN 2304-8158). This special issue belongs to the section "Food Microbiology".

Deadline for manuscript submissions: closed (10 January 2023) | Viewed by 22879

Special Issue Editor

Research Centre for Animal Production and Aquaculture (CREA-ZA), Council for Agricultural Research and Economics (CREA), Rome, Italy
Interests: cheese microorganisms; microbial isolation; molecular detection and characterization; lactic acid bacteria; starter cultures; functional cultures; dairy industry

Special Issue Information

Dear Colleagues,

Microorganisms play a fundamental role in cheese. Thanks to their metabolic activities, which take place both during production and ripening, they are involved in defining the quality and safety of the finished product. In addition, many microbial groups help promote health through the production of bioactive molecules, which either accumulate directly in the cheese or are the result of the transformation of its main milk components (sugars, proteins, and fats). Therefore, cheeses (especially artisanal ones) are a rich and still poorly explored source of useful microorganisms, which can be applied as both primary starters and secondary cultures. The isolation, molecular detection, and characterization of cheese-associated microorganisms (particularly the lactic acid bacteria) may have important implications for the dairy industry, which is continuously searching for new candidate strains for product diversification and process implementation.

Dr. Giorgio Giraffa
Guest Editor

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Keywords

  • cheese microorganisms
  • microbial isolation
  • molecular detection and characterization
  • lactic acid bacteria
  • starter cultures
  • functional cultures
  • dairy industry

Published Papers (9 papers)

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Research

Jump to: Review

13 pages, 443 KiB  
Article
Low-Level Clostridial Spores’ Milk to Limit the Onset of Late Blowing Defect in Lysozyme-Free, Grana-Type Cheese
by Domenico Carminati, Barbara Bonvini, Salvatore Francolino, Roberta Ghiglietti, Francesco Locci, Flavio Tidona, Monica Mariut, Fabio Abeni, Miriam Zago and Giorgio Giraffa
Foods 2023, 12(9), 1880; https://doi.org/10.3390/foods12091880 - 02 May 2023
Cited by 1 | Viewed by 1571
Abstract
The growth of clostridial spores during ripening leads to late blowing (LB), which is the main cause of spoilage in Grana Padano Protected Designation of Origin (PDO) cheese and other hard, long-ripened cheeses such as Provolone, Comté, and similar cheeses. This study aimed [...] Read more.
The growth of clostridial spores during ripening leads to late blowing (LB), which is the main cause of spoilage in Grana Padano Protected Designation of Origin (PDO) cheese and other hard, long-ripened cheeses such as Provolone, Comté, and similar cheeses. This study aimed to verify the cause–effect relationship between the level of clostridial butyric spores (BCS) in milk and the onset of the LB defect. To this end, experimental Grana-type cheeses were produced without lysozyme, using bulk milk with different average BCS content. The vat milk from the so-called “virtuous” farms (L1) contained average levels of BCS of 1.93 ± 0.61 log most probable number (MPN) L−1, while the vat milk from farms with the highest load of spores (L2), were in the order of 2.99 ± 0.69 log MPN L−1. Cheeses after seven months of ripening evidenced a strong connection between BCS level in vat milk and the occurrence of LB defect. In L2 cheeses, which showed an average BCS content of 3.53 ± 1.44 log MPN g−1 (range 1.36–5.04 log MPN g−1), significantly higher than that found in L1 cheeses (p < 0.01), the defect of LB was always present, with Clostridium tyrobutyricum as the only clostridial species identified by species-specific PCR from MPN-positive samples. The L1 cheeses produced in the cold season (C-L1) were free of defects whereas those produced in the warm season (W-L1) showed textural defects, such as slits and cracks, rather than irregular eyes. A further analysis of the data, considering the subset of the cheesemaking trials (W-L1 and W-L2), carried out in the warm season, confirmed the presence of a climate effect that, often in addition to the BCS load in the respective bulk milks (L1 vs. L2), may contribute to explain the significant differences in the chemical composition and some technological parameters between the two series of cheeses. Metagenomic analysis showed that it is not the overall structure of the microbial community that differentiates L1 from L2 cheeses but rather the relative distribution of the species between them. The results of our trials on experimental cheeses suggest that a low-level BCS in vat milk (<200 L−1) could prevent, or limit, the onset of LB in Grana-type and similar cheeses produced without lysozyme. Full article
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12 pages, 1321 KiB  
Article
Technological Characterization of Lactic Acid Bacteria Strains for Potential Use in Cheese Manufacture
by Fabrizio Domenico Nicosia, Alessandra Pino, Guilherme Lembi Ramalho Maciel, Rosamaria Roberta Sanfilippo, Cinzia Caggia, Antonio Fernandes de Carvalho and Cinzia Lucia Randazzo
Foods 2023, 12(6), 1154; https://doi.org/10.3390/foods12061154 - 09 Mar 2023
Cited by 4 | Viewed by 1794
Abstract
A total of 26 lactic acid bacteria isolates from both Italian and Brazilian cheeses were tested for their use in cheesemaking. Isolates were screened for salt tolerance, exopolysaccharide and diacetyl production, lipolytic, acidifying, and proteolytic activities. In addition, the aminopeptidase (Pep N and [...] Read more.
A total of 26 lactic acid bacteria isolates from both Italian and Brazilian cheeses were tested for their use in cheesemaking. Isolates were screened for salt tolerance, exopolysaccharide and diacetyl production, lipolytic, acidifying, and proteolytic activities. In addition, the aminopeptidase (Pep N and Pep X) activities, were evaluated. Most of the strains demonstrated salt tolerance to 6% of NaCl, while only two L. delbruekii (P14, P38), one L. rhamnosus (P50) and one L. plantarum (Q3C4) were able to grow in the presence of 10% (w/v) of NaCl. Except for 2 L. plantarum (Q1C6 and Q3C4), all strains showed low or medium acidifying activity and good proteolytic features. Furthermore, lipolytic activity was revealed in none of the strains, while the production of EPS and diacetyl was widespread and variable among the tested strains. Finally, regarding aminopeptidase activities, 1 L. delbrueckii (P10), 1 L. rhamnosus (P50), and 1 L. lactis (Q5C6) were considered as the better performing, showing high values of both Pep N and Pep X. Based on data presented here, the aforementioned strains could be suggested as promising adjunct cultures in cheesemaking. Full article
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22 pages, 3093 KiB  
Article
Growth and Biocontrol of Listeria monocytogenes in Greek Anthotyros Whey Cheese without or with a Crude Enterocin A-B-P Extract: Interactive Effects of the Native Spoilage Microbiota during Vacuum-Packed Storage at 4 °C
by Nikoletta Sameli and John Samelis
Foods 2022, 11(3), 334; https://doi.org/10.3390/foods11030334 - 25 Jan 2022
Cited by 8 | Viewed by 2201
Abstract
Effective biopreservation measures are needed to control the growth of postprocess Listeria monocytogenes contamination in fresh whey cheeses stored under refrigeration. This study assessed growth and biocontrol of inoculated (3 log10 CFU/g) L. monocytogenes in vacuum-packed, fresh (1-day-old) or ‘aged’ (15-day-old) Anthotyros [...] Read more.
Effective biopreservation measures are needed to control the growth of postprocess Listeria monocytogenes contamination in fresh whey cheeses stored under refrigeration. This study assessed growth and biocontrol of inoculated (3 log10 CFU/g) L. monocytogenes in vacuum-packed, fresh (1-day-old) or ‘aged’ (15-day-old) Anthotyros whey cheeses, without or with 5% of a crude enterocin A-B-P extract (CEntE), during storage at 4 °C. Regardless of CEntE addition, the pathogen increased by an average of 2.0 log10 CFU/g in fresh cheeses on day 15. Gram-negative spoilage bacteria also increased by an average of 2.5 log10 CFU/g. However, from day 15 to the sell-by date (days 35–40), L. monocytogenes growth ceased, and progressively, the populations of the pathogen declined in most cheeses. This was due to an unmonitored, batch-dependent natural acidification by spoilage lactic acid bacteria, predominantly Leuconostoc mesenteroides, which reduced the cheese pH to 5.5, and finally to ≤5.0. The pH reductions and associated declines in pathogen viability were greater in the CEntE-treated samples within each batch. L. monocytogenes failed to grow in cheeses previously ‘aged’ in retail for 15 days. Overall, high population levels (>7.5 log10 CFU/g) of psychrotrophic Enterobacteriaceae, particularly Hafnia alvei, were associated with an extended growth and increased survival of L. monocytogenes during storage. Full article
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19 pages, 2132 KiB  
Article
Antimicrobial Susceptibility of Lactobacillus delbrueckii subsp. lactis from Milk Products and Other Habitats
by Noam Shani, Simone Oberhaensli, Hélène Berthoud, Remo S. Schmidt and Hans-Peter Bachmann
Foods 2021, 10(12), 3145; https://doi.org/10.3390/foods10123145 - 18 Dec 2021
Cited by 5 | Viewed by 2883
Abstract
As components of many cheese starter cultures, strains of Lactobacillus delbrueckii subsp. lactis (LDL) must be tested for their antimicrobial susceptibility to avoid the potential horizontal transfer of antibiotic resistance (ABR) determinants in the human body or in the environment. To [...] Read more.
As components of many cheese starter cultures, strains of Lactobacillus delbrueckii subsp. lactis (LDL) must be tested for their antimicrobial susceptibility to avoid the potential horizontal transfer of antibiotic resistance (ABR) determinants in the human body or in the environment. To this end, a phenotypic test, as well as a screening for antibiotic resistance genes (ARGs) in genome sequences, is commonly performed. Historically, microbiological cutoffs (MCs), which are used to classify strains as either ‘sensitive’ or ‘resistant’ based on the minimal inhibitory concentrations (MICs) of a range of clinically-relevant antibiotics, have been defined for the whole group of the obligate homofermentative lactobacilli, which includes LDL among many other species. This often leads to inaccuracies in the appreciation of the ABR status of tested LDL strains and to false positive results. To define more accurate MCs for LDL, we analyzed the MIC profiles of strains originating from various habitats by using the broth microdilution method. These strains’ genomes were sequenced and used to complement our analysis involving a search for ARGs, as well as to assess the phylogenetic proximity between strains. Of LDL strains, 52.1% displayed MICs that were higher than the defined MCs for kanamycin, 9.9% for chloramphenicol, and 5.6% for tetracycline, but no ARG was conclusively detected. On the other hand, all strains displayed MICs below the defined MCs for ampicillin, gentamycin, erythromycin, and clindamycin. Considering our results, we propose the adaptation of the MCs for six of the tested clinically-relevant antibiotics to improve the accuracy of phenotypic antibiotic testing. Full article
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14 pages, 3900 KiB  
Article
Genomic and Metabolic Features of an Unexpectedly Predominant, Thermophilic, Assistant Starter Microorganism, Thermus thermophilus, in Chinese Inner Mongolian Cheese
by Lin Zhu, Zhaozhi Hou, Xinyu Hu, Xu Liu, Tian Dai, Xinyu Wang, Chunlin Zeng, Yuan Wang, Caizheng Wang, Shujing Yang, Henglin Cui and Wei Wei
Foods 2021, 10(12), 2962; https://doi.org/10.3390/foods10122962 - 02 Dec 2021
Cited by 4 | Viewed by 1696
Abstract
Inner Mongolian cheese is a traditional dairy product in China. It is produced without rennet, using naturally acidified milk that is simmered to achieve whey separation. In order to analyse the impact of simmering on the microbial community structure, high-throughput sequencing was performed [...] Read more.
Inner Mongolian cheese is a traditional dairy product in China. It is produced without rennet, using naturally acidified milk that is simmered to achieve whey separation. In order to analyse the impact of simmering on the microbial community structure, high-throughput sequencing was performed to obtain bacterial 16S rRNA sequences from cheeses from the Ordos (ES), Ulanqab (WS), Horqin (KS) and Xilingol (XS) grasslands of Inner Mongolia. The relative abundance of an unexpected microorganism, Thermus thermophilus, ranged from 2% to 9%, which meant that its dominance was second only to that of lactic acid bacteria (LABs). Genome sequencing and fermentation validation were performed in T. thermophilus N-1 isolated from the Ordos, and it was determined that T. thermophilus N-1 could ingest and metabolise lactose in milk to produce lactate during the simmering process. T. thermophilus N-1 could also produce acetate, propionate, citrate and other organic acids through a unique acetate production pathway and a complete propionate production pathway and TCA cycle, which may affect texture and flavour development in Inner Mongolian cheese. Simultaneously, the large amount of citrate produced by T. thermophilus N-1 provides a necessary carbon source for continuous fermentation by LABs after the simmering step. Therefore, T. thermophilus N-1 contributes to cheese fermentation as a predominant, thermophilic, assistant starter microorganism unique to Chinese Inner Mongolian cheese. Full article
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21 pages, 620 KiB  
Article
Assessment of the Spoilage Microbiota during Refrigerated (4 °C) Vacuum-Packed Storage of Fresh Greek Anthotyros Whey Cheese without or with a Crude Enterocin A-B-P-Containing Extract
by Nikoletta Sameli, Eleni Sioziou, Loulouda Bosnea, Athanasia Kakouri and John Samelis
Foods 2021, 10(12), 2946; https://doi.org/10.3390/foods10122946 - 30 Nov 2021
Cited by 5 | Viewed by 1836
Abstract
Although fresh whey cheeses are prone to rapid deterioration, mainly by psychrotrophic Gram-negative bacteria and lactic acid bacteria (LAB), data on the specific spoilage species in traditional Greek whey cheeses are scarce. Therefore, this study quantified growth and characterized the primary spoilage bacteria [...] Read more.
Although fresh whey cheeses are prone to rapid deterioration, mainly by psychrotrophic Gram-negative bacteria and lactic acid bacteria (LAB), data on the specific spoilage species in traditional Greek whey cheeses are scarce. Therefore, this study quantified growth and characterized the primary spoilage bacteria in fresh Anthotyros whey cheeses stored at 4 °C in a vacuum for 40 days, without or with an added 5% (v/w) of an enterocin A-B-P crude extract (CEntE). Psychrotrophic Pseudomonas spp., Aeromonas spp., Hafnia spp. and Serratia spp. grew faster than LAB during early storage. However, LAB outgrew the Gram-negative bacteria and prevailed by mid to late storage in all cheese batches, causing a strong or milder batch-dependent natural acidification. Two major non-slime-producing and two minor biotypes of Leuconostoc-like bacteria, all identified as Leuconostoc mesenteroides by 16S rRNA sequencing, dominated the LAB association (76.7%), which also included four subdominant Carnobacterium maltaromaticum biotypes (10.9%), one Leuconostoc lactis biotype (3.3%) and few Lactococcus (1.6%), mesophilic Lactobacillus (0.8%) and Enterococcus (0.8%). Growth and distribution of LAB and Gram-negative species were strongly batch-dependent and plant-dependent. The CEntE neither retarded growth nor altered the whey cheese spoilage association but enhanced LAB growth and the declines of Gram-negative bacteria by late storage. Full article
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9 pages, 1555 KiB  
Communication
Mycobiota Composition of Robiola di Roccaverano Cheese along the Production Chain
by Federica Biolcati, Ilario Ferrocino, Maria Teresa Bottero and Alessandra Dalmasso
Foods 2021, 10(8), 1859; https://doi.org/10.3390/foods10081859 - 11 Aug 2021
Cited by 3 | Viewed by 1794
Abstract
Robiola di Roccaverano is a Protected Designation of Origin (PDO) cheese from the Piedmont region of Italy. In this study, the mycobiota occurring during Robiola di Roccaverano production was elucidated. Samples of milk, Natural Milk Cultures (NMC), curd, 5- and 15-days ripened cheese [...] Read more.
Robiola di Roccaverano is a Protected Designation of Origin (PDO) cheese from the Piedmont region of Italy. In this study, the mycobiota occurring during Robiola di Roccaverano production was elucidated. Samples of milk, Natural Milk Cultures (NMC), curd, 5- and 15-days ripened cheese were collected from one dairy plant and the mycobiota was analyzed by the metataxonomic approach. Milk samples showed a high diversity and Cladosporium, Kluyveromyces marxianus, Geotrichum candidum and Debaryomyces hansenii were found with higher relative abundance. This mycobiota remains quite stable in NMC and curd matrices although the relative abundance of K. marxianus and G. candidum yeasts increased significantly and shaped the fungal composition of 5- and 15-day ripened cheese. Full article
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Review

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13 pages, 836 KiB  
Review
The Microbiota of Grana Padano Cheese. A Review
by Giorgio Giraffa
Foods 2021, 10(11), 2632; https://doi.org/10.3390/foods10112632 - 29 Oct 2021
Cited by 15 | Viewed by 2816
Abstract
Grana Padano (GP) is the most appreciated and marketed cheese with Protected Designation of Origin in the world. The use of raw milk, the addition of undefined cultures (defined as ‘sieroinnesto naturale’), the peculiar manufacturing proces, and the long ripening make the cheese [...] Read more.
Grana Padano (GP) is the most appreciated and marketed cheese with Protected Designation of Origin in the world. The use of raw milk, the addition of undefined cultures (defined as ‘sieroinnesto naturale’), the peculiar manufacturing proces, and the long ripening make the cheese microbiota play a decisive role in defining the quality and the organoleptic properties of the product. The knowledge on the microbial diversity associated with GP has been the subject, in recent years, of several studies aimed at understanding its composition and characteristics in order, on the one hand, to improve its technological performances and, on the other hand, to indirectly enhance the nutritional quality of the product. This review aims to briefly illustrate the main available knowledge on the composition and properties of the GP microbiota, inferred from dozens of studies carried out by both classical microbiology techniques and metagenomic analysis. The paper will essentially, but not exclusively, be focused on the lactic acid bacteria (LAB) derived from starter (SLAB) and the non-starter bacteria, both lactic (NSLAB) and non-lactic, of milk origin. Full article
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21 pages, 558 KiB  
Review
Artisanal Brazilian Cheeses—History, Marketing, Technological and Microbiological Aspects
by Ana Lucia Barretto Penna, Mirna Lucia Gigante and Svetoslav Dimitrov Todorov
Foods 2021, 10(7), 1562; https://doi.org/10.3390/foods10071562 - 06 Jul 2021
Cited by 13 | Viewed by 4799
Abstract
This review focused on the historical, marketing, technological, and microbiological characteristics of artisanal Brazilian cheese. Brazilian cheese production was introduced and developed from the influence of immigrants considering the combination of climate, races of the animals, quality and specificity of milk, technological cheese-making [...] Read more.
This review focused on the historical, marketing, technological, and microbiological characteristics of artisanal Brazilian cheese. Brazilian cheese production was introduced and developed from the influence of immigrants considering the combination of climate, races of the animals, quality and specificity of milk, technological cheese-making processes and environmental microbiology, among other factors. It resulted in cheese products with specific physicochemical, microbiological, and sensory quality, which represent the heritage and identities of the different Brazilian regions. The production of artisanal cheese increased in many Brazilian regions, mainly in the southeast, especially due to the traditional production and innovative development of new varieties of cheese. The microbiological quality and safety of raw-milk artisanal cheese continues to be a concern and many studies have been focusing on this matter. Special attention needs to be given to the cheeses produced by raw milk, since numerous reports raised concerns related to their microbiological safety. This fact requires attention and the implementation of strict hygiene practices on the production and commercialization, besides appropriate governmental regulations and control. However, more studies on the relationship between technological processes and microbiological properties, which results in a superior culinary quality and safety of artisanal Brazilian cheeses, are needed. Full article
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