An overview of the microbiological characteristics of the wooden shelves used in cheese ripening

Raimondo Gaglio, Massimo Todaro, Luca Settanni* An overview of the microbiological characteristics of the wooden shelves used in cheese ripening

Una panoramica delle caratteristiche microbiologiche delle assi in legno impiegate per la maturazione dei formaggi

Dipartimento Scienze Agrarie, Alimentari e Forestali, Ed. 5, Università degli Studi di Palermo, Viale delle Scienze, 90128, Palermo, Italy

*Corresponding author: Luca Settanni

Dipartimento Scienze Agrarie, Alimentari e Forestali, Ed. 5, Università degli Studi di Palermo, Viale delle Scienze, 90128, Palermo, Italy E-mail: luca.settanni@unipa.it Received: Apr 20, 2022 Accepted: Jul 04, 2022 DOI: 10.36138/STLC.04.2022.02

Abstract Cheese ripening is a complex phenomenon affected by several factors. Among these, the influence of the wooden shelves used for aging traditional cheeses is underrated. The surfaces of the wooden boards are in contact with cheese rind for the entire ripening period and develop a microbial community. The aims of this review article is to provide an overview on the microbiological approaches applied to study the biofilms associated to the wooden tools used for dairy productions and to summarize the studies specifically performed on the wooden shelves used to ripen traditional cheeses. A critical discussion is focused on the hygienic aspects and the safety of these systems applied in cheese production for centuries.

Keywords: J Cheese ripening J Lactic acid bacteria J Traditional cheeses J Wooden shelves Riassunto La maturazione dei formaggi è un fenomeno complesso che dipende da vari fattori. Tra questi, l’influenza delle scaffalature in legno impiegate per la maturazione dei formaggi tradizionali è un aspetto sottovalutato e poco studiato. Le superfici delle assi di legno sono a contatto con la crosta del formaggio per l’intero periodo di maturazione e sviluppano una comunità microbica. L’obiettivo di questo articolo è quello di fornire una panoramica sugli approcci microbiologici applicati per studiare i biofilm associati alle attrezzature in legno impiegate per le produzioni casearie e riassumere gli studi eseguiti nello specifico sugli scaffali di legno utilizzati per maturare i formaggi tradizionali. La discussione critica è focalizzata sugli aspetti igienici e sulla sicurezza di questi sistemi applicati per secoli nella produzione dei formaggi.

Parole chiave: J Maturazione dei formaggi J Batteri lattici J Formaggi tradizionali J Assi di stagionatura

INTRODUCTION

The dairy tradition is often firmly linked to the use of wood. Nowadays, wood represents the raw material for the manufacture of the equipment and utensils used in the dairy factories for many typical cheeses (1), especially those enjoying quality labels (2, 3). In the past, several containers in contact with foods were made of wood, mainly because this material was locally available at low cost (4). At that time, the importance of wood was considered very crucial for the production of cheeses (5). The use of wooden equipment for dairy productions in Europe has been almost replaced by stainless steel and plastic tools in several countries, because these materials are easy to clean. Basically, wooden equipment are still used in France and Italy to produce several traditional and typical cheeses thanks to the derogation of the European Commission (EC) no. 2074/2005 from the regulation EC no. 852/2004 for foods with traditional characteristics “as regards the type of materials of which the instruments and the equipment used specifically for the preparation, packaging, and wrapping of these products are made” (6). However, wood in dairy sector is still present in other continents, like South America (7) and North America (8). The issues related with the wood surface derive from its porous ultrastructure facilitating the absorption and trapping of bacteria determining the development of complex biofilms (9). A biofilm is an aggregate of microorganisms embedded into their extracellular polymeric substances (EPS) adhering to a solid surface (10). Traditional Sicilian cheeses, especially those enjoying a protection designation of origin (PDO) are in contact with wood during the entire production cycle, from milk collection until the end of ripening. The dairy tool better characterized for the microbiological aspects and safety is the wooden vat that is first used to collect milk and then for milk curdling (3). Several investigations demonstrated that the wooden vat represents a safe system to produce cheeses (3, 1115). Those works focused on the wooden vat biofilms evidenced the presence of lactic acid bacteria (LAB), responsible for curd acidification (starter LAB) and cheese ripening (non-starter LAB), and the absence of pathogenic species, in particular Listeria monocytogenes and Salmonella spp. taken as food safety criteria in light of the regulation EC no. 2073/2005 (16). Another well characterized dairy wooden tool is the open-topped table used for curd acidification or moulding, dominated by LAB (7, 17). Although the research on the microbiological characterization of the wooden shelves is more limited, in the last years, efforts are being made to determine firstly the safety of these tools widely used for cheese ripening. The present review article reports the main findings of the characterization of the wooden boards used to ripen traditional cheeses focusing on the microbiological aspects and highlighting the main strength points and drawbacks.

APPROACHES TO ANALYSE WOODEN SURFACE MICROBIOLOGY

The approaches applied to characterize the microbiological aspects of the wooden boards in contact with cheese during ripening are different, but the collection of biofilms from the wooden surface is necessary in any case. Basically a biofilm from a surface can be collected through destructive and non-destructive methods (1). So far, there is no standard collection method for wooden surfaces because their roughness and porousness determine difficulties in recovering microorganisms (18). In case of wooden surface biofilms, destructive methods for col-

Figure 1. Biofilm collection from wooden shelf surfaces. A, destructive method; B, non-destructive method.

lection consist on the cut (planning) of a restricted representative area by scalpel or cutter (17) (Fig. 1A) or planer or of a surface grinding (19). The non-destructive approach is mostly based on the collection of biofilms by a swab, gauze or brush within a given area delimited with sterile squares (Fig. 1B). Ismail and co-workers (19) stated that destructive methods recover wood associate microorganisms more efficiently than non-destructive methods. However, the last approach does not cause damages to the structure and can be routinely applied to monitor the hygienic characteristics of the wooden tools. Wooden biofilms can be visually inspected by microscopy. In order to produce a complete overview of the microorganisms developed into such a complex EPS matrix, scanning electron microscopy (SEM) is generally performed (20). The photograph taken during the analysis allow to determine microbial cell morphologies, the distribution of the different cells, the thickness of the polymeric substance in which they are embedded and the level of wood coverage by the biofilm. Of course, SEM can only be performed after the destructive collection of the biofilm, since wood splinters are sputter coated with a thin layer of gold before microscopic investigation (14). The biofilms collected by non-destructive methods are generally subjected to the classical analysis by plate count (culture-dependent approach) and/or analyzed by the modern high-throughput sequencing (HTS) of DNA of all microbial nucleic acids (shotgun sequencing) present in a given sample. The last (culture-independent) approach provides a complete overview of the microbial community composition of complex matrices (21).

MICROSCOPIC INVESTIGATION OF WOODEN SHELVES USED TO RIPEN CHEESE

One of the most recent study carried out to characterize the wooden boards used for cheeses ripening was reported by Settanni et al. (22). In that study, the wooden shelves analyzed were made of silver fir, chestnut, Swedish pine, stone pine and common beech, and were associated to the ripening of PDO Pecorino Siciliano, PDO Piacentinu Ennese, and agri-food product (TAP) Caciocavallo Palermitano which represent three typical cheeses made from raw milk in Sicily (south Italy). The wooden boards were sampled during cheese ripening (the contact with cheese lasted two months for PDO Piacentinu Ennese, while four to five months for the other two products) from 18 dairy facilities among different Sicilian provinces. The photographs taken from the wooden shelf splinters subjected to SEM analysis (Fig. 2) showed a different level of microbial coverage. In a few cases, the woody roughness was well visible and only a few cells adhered to the shelf surface, but for the majority of the samples the biofilms covered almost the entire surface. The lack of a continuous thick layer of EPS typical of bacterial biofilms detected on the wooden vats used for milk

Figure 2. Scanning electron microscopy (SEM) micrographs of wooden splinters from wooden boards used for traditional Sicilian cheese ripening.

curdling (11, 14) could not be imputable to a different cleaning procedure, because all shelves were cleaned once a year by a thorough brushing and a pressure washer, at the end of the summer season. Not even a different contact with cheese rind could explain the different distribution of the biofilms, since all shelves were sampled from the area just in contact with the cheeses. Regarding cell composition, the biofilms mainly hosted bacterial populations with different proportions of cocci and rods, even though the majority of samples were dominated by cocci. In some biofilms, yeast cells were detected at a high frequency and a few fungal hyphae showed the high level of complexity of the microbial community of the wooden boards used for Sicilian cheese ripening. Mariani et al. (23) focused on the characterization of the wooden shelves used to ripen PDO Reblochon de Savoie, a raw milk smear cheese produced in the French Alps. The shelves analyzed were made of spruce wood, had a different age (from <4 years to >8 years) and were sampled from eight farmhouses. SEM showed the presence of bacteria and yeasts mostly in cracks, apertures, and pores of the wooden surfaces. Also in this case, the microbial biofilms did not cover entirely the surface of the shelves. Geotrichum candidum cells were easily recognized thanks to their typical cylinder shape. SEM was also successfully applied to evaluate the efficacy of a cleaning-drying treatment of spruce wood shelves used to ripen PDO Reblochon de Savoie cheese (24). The procedure applied consisted of cleaning with a brushing machine using cold water and drying in air with dedicated machines and SEM analysis clearly showed that cleaning-drying removed consistently yeasts and bacteria from the wooden shelves. SEM analyses was also particularly useful to monitor the adhesion of bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and yeasts (Pichia membranifaciens) artificially inoculated onto the surface of three wood types: poplar, Norway spruce and European beech (25). Although not able to quantify microbial cells, SEM micrographs clearly showed the different adherence behaviour of the species included in the test and indicated that P. aeruginosa was the species that better adhered to all wood types.

CULTURE-DEPENDENT ANALYSYS OF WOODEN SHELVES

With the aim to investigate on the safety aspects of the wooden shelves used for cheese ripening, the determination of the level and the identification of the viable cells is of paramount importance. Mariani et al. (23) characterized the wooden shelves used for PDO Reblochon de Savoie smear cheeses also by plate counts. Wooden shelf community was dominated by micrococci - corynebacteria which accounted for 7.2-7.3 Log CFU/cm2 followed by yeasts and molds in the range 6.0-6.1 Log CFU/cm2. Leuconostocs, lactobacilli, enterococci, staphylococci, and pseudomonads were found at low levels (3.7, 2.2, 3.1, 3.2, and 3.0 Log CFU/ cm2, respectively). These results were not surprising because smear cheeses are inoculated with surface starters that are commercially available (26). Thus, the dominance of micrococci-corynebacteria and yeasts is an expected phenomenon. The wooden shelves analyzed within those used to ripen Sicilian cheeses were specifically investigated for 12 microbial groups (22). The differences found among the 18 shelves were particularly high. E.g. total mesophilic microorganisms (TMM) were in the range 2.4-7.8 Log CFU/cm2. LAB were investigated considering five main groups: mesophilic rods, thermophilic rods, mesophilic cocci, thermophilic cocci and enterococci. The group of mesophilic cocci was detected at the highest levels on all boards and the densities registered were comparable to those of TMM, indicating their dominance over the bacterial community of each sample. Enterococci were enumerated from all wooden shelves and their levels did not exceed 4.1 log CFU/cm2. Regarding the undesirable groups, members of the Enterobacteriaceae family were found on six wooden shelves, with coliforms counted only from four samples. Pseudomonads were in the range 2.5 - 5.8 log CFU/cm2 on 11 wooden shelves. Yeasts colonized all wooden boards in the range 1.8 - 6.8 log CFU/cm2, while molds developed from 13 biofilms and their level was not above 5.4 Log CFU/ cm2. Regarding microbiological and hygiene criteria for foodstuffs, barely three shelves were characterized by the presence of viable E. coli which, however, did not carry Shiga-toxigenic E. coli genes after PCR investigation, while coagulase-positive staphylococci, Salmonella spp., and L. monocytogenes were not detected at all. A multivariate statistical treatment of the microbial loads indicated a cheese typology effect of the wooden shelves used for Sicilian cheese ripening (27).

NEXT-GENERATION SEQUENCING APPROACH

Next-generation sequencing tools show a high degree of identification and discrimination among microbial populations. Thus, in the last years, HTS became quite common to investigate deeply the biodiversity of the microbial communities of complex matrices. These tools are used in combination or even as alternative to the classi-

cal culture-dependent methods to describe the microbial composition of food matrices (28, 29) and biofilms associated to the equipment surfaces in contact with foods (15, 17). Settanni et al. (22) applied Illumina technology to perform the taxonomic classification of the biofilms associated to the ripening of Sicilian cheeses. This approach allowed the identification of 137 bacterial groups from 93 families. The species identified most frequently from all wooden surfaces was Staphylococcus equorum, that reached a maximum abundance of 64.75%. This species is salt tolerant (30, 31) and represents a starter culture for surface-ripened semihard cheeses (32). Other cheese-surface-ripening bacteria such as Brevibacterium and Corynebacterium were detected in almost all biofilm samples. Illumina analysis also identified several halophilic (Halomonas, Tetragenococcus halophilus, Chromohalobacter, Salimicrobium, Marinococcus, Salegentibacter, Haererehalobacter, Marinobacter, and Idiomarinaceae) and moderately halophilic (Salinicoccus, Psychrobacter, and Salinisphaera) species, while LAB, represented by Leuconostoc, Lactococcus, Lactobacillus, Pediococcus, and Streptococcus were detected at low percentages. The same (Illumina) technology was used as sole tool to describe the microbiota of five wooden shelves used for aging artisan cheeses in three dairy facilities located in the United States (33). The boards were made of cedar, pine and spruce. Although also in this case a significant board-to-board variation was observed, the genera most frequently identified were Brevibacterium (2363%), Psychrobacter (~42%), Brachybacterium (11-23%), and Staphylococcus (11-16%), followed by low levels of Jeotgalicoccus, Nocardiopsis, and Corynebacterium. Those data suggested that cheese-ripening environment selects for salt- and cold-tolerant bacteria. Furthermore, Guzzon et al. (34) investigated the biofilms of the wooden shelves used to ripen smear Fontina cheese by 454 pyrosequencing, finding a cause-effect relationship between the dominant Actinobacteria and the red-brown pigmentation defect.

CONCLUSIONS

The combined approaches applied to study the microbial diversity of the wooden shelves used to ripen different cheeses throughout the world evidenced that the majority of microorganisms associated to these systems are typically associated with smear cheeses. Among bacteria, those most frequently identified belonged to the halophilic and halotolerant groups. In particular, in all works performed so far, food safety and process hygiene criteria showed that the wooden shelves do not represent microbiological risks for the human health. However, future works are necessary to better correlate microbial community of the wooden shelves and the final characteristics of the cheeses ripened onto their surfaces.

CONFLICT OF INTEREST DISCLOSURE

All authors declare that they have no conflict of interest inherent the present paper.

FUNDING

This work was supported by AGER 2 Project, grant no. 2017-1144.

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