Occurrence of Clostridium spp. in ovine milk and Manchego cheese with late blowing defect: Identification and characterization of isolates

Abstract

The presence of lactate-fermenting clostridial spores was investigated in 194 samples of raw bulk-tank ovine milk and in 57 Manchego cheeses with late blowing defect collected throughout one year from 55 factories. Spores were detected in 97% of milk samples analysed, with a most probable number (MPN) of 14.5 spores mL⁻¹ and significantly (P < 0.001) higher counts in summer milk. The highest incidence of late blowing defect was also recorded for Manchego cheeses produced in summer. Out of 223 isolates from spoiled cheeses, 78.9%, 10.3%, 9.0% and 1.8% were identified as Clostridium sporogenes, Clostridium beijerinckii, Clostridium tyrobutyricum and Clostridium butyricum, respectively. Among seven bacteriocins produced by lactic acid bacteria, nisin A and Z were the only ones able to inhibit the growth of all Clostridium isolates.

Introduction

Butyric acid fermentation is responsible for the late blowing defect caused by the outgrowth in cheese of species of the genus Clostridium capable of fermenting lactic acid with production of butyric acid, acetic acid, carbon dioxide and hydrogen. It is one of the major causes of spoilage in semi-hard and hard ripened cheeses, resulting in the appearance of texture and flavour defects (McSweeney & Fox, 2004), with an unfavourable economic impact on cheese production. Clostridium tyrobutyricum, a Gram-positive, anaerobic, spore forming bacterium, is considered the primary cause of late blowing in cheese. However, other clostridial species such as Clostridium sporogenes, Clostridium beijerinckii and Clostridium butyricum have also been shown to contribute significantly to the appearance of this defect in cheese (Cocolin et al., 2004, Klijn et al., 1995, Le Bourhis et al., 2005, Le Bourhis et al., 2007).

Clostridium spores present in cheese originate from milk, which becomes contaminated during the milking process. Silage of poor microbiological quality has been identified as the main source of raw milk contamination with spores that are able to survive the pasteurization process (Dasgupta and Hull, 1989, Julien et al., 2008, Te Giffel et al., 2002, Vissers et al., 2006, Vissers et al., 2007). Growth of Clostridium is critically influenced by lactic acid concentration, salt concentration, pH, moisture and fat content, ripening time and temperature, and the presence of other microorganisms in cheese (Goudkov & Sharpe, 1965). The size, shape and texture of the cheese also play a significant role (Aureli and Franciosa, 1999, Dasgupta and Hull, 1989).

The most common strategies to prevent late blowing defect are bactofugation and microfiltration of milk, and the addition of nitrate or lysozyme (van den Berg et al., 2004, Lodi, 1990, Stadhouders, 1990). However, reduction in the number of spores by bactofugation is often not sufficient to prevent late blowing in cheese. Microfiltration, which achieves about 99% reduction of spore forming bacteria relative to the 95% obtained by bactofugation, can be applied only to skim milk because the milk fat globules are too large to pass through the microfiltration membrane. The European Food Safety Authority (EFSA) recommends minimizing the use of nitrate and nitrite as food additives in order to keep the level of potentially carcinogenic nitrosamines as low as possible (EFSA, 2010). Lysozyme, obtained from hen egg white, could pose risks for allergic subjects (Fremont, Kanny, Nicolas, & Moneret-Vautrin, 1997). Moreover, there is an increasing demand from consumers for additive free food products. An alternative strategy to prevent late blowing defect is the use of bacteriocinogenic lactic acid bacteria (LAB) in cheese manufacture (Anastasiou et al., 2009, Martínez-Cuesta et al., 2010, Matijasic et al., 2007, Rilla et al., 2003).

Manchego cheese, the best known among the Spanish cheeses with a Protected Designation of Origin (PDO), is a semi-hard or hard variety made in the La Mancha region from raw or pasteurized ovine milk from the Manchega breed. Bovine rennet is used as milk coagulant, after inoculation with mesophilic starter cultures. The use of thermophilic starters as adjunct cultures tends to increase. Late blowing defect associated to the presence of Clostridium has been occasionally detected in some Manchego cheese factories, causing important losses in cheese production.

Information on the presence of Clostridium in ovine milk and cheeses is scarce. The objectives of the present work were 1) to investigate the occurrence of clostridial spores in ovine milk from the Manchega breed, 2) to identify Clostridium isolates from Manchego cheeses with late blowing defect, and 3) to characterize these isolates with respect to production of organic acids and sensitivity to LAB bacteriocins.