Outgrowth inhibition of Clostridium beijerinckii spores by a bacteriocin-producing lactic culture in ovine milk cheese Sonia Garde a, ⁎, Marta Ávila a , Ramón Arias b , Pilar Gaya a , Manuel Nuñez a a Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña km 7, 28040 Madrid, Spain b Centro Regional de Selección y Reproducción Animal (CERSYRA) JCCM, Avenida del Vino n° 6, 13300 Valdepeñas, Spain abstract article info Article history: Received 10 May 2011 Received in revised form 11 July 2011 Accepted 14 July 2011 Available online 22 July 2011 Keywords: Clostridium beijerinckii Cheese blowing Outgrowth inhibition Bacteriocin producer Nisin Lacticin 481 In the manufacture of model cheeses, ovine milk was deliberately contaminated with spores of Clostridium beijerinckii INIA 63, a wild isolate from Manchego cheese with late blowing defect, and inoculated with nisin- and lacticin 481-producing Lactococcus lactis subsp. lactis INIA 415 as starter, to test its potential to prevent the late blowing defect, or with L. lactis subsp. lactis INIA 415-2, a spontaneous mutant not producing bacteriocins. Cheeses made individually with the lactococcal strains, without clostridial spores, served as controls. Cheese made with clostridial spores and L. lactis subsp. lactis INIA 415-2 showed late blowing defect after 120 days of ripening. Spoilt cheese also showed lower concentrations of lactic acid, and higher levels of acetic, propionic and butyric acids, and of other volatile compounds such as 2-propanol and 1-butanol, than control cheese. In addition, cheese made with the bacteriocin producer did not show any late blowing symptoms, despite its spore counts similar to those of blown cheese, pointing to outgrowth inhibition of C. beijerinckii spores by bacteriocins. Besides, cheese made with the bacteriocin producer showed similar concentrations of lactic acid and volatile compounds than control cheese. Inclusion of L. lactis subsp. lactis INIA 415 in starter cultures seems a feasible method to prevent late blowing defect in cheese without altering its sensory characteristics. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Butyric acid fermentation, also known as late blowing defect, is one of the major causes of spoilage in semi-hard and hard ripened cheeses, resulting in the appearance of texture and flavor defects that generate severe economic losses in the cheese industry (McSweeney and Fox, 2004). Butyric acid fermentation in cheese is caused by the outgrowth of species of the genus Clostridium capable of fermenting lactic acid with production of butyric acid, acetic acid, carbon dioxide and hydrogen. Although Clostridium tyrobutyricum, an anaerobe Gram- positive sporeforming bacterium, is considered the primary cause of late blowing in cheese, other clostridial species such as Clostridium sporogenes, Clostridium beijerinckii and Clostridium butyricum also have been shown to significantly contribute to the appearance of this defect in cheese (Cocolin et al., 2004; Klijn et al., 1995; Le Bourhis et al., 2005, 2007). Clostridium spores coming from soil are able to develop in silage, contaminate milk during the milking process and survive pasteurization (DasGupta and Hull, 1989; Julien et al., 2008; Te Giffel et al., 2002; Vissers et al., 2006, 2007). Growth of Clostridium in cheese is critically influenced by lactic acid concentration, salt concentration, pH, moisture and fat content, ripening time and temperature, and the presence of other microorganisms (Goudkov and Sharpe, 1965). Bactofugation and microfiltration of milk, and the addition of nitrate or lysozyme are common strategies to prevent late blowing defect (Lodi, 1990; Stadhouders, 1990; van den Berg et al., 2004). However, the reduction in spore numbers achieved by bactofugation may be insufficient to prevent late blowing defect, microfiltration can be applied only to skim milk because milk fat globules are too large to pass through the microfiltration membrane, and the use of chemicals is precluded by the increasing demand for additive-free food products. An alternative strategy to prevent butyric acid fermentation is the inoculation of milk with bacteriocinogenic lactic acid bacteria (LAB) in cheese manufacture. Nevertheless, studies on the use of LAB for such aim are scarce, and none of the existing studies have been carried out in ovine milk cheeses, or investigated the inhibition of species other than C. tyrobutyricum. Nisin Z-producing Lactococcus lactis ssp. lactis IPLA 729 was successfully used to control the growth of C. tyrobutyricum CECT 4011 in Vidiago cheese, a semi-hard bovine milk cheese variety (Rilla et al., 2003), and bacteriocin-producing Lactobacillus gasseri K7 delayed blowing of cheeses artificially inoculated with C. tyrobutyricum (Bogovic Matijasic et al., 2007). Lacticin 3147-producing L. lactis IFPL 3593 controlled the outgrowth of Clostridium spores and late blowing in semi- hard cheese (Martínez-Cuesta et al., 2010). Some of these studies described the effect of Clostridium and LAB on organic acids but the effect on the volatile fraction of cheese remains unknown. International Journal of Food Microbiology 150 (2011) 59–65 ⁎ Corresponding author. Tel.: + 34 91 3474037; fax: + 34 91 3572293. E-mail address: sgarde@inia.es (S. Garde). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.07.018 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro