Jenny Milk as Inhibitor in Cheese Making

Late blowing defects on ripened semihard and hard cheese are an important problem with a high negative economic impact in dairy production. The causes depend on both technological and microbiological factors. Technological factors include milk quality, heat treatment, hygiene practices, manufacture technology, compositional parameters and ripening temperature/moisture, which influence cheese making and ripening (Bogovic´ Matijasˆic´ et al. 2007). Microbiological factors are the most difficult to control, because undesirable microorganisms, such as coliforms, yeasts, heterofermentative lactic acid bacteria and spore-forming bacteria, may cause early or late blowing defects in cheeses (Little et al. 2008;

G,omez-Torres et al. 2014). Late blowing defect in semihard and hard cheeses has

been attributed to the outgrowth of strains of Clostridium spp. (mainly C. butyricum and C. tyrobutyricum), capable of fermenting lactic acid with production of butyric acid, acetic acid, carbon dioxide and hydrogen (Garde et al. 2012). Clostridium spores are ubiquitous, much more resistant to heat, chemicals, irradiation and desiccation than vegetative cells, and its growth in cheese is affected critically by different factors such as salt concentration, pH, ripening time and temperature as well as by the presence of other microorganisms (Garde et al. 2011).

Many studies have attempted to prevent late blowing by physical treatments (bactofugation or microfiltration prior to processing), or by the use of additives (nitrate or lysozyme), or by the addition of strains of lactic acid bacteria (LAB) producing bacteriocins, active peptides displaying a bactericidal mode of action towards specific Gram-positive bacteria (Wasserfall and Teuber 1979; Vissers et al. 2007; Mart`ınez-Cuesta et al. 2010; Schneider et al. 2010).

Among the above prevention methods, the use of lysozyme as a commercial additive is the preferred one since 1983. Lysozyme, which is typically extracted from hen egg white (HEW, 3.5 % of the egg white proteins), has been approved as a preservative (E1105) in the entire European Community, according to the Directive No. 95/2/EC (quantum satis in ripened cheese) (Pellegrino and Tirelli 2000; Scharfen et al. 2007; Schneider et al. 2011). In Italy, the use of lysozyme is quite widespread: this enzyme has been employed in the process of making several cheeses, such as Grana Padano cheese, grated hard cheese mixtures (Iaconelli et al. 2008; Panari and Filippi 2009) and semihard goat and ewe cheeses (Dragoni et al. 2011; Schneider et al. 2011). The content of lysozyme from egg in cheese ranges from 50 to 350 μg/g of cheese, with the maximum of 400 μg/g of cheese depending on the type of cheese and the production process (Pellegrino and Tirelli

2000; A´ vila et al. 2014).

In recent years, the use of lysozyme from egg as a prevention agent has waned, since some studies have shown its allergenic effect in consumers allergic to egg, due to its content in ovomucoid, ovalbumin and conalbumin (Fre´mont et al. 1997; Pe´rez-Caldero et al. 2007). In the last decade, a number of severe allergic reactions have been recorded due to the presence of lysozyme E1105 in semihard cheeses. Fre´mont et al. (1997) found patients allergic to eggs to show a severe reaction after eating Gruyere cheese. Kerkaert et al. (2010) reported that 5 out of 21 case studies of allergic reactions to eggs were attributed to the presence of this additive in cheese and that this additive was likely responsible for episodes of severe edema (Pe´rezCaldero et al. 2007). For these reasons, in the recently changed EC legislation, the use of lysozyme as an additive has to be declared on the label (EC legislation in Europe 2003/89/EC, Directive 2000/13/EC).

Jenny milk may be a possible alternative to the use of lysozyme from egg during cheese making. In fact, jenny milk is characterised by a high lysozyme content, ranging from 1.0 to 3.7 mg/mL, according to the lactation stage and the production season (Zhang et al. 2008; Vincenzetti et al. 2012). The content of lysozyme in jenny milk is much higher than cow (0.13 μg/mL), ewe (0.20 μg/mL) or goat milk (0.25 μg/mL) (Fratini et al. 2006; Scharfen et al. 2007; Cosentino and Paolino 2012). Moreover, in jenny milk, lysozyme shows the highest activity at an optimum temperature of 37 oC and is stable up to a temperature of 50 oC, decreasing to 50 % of activity at 70 oC. Recently, Galassi et al. (2012) described the addition of jenny milk as a substitute for egg lysozyme to prevent late blowing in Grana Padano cheese. The authors found that the addition of 10 L of jenny milk in 500 L of cow milk reduced significantly physico-chemical and microbiological defects of cheese. Cosentino and Paolino (2012) studied the effect of lysozyme from jenny milk on blowing defects in artisanal ewe cheese caused by clostridia and coliforms, usually present in ewe cheese produced in traditional cheese factories. When adding jenny milk to ewe milk, no late blowing defect on cheese was observed (Fig. 3). Therefore, lysozyme contained in jenny milk was found to be an important inhibitor agent against coliform bacteria, although its addition to ewe milk did not affect the number of Clostridium butyricum spores (Cosentino and Paolino 2012; Cosentino et al. 2013b). The lower content of coliforms in treated ewe cheese was in agreement with results from the literature on reduced growth of C. butyricum in Grana Padano (Iaconelli et al. 2008; Dragoni et al. 2011) and Gouda cheese (Bester and Lombard 1990). Mart`ınez-Cuesta et al. (2010) observed a higher contamination of Clostridium in Manchego control cheese compared with that treated with lysozyme HEW.

Cosentino et al. (unpublished data) also evaluated whether increasing additions of jenny milk to pasteurised cow milk reduced the late blowing defect in semihard cheese caused by C. tyrobutyricum. To verify this hypothesis, the authors made two types of cheeses, control and treated, with the latter being deliberately contaminated with approximately 3 log spores/mL milk of C. tyrobutyricum CLST01, in order to induce butyric acid fermentation and consequent blowing defect. Both control and treated cheeses were made by adding different aliquots of jenny milk to cow milk. The addition of jenny milk resulted in a sporostatic effect on both control and treated cheeses. Visual and odour inspections during ripening demonstrated that all cheeses contaminated with C. tyrobutyricum developed signs of late blowing

Fig. 3 Signs of late blowing defects in ewe cheese (from first to third row) and cow cheese (from fourth to sixth row) at the end of ripening

defect, except the cheese containing the highest aliquot of jenny milk. This product presented a uniform texture without cracks and splits (Fig. 3). The content of

lysozyme was high in both control and treated cheeses (1.57 and 1.52 mg/kg, respectively). A´ vila et al. (2014) found that 40 μg/mL of lysozyme was the

concentration required to completely inhibit the growth of vegetative cells of

C. tyrobutyricum strains. Cosentino et al. (unpublished data) also found that the acceptability of cheeses was not affected by the addition of jenny milk, since consumers did not found differences between the products made with only cow milk and those made also with jenny milk. These results are in line with the findings of Galassi et al. (2012).

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