Investigation of the protective effect of whey proteins on lactococcal phages during heat treatment at various pH
Introduction
Whey represents a significant byproduct of cheese-making, and its availability is growing worldwide (Siso, 1996). Whey, which separates from the curd during the manufacture of cheese, was historically considered an undesirable waste (Smithers et al., 1996). However, years of research shed light on the nutritional and biological value of its proteins. As such, whey is now recognized as a valuable component in various industries (Madureira et al., 2007). The main whey proteins are β-lactoglobulin (BLG), α-lactalbumin (LAC), bovine serum albumin (BSA), immunoglobulins (Igs) and lactoferrin (LF) (Korhonen and Pihlanto, 2007).
Whey proteins represent 20% of the total milk protein and their addition to milk can lead to significant increase in cheese yield (Banks et al., 1994). As such, different strategies have been tested to incorporate whey proteins into cheese (Brown and Ernstrom, 1982, Hinrichs, 2001, Lawrence, 1989, Lawrence, 1993, Lee et al., 2013, Lelievre and Lawrence, 1988, Punidadas et al., 1999). Most of these strategies are based on the formation of whey protein microparticles (or whey protein aggregates) prior to their addition to milk. For example, fat-free whey is first pasteurized in order to inactivate the remaining starter bacteria and concentrated by ultrafiltration, leading to whey protein concentrate (WPC) of 10% or more protein. Then, a combination of heating and shearing is applied to the WPC before being added to new cheese milk batches (Atamer et al., 2013, Byylund, 1995).
However, such recycling process is risky because it may not be sufficient to guarantee phage destruction, even after the concentration of these proteins and the heat treatments applied (Atamer et al., 2009, Atamer et al., 2013, Atamer and Hinrichs, 2010, Guglielmotti et al., 2011). To manufacture cheese, lactic acid bacteria (LAB) starter cultures are added to heat-treated milk to control the fermentation process and to ensure high-quality cheese products (Garneau and Moineau, 2011). For example, strains of Lactococcus lactis are used extensively for the production of cheddar cheese (Bissonnette et al., 2000). In the non-sterile environment of heat-treated milk, the added lactococcal cells will come into contact with virulent phages that are ubiquitous in milk (Moineau and Lévesque, 2005) and dairy environs (Verreault et al., 2011). Phage population can increase rapidly by replicating and lysing the phage-sensitive cells present in the starter culture. Therefore, phage contamination represents a significant risk to the cheese manufacturing process as it may lead to production delays, lower quality product, or, in the worst cases, total loss (Samson and Moineau, 2013). Because virulent lactococcal phages can be found in whey-derived products (Labrie and Moineau, 2000), they represent an additional risk if WPC are added to milk to make cheese.
All currently known L. lactis phages belong to Caudovirales order (double-stranded DNA genome, tailed phages) and they are mostly members of the Siphoviridae family (long non-contractile tail) (Deveau et al., 2006). Most isolated lactococcal phages belong to 936 group (Mahony et al., 2012, Moisan and Moineau, 2012). The International Dairy Federation (IDF) previously suggested that a heat treatment of 15 min at 90 °C led to phage inactivation (Svensson and Christiansson, 1991). However, in the last years, some phages were found to be resistant to the above mentioned conditions (Capra et al., 2009, Capra et al., 2013). For example, the lactococcal phage P1532 (936 group) remained infectious in skim milk after heating at either 90 °C for 20 min or 97 °C for 5 min (Atamer et al., 2009). Thereafter, a prolonged heating-time of 45 min at 90 °C was proposed (Capra et al., 2013). The suspension media also influence the thermal inactivation of phages and a protective effect of whey proteins on lactococcal phages has been reported previously (Daoust et al., 1965). However, such protective effect of whey proteins on phages has yet to be explained.
To reduce the risk of fermentation failure due to phages and to maximize whey recycling, there is a critical need for understanding the nature of this protective effect. The objectives of this study were to: 1) investigate the thermal inactivation of a thermo-resistant lactococcal virulent phage (P1532) in WPC and in individual whey components β-lactoglobulin, α-lactalbumin and bovine serum albumin under different heat treatments and pH conditions; 2) relate phage inactivation to molecular structural changes monitored by Fourier transform infrared (FTIR) spectroscopy as a function of the above mentioned conditions. FTIR has been previously used to follow the conformational modifications of proteins in many tested conditions (Allain et al., 1999, Subirade et al., 1994), giving information on the denaturation, aggregation and gelation of globular proteins (Lefevre and Subirade, 2000).
Section snippets
Bacterial strain, phage and culture conditions
The lactococcal phage P1532 used in this study is stored at the Félix d'Hérelle Reference Center for Bacterial Viruses (www.phage.ulaval.ca). Its bacterial host, L. lactis 7–18, was grown at 30 °C in M17 broth (Oxoid Ltd., Basingstoke, Hampshire, England) supplemented with 0.5% glucose (GM17) (Terzaghi and Sandine, 1975). For phage amplification, L. lactis 7–18 was grown in GM17 until an optical density (600 nm) of 0.1. Then, 10 mM of CaCl2 was added as well as 105 PFU/ml of phage P1532. The
Thermal inactivation of P1532 in WPC at various pH
To study the influence of pH on the whey protective effect during heat treatment (5, 10 or 20 min), phage-containing WPC (5% protein) samples were adjusted to various pH (6.4, 5 and 4). pH adjusted GM17 medium was used as control. Phage titers were determined before and after treatments. Fig. 1 shows the inactivation of phage P1532 expressed as log10 units reduction after heat treatment at 95 °C. At pH 6.4, phages were detected after all three heating times. However, phage inactivation was more
Conclusions
The protective effect of whey proteins and three of its components during thermal inactivation of phage P1532 at various pH was investigated. From our results the following conclusions can be drawn:
- i.
The thermal stability of phage P1532 was confirmed. Moreover, phage P1532 was protected in whey protein suspensions.
- ii.
The protective effect of WPC against thermal inactivation of phage P1532 was dependent of pH and the duration of the treatment at 95 °C. The protective effect of WPC decreases after
Acknowledgments
We thank Molly Dussault for technical assistance at the beginning of the project. We are grateful to Horst Neve for the phage P1532 and its host as well as Manon Duquenne for the WPC. The authors acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC) through Strategic and Discovery grants. SM holds a Tier 1 Canada Research Chair in Bacteriophages.
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