Wageningen Academic P u b l i s h e r s
Beneficial Microbes, 2015; 6(3): 381-386
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0038 - Friday, September 22, 2017 12:16:50 AM - Göteborgs Universitet IP Address:130.241.16.16
Impact of different cryoprotectants on the survival of freeze-dried Lactobacillus rhamnosus and Lactobacillus casei/paracasei during long-term storage A. Jofré*, T. Aymerich and M. Garriga IRTA-Food Safety Programme, Finca Camps i Armet, 17121 Monells, Spain; [email protected] Received: 3 April 2014 / Accepted: 27 August 2014 © 2014 Wageningen Academic Publishers
RESEARCH ARTICLE Abstract The production of long shelf-life highly concentrated dried probiotic/starter cultures is of paramount importance for the food industry. The aim of the present study was to evaluate the protective effect of glucose, lactose, trehalose, and skim milk applied alone or combined upon the survival of potentially probiotic Lactobacillus rhamnosus CTC1679, Lactobacillus casei/paracasei CTC1677 and L. casei/paracasei CTC1678 during freeze-drying and after 39 weeks of storage at 4 and 22 °C. Immediately after freeze-drying, the percentage of survivors was very high (≥94%) and only slight differences were observed among strains and cryoprotectants. In contrast, during storage, survival in the dried state depended on the cryoprotectant, temperature and strain. For all the protectants assayed, the stability of the cultures was remarkably higher when stored under refrigeration (4 °C). Under these conditions, skim milk alone or supplemented with trehalose or lactose showed the best performance (reductions ≤0.9 log units after 39 weeks of storage). The lowest survival was observed during non-refrigerated storage and with glucose and glucose plus milk; no viable cells left at the end of the storage period. Thus, freeze-drying in the presence of appropriate cryoprotectants allows the production of long shelf-life highly concentrated dried cultures ready for incorporation in high numbers into food products as starter/potential probiotic cultures. Keywords: carbohydrates, lyophilisation, probiotic bacteria, skim milk, sublethals
1. Introduction The industrial use of lactic acid bacteria (LAB) as starter cultures in fermented products or as probiotics and their direct inclusion in food or dietary supplements require the delivery of stable cultures in terms of viability and functionality. Thus, the maximisation of survival of LAB cultures during drying and subsequent long-term storage under refrigeration or room temperature is of vital importance at the technological and economical level (Poddar et al., 2012; Selmer-Olsen et al., 1999; Tripathi and Giri, 2014). Freeze-drying is commonly used for the preservation and long-term storage of LAB, as it allows easy and inexpensive shipping and handling. Conversely, it produces undesirable side effects decreasing the viability of cultures (Carvalho et al., 2002; Morgan et al., 2006; Tripathi and Giri, 2014). The mechanisms of damage during freeze-drying are
complex and not fully understood to date but they can be attributed primarily to ice crystal formation, high osmolarity due to high concentrations of internal solutes with membrane damage, macromolecule denaturation, and the removal of water, which affect properties of many hydrophilic macromolecules in the cells (Santivarangkna et al., 2008; Zayed and Roos, 2004). Although most reports focused on survival during the process of drying, a low inactivation rate during subsequent storage is equally or even more important when a very long storage period is required (Santivarangkna et al., 2008). During storage, low water activity, storage temperature and packaging material keeping the moisture content low enough to maintain the dried culture in a glassy state and protect it from oxygen have shown to affect viability (Carvalho et al., 2004b; Higl et al., 2007; Poddar et al., 2014; Santivarangkna et al., 2008). To minimise inactivation during drying and improve the stability of the dried product, it is a common procedure
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014. 0038381
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0038 - Friday, September 22, 2017 12:16:50 AM - Göteborgs Universitet IP Address:130.241.16.16
A. Jofré et al.
to create a protective amorphous sugar matrix around the individual bacteria by adding sugars to the cell suspension before the drying process (Bergenholtz et al., 2012; Carvalho et al., 2004a; Higl et al., 2007). In LAB, different levels of protection have been observed with different sugars (e.g. glucose, fructose, lactose, mannose and sucrose), sugar alcohols (e.g. sorbitol and inositol) and non-reducing sugars (e.g. trehalose) (Carvalho et al., 2002; Higl et al., 2007; Miao et al., 2008; Pehkonen et al., 2008; Strasser et al., 2009; Zamora et al., 2006). Skim milk applied alone or in combination with other cryoprotectants is also widely applied in the starter culture industry (Morgan et al., 2006). Additionally, microrganisms greatly vary in their tolerance to freeze-drying. In general, Gram-positive bacteria show a higher tolerance than Gram-negative bacteria, which is probably related to their genetic constitution and cell wall and membrane composition (Carvalho et al., 2004b; Morgan and Vesey, 2009). However, there are considerable differences in the freeze-drying tolerance of closely related organisms; different strains of the same species can behave completely different. Thus, the freeze-drying process must be optimised for specific strains to achieve maximum viability and stability. The major problem to overcome seems to be the lack of generic theories for all bacterial strains (Morgan and Vesey, 2009; Morgan et al., 2006). Lactobacillus rhamnosus and Lactobacillus casei/paracasei are important in a number of industrial applications and used as starter cultures in traditional and functional fermented products (Leroy et al., 2006). Recently, we isolated and characterised from infants’ faeces three strains, i.e. L. rhamnosus CTC1679, L. casei/paracasei CTC1677 and L. casei/paracasei CTC1678, with putative probiotic properties suitable as starter cultures in fermented sausages (Rubio et al., 2014a,b,c). As the industrial application of a starter culture requires highly concentrated dried cultures, we evaluated the protective effect of various sugars previously reported to have protective capacity during freeze-drying (glucose, lactose and trehalose), applied alone or in combination with skim milk, upon survival of the three putative probiotic strains during freeze-drying and subsequent storage for 39 weeks at 4 and 22 °C to find the most effective protectant(s) for the production of cultures containing high levels of viable cells after a longterm storage period.
2. Material and methods Freeze-drying
(4.5 ml) and let 30 min at room temperature to allow equilibration between cells and the compound added: T (5% trehalose), G (10% glucose), L (7% lactose), M (11% skim milk), TM (5% trehalose+11% skim milk), GM (10% glucose+11% skim milk) and LM (7% lactose+11% skim milk). Subsequently, aliquots of 0.5 ml were prepared in freeze-drying vials, frozen at -80 °C for 2 h and subsequently freeze-dried in a Christ Alpha 1-4 (B. Braun Biotech Int., Melsungen, Germany). To determine the weight of the dry culture, vials were weight empty and after freeze-drying. Two independent experiments were performed on different days.
Storage After freeze-drying, vials containing dried 0.5 ml aliquots were tightly capped and individually vacuum packed in metallic pouches (PET/MET+PE), oxygen permeability
Beneficial Microbes, 2015; 6(3): 381-386
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0038 - Friday, September 22, 2017 12:16:50 AM - Göteborgs Universitet IP Address:130.241.16.16
Impact of different cryoprotectants on the survival of freeze-dried Lactobacillus rhamnosus and Lactobacillus casei/paracasei during long-term storage A. Jofré*, T. Aymerich and M. Garriga IRTA-Food Safety Programme, Finca Camps i Armet, 17121 Monells, Spain; [email protected] Received: 3 April 2014 / Accepted: 27 August 2014 © 2014 Wageningen Academic Publishers
RESEARCH ARTICLE Abstract The production of long shelf-life highly concentrated dried probiotic/starter cultures is of paramount importance for the food industry. The aim of the present study was to evaluate the protective effect of glucose, lactose, trehalose, and skim milk applied alone or combined upon the survival of potentially probiotic Lactobacillus rhamnosus CTC1679, Lactobacillus casei/paracasei CTC1677 and L. casei/paracasei CTC1678 during freeze-drying and after 39 weeks of storage at 4 and 22 °C. Immediately after freeze-drying, the percentage of survivors was very high (≥94%) and only slight differences were observed among strains and cryoprotectants. In contrast, during storage, survival in the dried state depended on the cryoprotectant, temperature and strain. For all the protectants assayed, the stability of the cultures was remarkably higher when stored under refrigeration (4 °C). Under these conditions, skim milk alone or supplemented with trehalose or lactose showed the best performance (reductions ≤0.9 log units after 39 weeks of storage). The lowest survival was observed during non-refrigerated storage and with glucose and glucose plus milk; no viable cells left at the end of the storage period. Thus, freeze-drying in the presence of appropriate cryoprotectants allows the production of long shelf-life highly concentrated dried cultures ready for incorporation in high numbers into food products as starter/potential probiotic cultures. Keywords: carbohydrates, lyophilisation, probiotic bacteria, skim milk, sublethals
1. Introduction The industrial use of lactic acid bacteria (LAB) as starter cultures in fermented products or as probiotics and their direct inclusion in food or dietary supplements require the delivery of stable cultures in terms of viability and functionality. Thus, the maximisation of survival of LAB cultures during drying and subsequent long-term storage under refrigeration or room temperature is of vital importance at the technological and economical level (Poddar et al., 2012; Selmer-Olsen et al., 1999; Tripathi and Giri, 2014). Freeze-drying is commonly used for the preservation and long-term storage of LAB, as it allows easy and inexpensive shipping and handling. Conversely, it produces undesirable side effects decreasing the viability of cultures (Carvalho et al., 2002; Morgan et al., 2006; Tripathi and Giri, 2014). The mechanisms of damage during freeze-drying are
complex and not fully understood to date but they can be attributed primarily to ice crystal formation, high osmolarity due to high concentrations of internal solutes with membrane damage, macromolecule denaturation, and the removal of water, which affect properties of many hydrophilic macromolecules in the cells (Santivarangkna et al., 2008; Zayed and Roos, 2004). Although most reports focused on survival during the process of drying, a low inactivation rate during subsequent storage is equally or even more important when a very long storage period is required (Santivarangkna et al., 2008). During storage, low water activity, storage temperature and packaging material keeping the moisture content low enough to maintain the dried culture in a glassy state and protect it from oxygen have shown to affect viability (Carvalho et al., 2004b; Higl et al., 2007; Poddar et al., 2014; Santivarangkna et al., 2008). To minimise inactivation during drying and improve the stability of the dried product, it is a common procedure
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014. 0038381
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0038 - Friday, September 22, 2017 12:16:50 AM - Göteborgs Universitet IP Address:130.241.16.16
A. Jofré et al.
to create a protective amorphous sugar matrix around the individual bacteria by adding sugars to the cell suspension before the drying process (Bergenholtz et al., 2012; Carvalho et al., 2004a; Higl et al., 2007). In LAB, different levels of protection have been observed with different sugars (e.g. glucose, fructose, lactose, mannose and sucrose), sugar alcohols (e.g. sorbitol and inositol) and non-reducing sugars (e.g. trehalose) (Carvalho et al., 2002; Higl et al., 2007; Miao et al., 2008; Pehkonen et al., 2008; Strasser et al., 2009; Zamora et al., 2006). Skim milk applied alone or in combination with other cryoprotectants is also widely applied in the starter culture industry (Morgan et al., 2006). Additionally, microrganisms greatly vary in their tolerance to freeze-drying. In general, Gram-positive bacteria show a higher tolerance than Gram-negative bacteria, which is probably related to their genetic constitution and cell wall and membrane composition (Carvalho et al., 2004b; Morgan and Vesey, 2009). However, there are considerable differences in the freeze-drying tolerance of closely related organisms; different strains of the same species can behave completely different. Thus, the freeze-drying process must be optimised for specific strains to achieve maximum viability and stability. The major problem to overcome seems to be the lack of generic theories for all bacterial strains (Morgan and Vesey, 2009; Morgan et al., 2006). Lactobacillus rhamnosus and Lactobacillus casei/paracasei are important in a number of industrial applications and used as starter cultures in traditional and functional fermented products (Leroy et al., 2006). Recently, we isolated and characterised from infants’ faeces three strains, i.e. L. rhamnosus CTC1679, L. casei/paracasei CTC1677 and L. casei/paracasei CTC1678, with putative probiotic properties suitable as starter cultures in fermented sausages (Rubio et al., 2014a,b,c). As the industrial application of a starter culture requires highly concentrated dried cultures, we evaluated the protective effect of various sugars previously reported to have protective capacity during freeze-drying (glucose, lactose and trehalose), applied alone or in combination with skim milk, upon survival of the three putative probiotic strains during freeze-drying and subsequent storage for 39 weeks at 4 and 22 °C to find the most effective protectant(s) for the production of cultures containing high levels of viable cells after a longterm storage period.
2. Material and methods Freeze-drying
(4.5 ml) and let 30 min at room temperature to allow equilibration between cells and the compound added: T (5% trehalose), G (10% glucose), L (7% lactose), M (11% skim milk), TM (5% trehalose+11% skim milk), GM (10% glucose+11% skim milk) and LM (7% lactose+11% skim milk). Subsequently, aliquots of 0.5 ml were prepared in freeze-drying vials, frozen at -80 °C for 2 h and subsequently freeze-dried in a Christ Alpha 1-4 (B. Braun Biotech Int., Melsungen, Germany). To determine the weight of the dry culture, vials were weight empty and after freeze-drying. Two independent experiments were performed on different days.
Storage After freeze-drying, vials containing dried 0.5 ml aliquots were tightly capped and individually vacuum packed in metallic pouches (PET/MET+PE), oxygen permeability
