Effect of protective agents on the viability of Lactococcus lactis subjected to freeze-thawing and freeze-drying

The effect of different protectants and the impact of the initial cell density on the viability of Lactococcus lactis Sr. 3.54 subjected to freeze-thawing and freezedrying was studied. Several additives were tested as protective agents against freezing and drying injuries. Maximum viability of the cells after freeze-thawing was obtained with sucrose and skim milk mixtures as protective agents (78% viability). Freeze-drying with protectants based on skim milk or MRS-broth were most effective (survival levels >60%). The effect of the initial cell load on the final recovery was dependent on the protectant. Every sample showed an increase in viability when a high initial cell concentration (10cfu ml) was used. The blank showed a 1500 fold increase, skim milk/sucrose based lyophilisates an 1,7 fold increase in viability when the initial cell load was changed from 10 to 10cfu ml. The use of 10cfu ml as initial cell concentration and sucrose/skim milk as protectant yielded a lyophilisate with 71% viability. Results suggest the possibility of producing freeze dried powders of Lactococcus lactis with high viability for the food industry.


Introduction
Adequate shelf life, high cell viability and activity are ther equirements for bacterial formulations.L actic acid bacteria and other biological materials used as starter cultures in the dairyi ndustry or as silage preservantsa re oftenp reserved by freezing and/or freezedrying resulting in products with excellentl ong-terms tabilityi nm ost cases [ 1].
Lyophilisation/freeze-dryingisdefinedasastabilizing process in which the substancesare firstf rozen followed by as ublimation( primary drying) and desorption (secondary drying) step in order to reduce the water content to levels that willn ol onger support biological growth or chemical reactions [2,3].Dried cultures are favoured over frozen cultures becauseoflower transport and storagecosts.
However, freeze-drying has some undesirable side effects, such as denaturation of sensitive proteins leading to decreased viability or activity.Freeze-drying subjects bacteria to twod ifferent kindso fs tress.Firstt of reezing, wherebyo nlyt he free accessable wateri s displaced, and subsequentlyt od rying, where even nonfreezeable water can be removed.
The mechanism of protection is different for both situations [4,5].
The combined damage caused by freeze-drying can be attributed to two primary causes: changes in the physical state of the membrane lipids and protein damage or proteind enaturation [ 6,11].As wateri sr emoved from theb iomembrane,t he headgroups of thel ipidsa re broughtc losert ogethera nd this results in increased van der Waals interaction between the acyl chains.This forces the biomembrane into the gel phase at room temperature.W hen rehydrated the membrane undergoes ap hase transition and as ar esult it can become leaky.It has been suggestedt hat sugars depress the phase transition in dry phospholipidsb yf orming hydrogen bounds witht he polar headgroups.
Thisi sk nown as the water replacement hypothesis.Thea bility of different protectants to shelterp roteins from denaturation could also be due to the abilityo ff ormingh ydrogen bonds with the protein when water is removed.Also, the changes in solution properties during freezing and thawing, such as the change in pH value at the eutectic point, can cause harm.The major damagesr esulting fromf reezing are fusion and thermotropic phase transitions of the cellmembranes.Cryoprotectants can preventthisvia the increase in the charged ensity around the headgroups of the biolayer and therefore inhibiting fusion by electrostatic repulsion, or they can limit fusion by steric hinderance.Beside the used protectivea gents the initialc elll oad, the rehydration media [18], as well as the growing conditions [ 19] influence the resulting viability after freeze-drying.Another important parameter affecting the stability of the producti st he value of the water activity (a w -value).
This valued epends on thep rotectanta sw ella so nt he parameters of the drying process (e.g.time) and can be adjusted to acertaindegree after freeze-drying.By definitionthe a wvalue is the partial pressure of water in the product divided by that of pure water.At high a w -values, the dry cell canb ed estroyed because solutes can diffuse in water and therefored amage the cell by an osmotic effect [20].T he aim of this studyw as to investigate the effect of different cryo-and lyoprotective agents on Lactococcusl actis Sr.
3.54, as train used in ensiling and in the dairy industry,a nd to study the influence of the initial cell concentrationo nt he viabilityo ft his special strain.As the potency of different protectants depends on the bacterial strain, there could be variations in results to other strains.This study is assessing potential protectants using only one strain of Lactococcus lactis.

Rehydration and determinationofthe colony forming units (cfu)
Allsamples were stored at -80°C for a7days prior to freeze drying or thawing.
Afterf reeze-drying thes amples wereb rought to their original volume with sterile physiological NaCls olution, incubated for 20 minutes at 30°C.As amplew as decimallyd iluted in physiological NaCl and subsequently planted onto MRS-agar.
To estimate the protection provided for freeze-thawing, samples were thawed at 4°C overnight.Prior to counting, the samples were brought to room temperature.
Each presented value is the mean of 5d ifferent experiments.The estimated error was lessthanhalf an order of magnitude.

Determination of the water activity (a w -value)
The a w -value was measured with ameasuring unit AWX 3000/AWX 3001 (ebro electronic GmbH, Ingolstadt, D).Directly after freeze-drying the samples were put into the measurement chamberfor 90 minutes at 25°C.

Viability after freeze-drying
Thereforet he viabilityc ould be note stimated.N ot only thev iability buta lsot he physical structureo ft he lyophilisate is important.On the one hand, all skim milk lyophilisatesp rovidedaf reezed ried material with al ight andp orouss tructure that facilitadedr ehydration.M annitola nd PVPK 25,D extran 4a sw ella sN aCla lso yielded well-restorable products.On the other hand, sugarsyielded glasslike, highly hydroscopic masses.The hydroscopicity of sugars is also important with respect to the watera ctivity value whichg reatlyi nfluences thes helf life of microbial products.
To improve not onlyt he viability but also the physical structure, mixtures of good bulking agents( e.g.skim milk, mannitol) and good lyoprotectants (sugars) were used.Figures 1a nd 2s how thee ffect of freeze-thawing andf reeze-drying.Fig. 1 shows the protection given by sugars and some inorganicc ompounds.Usinga wide rangeo fp rotectants,s ignificant differencesi nt he viabilityo fc ells of Lactococcus lactis afterf reeze-thawing and freeze-drying were observed, dependingo nt he protectant used.W hen using sugars, disaccharidesg ave better viabilitiest hanm onosaccharides.1 5% sucrose( 39%v iability)g avet he best viability.Fig. 2s howst he results yielded by complex media.The most effective mixtures as protective agents were 10% skim milk/sucrose (62% viability), 20% skim milk/sucrose (60% viability) as well as MRS-broth+5 %s kimm ilk/sucrose (62% viability) and MRS-broth +5 %m annitol (62% viability).These mixtures combine good bulking agentswith sugars.In general,t he viability obtained when skimm ilk/sucrosem ixturesw ereu sed as protective agents was higher than that obtained with MRS-broth or sugars alone as protectors.Moreover, resultsshowed that the effect of initial concentration had a marked dependence on the type of protectantsu sed.For good protectants, the improvement attributed to higher cell concentrations was smallw hereas the increase of viabilityf or less suitable protectants was noteworthy.Especially the blank (sample without protectants) yielded a1 500 fold (from 0,02% to 30%) higher viabilitywhen10 10 cfu ml -1 as initial cell load was used instead of 10 09 cfu ml -1 .10%skim milk/sucrose yieldedo nlya n1 ,7 fold increase of viability( from 62%t o7 1%)

Effect of initialcellcount on survival during lyophilisation
The resultsc an be seen in Fig. 3.The protective effect of ah igher cell count in the solution subjected to freeze-drying can be seen best when compared to the effect on bacterialyophilised without protective agents (=blank).
The error barsshow the standard deviation.
For some samples, thea w -value after lyophilisation was estimated.As seen in Fig. 4t he a w -value is highly dependant on the nature of the protectant used.This is alsoo fi nterest for optimizingt he shelf lifeo ft he lyophilisated product.Ongoing investigations suggest that the shelf life depends to ac ertain degree, on the a wvalue.The higher thea w -value after lyophilisation the lower the shelf life stability.
Thep rotectanti tselfi sm uchm orei mportant forg oods tability.T he influenceo f different a w -values after lyophilisation using the same protectant have not been estimatedyet.

Fig. 1 .
Fig. 1 .E ffect of different cryo-and lyoprotectants on the viability after freezethawing and freeze-drying: sugarsa nd simple inorganicc ompounds.If not other indicated 10%(w/w) solutions were used.The error bars show the standarddeviation.

Fig. 2 .
Fig. 2 .E ffect of different cryo-and lyoprotectants on the viability after freezethawing and freeze-drying: complex media;u nlessi ndicated otherwise 10%(w/w) solutions were used.The error bars show the standard deviation.