CRYOBIOLOGY
27, 569-575 (1990)
Freeze-Drying of Streptococcus thermophilus: A Comparison between the Vacuum and the Atmospheric Method E. WOLFF, B. DELISLE, I.N.R.A.
Laboratoire
de G&k
G. CORRIEU, AND H. GIBERT*
des Procidt!s Biotechnologiques Agro-alimentaires, F 78850 Thiverval Grignon, France
and *I.N.A.P.G.,
Frozen suspensions of Streptococcus thermophilus were 6eeze-dried in a vacuum or a Ruidized adsorbent bed at atmospheric pressure. Optimum operating conditions for each process were defined. For the duration of processing and survival rate of bacteria, in each case vacuum freeze-drying seemed more satisfactory than atmospheric pressure freeze-drying. The use of reconstituted skimmed milk as a suspension medium provides good protection for S. thermophilus. o 1990~cadcmic PESS, IIIC.
Over the last few years, the freeze-dried microorganism market has grown considerably, especially for lactic acid starters. The dairy industry is keen to improve standardization of its fermented products while minimalizing the risk of contamination. Freezedried lactic acid bacteria seems to be an adequate answer. The use of active cultures has been replaced by the use of concentrated cultures frozen at - 20 or - 40°C in diluted glycerol (1) and at - 196°C in liquid nitrogen (8). At the same time, the desire for easy conservation of microorganism strains initiated the search for suitable dehydration techniques such as vacuum drying (8), spraydrying (l), or freeze-drying. Of these techniques, freeze-drying, the use of which has increased steadily since 1970, gives the best survival rates (5). Freeze-dried “powder” is easy to employ and usable directly in the process tanks (direct inoculation), and the properties of freeze-dried strains are well preserved (6). Over the past 10 years, a new process of freeze-drying using a fluidized adsorbent bed has been suggested by Gibert (3) and developed in later studies (4, 9, 12); it seems to offer considerable economic Received March 13, 1989; accepted September 27, 1989.
promise (lo), including a one-third saving on energy costs compared to the traditional vacuum freeze-drying process. This process is based on a decrease of partial pressure of water vapor not by working under vacuum, but by adsorption in a fluidized adsorbent bed. The frozen product is closely mixed together with fine adsorbing particles (pregelatinized starch). All water vapor emitted by sublimation or left in the apparatus is then caught by these particles. It is thus possible to sublimate the ice with air at atmospheric pressure (10, 11). This paper considers the advantages of this new process for freeze-drying of lactic acid bacteria. To this end, comparative tests using traditional vacuum freezedrying and atmospheric pressure freezedrying were carried out under varying experimental conditions, with a thermophilic lactic acid bacterium (Streptococcus thermophilus) which is used inter alia to manufacture yogurt. APPARATUS AND METHOD
Preparation of Active Bacteria Frozen Samples
and
A strain of S. thermophilus (CNRZ 404) was produced in a 15-liter automatic Biolafitte fermenter. pH was maintained at 6.5, temperature at 41”C, and stirring speed at 569 0011-2240190$3.00 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in any form rcscrved.
570
WOLFF
200 rpm. The fermentation medium was composed of lactoserum (60 g/liter) (Prolabo), lactose (40 g/liter) (Prolabo), bactopeptone (5 g/liter) (Difco), yeast extract (5 g/liter) (D&o), and antifoam silicone 426R (1 g/liter) (Prolabo). This medium was inoculated with a reference freeze-dried sample of 5. thermophilus (I g/IO liters) from a homogeneous batch of the same strain. Fermentation was halted at the end of the log phase of bacterial growth. Using sterile handling, the medium was placed in 500-ml centrifuge vessels, cooled to 4°C (30 min) and then centrifuged at 4°C for 15 min at ll,OoOg. The centrifuge residues were mixed and made into a suspension with an equal weight of protectant medium made up of reconstituted skimmed milk (Elle & Vire Dairy) enriched with currently used protectant molecules. Five formulae were tested: -skimmed milk, -skimmed milk with added glycerol (1%) (Prolabo), -skimmed milk with added glycerol (1%) and glucose (5%) (Prolabo), -skimmed milk with added dimethyl sulfoxide (DMSO) (1%) (Prolabo), and -skimmed milk with added polyethylene glycol (PEG) (l%), molecular weight 1500 (Prolabo). Concentrates thus obtained were placed in flasks for freeze-drying (10 ml in a 50-ml flask for vacuum freeze-drying) or formed into 0.16-ml pellets (for atmospheric freezedrying). These pellets were made in an aluminum matrix grooved with small cylinders 3 mm in thickness and 1 cm in diameter. Samples were then frozen at - 75°C. Three fermentations carried out under simiIar conditions, labeled Fl, F2, and F3, were needed to produce all the samples. The concentrates were cooled from +2O”C to -75°C at an average speed of - 0.4”C/min. The incipient freezing point, determined from the evolution of the product temperature during the freezing step,
ET AL.
was - 1.3”C for concentrates in pure milk, - 1.7”C in milk with added glycerol, and -2.5”C for milk with added glycerol and glucose. Supercooling was observed in all cases. The incipient melting point, determined from the evolution of the product temperature during the melting step of some samples, was much more difficult to determine precisely. It was - 2.6 + 0.4”C for concentrates in pure milk, -2.9 * 0.5”C in milk with added glycerol, and -4.l”C ? 0.4”C for milk with added glycerol and glucose. Freeze-Drying Processes Vacuum freeze-drying of the flask samples was carried out in a SMHl5 freezedrier from the USIFROID range (USIFROID, rue Claude 3emard, F 78312 Maurepas Cedex, France). Flasks were placed on a heating plate in a vacuum chamber containing a condenser cooled to -65°C. The equipment could be adjusted to control the heating plate temperature and the working pressure. Apart from the composition of the protectant medium, the influence of the heating plate temperature and the working pressure on survival of S. thermophilus was also studied. Heating plate temperatures studied were - 15, 0, and 15°C. In order to avoid melting, pressure was never above 300 Pa: we chose working pressures of 1, 100, and 300 Pa. These values correspond to mean values of the product temperature in the frozen core of - 40, - 18, and - 8”C, respectively. Under these temperature and pressure conditions, approximateIy 8 hr of treatment was required to reach water content IeveIs below 0.20 kg water/kg dry matter. Experiments were therefore run for 8 hr periods. The atmospheric pressure freeze-drying process suggested by Gibert in 1976 has been used in this study. The freeze-drying pilot plant is described by Wolff (10, 11). The main feature of this process is that the frozen product is mixed thoroughly with small particles of adsorbent (under 200 pm)
FREEZE-DRYING
571
OF Streptococcus thermophilus
in a column. It is then possible to work with air at atmospheric pressure. The effect of temperature of the bed of adsorbent and the composition of the protectant medium on survival of S. thermophillts was studied. The working temperature had to be below the incipient melting point of the product and therefore the study focused on two working temperatures, - 5°C and - WC, corresponding approximately to the same temperature for the frozen product. To obtain the required level of dehydration, freeze-drying was carried out for 15 hr.
ucts used for counting was made in reconstituted skimmed milk at 30°C. -The comparison of the different parameters studied was based statistically on the Student-t test applied to the mean value of survival rates obtained in each case. RESULTS
AND
DISCUSSION
Preliminary Remarks In the three fermentations carried out to produce the samples needed for this study, the initial number of CFU per milliliter was multiplied by 70, 300, and 1600 to reach a final number between 10’ and 10” CFU/ml. Analysis Methods The average number of bacteria forming a Results are reported with the following in colony, observed under a microscope, increased by 1.8 to 4.5 during culture. In all mind: -The water content of freeze-dried cases, diplococci made up about 50% of the products was obtained by weighing them colonies . After centrifuging, the average number of before and after 24 hr in a heating chamber at 100°C. The results are expressed in kilo- CFU per gram of DM was multiplied by 5 grams of water per kilograms of dry matter compared to nonconcentrated active bacteria, since all soluble matter was eliminated (DW. -The survival rate of S. thermophilus in the supernatant. After adding the prowas determined by counting the colony tectant medium to the same weight as the forming units (CFU) in a petri dish inocu- residue, we obtained about 5.10” CFUlml lated using the spiral method on an agar corresponding to 6.10” CFUlg DM. Ml7 (Biokar) type medium. The counting was carried out twice for two different di- Influence of Freezing and Storage on the Survival of S. thermophilus lutions. The average of the four values thus The selected suspension medium (reconobtained expressed in CFU per milliliter or in CFU per gram of DM suffered a margin stituted skimmed milk) and the freezing technique gave a survival rate of nearly of error of about 30%. -The thawing method used to count fro- 100%for S. thermophilus after freezing (see zen samples consisted of warming up the Table 1). An increase in cell concentration was in fact observed, which could be exproduct in a waterbath at 30°C. -The rehydration of freeze-dried prod- plained by the breaking of some chains durTABLE 1 Influence of the Suspension Media on Cellular Concentration (CFUlg DM) before and after Freezing at - 75°C of Streptococcus thermophib4s
Suspension medium
Skimmed milk (CFUlg DM)
Before freezing After freezing
4.3 x LO”
6.1 x 10”
Skimmed milk + glycerol (CFUlg DM)
Skimmed milk + glycerol + glucose (CFUlg DM)
6.2 x 10” 7.8 x 10”
6.2 x 1O’l 8.6 x to”
572
WOLFF
ET AL.
TABLE 3 ing freezing, thereby increasing the number of CFU counted, Pure reconstituted skim- Water Content after Vacuum Freeze-Drying (8 hr) of a Streptococcus thermophilus Suspension med milk therefore seemsto provide a satisfactory protectant medium. Water Content (kg Water/kg DM) Products frozen in this manner were not Pressure: 1 Pa lOOpa 300 Pa all used immediately for freeze-drying. We Temperature (“C) studied their mortality during frozen stor-15 0.08 0.16 age. After 15 weeks in storage at -75°C 0 0.14 1.11 0.15 the survival rate was almost 100%(see Ta15 0.11 ble 2), The standard deviation obtained per column was rather high compared to the average which led us to interpret results water/kg DM. It should be noted that some with considerable caution. authors report experiments where dehydration is continued to 0.01 kg water/kg DM (2) Influence of Vacuum Freeze-Drying on or even 0.001 kg water/kg DM, without, the Dehydration and SurvivaI of however, stating the freeze-drying condiS. thermophilus tions (pressure, plate temperature, and duWater content (Table 3). For a pressure ration). The addition of cryoprotectants, whatof 300 Pa, the residual water content after 8 hr of freeze-drying was close to 1 kg water/ ever they were, did not significantly influkg DM, which is too high for the proper ence the final water content. Survival. Results relative to the two ferconservation of bacteria. At this pressure, the low diffusivity of water vapor militates mentations Fl and F2 expressed as cell against transfer of this vapor, and the de- concentrations and as corresponding survival rates are shown in Table 4. For a plate hydration time is therefore far too long. As for the other conditions of the exper- temperature of lS”C, the survival rate was iment, the water content stabilized around very low. This operating parameter there0.15 kg water/kg DM. At 1 Pa and - 15”C, a fore did not warrant further study. As for the effects of temperature and residual water content of 0.08 kg water/kg DM was achieved after 8 hr. The reference pressure, the homogeneity of the results for freeze-dried sample (CNRZ 404) used was Fl and F2 (Table 4 except results at 15°C characterized by a water content of 0.14 kg and at 1 Pa for Fl) has been outlined staTABLE 2 Influence of Conservation at - 75°C on Cellular Concentration (CFU/g DM) of Streptococcus
Time (weeks) 0 1 2 3 4 6 15 Average value Standard deviation
Milk (CFU/g DM) 6.1 x 3.6 x 6.4 x 3.7 x 3.0 x 2.7 x 6.5 x 4.6 X 1.7 x
10” 10” 10” 10” 10” LO” 10” 10” 10”
Milk + glycerol (CFU/g DM) 7.8 4.4 9.2 5.8 5.2 6.0 6.0 6.3 I.6
x x x x x x x x x
10” 10” IO” 10” 10” 10” IO” 10” 10”
thermophilus
Milk + glycero1 + glucose (CFU/g DM) 8.6 5.1 9.1 3.9 6.0 7.5 6.4 6.7 1.9
x x x x x x x x x
10” 10” 10” 10” IO” 10” 10” 10” 10”
FREEZE-DRYING
573
OF Streptococcus thermophilus
TABLE 4 Influence of Vacuum Freeze-Drying (8 hr) on Cellular Concentration (CFU/g DM) and Survival Rate (%) of Streptococcus Pressure:
Protectivemedium: Initial coucn: Temperature(“C)
1 Pa Milk + glycerol 2.4 x 10”
thermophilus
FermentationFl 100Pa loo Pa Milk + glycerol Milk + PEG 2.4 x 10” 2.3 x 10”
15
loo Pa Milk + DMSO 1.7 x 101’
300Pa Milk + glycerol 2.4 x lOI’
1.2 x 101’ 52%
I.1 x 10’1 46%
1.4 x 10’0
-15
1.2 ZD/Oll
0
1.1 x 10’0 5%
Pressure:
1 Pa Milk
9.55Z010 3%
6.9 x 10’0 30%
FermentationF2 1 Pa Milk Milk + glycerol + glycerol
Initial ceucn: Temperature(9C)
5.5 x IO”
6.9 x IO”
7.3 x IO”
5.5 x IO”
6.9 x IO”
100Pa Milk + glycerol t glucose 7.3 x 10”
- 15
3.6 x 10” 65% 3.8 x 10” 49%
3.6 x 10” 52% 3.5 x 10” 51%
3.5 x 10”
3.5 x IO” 44% 4.0 x 10” 73%
3.1 x IO” 45% 3.8 x 10” 55%
3.1 x 10” 42% 3.6 x 10” 4%
Protectionmedium:
lpa
+ glucose
0
3.4ZOt’ 47%
tistically. It was shown that no statistical difference can be observed at a 5% risk level. The mean value of the survival rate did not depend on either the temperature of the heating plate, so long as it was below or equal to O’C, or the pressure. The survival rates of concentrates from F2 fermentations allow comparison between the various protectant media. No statistically significant differences (level of risk, 5%) can be observed between samples in pure milk or in milk with added glycerol and glucose. The low concentration of the cryoprotectant molecules used is probably part of the explanation of this fact.
100Pa Milk
looPa Milk
+ glycerol
The average survival rates were 55% for F2 and 40% for Fl . This difference could be due to the initial state of the microorganisms which could be different at the end of the two fermentations. Influence of Atmospheric Pressure Freeze-Drying on the Dehydration and Survival of S. thermophilus The results of the experiments carried out at - 5°C and - 15°C in relation to water content and survival rates are shown in Table 5. Water content. The water content obtained after 15 hr of freeze-drying at - 5°C
TABLE 5 Influence of Atmospheric Freeze-Drying (15 hr) on Cellular Concentration fCFU/g DM) and Survival Rate (%) of Streptococcus thcrmophilus luitial concentration (CFU/g DM) FI: 2.4 x 10” F3: 5.0 x 10” F3: 5.0 x IO”
Temperature w”c) -5 -15 - 15
Suspension medium milk + glycerol milk + glycerol milk + glycerol + glucose
Water content (kg water/ kg DM) 0.17 0.21 0.21
Concn. (CFUlg DM) 6.3 x lOa 1.7 x 10” 1.5 x 10”
Survival rate (%I 3 35 31
574
WOLFF
was 0.17 kg water/kg DM; at - 15°C it was 0.21 kg water/kg DM. These results were higher than those obtained by vacuum freeze-drying (8 hr). Atmospheric freezedrying is thus slower than vacuum freezedrying. Survival. Freeze-drying at - 15°C gave final bacterial concentration and survival rates much higher than those obtained after freeze-drying at - 5°C (Table 5). Probably, at - 5”C, the freeze-drying temperature was too close to the melting temperature of the ice in the product and this may be detrimental for the bacteria. This difference was obtained from tests on different fermentations. Different initial states of the bacteria could also explain these differences but the temperature factor certainly had a predominant effect in the differences obtained. The mean value of the survival rate obtained at - 15°C was about 33%. Pure milk, as shown above, seemed to be a good protectant medium. Comparison
of the Two Processes
Technology, Vacuum freeze-drying is a technology that is easy to carry out but it has three main disadvantages: 1. An air-tight chamber is necessary to work under vacuum and makes it difficult to obtain high intensity of heat transfer and to work in a continuous way. 2. The ice condensers must be cooled at a very low temperature and defrosted reg ularl y . 3. Heating by heating plates is not efficient because the contact resistance to heat transfer between solid particles at these pressures is very high. Atmospheric freeze-drying is also very simple to use. It can be driven in a continuous way but has two different disadvantages: 1. At the end of the freeze-drying step, it is necessary to regenerate the adsorbent. 2. Some small particles of adsorbent (starch) may spoil the freeze-dried product. Energy aspects. Freeze-drying is well
ET
AL.
known to be an expensive technique. For the vacuum freeze-drying process, the authors generally adopt the following order of magnitude for the energy cost (7): cooling request, 3600 kJ/kg of ice; and heating request, 4900 kJ/kg of ice. Under atmospheric pressure it has been shown that these costs can in practice be reduced in a continuous duty atmospheric pressure freeze-drying facility, including the adsorbent regeneration cost, at (10, 11): cooling request, 2250 W/kg of ice; and heating request, 3250 kJ/kg of ice. A 35% savings on energy costs may be expected. But it has also been shown that the freeze-drying time in this new process is approximately twice that under vacuum. Survival rate of lactic acid bacteria. Taking into account F2 and F3 fermentations where the initial concentrations of bacteria were comparable, the survival rate obtained for the vacuum freeze-drying of S. thermophilus was 55% and only 33% at atmospheric pressure. This shows that the survival rate is greater in the traditional freeze-drying process than in the new process, at least under the experimental conditions tested, If there are new improvements in atmospheric freeze-drying, it is possible that this difference can be reduced. However, it is too early to exclude this process at this step of the research. CONCLUSION
Whatever freeze-drying process was used, it was possible to obtain good quality freeze-dried products (cell concentration between 10” and 3.10” CFU/g DM for residual water contents of the order of 0.1 kg water/kg DM). These properties are equivalent to those of freeze-dried products currently on the market. Of the operating conditions investigated, the freeze-drying conditions that gave the best results were -Pressure between 1 and 100 Pa and heating temperature of the lower plate below O”C, for vacuum freeze-drying,
FREEZE-DRYING
OF Streptococcus thermophilus
--Operating temperature rather low (- WC) for atmospheric freeze-drying. -The addition of reconstituted skimmed milk as cryoprotectant suspension medium. At first, vacuum freeze-drying seemed to be best suited to the dehydration of concentrated suspensions of S. thermophilus. In fact, atmospheric pressure freeze-drying called for double the processing time (15 hr instead of 8 hr) and half the survival rate (33% instead of 55%). However, the energy costs can be decreased by one-third compared with those of vacuum freeze-drying. The initial state of microorganisms seems to have an effect on survival rates. It is therefore important to control, indeed to optimize, each of the stages implemented before freezing and freeze-drying, i.e., conditions of fermentation, time of sampling, process, and level of concentration. REFERENCES
I. Barbour, E. A., and Priest, F. G. The preservation of Lnctobacilli: A comparison of three methods. Left. Appl. Microbial. 2, 69-71 (1986). 2. Bousfield, I. J., and Mackensie, A. R. Inactivation of bacteria by freeze-drying. In “The Society for Applied Bacteriology,” (F. D. Skinner
$75
and W. B. Hugo, Eds.), Symposium series No. 5, Academic Press, London, 1976. 3. Gibert, H. French Patent ANVAR 76,39,864; U.S. Patent 4,175,334 (1976). 4. Kijanapanich, P. “Lyophilisation de de&es alimentaires en couche fluidiske sous pression atmosph6rique: influence de la nature de l’adsorbant.” Thesis, INPT, Toulouse, 1981. 5. Martin, M. Les levains concentrds lyopbili& La technique l&Pm 976, 45-49 (1983). 6. Robinson, R. K. Freeze-dried starter concentrates. Dairy I&. Inr. 46(10), 15-22 (1981). 7. Simatos, D., Blond, G., Dauvois, Ph., and Sauvageot, F. “La lyophilisation: principe et applications.” Collection de I’ANRT, Paris, 1974. 8. Tamine, A. Y., and Deeth, H. G. Yogurt: Technology and biochemistry. J. Food Pmt. 43(12), 939-477 (1980). 9. Wachet, J. N. “Etude d’un pro&de de lyophilisation en couche fluidiste sous pression atmosphkique.” Thesis, USTL, Montpellier, 1978. 10. Wolff, E., and Gibert, H. Freeze-drying at atmospheric pressure: Design and energy considerations. In “Drying ‘86” (A. S., Mujumdar, Ed.). Hemisphere, Washington, 1986. 11. Wolff, E. “Cinktique et mod&sation de la lyophilisation sous vide et de la lyophilisation & pression atmosphkrique.” Thesis, INPT, Toulouse, France (1988). 12. Yassin, K. E. E. “Lyaphilisation sous pression atmosphtrique: mise en oeuvre d’un adsorbant alimentaire.” Thesis, INPT, Toulouse, France (1984).
27, 569-575 (1990)
Freeze-Drying of Streptococcus thermophilus: A Comparison between the Vacuum and the Atmospheric Method E. WOLFF, B. DELISLE, I.N.R.A.
Laboratoire
de G&k
G. CORRIEU, AND H. GIBERT*
des Procidt!s Biotechnologiques Agro-alimentaires, F 78850 Thiverval Grignon, France
and *I.N.A.P.G.,
Frozen suspensions of Streptococcus thermophilus were 6eeze-dried in a vacuum or a Ruidized adsorbent bed at atmospheric pressure. Optimum operating conditions for each process were defined. For the duration of processing and survival rate of bacteria, in each case vacuum freeze-drying seemed more satisfactory than atmospheric pressure freeze-drying. The use of reconstituted skimmed milk as a suspension medium provides good protection for S. thermophilus. o 1990~cadcmic PESS, IIIC.
Over the last few years, the freeze-dried microorganism market has grown considerably, especially for lactic acid starters. The dairy industry is keen to improve standardization of its fermented products while minimalizing the risk of contamination. Freezedried lactic acid bacteria seems to be an adequate answer. The use of active cultures has been replaced by the use of concentrated cultures frozen at - 20 or - 40°C in diluted glycerol (1) and at - 196°C in liquid nitrogen (8). At the same time, the desire for easy conservation of microorganism strains initiated the search for suitable dehydration techniques such as vacuum drying (8), spraydrying (l), or freeze-drying. Of these techniques, freeze-drying, the use of which has increased steadily since 1970, gives the best survival rates (5). Freeze-dried “powder” is easy to employ and usable directly in the process tanks (direct inoculation), and the properties of freeze-dried strains are well preserved (6). Over the past 10 years, a new process of freeze-drying using a fluidized adsorbent bed has been suggested by Gibert (3) and developed in later studies (4, 9, 12); it seems to offer considerable economic Received March 13, 1989; accepted September 27, 1989.
promise (lo), including a one-third saving on energy costs compared to the traditional vacuum freeze-drying process. This process is based on a decrease of partial pressure of water vapor not by working under vacuum, but by adsorption in a fluidized adsorbent bed. The frozen product is closely mixed together with fine adsorbing particles (pregelatinized starch). All water vapor emitted by sublimation or left in the apparatus is then caught by these particles. It is thus possible to sublimate the ice with air at atmospheric pressure (10, 11). This paper considers the advantages of this new process for freeze-drying of lactic acid bacteria. To this end, comparative tests using traditional vacuum freezedrying and atmospheric pressure freezedrying were carried out under varying experimental conditions, with a thermophilic lactic acid bacterium (Streptococcus thermophilus) which is used inter alia to manufacture yogurt. APPARATUS AND METHOD
Preparation of Active Bacteria Frozen Samples
and
A strain of S. thermophilus (CNRZ 404) was produced in a 15-liter automatic Biolafitte fermenter. pH was maintained at 6.5, temperature at 41”C, and stirring speed at 569 0011-2240190$3.00 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in any form rcscrved.
570
WOLFF
200 rpm. The fermentation medium was composed of lactoserum (60 g/liter) (Prolabo), lactose (40 g/liter) (Prolabo), bactopeptone (5 g/liter) (Difco), yeast extract (5 g/liter) (D&o), and antifoam silicone 426R (1 g/liter) (Prolabo). This medium was inoculated with a reference freeze-dried sample of 5. thermophilus (I g/IO liters) from a homogeneous batch of the same strain. Fermentation was halted at the end of the log phase of bacterial growth. Using sterile handling, the medium was placed in 500-ml centrifuge vessels, cooled to 4°C (30 min) and then centrifuged at 4°C for 15 min at ll,OoOg. The centrifuge residues were mixed and made into a suspension with an equal weight of protectant medium made up of reconstituted skimmed milk (Elle & Vire Dairy) enriched with currently used protectant molecules. Five formulae were tested: -skimmed milk, -skimmed milk with added glycerol (1%) (Prolabo), -skimmed milk with added glycerol (1%) and glucose (5%) (Prolabo), -skimmed milk with added dimethyl sulfoxide (DMSO) (1%) (Prolabo), and -skimmed milk with added polyethylene glycol (PEG) (l%), molecular weight 1500 (Prolabo). Concentrates thus obtained were placed in flasks for freeze-drying (10 ml in a 50-ml flask for vacuum freeze-drying) or formed into 0.16-ml pellets (for atmospheric freezedrying). These pellets were made in an aluminum matrix grooved with small cylinders 3 mm in thickness and 1 cm in diameter. Samples were then frozen at - 75°C. Three fermentations carried out under simiIar conditions, labeled Fl, F2, and F3, were needed to produce all the samples. The concentrates were cooled from +2O”C to -75°C at an average speed of - 0.4”C/min. The incipient freezing point, determined from the evolution of the product temperature during the freezing step,
ET AL.
was - 1.3”C for concentrates in pure milk, - 1.7”C in milk with added glycerol, and -2.5”C for milk with added glycerol and glucose. Supercooling was observed in all cases. The incipient melting point, determined from the evolution of the product temperature during the melting step of some samples, was much more difficult to determine precisely. It was - 2.6 + 0.4”C for concentrates in pure milk, -2.9 * 0.5”C in milk with added glycerol, and -4.l”C ? 0.4”C for milk with added glycerol and glucose. Freeze-Drying Processes Vacuum freeze-drying of the flask samples was carried out in a SMHl5 freezedrier from the USIFROID range (USIFROID, rue Claude 3emard, F 78312 Maurepas Cedex, France). Flasks were placed on a heating plate in a vacuum chamber containing a condenser cooled to -65°C. The equipment could be adjusted to control the heating plate temperature and the working pressure. Apart from the composition of the protectant medium, the influence of the heating plate temperature and the working pressure on survival of S. thermophilus was also studied. Heating plate temperatures studied were - 15, 0, and 15°C. In order to avoid melting, pressure was never above 300 Pa: we chose working pressures of 1, 100, and 300 Pa. These values correspond to mean values of the product temperature in the frozen core of - 40, - 18, and - 8”C, respectively. Under these temperature and pressure conditions, approximateIy 8 hr of treatment was required to reach water content IeveIs below 0.20 kg water/kg dry matter. Experiments were therefore run for 8 hr periods. The atmospheric pressure freeze-drying process suggested by Gibert in 1976 has been used in this study. The freeze-drying pilot plant is described by Wolff (10, 11). The main feature of this process is that the frozen product is mixed thoroughly with small particles of adsorbent (under 200 pm)
FREEZE-DRYING
571
OF Streptococcus thermophilus
in a column. It is then possible to work with air at atmospheric pressure. The effect of temperature of the bed of adsorbent and the composition of the protectant medium on survival of S. thermophillts was studied. The working temperature had to be below the incipient melting point of the product and therefore the study focused on two working temperatures, - 5°C and - WC, corresponding approximately to the same temperature for the frozen product. To obtain the required level of dehydration, freeze-drying was carried out for 15 hr.
ucts used for counting was made in reconstituted skimmed milk at 30°C. -The comparison of the different parameters studied was based statistically on the Student-t test applied to the mean value of survival rates obtained in each case. RESULTS
AND
DISCUSSION
Preliminary Remarks In the three fermentations carried out to produce the samples needed for this study, the initial number of CFU per milliliter was multiplied by 70, 300, and 1600 to reach a final number between 10’ and 10” CFU/ml. Analysis Methods The average number of bacteria forming a Results are reported with the following in colony, observed under a microscope, increased by 1.8 to 4.5 during culture. In all mind: -The water content of freeze-dried cases, diplococci made up about 50% of the products was obtained by weighing them colonies . After centrifuging, the average number of before and after 24 hr in a heating chamber at 100°C. The results are expressed in kilo- CFU per gram of DM was multiplied by 5 grams of water per kilograms of dry matter compared to nonconcentrated active bacteria, since all soluble matter was eliminated (DW. -The survival rate of S. thermophilus in the supernatant. After adding the prowas determined by counting the colony tectant medium to the same weight as the forming units (CFU) in a petri dish inocu- residue, we obtained about 5.10” CFUlml lated using the spiral method on an agar corresponding to 6.10” CFUlg DM. Ml7 (Biokar) type medium. The counting was carried out twice for two different di- Influence of Freezing and Storage on the Survival of S. thermophilus lutions. The average of the four values thus The selected suspension medium (reconobtained expressed in CFU per milliliter or in CFU per gram of DM suffered a margin stituted skimmed milk) and the freezing technique gave a survival rate of nearly of error of about 30%. -The thawing method used to count fro- 100%for S. thermophilus after freezing (see zen samples consisted of warming up the Table 1). An increase in cell concentration was in fact observed, which could be exproduct in a waterbath at 30°C. -The rehydration of freeze-dried prod- plained by the breaking of some chains durTABLE 1 Influence of the Suspension Media on Cellular Concentration (CFUlg DM) before and after Freezing at - 75°C of Streptococcus thermophib4s
Suspension medium
Skimmed milk (CFUlg DM)
Before freezing After freezing
4.3 x LO”
6.1 x 10”
Skimmed milk + glycerol (CFUlg DM)
Skimmed milk + glycerol + glucose (CFUlg DM)
6.2 x 10” 7.8 x 10”
6.2 x 1O’l 8.6 x to”
572
WOLFF
ET AL.
TABLE 3 ing freezing, thereby increasing the number of CFU counted, Pure reconstituted skim- Water Content after Vacuum Freeze-Drying (8 hr) of a Streptococcus thermophilus Suspension med milk therefore seemsto provide a satisfactory protectant medium. Water Content (kg Water/kg DM) Products frozen in this manner were not Pressure: 1 Pa lOOpa 300 Pa all used immediately for freeze-drying. We Temperature (“C) studied their mortality during frozen stor-15 0.08 0.16 age. After 15 weeks in storage at -75°C 0 0.14 1.11 0.15 the survival rate was almost 100%(see Ta15 0.11 ble 2), The standard deviation obtained per column was rather high compared to the average which led us to interpret results water/kg DM. It should be noted that some with considerable caution. authors report experiments where dehydration is continued to 0.01 kg water/kg DM (2) Influence of Vacuum Freeze-Drying on or even 0.001 kg water/kg DM, without, the Dehydration and SurvivaI of however, stating the freeze-drying condiS. thermophilus tions (pressure, plate temperature, and duWater content (Table 3). For a pressure ration). The addition of cryoprotectants, whatof 300 Pa, the residual water content after 8 hr of freeze-drying was close to 1 kg water/ ever they were, did not significantly influkg DM, which is too high for the proper ence the final water content. Survival. Results relative to the two ferconservation of bacteria. At this pressure, the low diffusivity of water vapor militates mentations Fl and F2 expressed as cell against transfer of this vapor, and the de- concentrations and as corresponding survival rates are shown in Table 4. For a plate hydration time is therefore far too long. As for the other conditions of the exper- temperature of lS”C, the survival rate was iment, the water content stabilized around very low. This operating parameter there0.15 kg water/kg DM. At 1 Pa and - 15”C, a fore did not warrant further study. As for the effects of temperature and residual water content of 0.08 kg water/kg DM was achieved after 8 hr. The reference pressure, the homogeneity of the results for freeze-dried sample (CNRZ 404) used was Fl and F2 (Table 4 except results at 15°C characterized by a water content of 0.14 kg and at 1 Pa for Fl) has been outlined staTABLE 2 Influence of Conservation at - 75°C on Cellular Concentration (CFU/g DM) of Streptococcus
Time (weeks) 0 1 2 3 4 6 15 Average value Standard deviation
Milk (CFU/g DM) 6.1 x 3.6 x 6.4 x 3.7 x 3.0 x 2.7 x 6.5 x 4.6 X 1.7 x
10” 10” 10” 10” 10” LO” 10” 10” 10”
Milk + glycerol (CFU/g DM) 7.8 4.4 9.2 5.8 5.2 6.0 6.0 6.3 I.6
x x x x x x x x x
10” 10” IO” 10” 10” 10” IO” 10” 10”
thermophilus
Milk + glycero1 + glucose (CFU/g DM) 8.6 5.1 9.1 3.9 6.0 7.5 6.4 6.7 1.9
x x x x x x x x x
10” 10” 10” 10” IO” 10” 10” 10” 10”
FREEZE-DRYING
573
OF Streptococcus thermophilus
TABLE 4 Influence of Vacuum Freeze-Drying (8 hr) on Cellular Concentration (CFU/g DM) and Survival Rate (%) of Streptococcus Pressure:
Protectivemedium: Initial coucn: Temperature(“C)
1 Pa Milk + glycerol 2.4 x 10”
thermophilus
FermentationFl 100Pa loo Pa Milk + glycerol Milk + PEG 2.4 x 10” 2.3 x 10”
15
loo Pa Milk + DMSO 1.7 x 101’
300Pa Milk + glycerol 2.4 x lOI’
1.2 x 101’ 52%
I.1 x 10’1 46%
1.4 x 10’0
-15
1.2 ZD/Oll
0
1.1 x 10’0 5%
Pressure:
1 Pa Milk
9.55Z010 3%
6.9 x 10’0 30%
FermentationF2 1 Pa Milk Milk + glycerol + glycerol
Initial ceucn: Temperature(9C)
5.5 x IO”
6.9 x IO”
7.3 x IO”
5.5 x IO”
6.9 x IO”
100Pa Milk + glycerol t glucose 7.3 x 10”
- 15
3.6 x 10” 65% 3.8 x 10” 49%
3.6 x 10” 52% 3.5 x 10” 51%
3.5 x 10”
3.5 x IO” 44% 4.0 x 10” 73%
3.1 x IO” 45% 3.8 x 10” 55%
3.1 x 10” 42% 3.6 x 10” 4%
Protectionmedium:
lpa
+ glucose
0
3.4ZOt’ 47%
tistically. It was shown that no statistical difference can be observed at a 5% risk level. The mean value of the survival rate did not depend on either the temperature of the heating plate, so long as it was below or equal to O’C, or the pressure. The survival rates of concentrates from F2 fermentations allow comparison between the various protectant media. No statistically significant differences (level of risk, 5%) can be observed between samples in pure milk or in milk with added glycerol and glucose. The low concentration of the cryoprotectant molecules used is probably part of the explanation of this fact.
100Pa Milk
looPa Milk
+ glycerol
The average survival rates were 55% for F2 and 40% for Fl . This difference could be due to the initial state of the microorganisms which could be different at the end of the two fermentations. Influence of Atmospheric Pressure Freeze-Drying on the Dehydration and Survival of S. thermophilus The results of the experiments carried out at - 5°C and - 15°C in relation to water content and survival rates are shown in Table 5. Water content. The water content obtained after 15 hr of freeze-drying at - 5°C
TABLE 5 Influence of Atmospheric Freeze-Drying (15 hr) on Cellular Concentration fCFU/g DM) and Survival Rate (%) of Streptococcus thcrmophilus luitial concentration (CFU/g DM) FI: 2.4 x 10” F3: 5.0 x 10” F3: 5.0 x IO”
Temperature w”c) -5 -15 - 15
Suspension medium milk + glycerol milk + glycerol milk + glycerol + glucose
Water content (kg water/ kg DM) 0.17 0.21 0.21
Concn. (CFUlg DM) 6.3 x lOa 1.7 x 10” 1.5 x 10”
Survival rate (%I 3 35 31
574
WOLFF
was 0.17 kg water/kg DM; at - 15°C it was 0.21 kg water/kg DM. These results were higher than those obtained by vacuum freeze-drying (8 hr). Atmospheric freezedrying is thus slower than vacuum freezedrying. Survival. Freeze-drying at - 15°C gave final bacterial concentration and survival rates much higher than those obtained after freeze-drying at - 5°C (Table 5). Probably, at - 5”C, the freeze-drying temperature was too close to the melting temperature of the ice in the product and this may be detrimental for the bacteria. This difference was obtained from tests on different fermentations. Different initial states of the bacteria could also explain these differences but the temperature factor certainly had a predominant effect in the differences obtained. The mean value of the survival rate obtained at - 15°C was about 33%. Pure milk, as shown above, seemed to be a good protectant medium. Comparison
of the Two Processes
Technology, Vacuum freeze-drying is a technology that is easy to carry out but it has three main disadvantages: 1. An air-tight chamber is necessary to work under vacuum and makes it difficult to obtain high intensity of heat transfer and to work in a continuous way. 2. The ice condensers must be cooled at a very low temperature and defrosted reg ularl y . 3. Heating by heating plates is not efficient because the contact resistance to heat transfer between solid particles at these pressures is very high. Atmospheric freeze-drying is also very simple to use. It can be driven in a continuous way but has two different disadvantages: 1. At the end of the freeze-drying step, it is necessary to regenerate the adsorbent. 2. Some small particles of adsorbent (starch) may spoil the freeze-dried product. Energy aspects. Freeze-drying is well
ET
AL.
known to be an expensive technique. For the vacuum freeze-drying process, the authors generally adopt the following order of magnitude for the energy cost (7): cooling request, 3600 kJ/kg of ice; and heating request, 4900 kJ/kg of ice. Under atmospheric pressure it has been shown that these costs can in practice be reduced in a continuous duty atmospheric pressure freeze-drying facility, including the adsorbent regeneration cost, at (10, 11): cooling request, 2250 W/kg of ice; and heating request, 3250 kJ/kg of ice. A 35% savings on energy costs may be expected. But it has also been shown that the freeze-drying time in this new process is approximately twice that under vacuum. Survival rate of lactic acid bacteria. Taking into account F2 and F3 fermentations where the initial concentrations of bacteria were comparable, the survival rate obtained for the vacuum freeze-drying of S. thermophilus was 55% and only 33% at atmospheric pressure. This shows that the survival rate is greater in the traditional freeze-drying process than in the new process, at least under the experimental conditions tested, If there are new improvements in atmospheric freeze-drying, it is possible that this difference can be reduced. However, it is too early to exclude this process at this step of the research. CONCLUSION
Whatever freeze-drying process was used, it was possible to obtain good quality freeze-dried products (cell concentration between 10” and 3.10” CFU/g DM for residual water contents of the order of 0.1 kg water/kg DM). These properties are equivalent to those of freeze-dried products currently on the market. Of the operating conditions investigated, the freeze-drying conditions that gave the best results were -Pressure between 1 and 100 Pa and heating temperature of the lower plate below O”C, for vacuum freeze-drying,
FREEZE-DRYING
OF Streptococcus thermophilus
--Operating temperature rather low (- WC) for atmospheric freeze-drying. -The addition of reconstituted skimmed milk as cryoprotectant suspension medium. At first, vacuum freeze-drying seemed to be best suited to the dehydration of concentrated suspensions of S. thermophilus. In fact, atmospheric pressure freeze-drying called for double the processing time (15 hr instead of 8 hr) and half the survival rate (33% instead of 55%). However, the energy costs can be decreased by one-third compared with those of vacuum freeze-drying. The initial state of microorganisms seems to have an effect on survival rates. It is therefore important to control, indeed to optimize, each of the stages implemented before freezing and freeze-drying, i.e., conditions of fermentation, time of sampling, process, and level of concentration. REFERENCES
I. Barbour, E. A., and Priest, F. G. The preservation of Lnctobacilli: A comparison of three methods. Left. Appl. Microbial. 2, 69-71 (1986). 2. Bousfield, I. J., and Mackensie, A. R. Inactivation of bacteria by freeze-drying. In “The Society for Applied Bacteriology,” (F. D. Skinner
$75
and W. B. Hugo, Eds.), Symposium series No. 5, Academic Press, London, 1976. 3. Gibert, H. French Patent ANVAR 76,39,864; U.S. Patent 4,175,334 (1976). 4. Kijanapanich, P. “Lyophilisation de de&es alimentaires en couche fluidiske sous pression atmosph6rique: influence de la nature de l’adsorbant.” Thesis, INPT, Toulouse, 1981. 5. Martin, M. Les levains concentrds lyopbili& La technique l&Pm 976, 45-49 (1983). 6. Robinson, R. K. Freeze-dried starter concentrates. Dairy I&. Inr. 46(10), 15-22 (1981). 7. Simatos, D., Blond, G., Dauvois, Ph., and Sauvageot, F. “La lyophilisation: principe et applications.” Collection de I’ANRT, Paris, 1974. 8. Tamine, A. Y., and Deeth, H. G. Yogurt: Technology and biochemistry. J. Food Pmt. 43(12), 939-477 (1980). 9. Wachet, J. N. “Etude d’un pro&de de lyophilisation en couche fluidiste sous pression atmosphkique.” Thesis, USTL, Montpellier, 1978. 10. Wolff, E., and Gibert, H. Freeze-drying at atmospheric pressure: Design and energy considerations. In “Drying ‘86” (A. S., Mujumdar, Ed.). Hemisphere, Washington, 1986. 11. Wolff, E. “Cinktique et mod&sation de la lyophilisation sous vide et de la lyophilisation & pression atmosphkrique.” Thesis, INPT, Toulouse, France (1988). 12. Yassin, K. E. E. “Lyaphilisation sous pression atmosphtrique: mise en oeuvre d’un adsorbant alimentaire.” Thesis, INPT, Toulouse, France (1984).
