Effects of Additives on the Survival of Lactic Streptococci in Frozen Storage

Three single-strain cultures, Streptococcus lactis C₂, S. cremoris R₁, and S. diacetilactis DRC₂, were frozen and stored in skim milk, in skim milk containing apple juice, and in skim milk containing one of the following additives: glycerol (10%, v/v), dimethyl sulfoxide (10%, v/v), l-malic acid (0.5 and 2.0%, w/v), acetamide (0.5 and 2.0%, w/v), or succinimide (0.5 and 2.0%, w/v). Cultures were frozen and stored at -23.3 C, frozen and stored at -196 C in liquid nitrogen, or frozen at -196 C and stored at -23.3 C. Cultures frozen and stored at -196 C in liquid nitrogen gave the greatest recovery of viable cells. The number of cells surviving after storage at -23.3 C was greater when the cells had been frozen in liquid N₂ than when they had been frozen at -23.3 C. All strains stored at -23.3 C showed a decrease in numbers of surviving cells; additives, particularly l-malic acid and apple juice, were advantageous in preserving the viability of the S. lactis C₂ and S. cremoris R₁ strains, but had little or no effect on the survival of S. diacetilactis DRC₂. l-Malic acid and apple juice stimulated acid production for all cultures in activity tests following incubation after thawing, whereas glycerol and dimethyl sulfoxide retarded its development.     

Cryopreservation studies of Campylobacter

Seven strains of Campylobacter fetus ss. fetus, one of Campylobacter fetus ss. venerealis, and one of Campylobacter jejuni were preserved using a variety of cryopreservation methods. Organisms were frozen to -150 degrees C in a liquid nitrogen refrigerator, in the freezer compartment of a refrigerator (-20 degrees C), and in a mechanical freezer (-65 degrees C). In the latter two cases, viabilities of the organisms were compared after being frozen in Brucella Albimi broth and 10% glycerol. Viabilities were also examined after Campylobacter species were freeze-dried using rapid or slow cooling, using sucrose or skim milk as cryoprotective agents and in bulb-type vials on a manifold or batch vials. Preservation in liquid nitrogen resulted in no loss in viability after 4 years storage. When Campylobacter species were frozen at -20 degrees C, no cells were recovered after 1 month storage in Brucella Albimi broth or seven months in glycerol. A 6.5 log decrease in viability resulted after organisms were frozen at -65 degrees and subsequently stored at the same temperature for 2 years. In this case, glycerol had no protective advantage over Brucella Albimi broth. Postpreservation viability of organisms cooled slowly was two logs higher than those cooled rapidly prior to freeze-drying. When skim milk or sucrose were employed as cryoprotective agents during freeze-drying, equal viabilities resulted. Equivalent viabilities were also demonstrated when the bulb type or "batch" vials were utilized for freeze-drying. No significant differences were observed between the viabilities of the three species when a given cryopreservation method was employed.     

Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying

The effect of initial cell density, protective agents and rehydration media on the viability of biocontrol agent Pantoea agglomerans CPA-2 when subjected to freeze-drying was studied. Several additives were tested as protective agents against freeze-drying injury. Maximum viability of the bacterial cells was obtained with disaccharides (survival levels > 60%). Freeze-dried samples were rehydrated with several media; the highest percentage viability was obtained with 10% non-fat skim milk (100%+). The effect of initial bacterial load on the final recovery was dependent on protectant but not on rehydration media. Sucrose was an effective protectant when a high initial concentration (10(10) cfu ml(-1) was used; the opposite occurred with non-fat skim milk. The use of 10(10) cfu ml(-1) as an initial concentration, sucrose as a protectant and non-fat skim milk as a rehydration medium enabled 100% of P. agglomerans viability to be conserved after freeze-drying. Results suggest the possibility of achieving a good formulation system for the studied biocontrol agent with a high number of viable cells to be used toward pathogens, which is desirable for the industrial development of the product.     

Combined effects of glycerol concentration, cooling velocity, and osmolality of skim milk diluents on cryopreservation of ram spermatozoa

The interaction of glycerol concentration from 0 to 16% and cooling velocity from 1 to 100 degrees C/min on freeze-thaw survival of ram spermatozoa was studied using a diluent based on 15% skim milk (450 mOs/kg water). Optimal spermatozoa survival (percentage motility and rating) was obtained with 4 to 6% glycerol and freezing rates of 10 to 100 degrees C/min. Similar results were obtained with 8% glycerol at freezing rates of 5 to 30 degrees C/min. Although the ram spermatozoa tolerated several cooling velocities at each glycerol concentration, increasing the concentration of glycerol resulted in a downshift in the range of optimal cooling velocities. Glycerol concentrations above 8% were toxic and contributed greatly to the progressive decrease in spermatozoa survival. Comparison of the 15% skim milk diluent (450 mOs/kg water) with a 19% skim milk diluent (600 mOs/kg water) showed that optimal cryosurvival was obtained with 4 to 6% glycerol and freezing rates of 10 to 100 degrees C/min with both diluents.     

Growth of Salmonella typhimurium in Skim Milk Concentrates

The influence of various levels of skim milk solids and temperature on the duration of lag phase, growth rate, and extent of growth of Salmonella typhimurium was investigated. The effect on growth of salmonellae (and a strain of Escherichia coli) of reduced pressure at a constant solids level and under conditions simulating vacuum condensation of skim milk was also studied. S. typhimurium grew when inoculated into skim milk solutions ranging from 10 to 60% solids and over a temperature range of 23 to 44 C. At 10 to 12 C, growth was evident only in the 10% skim milk. As the total solids level was increased or incubation temperature was deviated from the optimum, or both, there was an increase in the lag phase and generation time of salmonellae. A lower cell population also resulted. The generation time at 37 C of S. typhimurium incubated at atmospheric pressure was approximately one-half that in skim milk concentrates held under reduced pressure. In addition, a slightly longer lag phase and lower cell yield characterized the growth under reduced pressure. Concentration of skim milk had little or no effect on viability of salmonellae or E. coli when the vapor temperature in the vacuum pan was below the maximum growth temperature for salmonellae. Increasing the vapor temperature to 48 C caused a two-log reduction in viable organisms during the concentrating period (65 min).     

Comparative study of the efficiency of some additives in protecting lactic acid bacteria against freeze-drying

Cultures of 14 lactic acid bacteria species were freeze-dried in 10 or 20% non-fat skim milk and in distilled water containing one of the following additives: bovine albumin, glycogen, dextran, polyethylene glycol (PEG) 1000, PEG 4000, PEG 6000, glycerol, beta-glycerophosphate, sodium glutamate, asparagine, or cysteine. Each of the potential protective agents tested exhibited marked variations in the protection afforded to different species, none of them was effective for the preservation of viability of thermophilic lactobacilli. However, glycerol provided effective protection for L. leichmannii ATCC 4797 (90% survival), while L. bulgaricus ATCC 11842 reached a viability of 78% with 0.04 M cysteine.     

Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa

Spermatozoa from cauda epididymis of mature mice were suspended in preservation solution (Dulbecco's PBS containing raffinose in combination with glycerol, DMSO or skim milk as freezing protective agents). The suspension was frozen by the dry ice-alcohol method and preserved for 1-120 days in liquid nitrogen (-196 degrees C). Highest sperm viability after thawing was obtained with a combination of 10% raffinose and 5% glycerol or with a combination of 10% raffinose and 10% DMSO. These frozen thawed sperm were found to have fertilizing capacity when used for in vitro fertilization. The 2-cell embryos obtained through the above procedures developed into normal pups at a high rate when transferred into the oviducts of pseudopregnant female mice.     

Heat Resistance of Salmonellae in Concentrated Milk

The heat resistance of Escherichia coli, Salmonella typhimurium, and Salmonella alachua in milk solutions containing 10, 30, 42, and 51% (w/w) skim milk for total solids was determined. Increased milk-solids level effected a significant increase in the heat resistance of each organism. Although E. coli was more heat-resistant than both strains of Salmonella in 10% milk, the situation was reversed in 42 and 51% milk. Prior growth temperature was found to exert a profound effect on the heat resistance of S. typhimurium. Growth of S. typhimurium in 42% milk solids for 24 hr did not greatly enhance the thermal resistance of the organism when heated in a fresh 42% solids concentrate. Application of a partial vaccum during heating greatly diminished the decimal reduction times of S. typhimurium and E. coli and, in addition, virtually eliminated the protective effect of increased solids level.     

Optimization of a protective medium for enhancing the viability of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus based on response surface methodology

Response surface methodology (RSM) was used to optimize a protective medium for enhancing the cell viability of Lactobacillus delbrueckii subsp. bulgaricus LB14 during freeze-drying. Using a previous Plackett–Burman design, it was found that sucrose, glycerol, sorbitol and skim milk were the most effective freeze-drying protective agents for L. bulgaricus LB14. A full factorial central composite design was applied to determine the optimum levels of these four protective agents. The experimental data allowed the development of an empirical model (P<0.0001) describing the inter-relationships between the independent and dependent variables. By solving the regression equation, and analyzing the response surface contour and surface plots, the optimal concentrations of the agents were determined as: sucrose 66.40 g/L, glycerol 101.20 g/L, sorbitol 113.00 g/L, and skim milk 130.00 g/L. L. bulgaricus LB14 freeze-dried in this medium obtained a cell viability of up to 86.53%.     

Lipase activity ofPseudomonas fluorescens in cold raw skim milk with different lipid supplements

Different dairy-like fat fractions were added to raw skim milk in order to determine their effect on lipase production byPseudomonas fluorescens growing on raw milk at 7°C. Supplementation with preparations of fat globule membranes, mixtures of capric acid, glycerol monocaprinate, and glycerol dicaprinate, cholesterol, or lecithin at concentrations similar to those occurring in whole milk had only slight effects on lipase production, whereas supplementation with 0.9% glycerol trioleate decreased lipase activity in milk by 90 %.     

Fertilizing potential of mouse spermatozoa cryopreserved in a medium containing whole eggs

Experiments were conducted to improve survival of mouse spermatozoa through the cryopreservation process. In the first experiment, percentages of motile spermatozoa and fertilizing capacities of spermatozoa were evaluated when mouse spermatozoa were cryopreserved using three previously reported cryopreservation media: (1) 18% raffinose in 3% skim milk; (2) Tes/Tris medium containing 25% egg yolk and 1.25% glycerol; and (3) PBS containing 18% raffinose and 1.75% glycerol, each at three different cooling rates (-3, -10, and -50 degrees C/min). Spermatozoa frozen in the skim milk/raffinose medium exhibited the highest percentage of motile spermatozoa (39%) when cells were frozen at -10 degrees C/min (P<0.05). The second experiment evaluated the effects of modifying the Tes/Tris/egg yolk medium, comparing different concentrations of egg yolk, BSA, and sodium dodecyl sulfate. Reducing egg yolk from 25% of the medium volume to 5%, increased percentages of motile spermatozoa after cryopreservation from 29 to 36% (P<0.05). Addition of 1% BSA and sodium dodecyl sulfate to medium containing 5% egg yolk further improved percentages of motile spermatozoa after freezing. In the final experiment, 20% whole egg was substituted for 5% egg yolk and 1% BSA used in previous experiments and resulted in percentages of motile spermatozoa (51%) equal to that of the skim milk-raffinose medium. However, fertility rates were higher (68%) than for spermatozoa frozen in the skim milk-raffinose medium (P < 0.05) and were comparable to the fertility rates of fresh spermatozoa (77%; P>0.05). In conclusion, freezing mouse spermatozoa in a medium containing 20% whole egg, 0.035% sodium dodecyl sulfate, and 1.25% glycerol using a cooling rate of -10 degrees C/min preserves the motility and fertilization capacity of mouse spermatozoa.     

Repair and enterotoxin synthesis by Staphylococcus aureus after thermal shock.

To study repair and enterotoxin synthesis, four staphylococcal strains (FRI-100, FRI-137, FRI-472, and S6) were subjected to sublethal heat treatment, transferred to four liquid repair media (1% powdered skim milk in distilled water, complex medium, M9 minimal salt medium, and saline solution), and then incubated at different temperatures. Powdered skim milk proved to be the most efficient medium for promoting the repair of injured cells, particularly at 37 degrees C. Minimal salt medium also gave good results. Salt tolerance also increased at 4 degrees C, although it did not reach normal values. After 6 h of incubation at 37 degrees C in powdered skim milk, strain FRI-100 synthesized detectable amounts of enterotoxin A. After 10 h of incubation in the same medium at the same temperature, enterotoxins were detected in all of the strains.     

Improving survival and storage stability of bacteria recalcitrant to freeze-drying: a coordinated study by European culture collections

The objective of this study is to improve the viability after freeze-drying and during storage of delicate or recalcitrant strains safeguarded at biological resource centers. To achieve this objective, a joint experimental strategy was established among the different involved partner collections of the EMbaRC project (www.embarc.eu). Five bacterial strains considered as recalcitrant to freeze-drying were subjected to a standardized freeze-drying protocol and to seven agreed protocol variants. Viability of these strains was determined before and after freeze-drying (within 1 week, after 6 and 12 months, and after accelerated storage) for each of the protocols. Furthermore, strains were exchanged between partners to perform experiments with different freeze-dryer-dependent parameters. Of all tested variables, choice of the lyoprotectant had the biggest impact on viability after freeze-drying and during storage. For nearly all tested strains, skim milk as lyoprotectant resulted in lowest viability after freeze-drying and storage. On the other hand, best freeze-drying and storage conditions were strain and device dependent. For Aeromonas salmonicida CECT 894^T, best survival was obtained when horse serum supplemented with trehalose was used as lyoprotectant, while Aliivibrio fischeri LMG 4414^T should be freeze-dried in skim milk supplemented with marine broth in a 1:1 ratio. Freeze-drying Campylobacter fetus CIP 53.96^T using skim milk supplemented with trehalose as lyoprotectant resulted in best recovery. Xanthomonas fragariae DSM 3587^T expressed high viability after freeze-drying and storage for all tested lyoprotectants and could not be considered as recalcitrant. In contrary, Flavobacterium columnare LMG 10406^T did not survive the freeze-drying process under all tested conditions.     

Destruction of Microbacterium lacticum, Escherichia coli and Staphylococcus aureus in milk by spray-drying. I. Selective count of surviving bacteria]

In this paper a method which allows the measure of microbial death rate during spray-drying by means of a streptomycin-resistant mutant that can be grown on a streptomycin-containing agar is described. Plate counts of Microbacterium lacticum, Escherichia coli, and Staphylococcus aureus recovered from skim milk powders were done on plate count agar in the presence and absence of streptomycin and on various selective media. The powders were produced from evaporated milk previously inoculated with those organisms. Our results showed that the proposed method allows the recovery of 78% of M. lacticum, 61% of E. coli, and 100% of S. aureus that survived spray-drying. Recoveries of surviving E. coli on violet bile agar and brilliant green bile 2% were 34% and 29% respectively. Baird-Parker and mannitol salt agar media allow the recovery of all surviving S. aureus, thus showing that S. aureus cells did not lose their ability to grow in media containig 7.5% NaCl. Our results show that physiological injury of the cells during spray-drying differs from injury due to heating only.     

Production of lyophilized culture ofLactobacillus acidophilus with preserving cell viability

Optimal lyophilization process was developed for manufacturing the dried product ofLactobacillus acidophilus with high cell viability. Three major factors, freezing rate, specific surface area of samples, and stabilizer type and their synergy were shown to play a crucial role in the development of an effective lyophilization process. Finally we found an optimal combination among three process parameters mentioned above: an exceptionally high cell survival percentage of 90% was achieved using the 8.28 cm⁻¹ specific surface area of samples, slow freezing rate, and a stabilizer composition of 4% skim milk+1% glycerol +0.1% calcium chloride.     

Effect of the rehydration medium on the recovery of freeze-dried lactic acid bacteria.

Sixteen cultures of lactic acid bacteria were freeze-dried in 10% nonfat skim milk plus 0.75 M adonitol and rehydrated by using different rehydration media. Marked variations in their capacity to repair cellular damage after freeze-drying were observed among the species and strains under consideration.     

Effect of the rehydration medium on the recovery of freeze-dried lactic acid bacteria

Sixteen cultures of lactic acid bacteria were freeze-dried in 10% nonfat skim milk plus 0.75 M adonitol and rehydrated by using different rehydration media. Marked variations in their capacity to repair cellular damage after freeze-drying were observed among the species and strains under consideration.     

Isolation of salmonellas by immunomagnetic separation.

Magnetisable particles, coated with anti-salmonella serum, were used to isolate Salmonella livingstone from pure cultures, mixed cultures and food samples. Beads (10(7] were generally incubated with 10(4) Salm. livingstone cells/ml for 60 min at room temperature. The incubation and washing medium (0.01 mol/l phosphate-buffered saline; PBS) contained 0.1% bovine serum albumin (BSA) and 0.1% Tween 20, respectively. This method gave a recovery for Salm. livingstone of 51.0 +/- 7.8%. However, other micro-organisms such as Aeromonas hydrophila interfered with this test because of non-specific reactions (recovery 50.9 +/- 12.7%). These non-specific reactions could be decreased by using 4% skim milk instead of 0.1% BSA in the incubation medium. The ratio of the recovery of Salm. livingstone relative to the recovery of Aer. hydrophila changed from 0.9 when PBS with 0.1% BSA was used, to 13.4 when PBS with 4% skim milk was used. Immunomagnetic separation of Salmonella spp. from food samples offers good prospects for concentrating salmonella cells from heterogeneous bacterial suspensions, such as enrichment broths.     

C-390 as sole selective agent for isolation of Pseudomonas aeruginosa from hospital waste water

Pseudomonas aeruginosa was recovered (in numbers ranging from 10(2) to 10(5) colony-forming units per millilitre) from heavily contaminated hospital waste water when grown at 41.5 degrees C on a differential medium agar containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390) at a final concentration of 30 micrograms/mL. The medium appeared to be highly selective for P. aeruginosa with 95-100% of all colonies isolated from four different hospital waste waters being identified as P. aeruginosa. Many strains of P. aeruginosa isolated from hospital waste waters failed to hydrolyse casein when grown on skim milk agar and this medium appeared to restrict pigment production to only pyoverdin (detectable only under ultraviolet light). However, most strains were capable of casein hydrolysis when grown on a modified skim milk medium.     

Thermal Resistance of Staphylococcus aureus in Milk, Whey, and Phosphate Buffer

The thermal resistance of four strains of coagulase-positive Staphylococcus aureus was determined in phosphate buffer, whole milk, skim milk, and Cheddar cheese whey. The logarithmic order of death prevailed until about 99.99 to 99.999% of the organisms were destroyed, after which there was a decline in the rate of destruction. The organisms were more resistant in skim milk and Cheddar cheese whey than in phosphate buffer and whole milk. Thermal resistance varied among strains of S. aureus but was consistent with individual strains. As the age of cultures of strain B-120 increased from 12 to 228 hr, the D(55) values increased from 0.95 to 3.0. The thermal resistance of cultures obtained from survivors to partial thermal destruction was similar to that of the parent cultures.