Lyophilized Lactic Acid Starter Culture Concentrates: Preparation and Use in Inoculation of Vat Milk for Cheddar and Cottage Cheese1

tyophilized Lactic Acid Starter Culture Concentrates: Preparation and Use in Inoculation of Vat Milk for Cheddar and Cottage Cheese1 C. A. SPECKMAN, W. E. SANDINE, and P. R. ELLIKER Department of Microbiology Oregon State University, Corvallis 97331 Abstract

mental to bacterial cells (15, 21, 2£), with eell death occurring during the freezing, drying, storage, and rehvdration of the cultures. Dry concentrated starter bacteria would provide obvious advantages, e.g. direct inoculation of vat milk f:~r cheese manufacture. We developed a method of preparing active, dry concentrates of lactic starter cultures which were used to inoculate directly vats of milk for cheese manufaeture.

A method to concentrate and lyophilize milk-grown cultures of lactic streptococci which produced cells active in fermentation is described. Inoculated nonfat milk was maintained at pH 6.3 by continuous addition of 30% aqueous sodium carbonate. After growth for 15 to 18 h at 30 C, the pH was adjusted to 6.9, and 4.5% sodium citrate was added to allow recovery of the cells by eentrifugation. The cells were resuspended in a medium of 10% sucrose, 5% sodium citrate, 5% gelatin, and 2% monosodittrn glutamate to 1/20th the original culture volume, and were frozen and lyophilized. Vials containing the dr), concentrate were sealed under vacuum, stored at --22 C and + 2 2 C, and examined for cell viability and activity, Activity was assayed by simulating cottage and Cheddar cheese making procedures and measuring the final pH. Several lyophilized cultures maintained activity similar to conventional culture for storage of up to 3 mo. Cheddar and cottage cheese were made in 363-kg capacity vats with lyophilized and conventional cultures, and the dry concentrates performed as well as conventional cultures.

Methods and Materials

Introduction

Frozen, concentrated lactic starter eul~res were first used commercially by the dairy fermentation industry in 1963 (17). Several studies have been made of culture concentrates (1, 4, 5, 6, 9, 20, 23, 24, 25, 26, 31, 32, 34, 35, 39, 41, 43, 44), and these were recently reviewed (30). However, few (2, 3) have considered lyophilization of concentrates to avoid the expense of shipping and maintaining in a frozen state. Freeze-drying, however, is detriReceived January 23, 1973. Technical Paper No. 3477. Oregon Agricultural Experiment Station.

Bacterial cultures. Cultures obtained from Barbara Keogh, Commonwealth Scientific and Industrial Research Organization, Highett, Victoria, Australia, were Streptococcus lactis C2, el0, and Streptococcus cremoris EB2, EB4, EB7, EB9, E8, HIP, e l , C3, Cll, C13, and ML1; those obtained from Dr. Lindsay Pearce, New Zealand Dairy Research Institute, Palmerston North, New Zealand, were S. lactis ML8, WM1, and S. cremoris AM1, AM2; and P2. S. cremoris strains 163, 220, 459, 819, and 990 were from the Oregon State University culture collection maintained in the Department of Microbiology. The identity of the cultures was confirmed by taxonomic tests described by Sandine et al. (40). Culture media. Stock cultures were maintained frozen (--20 C) in sterile nonfat milk (NFM - 11% solids) containing 15% (v/v) glycerol. Cultures were inoculated (10%) into this medium and immediately frozen without incubation. When a subculture was desired, the frozen stock was thawed at 30 C and incubated at 22 C for 18 h. From this mature culture, at least three successive transfers were made similarly with a 1% (v/v) inoculum before the culture was used in a scheduled experiment. A 14-1iter Fermentation Design fermentor or a l-liter Scientific Products Micro-fermentor was used in growth studies and for production of cells for the lyophilization studies, pH was monitored and maintained by a Fermentation Design pH control module with an Ingold pH electrode. The base was prepared at 30% (w/v) concentration and sterilized by autoelaving. Base was added automatically to the

165

166

SPECKMAN ET AL

culture medium by peristaltic pumps controlled by the pH module.

Harvesting of cells and preparation of concentrate. Cells were harvested from NFM cultures at the end of logarithmic phase of growth by the procedure of Stadhouders et al. (44). The culture was first adjusted to pH 6.9 with 30% sodium carbonate. Sodium citrate was added to a final concentration of 4.5% with stirring for 10 min. The culture was centrifuged at 7,970 )< g for 1 h in a model RC2 Sorvall refrigerated centrifuge. Sedimented cells were reconstituted in the specified suspension fluid and adjusted to pH 7.0 with sterile 1N sodium hydroxide. This cell suspension was the cell concentrate. Numbers of viable cells in various culture preparations were determined by plating on lactic agar with incubation at 30 C for 48 h (16). Acid-producing activity. Activity of different culture preparations was determined by inoculating cells into 11% NFM which had been steamed (80 C) for 30 min. The inoculated milk was incubated at 30 C or 37 C for 4 h, then placed in ice. The milk was titrated with .1 N sodium hydroxide to pH 8.3 with a Fisher Automatic Titrimeter. Activity was expressed as percent lactic acid. Lyophilization. The suspending solution (GCGS) of Lagoda and Bannikova (27) contained 5% gelatin, 5% sodium citrate, 2% monosodium glutamate (MSG), and 10% sucrose. Cells from a 700-ml batch culture were hmwested and resuspended in GCGS solution to a final volume of 30 ml in a screw cap test tube. A homogenous mixture of cells and suspending medium was made by agitation on a Vortex mixer, and a final pH of 7.0 was obtained with i N NaOH. One-milliliter aliquots were placed in 10-ml rubber-stoppered serum bottles and frozen in dry ice-acetone. The stoppers were loosened, and the contents were lyophilized in a Refrigeration for Science (Atmo-Vac Model). Vials were sealed under vacuum by a plunger forcing the rubber stoppers down on the vials in the vacuum pan. An aluminum cap was then crimped over the rubber stoppers to hold them secure. The vials were divided into two groups and stored at 22 C and at - 2 2 C. Method of rehydration. The dry starter concentrate was rehydrated by removing the stopper and adding 10 ml of sterile rehydration medium to each vial at room temperature (25 C). The vials were restoppered and allowed to rehydrate until complete dispersion of the concentrate. Different media were used for rehydration including water, 10% NFM, JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 2

and a special rehydration medium (RM) containing 20% lactose and 1.0% stimilac (Marschall Division of Miles Laboratories, Inc., Madison, Wisconsin). Simulated product activity tests. A modification of the activity test described by Pearce (33) was used for evaluation of cultures for Cheddar cheese manufacture. The test was in dilution bottles containing 100 ml of 11% NFM previously steamed 30 min and chilled in an ice bath. Milk in the bottles was inoculated with either a 2-ml quantity of a 15-h conventional NFM culture or .2 ml of the rehydrated 10 ml culture. This provided an approximately equal cell number inoculum and facilitated comparison. Each bottle was shaken, placed in a water bath at 31 C for 20 min, and 1.0 ml of a 1:50 dilution of rennet was added to each bottle. An additional 50 rain of incubation was allowed before the cooking period which consisted of raising the water bath temperature to 39 C slowly over 30 min. Following cooking, the temperature was slowly decreased to 37 C for 30 min and maintained at 37 C for 130 min. Then the temperature was set at 32 C for the final 40 min. After 5 h the curd was macerated by vigorous shaking of the bottles. The bottles were placed in an ice bath overnight, and pH was measured the following morning. The cottage cheese activity test consisted of inoculating 100 ml of steamed milk in the same manner and incubating at 23 C for 11 h. This was followed by maceration of the curd, holding at 4 C overnight, and measuring pI-I the following morning. Cheese making. Cheese was made in two vats each containing 363 kg of milk. One vat was inoculated with a fresh, conventional 15-h NFM culture while the other vat was inoculated with an equal-cell-number inoeulum of lyophilized concentrate rehydrated in RM. Methods of manufacture for Cheddar (regular or Colby type) and cottage cheese were conventional (45). Results

Preliminary studies (7) established optimum conditions for concentrated culture preparation following controlled growth in sterile 11% NFM, i.e., 30 C, flushing with nitrogen for 5 min before inoculation, agitation at 100 rpm, and maintenance of pH at 6.3 with 30% sodium carbonate. The approximate number of cells in the concentrated, frozen-concentrated, and lyophilized-concentrated cultures compared to conventional culture, a frozen conventional culture, and a lyophilized conven-

LYOPHILIZED STARTER

167

TABLE 1. Comparison of the numbers of ceils obtained by different types of cultures of lactic streptococeL Growth results (cfu/ml × l&) Type of culture Conventional eul~re ~ Frozen conventional culture

ML8

S. lactis C2

2.2 2.0

1.8 1.0

Lyophilized conventional culture

.25

Concentrated culture Frozen and thawed concentrated culture Lyophilized and rehydrated concentrated culture

C10

HP

2.8 2.6

.17

S. cremoris C1

1.1 .79

.20

1.6 1.0

.28

E8 1.2 .8

.19

.4

180.0 170.0

160.0 150.0

250.0 250.0

90.0 90.0

125.0 135.0

95.0 75.0

180.0

160.0

250.0

80.0

125.0

75.0

~'Nonfat milk (NFM) culture grown 15 h at 22 C.

tional culture indicates that the cryoprotective medium (GCGS) used to prepare the cell concentrates protects them from the loss sustained when the conventional culture was frozen and thawed or lyophilized (Table 1). However, because cell numbers alone is a poor eriterion for evaluating culture performance, activity was tested. Results of activity tests at different stages of preparation in comparison to activity of cells taken from a conventional NFM culture are shown (Table 2). Effect of additive carry-over from GCGS or RM was negligible; tests with one culture (C1) revealed no stimulation in

acid production by adding the appropriate amount carried over when NFM was inoculated from concentrated or rehydrated cells. The lyophilized concentrate was as active as the conventional culture in acid production at either 30 C or 37 C. Cells harvested from 17-h NFM cultures grown at 32 C and maintained at pH 6.3 withstood freezing and lyophilization better than cells harvested earlier. Concentrates of cells from cultures grown longer than 17 h did not show increased activity. Therefore, these conditions (time, temperature, pH, and medium) were used for preparing and evaluating S. lactis and

TAnLE 2. Activitya of various culture preparations after incubation of indieated cell numbers for 4 h in steamed nonfat milk (NFM). Number of cells/100 ml (109) C1 C10 ML8 E8 Conventionalb Neutralized° Concentrated Lyophilized rehydrated concentratee

C1

Percent lactic acid in medium 30 C 37 C C10 ML8 E8 C1 C10 ML8

E8

3.4 3.6 4.5

2.9 2.5 2.2

3.1 1.8 1.6

2.0 5.5 2.6

.49 .59 .55

.44 .34 .38

.41 .40 .40

.34 .39 .45

.59 .70 .63

.52 .42 .45

.52 .52 .52

.34 .49 .48

3.0

2.5

1.5

4.4

.57

.42

.38

.42

.65

.47

.49

.49

" Expressed as ~ lactie acid determined by titrating a 9-ml sample to pH 8.3 with .1 N alkali. b NFM culture grown 15 h at 22 C. c pH maintained at 6.3 with 30% sodium carbonate during growth for 17 h at 22 C. d The 17-h neutralized culture was adjusted to pH 6.9 with 30% sodium carbonate, and then sodium citrate was added to 4.5%; cells from 750 ml were harvested by centrffugation and suspended to 30 ml volume in GCGS (1:1 dilution); 1.0 ml of a 1:100 dilution was used to inoculate 100 rnl of steamed NFM tempered to 30 or 37 C. e An aliquot (1.0 ml) of the cell concentrate described in footnote 'd' was lyophilized and rehydrated to 10.0 ml rehydration medium (RM); .1 ml was then used to inoculate 100 ml of steamed NFM tempered to 30 or 37 C. JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 2

168

SPECKMAN E T AL

TABLg 3. Number of cells at different steps in the process of making a starter culture concentrate of S. cremoris CI.

Vohnne (ml)

Step Milk culture at 17 h After citrate addition Supernatant Suspended cells ~ Frozen and thawed concentrate Rehydratedb

Counts (cfu X 109/ml)

Total counts (cfu × 101~)

Cells remaining (~)

8.8 4.3 .4 125 135 125

66 45 4 37 40 37

100 68 6 50 61 56

750 1,050 1,040 30 30 30

Suspended in GCGS medium. b Rehydrated in rehydration medium (RM). S. cremoris culture concentrates.

Culture populations during cell concentration are in Table 3. Some loss of cells occurred after citrate addition, but thereafter little loss occurred. The frozen concentrate represented approximately a 100-fold concentration of cells compared to conventional 15-h bulk culture. Lyophilization further reduced the weight of a 30-ml concentrate preparation from 32 to approximately 5 g. Storage. Storage stabilitT of the lyophilized concentrates is summarized in Table 4. Loss of vacuum during storage permitted extensive cell death regardless of storage temperature. Temperatures (9_,2 C and --22 C) in storage tests were chosen for practicability, i.e. room temperature or deep freeze storage. Stability in activity of cells stored at the two different temperatures was approximately the same provided the organisms were reconstituted with the RM. This was not in earlier work (7) when rehydration was accomplished by use of sterile .1% NFM and there was decreased cell activity indicating increasing injury with storage. Activity of strains sensitive to lyophilization as concentrates (strain ML1) was not im-

proved by an equal number of heat-killed cells which were highly active as freeze-dried concentrates. The RM significantly increased culture activity. This rehydration effect became more pronounced with time during culture storage (Table 5) indicating that a suitable rehydration environment was required to allow the ceils to repair and maintain maximum activity. Only S. lactis ML8 did not require RM to maintain maximum activity during storage, though activity maintained was not as great as S. lactis C10. Rehydration with RM, therefore, made it possible to maintain lyophflized cultures for 3 mo with minimum loss of activity. Duration of rehydration depended on complete dispersion of the dry concentrate, and, normally, rehydration was accomplished within 15 to 25 min. Longer rehydration periods, i.e. 1 h, did not affect culture activity. Concentrations of the cells higher than detailed in Methods and Materials produced a matrix that was difficult to rehydrate. Cheddar cheese simulated activity tests for lyophilized culture concentrates are compared

TAnLE 4. pH values obtained when lyophilized lactic streptococcal starter concentrates were tested by the Cheddar cheese activity test. Culture S. S. S. S. S. S. S.

lactis C2 lactis ML8 lactis C10 cremoris EB2 cremoris EB9 cremoris C1 cremoris E8

pH after Cheddar cheese activity test :' i mob 2 mo 3 mo --22 C q- 22 C -22 C +22 C --22 C q- 9.2 C 5.6 5.5 5.3 5.0 5.6 5.0 5.8

5.6 5.6 5.3 5.6 5.6 5.0 5.8

5.7 5.7 5.3 5.7 5.6 5.0 5.8

5.7 5.7 5.3 5.7 5.7 5.0 5.8

5.7 5.7 5.4 5.7 5.7 5.1 5.8

5.8 5.8 5.4 5.7 5.7 5.1 5.8

pH value of 5.4 or lower insured sufficient activity for Cheddar cheese manufacture by direct vat milk inoculation within 5.5 h. bActivity after 1 mo of storage was the same for all culhlres as found immediately after concentrate preparation. .JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 2

169

LYOPHILIZED STARTER

TABLE 5. Effect of different rehydration media (RM) on the activity~ of lyophilized lactic streptococci stored 1 to 4 me at 22 C.

Culture S. S. S. S. S. S. S. S. S.

lactis C10 cremoris EB2 cremoris E8 lactis ML8 lactis C2 lactis C2 q- S. lactis C2 q- S. lactis C2 + S. lactis C2 q- S.

Storage time ( me ) 1 1 1 2 3 1 3 3 4

cremor/s E8 cremoris E8 cremoris EB1 cremoris ML1b

Water

Final pH 10% nonfat miJk

Rehydration medium

5.5 5.7 6.0 5.7 5.7 6.0 6.3 6.2 6.5

5.4 5.6 5.9 5.7 5.7 5.9 6.0 6.1 6.3

5.3 5.6 5.8 5.7 5.6 5.9 5.9 5.9 6.0

Cheddar cheese activity test as described in Methods. b Strain ML1 alone was unsatisfactory in activity performance even in RM (pH ~ 6.5) after storage for 1 wk at --22 C. to conventional cultures (Table 6). A difference of .1 pH unit represented approximately 10 min of cheese making time (33), and cultures, especially S. cremoris C1 and C10, appeared satisfactory as dry concentrates. Evaluation of cultures for cottage cheese was under long set conditions (23 C) rather than the short set method because a smaller amount (2%) of inoculum was required. The large amount of bulk culture (5% v / v ) required for short set cottage cheese was sufficient to lower the pH of the milk .5 pH unit upon inoculation. The lyophilized starter concentrate was at a neutral pH and did not give an initial pH drop; otherwise, a false indication of an apparent lag in pH would be given, making accurate comparison difficult. With the possible exception of S. lactiz' strains C2 and MLS, activities of the two culture types compared favorably. Typical results (Tables 8, 9, and 10) of cheese-making trials designed to compare lyophilized concentrated cultures with convenTABLE 6. Comparison of the activity of conventional and lyophilized, concentrated lactic streptococcal cultures as determined by final pH achieved after the Cheddar cheese activity test.

Culture S. cremoris C1 S. Iactis C10 S. lactis ML8

S. /act/s C2 S. cremoris EB2 S. crewmris EB9 S. cremoris E8

Final pH Conventional Lyophilized 15-h culture concentrate 5.0 5.3 5.3 5.5 5.5 5.7 5.7

5.0 5.3 5.5 5.6 5.6 5.6 5.8

tional 15-h bulk type cultures are in Tables 8 to 10. The conventional culture and lyophilized concentrate had almost the same activity in the vat for cottage cheese (Table 8). For Cheddar cheese (Table 9) both vats had similar increases in acidity at each phase. No difference was noted between activity of the lyophilized and conventional bulk cultures. The same was noted for Colby type cheese (Table ]0). Discussion

Frozen concentrates of lactic streptococcal starter bacteria available for use in dairy fermentations are grown in nonmilk media to facilitate removal of cells by eentrifugation. Since milk-grown cells likely would be better suited to initiate milk fermentations with a minimum lag, our study was confined to cells harvested from milk. In this regard, Cowman TABLE 7. Comparison of the activity of conventional and lyophilized concentrated lactic streptococcal cultures as determined by the final pH achieved after the cottage cheese activity test. Final pH Conventional Lyophilized 15-h culture concentrate

Culture S. cremoris C1 S. lactis C10

S. eremor/v EB2 cremoris EB9 cremoris E8 lactis C2 lactis ML8 lactis C2 & S. cremoris E8

S. S. S. S. S.

4.4 4.7 4.7 4.7 4.7 4.7 4.8

4.4 4.7 4.7 4.8 4.8 4.9 5.1

5.0

5.1

JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 2

170

SPECKMAN ET AL

TABLE 8. Comparison of titratable acidities found during manufacture of cottage cheese from milk inoculated with a conventional or lyophilized concentrated culture of S. lactis C10.

Step Addition of 15 starter Ripening Ripening Cutting"

Time (h)

Conventional 15-h oflture Temp Acidity

0 4 5 13

23 26 24 23

Lyophilized concentrate Temp Acidity

.18 .23 .28 .58

23 23 23 23

.18 .20 .24 .55

" After cutting, both vats were cooked out to 52 C and normal cheese resulted in both cases. et al. (10, 11, 12, 13, 14) described a membrane and an intracellular proteinase from lactic streptococci whose activities were required to provide cells growing in milk with organic nitrogen. Cells grown in nonmilk media lacking casein may have impaired ability to initiate growth rapidly and to produce acid in milk, especially if they are injured during aging (11). Rogers (38) in 1914 observed the marked increase in stability of lyophilized cultures stored under vacuum; we confirmed this. The cause for the deleterious effect of air is not known. Webb (46) suggested that oxygen inactivates a membrane-bound system necessary to repair injury occurring during lyophilization. Lion and Bergmann (28), Lion et al. (29), Dimmick and Heckly (15), and Heckly et al. (22) have shown a correlation between free radical formation in lyophilized bacteria, measured by electron paramagnetic resonance, and viability. In this study, strain selection was critical because some strains within a species lacked the ability to withstand the stress of freezing and lyophilization. This could cause a strain balance shift in dry concentrates of mixed cultures such as that described by Gilliland (19) in frozen cultures. Therefore, to collect different lyophilized concentrates for routine use, we needed to screen a large

number of strains to find those that could withstand freezing and lyophilization. Gibson et al. (18) have shown in studies of frozen storage of the lactic streptococci, considerable variation among strains of the same species. Bergere (4) found S. crenurris more sensitive to lactate, and Stadhouders et al. (43) found that coneentrates of mixed cultures containing mostly S. cremoris achieved lower final populations than those reached by S. lactis C10. Attempts to understand differences in susceptibility among strains, species, or genera of bacteria are further complicated by an inadequate understanding of the mechanisms involved in loss of viability during lyophilization and storage in the dried state. Differences in the susceptibility of the cell wall to stress or of key enzymes to denaturation may account for differences among different organisms. The GCGS medium afforded maximum protection of cells during freezing, lyophilization, and storage compared to other suspending media. Maintenance of vacuum in the vials was the most critical factor during storage. With no vacuum, the lyophilized concentrate had a minimum of activity. Loss of activity was due mainly to cell death during storage as evidenced by low plate counts and failure of the culture to respond to different resuspension media. The small decreases in activity

TABLE 9. Comparison of titratable acidities during manufacture of Cheddar cheese from milk inoculated with a conventional or lyophilized concentrated culture of S. lactis C10. Step Starter addition Rennet addition Cutting Start cooking End cooking Start draining Milling Salting pH at 60 days

Conventional Culture Time Acidity

Lyophilized Concentrate Time Acidity

0 1:15 1:55 2:10 2:40 3:30 6:00 6:10 ...

0 1:15 1:55 2:05 2:40 3:25 5:15 5:20

JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 2

.18 .19 .11 ... ~17 .45

.17 .17 .12 ... ".i7 .46

LYOPHILIZED STARTER

TABLE 10. Comparison of titratable acidities during manufacture of Colby type cheese from milk inoculated with a conventional or lyophflized concentrated mixed strain starter culture".

Step

Time

Starter addition Rennet addition Cutting Start cooking End cooking Start draining pH off press

0 1: 00 1:35 2:00 2: 30 2:45 ...

Acidity LyophiConvenlized tional concenculture trate .175 .185 .105 .125 ...... . 5.05

.170 .180 .105 .125 . 5.t);

Commercially available mJ,xed strain starter culture grown and made into concentrate; final pH in the Cheddar activity test was 5.2. with storage of the culture may in reality be due to slight vacuum losses in the vials. The stress of freezing and drying bacterial cells causes injury, and, therefore, rehydration becomes critical in achieving maximum cellular activity. Rehydration conditions should decrease osmotic shock and also provide an environment for repair of cellular damage (8). According to Ray et al. (36, 37) cell viability and lag of cell growth in Salmonella after rehydration was related to composition of rehydration media. Cowman et al. (10) reported probable freezing injury to the membrane proteinase system of S. lactis which prevented cells from obtaining sufficient growth peptides after thawing. Realizing that osmotic shock may cause further injury to cells upon rehydration (42), a rehydration medium minimizing cell stress was sought in the present work. The lactose-stimilac containing medium provided such an environment and enabled cells to begin optimum activity upon inoculation in the vat. About 2% water was ha the dry concentrates prepared as described. Studies by Bannikova et al. (3) indicated that residual moisture in lyophilized bacteria should neither be too low or too high if maximum stability is to be expected. More studies on this factor, where lyophilized lactic starter cultures are concerued, are warranted. Comparison of culture concentrates in simulated cheese making conditions permitted realistic evaluation of different cultures. Under these criteria the lyophilized concentrates compared favorably with the conventional starters. The lyophilized concentrates maintained high activity without special storage conditions

171

and were ready for inoculation within half an hour after rehydration. The economics of manufacture of such cultures on a commercial scale was not considered. Lyophilization would be a major expense item as well as ingredients of the eryoprotective and rehydration media. The present successful use of frozen concentrates by the dairy fermentation industry might prevent use of dry cell concentrates in the immediate future, especially until an economic study is conducted. Nevertheless, lyopbilized lactic acid starter culture concentrates can be expected to perform well under industrial conditions. More strains able to perform well under these conditions will be needed to allow eul~re rotation. Lyophilized concentrates should be able to replace the more conventional culture and eliminate many of the problems of culture handling. The methods described herein have been applied successfully to cultures for yogurt, Swiss and Italian cheese, and the ffndings will be reported in another paper.

Acknowledgments This research was supported in part by the Dairy and Food Industries Supply Association and by training grant 5 TOl GM007O4-1O from the National Institute for General Medical Sciences. The authors also express appreciation to Microlffe Technics of Sarasota, Florida, and Dr. L. L. McKay and Dr. E. A. Zotolla of the University of Minnesota for collaborative evaluation of the lyophilized, concentrated starters. References (1) Accolas, J. P., and J. Auclair. 1967. Storage of highly concentrated suspensions of lactic acid bacteria in the frozen state. Le Lait 47:253. (2) Bannikova, L., and I. V. Lagoda. 1970. Production of dried starter cultures from bacterial concentrate. VIII Int. Dairy Congr. IE:277. (3) Bannikova, L., I. Pyatnitsyna, and L. Kazantseua. 1964. Dried cultures from bacterial concentrate. MoL Prom. 25:39. (4) Bergere, J. R. 1968. Mass production of cells of lactic streptococci. I. General methods of study and factors influencing the growth of Streptococcus lactis C10. Le Lait 48:1. (5) Bergere, ~. R. 1968, Mass prodnction of cells of lactic streptococci. III. Production of different strains during culture at a constant pH. Le Lair 48:131. (6) Bergere, J. R., and J. Hermier. 1968. Mass production of cells of lactic streptococci. II. Growth of Streptococcue lactis in meJOURNAL OF DAIRY SCIENCE VOL. 57, NO. 2

172

SPECKMAN ET AL

dium at constant pH. Le Lair 48:13. (7) Blaine, J. W. 1972. Preparation and preservation of lactie acid starter eulture concentrates. Ph.D. Thesis. Oregon State University, Corvallis. (8) Choatc, R. V., and M. T. Alexander. 1967. The effect of the rehydration temperature and rehydration medium on the viability of freeze-dried Spirillum atlanticum. Cryobiology 3:419. (9) Christensen, W. V. 1971. Production of cell culture concentrates. U. S. Pat. No. 3,592,740. 10) Cowman, R. A., and M. L. Speck. 1967. Low temperature as an environmental stress on microbial enzymes. Cryobiology 6:291. 11 ) Cownmn, R. A., and M. L. :Speck. 1967. Proteinase enzyme system of lactic streptococci. I. Isolation and partial characterization. Appl. Microbiol. 15:851. 12 ) Cowman, R. A., and H. E. Swaisgood. 1966. Temperature dependent association-dissociation of Streptococcus lactus intracellular proteinase. Biochem. Biophys. Res. Commun. 23:799. 13) Cowman, R. A., D. C. Westhoff, H. E, Swaisgood, and M. L. Speck. 1970. Proteinase system of lactic streptococci. IV. Relationship between proteinase activity and growth at 32 C. J. Dairy Sci. 53:126. (14) Cowman, R. A., S. Yoshimura, and H. E. Swaisgood. 1968. Proteinase enzyme system of lactic streptococci. III. Substrate specificity of Streptococcus lactis intracellular proteinase. J. Bacteriol. 95:181. (15) Dimmiek, R. L., and R. J. Heckly. 1961. Free radical formation during storage of freezedried Serratia marcescens. Nature 192: 776. (16) Elliker, P. R., A. W. Anderson, and G. Hannesson. 1956. An agar culture medium for lactic acid streptococci and lactobacilli. J. Dairy Sei. 39:1611. (17) Farr, S. M. 1969. Milk fermenting product and method of making same. U. S. Pat. No. 3,420,742. (18) Gibson, C. A., G. B. Landerkin, and P. M. Morse. 1965. Survival of strains of lactic streptococci during frozen storage. J. Dairy Res. 32:151. (19) Gilliland, S. E. 1971. Strain balance of multiple strain lactic streptococcus coneentrated cultures. J. Dairy Sei. 54:1129. (20) Gilliland, S. E., E. D. Anna, and M. L. Speck. 1970. Concentrated cultures of Leuconostoc citrovorum. Appl. Microbiol. 19:890. (21) Heckly, R. J. 1961. Preservation of bacteria by lyophilization. Adv. Appl. Microbiol. 3:1. (22) Heckly, R. J., R. L. Dimmick, and J. j. j. Windle. 1963. Free radical formation and survival of lyophilized microorganisms. J. Bacteriol. 85:961. (23) Keen, A. R. 1972. Growth studies on the lactic streptococci. I. A laboratory apJOURNAL OF DAIRY SCIENCE VOL. 57, NO. 2

(24)

(25)

(26) (27) (28)

(29)

(30 (31

(32)

33) 34)

35)

(36)

(37) (38) (39)

(40)

paratus suitable for fermentation studies using skim-milk medium. J. Dairy Res. 39:133. Keen, A. R. 1972. Growth studies on the lactic streptococci. II. The effect of agitation of the growth characteristics of Streptococcus lactis ML~ in batch culture. J. Dairy Res. 39:141. Keen, A. R. 1972. Growth studies on the lactic streptococci. III. Observations on continuous growth behavior in reconstituted skim-milk. J. Dairy Re*. 39:151. Keen, A. R. 1972. Growth studies on the lactic streptococci. IV. Some observations on redox potentials. J. Dairy Res. 39:161. Lagoda, I. V., and L. A. Bannikova. 1970. Some factors affecting quality and storage life of dried starters. Mol. Prom. 31:11. Lion, M. B., and E. D. Bergmann. 1961. Substances which protect lyophilized E~cherichia coli against the lethal effect of oxygen. J. Gen. Microbiol. 25:291. Lion, M. B., J. S. Kirby-Smith, and M. L. Randolph. 1961. Electron spin resonance signals from lyophilized bacterial cells exposed to oxygen. Nature 192:34. Lloyd, G. T. 1971. New developments in starter technology. Dairy Sci. Abstr. 33:411. Lloyd, G. T., and E. G. Pont. 1973. An experimental continuous culture unit for the production of frozen concentrated cheese starters. J. Dairy Res. 40:149. Lloyd, G. T., and E. G. Pont. 1973. Some properties of frozen concentrated starters produced by continuous culture. J. Dairy Res. 40:157. Pearce, L. E. 1969. Activity test for cheese starter cultures. N. Z. J. Dairy Technol. 4:246. Peebles, M. M., S. E. Gilliland, and M. L. Speck. 1969. Preparation of concentrated lactie streptococcus starters. Appl. Microbiol. 17:805. Pont, E. G., and Gwenda L. Holloway. 1968. A new approach to the production of cheese starter. Australian J. Dairy Teeh. 23:22. Ray, B., J. J. Jezeski, and F. F. Busta. 1971. Effect of rehydration on recovery, repair and growth of injured freeze-dried Salmonella anatum. Appl. Microbiol. 22:184. Ray, B., J. J. Jezeski, and F. F. Busta. 1971. Repair of injury in freeze-dried Salmonella anatum. App1. Microbiol. 22:401. Rogers, L. A. 1914. The preparation of dried cultures. J. Infect. Diss. 14:100. Rousseaux, P., L. Vessal, E. Valles, J. Auclair and G. Mocquot. 1968. The use of concentrated frozen suspensions of thermophilie lactic acid bacteria in making Gruyere cheese. Le Lair 48:241. Sandine, W. E., P. R. Elliker, and H. Hays. 1962. Cultural studies on Streptococcus

LYOPHILIZED STARTER

diacetilactis and other members of the lactic streptococcus gxoup. Can. J. Mierobiol. 8:161. (41) Sherman, J. K. 1967. Freeze thaw induced latent injury as a phenomenon in Cryobiology. Cryobiology 3:407. (42) Sherman, J. K., and K. S. Kim. 1967. Correlation of cellular ultrastructure before freezing, while frozen and after thawing in assessing freeze thaw induced injury. Cryobiology 4:61. (43) Stadhouders, J., G. Hup, and L. A. Jansen. 1971. A study of the optimum conditions

173

of freezing and storing concentrated mesophilie starter. Neth. Milk Dairy J. 25:229. (44) Stadhouders, J., L. A. Jansen, and G. Hup. 1969. Preservation of starters and mass production of starter bacteria. Neth. Milk DailT J. 23:182. (45) Van Slyke, L. L., and W. V. Price. 1949. Cheese. Orange Judd Publishing Co., New York. (46) Webb, S. J. 1969. The effect of oxygen on the possible repair of dehydration damage by Escherichia coll. ]. Cert. Microbiol. 58: 317.

JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 2