Scandinavian Ropy Milk — Identification and Characterization of Endogenous Ropy Lactic Streptococci and Their Extracellular Excretion1

Scandinavian Ropy Milk - Identification and Characterization of Endogenous Ropy Lactic Streptococci and Their Extracellular Excretion 1 D R A G A N M A C U R A and P. M. T O W N S L E Y University of British Columbia Department of Food Science Vancouver V6T 2A2 ABSTRACT

Four bacterial strains isolated from Swedish ropy edible milk were subjected to tests for differentiating group N streptococci. One of the cultures was a variant of Streptococcus lactis, previously named Streptococcus lactis longi, and the other three were variants of Streptococcus cremoris with the suggested name Strep-

tococcus cremoris longi. Viscosity of dairy products produced with Streptococcus cremoris longi was unstable. At optimum growth temperatures stability depended upon the number of serial transfers and on the length of culture incubation. The viscous material produced by Streptococcus cremoris longi was most likely a glycoprotein. It consisted of 47% total protein, 20% methyl pentoses, 9.3% protein-bound hexose-like material, and a 2.8% sialic acid. A substantial portion of the molecule remains to be identified. INTRODUCTION

Cultured milk products always have been an important part of the human diet. They often are credited with variable therapeutic or prophylactic value (1, 3, 7, 17, 18, 19, 21, 22, 30). Cultured milk is easier to digest than fresh milk (3, 22). Many ethnic and research groups recommend cultured milk products for treating intestinal disorders (1, 15, 22, 30). Microflora of cultured milks may vary according to the area of origin and method of preparation. However, common characteristics of all bacteria in cultured milk products are that they sour milk and usually produce various

Received February 22, 1983. ~Authors acknowledge the Canadian Dairy Commission for financial support of this project. 1984 J Dairy Sci 67~735-744

amounts of flavoring and antibiotic substances. Bacteria in cultured milk production usually belong to the lactic bacilli or cocci, some of which are members of intestinal microflora (1, 4, 6, 30). In addition to bacteria, yeasts are frequently in some of these products. Yeasts usually contribute to effervescence, flavor, and hydrolysis of milk sugar. Well-known examples of cultured milk products are: yogurt, biogarde, kefir, buttermilk, cultured sour cream, and koumiss. Less well-known in North America is Scandinavian ropy sour milk. This low temperature fermentation has a consistency that resembles a stiff, extremely viscous dough (25), whose final thickness depends on the culture strain and milk fat content of the milk. It keeps longer than many fermented milks cultivated under the same conditions (23). Its characteristic ropy consistency is caused by slime that is excreted into the milk during the exponential growth phase of the microflora. This slime acts like a food stabilizer, preventing syneresis and graininess and providing a product with natural thickness. The composition of the slime was analyzed by Sundman (24) and by Nilsson and Nilsson (13). Both found that it contained a protein fraction, but they disagreed on its amino acid composition. This type of ropy milk is differentiated from problem ropy milk caused by other than lactic streptococci, as described by Olsen-Sopp (12) and Thomas et al. (26, 27). In spite of favorable characteristics, bacteria from Scandinavian ropy milk are not used extensively in the dairy industry, perhaps because of culture instability. Wolferstetter (32) stated that ropy milk was easy to sell in Sweden but that it was difficult to produce commercially. The difficulty most likely stemmed from a gradual loss of the typical ropy character as the culture lost its ability to produce slime. He referred to a critical balance of 60 to 70% ropy cells to 30 to 40% n o n r o p y

735

736

MACURA AND TOWNSLEY

cells in a good product. This culture ratio was difficult to maintain, especially at high growth temperature, i.e., 27°C. Wolferstetter recommended a growth temperature of 18 to 20°C, to sustain better constant slime production in the product. Microflora of Scandinavian ropy milk have been a major subject of debate within the limited literature. In 1899, Troili-Petersson (28) first studied microorganisms of Swedish ropy milk, "langmjolk". She isolated the slime-producing bacterium, which she named Bacterium lacticus longi because it closely resembled Bacterium lacticus acidi except for its ability to produce slime. Olsen-Sopp (14) studied the microorganisms in a Finnish ropy milk "taettemelk" and found that good taette was produced by a symbiotic relationship of certain Streptobacillus and Streptococcus with a yeast of genus Saccharomyces. When grown in pure culture separately, none of these microorganisms produced good taette; however, when they were grown together, a desirable milk product was obtained. Macy (10) isolated a slime-producing strain of Streptococcus, which closely resembled S. lactis var. hollandicus and S. taette, from Finnish "filli" or "piima". He described in great detail the morphology, cultural characteristics, and physiology of these bacteria and suggested that they be a new species, Streptococcus piima. Subsequent study by Nilsson (11) investigated the microflora of Swedish tatmjolk and reported that the slime-producing bacterium was identical to S. lactis except for the ability to produce slime. These researchers named it S. lactis longi. Further study of ropy bacteria by Sundman (23) revealed a ropy S. lactis variant in Swedish ropy milk, but she concluded that the ropy variant of S. cremoris was the predominanat microorganism in this product. Most recent extensive study of the microflora of Scandinavian ropy milk was by Forsen (5). In this investigation of Finnish "long milk", the slime was produced by the variants of S. cremoris, S. lactis, S. diacetylactis, and Leuconostoc sp. Streptococcus cremoris and S. lactis variants were most predominant and in equal proportions, followed by S. diacetylactis and

Leuconostoc sp. It appears that there are problems that need to be investigated before ropy cultures from Scandinavian ropy milk can be reliable cornJournal of Dairy Science Vol. 67, No. 4, 1984

mercial stocks. First, the ropy strains should be identified for better control of the stocks. Second, the slime-producing ability should be stabilized so that constant ropiness can be produced. And finally, it should be substantiated whether the slime produced by these bacteria is a glycoprotein or a polysaccharide. This is important for evaluation of functional properties and nutritional value of the final product. In this communication we report on the identification of pure strains of ropy streptococci isolated from the Scandinavian ropy milk, on their growth characteristics in skim milk, on the stability of slime production, and on a tentative identification of slime components.

MATERIALS AND METHODS Source of Cultures and Culture Maintenance

Ropy strains 701, 705, and LLF were supplied by the F o o d Research Institute, Ottawa. Ropy L415 was obtained from Central Laboratory of the Swedish Dairies Association, Malmo, Sweden. From this strain a more stable slime-producing strain was selected and called L416. Streptococcus lactis ATCC 19435 and Streptococcus cremoris ATCC 19257 were supplied by the American Type Culture Collection. All cultures were stored in liquid nitrogen or in a deep-freeze at - 6 5 ° C . Working culture volumes usually were developed from an isolate from a single ropy colony on t o m a t o or skim milk agar plates. Culture identification

Action in litmus milk was tested in Difco dehydrated litmus milk (B 107) prepared as per manufacturer's instruction. Tubes were incubated in triplicate at 18°C for 701,705, and L416 and at 22°C for LLF. Standards were incubated at 30°C. All readings were at 48 h. Ammonia production from arginine was tested in arginine broth and on bromocresol purple agar (BCPA) plates. Arginine broth was prepared as per Harrigan and McCance (8). Nessler's reagent was an indicator of ammonia production after 48 h of incubation at above indicated growth temperatures. Bromocresol purple agar was prepared per Reddy et al. (16). Results were recorded after 1 wk of incubating

ROPY LACTIC STREPTOCOCCI the plates at the growth temperatures listed. All samples were in triplicate. Growth at pH 9.2 was tested in casein digest medium (5) whose pH was adjusted to 9.2 with 1 N NaOH followed by sterilization and subsequent pH readjustment. Triplicate 5-ml samples of the medium were dispensed into culture tubes, loop inoculated from 24 h skim milk cultures, and then incubated at their respective growth temperatures. Casein digest medium at pH 6.8 was a control. Positive growth was recorded if absorbance at 520 nm increased by more than .27 units. This value was chosen arbitrarily, as it corresponded to visual detection of bacterial growth. Growth at 40°C was tested in skim milk. Cultures were grown in triplicate in pasteurized skim milk at 5% (vol/vol) of 24 h skim milk inoculum for 24 h at 40°C. Acid clot formation was used as an indication of growth. Growth in 4% NaCl was tested in casein digest medium (5) to which 4% NaC1 (wt/vol) was added. Five milliliters of this preparation in culture tubes was loop inoculated in triplicate from 24-h cultures grown in the same medium without NaCI and incubated at the respective growth temperatures of the test organisms for 48 h. Positive growth was recorded if absorbance at 520 nm increased more than .27 units. Casein digest medium with no added NaCl was used as a control. Gas production from citrate was tested in semi-solid citrate milk agar that was prepared and inoculated with test organisms according to the method of Crawford as reported by Harrigan and McCance (8). Tubes were incubated in triplicate at the respective growth temperatures for 48 h. After 4 days they were inspected for slits and fissures in the curd as indication of gas production from citrate. Growth Curve of

Streptococcus cremoris longi L416 Strain L416 was used to demonstrate the type of the growth curve most often observed when ropy bacteria are grown in skim milk. This culture was grown in 95 ml autoclaved (8 min at 121°C) commercial skim milk at 18°C. Measurements of viscosity, colony forming units, and pH were recorded in separate duplicate flasks at designated time intervals (see Figure 1).

30

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Figure 1. Growth curve of Streptococcus cremoris longi L416 in skim milk at 18°C. vpH (average of two readings), • apparent viscosity at constant shear rate of 500 per s. (average of four readings), • log of colony forming units (CFU)/ml (average of the counts on eight TJA plates).

Viscometric analysis was recorded with a coaxial rotovisco viscometer, Brabender Rheotron, with torsion dinamometer spring A and coaxial attachment A1 or C4. Duplicate readings of each sample were taken at an equalibrated shear stress at a constant shear rate of 500/s, and average apparent viscosity readings were calculated. Total plate count was on tomato juice agar plates. An appropriate dilution of each duplicate sample was surface spread on four TJA plates. Plates were incubated at 18°C. Colonies were counted after 4 days of incubation and the average number of colony forming units was determined. Acid development was monitored by measuring the pH of duplicate samples with a Fisher Accumet model 230 pH meter. Stability of Slime Production

Slime stability in ropy cultures was studied in skim milk. Ropy colonies of cultures 705, 701, L416, and LLF were propagated in autoclaved skim milk at their respective growth temperatures (18°C for 705, 701, and L416 and 22°C for LLF). After 24 h of incubation they were transferred to fresh milk at a 5% (vol/vol) inoculum and incubated further. The subsequent 10 serial transfers were after every 48 h of incubation. After each transfer, appropriate sterile dilutions were made, and the bacteria were surface plated on 10 skim milk Journal of Dairy Science Vol. 67, No. 4, 1984

738

MACURA AND TOWNSLEY

agar plates. Plates were incubated at 18~C for cultures 705, 701, and L416 and at 22°C for LLF. They were counted after 4 days of incubation or when the colonies could be visually evaluated. During counting, each colony was touched with a straight wire and recorded as ropy or nonropy. For further slime stability studies the same materials were used. This experiment was done in the same manner as described except: 1) only culture LLF was used, 2) all serial transfers were at exactly 50 h of incubation, 3) each plate was counted at exactly 48, 96, and 144 h of incubation and recorded as 2, 4, and 6 day growths, respectively.

Slime Composition

Production and purification of slime was according to the following procedure. Fresh cottage cheese whey, obtained from a local dairy, was used as a substrate. Its pH was adjusted to between 6.8 and 7.0 with 2N NaOH. The precipitate thus formed was separated by centrifuging at 13,200 x g for 10 min. The whey then was deproteinized with an ultrafiltrate unit and a Millipore membrane with an exclusion molecular weight of 10,000. The retentate was discarded, and the filtrate was filter-sterilized with a sterile Millipore filter of .45 /2m pore size. The filtrate was then inoculated at 5% (vol/vol) with a S. cremoris longi culture (L416) previously grown in the same medium for 24 h. The inoculum was developed from a ropy colony that was transferred from a milk agar plate to 5 ml of deproteinized sterile whey and further propagated at 5% inoculum. The inoculated bulk culture was incubated at 18°C, without shaking, for 24 h, then transferred to a cold room at 4 to 6°C for additional 24 h. The fermented whey changed from a clear light green solution to an opalescent, green, heavy egg while-like slime. When swirled in a flask or poured into a different container, it behaved like a heavy, viscous mucus, resisting flow at first and then "falling" out of the flask. The viscous mucous was centrifuged at 13,200 x g for 30 min to separate cells. To the supernatant fluid, an equal volume of 95% ethanol was added slowly with constant stirring. The slime precipitated as a stringly flocculant precipitate that was Journal of Dairy Science Vol. 67, No. 4, 1984

collected by either centrifuging at 13,200 × g for 5 min or by filtration. Precipitated slime then was dissolved in distilled water by stirring overnight at 5°C. An equal volume of cold 25% trichloracetic acid (TCA) was added to the water solubilized viscous sample. The sample was shaken vigorously by hand and left to stand for 15 rain. It then was centrifuged at 13,200 x g for 30 min and filtered with .45/am Millipore filter to separate the TCA-precipitated impurities and any remaining cells. The TCA was separated from the filtrate by mixing with ethyl ether. The filtrate containing TCA was mixed with an equal volume of ethyl ether and shaken vigorously by hand. The ether layer ~vas discarded and the same procedure repeated. The residual ether then was removed from aqueous filtrate by purging with nitrogen gas. The water soluble samjale was dialyzed against running tap water at 5vC for 48 h and freeze-dried. This material was subjected to the adopted tests for serum glycoproteins, amino acid analysis, and total protein content. Total protein content of the slime was determined by the method of Lowry et al. (9). Crystalline (4 x crystallized) bovine serum albumin (BSA) was used to construct the standard curve. The test sample was run in quadruplicate and results averaged. Amino acid analysis of the freeze-dried slime was by the methanesulfonic acid method (20). Cysteine was not analyzed. The single column system (Duran Chem. Corp., Palo Alto, CA) attached to a Phoenix model M6800 amino acid analyzer (Phoenix Precision Instrument Co., Philadelphia, PA) was used for amino acid analyses. Quantitative and qualitative amounts of each amino acid were determined with the aid of a Monroe programmable calculator. Summation of the quantitative amounts of each amino acid was a more reliable determination of the total protein content of the slime sample. Protein-bound hexoses was determined by the orcinol-sulfuric acid method (31) with an equal mixture of D-galactose and D-mannose for a standard. Samples were treated in duplicate and results averaged. An adaptation of the Dische and Shettles method as reported by Winzler (31) was used to determine the total methylpentose in the dry slime sample. Rhamnose was a methylpentose standard. The experimental sample was in duplicate, and results were averaged.

ROPY LACTIC STREPTOCOCCI H e x o s a m i n e in the dried slime was estimated by the m e t h o d of Morgan and Elson as described by Winzler (31) with glucosamine h y d r o c h l o r i d e as a standard. Five replicates of the experimental sample were analyzed and results averaged. Sialic acid in the dried slime sample was measured by thiobarbituric acid assay (29) with N-acetylneuraminic acid as a standard. T w o replicates o f the slime sample were run simultaneously and results averaged.

73 9

pH 9.2 whereas S. cremnoris does n o t (8). A m m o n i a p r o d u c t i o n from arginine was tested in two media arginine broth and BCPA. In the f o r m e r m e d i u m , d e t e c t i o n o f alkaline reaction was with Nessler's reagent; BCPA detects p H change• The BCPA is normally purple or dark blue; however, when it comes in c o n t a c t with acid ( p H < 5.2) it turns yellow. Thus, all colonies appear white at first. T h e y all form yellow zones within the first 2 days of growth, and then arginine utilizing bacteria (S. lactis) eliminate the yellow zones, indicating an alkaline reaction. Table 1 shows that of the strains tested, L L F appears to be a variant of S. lac'tis, and strains L416, 701, and 705 appear to be r o p y variants of Streptococcus cremoris. Because the r o p y S. lactis has been n a m e d Streptococcus lactis lougi (11, 28), it seems appropriate to n a m e the ropy S. cremoris variants Streptococcus cremoris longi.

RESULTS A N D DISCUSSION

Identification of the r o p y streptococci was with the tests for differentiating group N streptococci. O f the tests, key differentiating factors were p r o d u c t i o n of a m m o n i a and o t h e r alkaline substances such as amines from arginine and growth at pH 9.2. Streptococcus lactis releases a m m o n i a f r o m arginine and grows at

TABLE 1. Identification of ropy strains isolated from Scandinavian ropy milk (all results are triplicate readings). Biochemical tests Litmus milk Arginine broth BCPA k Citrate agar pH 9.2 4% NaC1 40Oc Identification of ropy strains

LLF

Ropy strains L416 701

705

Controls ATCC 19435 a ATCC 19257 b

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as. lactis standard culture (American Type Culture Collection). bs. cremoris standard culture (American Type Culture Collection). CAcid, curd, reduction. dAmmonia production from arginine. eNo ammonia production from arginine. fAlkaline reaction from arginine. gNo alkaline reaction from arginine. hNo gas production from citrate. iGrowth, as recorded by absorbance >~ .27 units at 520 nm. JNo growth, as recorded by absorbance at 520 nm. kBromoeresol purple agar. 1Deviates from Bergey's Manual of Determinative Bacteriology, 8th ed. Journal of Dairy Science Vol. 67, No. 4, 1984

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MACURA AND TOWNSLEY

In accordance with the commercial potential of these cultures it is useful to known their growth characteristics. Thus, the growth curve of ropy S. crernoris variant L416 (Figure 1) shows that in skim milk most acid production and cell growth occur during the first 16 to 20 h of incubation. Viscosity development increases up to 24 h of cell growth, stays constant for the next 24 h, and then gradually decreases. Rate or viscosity decrease varies with the strain of ropy bacteria (data not shown). Even though Figure 1 shows a small decrease of viscosity after 48 h (72 to 96 h region on the curve), visual observations showed that after 6 days of incubation at room temperature all ropy cultures became less viscous. The ropy nature of the product also was lost. Further investigation of culture instability revealed that the loss of viscosity was a function of time of incubation and number of serial transfers. Table 2 shows that after 10 serial transfers, percent ropy colonies decreased for all strains, regardless of the length of time of plate incubation. This experiment also suggested that for most serial transfers, the percent viscous colonies could be decreased to zero if the plates were incubated long enough at growth temperature• For example, ropy colonies were 100% for LLF at 146 h of plate incubation during transfer 1 whereas only 19% were ropy colonies when the same plates were left at incubation temperature for 265 h. Also, during transfer 5, the proportion of LLF colonies was 41% after 74 h of incubation, but it dropped to 0% when the same plates were incubated for a total of 288 h (Table 2). Thus, the second part of this experiment was designed so that the percent ropy colonies was determined at exactly 2, 4, and 6 days of incubation (Table 3). Each serial transfer was after 50 h of incubation. Ropy S. lactis variant LLF, which was noted for its slime instability (Table 2), lost most of its viscosity producing cells after six serial transfers or within 2 to 4 days of incubation, regardless of how many times the culture was transferred. Similar disappearance of viscosity was observed when the cultures were grown in skim milk, thus suggesting some degrading agent, possibly a glycoprotein hydrolase, that destroyed the slime within a few days of its maximum production. Rate of viscosity loss varied among culture strains, being generally higher for ropy S. lactis variant than for ropy variants of S. Journal of Dairy Science Vol. 67, No. 4, 1984

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aSerial t r a n s f e r i n t o s k i m m i l k e v e r y 4 8 h. b s u r f a c e s p r e a d p l a t e i n c u b a t i o n time• CNot c o u n t e d • dpetri plates incubated at 22°C. e p e t r i p l a t e s i n c u b a t e d at 1 8 ° C . fTotal number of colonies counted. g T o t a l n u m b e r o f r o p y c o l o n i e s in f.

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TABLE 3. Stability of slime production by S. lactis longi LLF in liquid skim milk as the percent of the ropy colony forming units on skim milk agar. Days of incubation at 22°C Transfer number a 0 1 2

Total b 482 1032 542

3

4 5 6 7 8 9 10

2 Ropy c

% Ropy

Total

455 983 441

94 86 81

516 946 550

290 71 90 61 44 42

50" 10 10 10 6 10

621 643 996 590 691 356

4 Ropy 6 0 3

% Ropy

Total

6 Ropy

% Ropy

1 0 1

350 969 520

3 0 0

1 0 0

d

576 691 944 633 688 438

. . . . . . . . . . . . . . . . 0 0 660 0 0 0 677 0 0 0 894 0 0 0 812 0 49 7 832 0 12 3 344 0

0 0 0 0 0 0

aTransfers into fresh skim milk were made at exactly 50-h intervals with a 5% (vol/vol) inoculum. bTotal number of colonies counted. CTotal number of ropy colonies in b. dNot determined.

cremoris. It was considerably slower at refrigeration temperatures, i.e., < 1 0 ° C . Thus, it appears that the decrease of viscosity of the culture on serial transfer may be due to the loss of e x t r a c h r o m o s o m a l d e o x y r i b o n u c l e i c acid, e.g., plasmid material. Plasmids are c o m m o n l y f o u n d within lactic acid bacteria and are responsible for a n u m b e r of cell reactions (2). The loss o f viscosity due to prolonged storage o f the culture at the growth t e m p e r a t u r e m a y be associated with p r o t e o l y t i c activity of these cultures. These points deserve careful investigation as t h e y are the key factors in the p e r m a n e n t solution of culture instability. C o m p o s i t i o n of the viscous material was studied by two research groups (13, 24). Both f o u n d that, even though the slime was difficult to rid of milk proteins, it contained a protein c o m p o n e n t . Our research c o n f i r m e d these findings and showed that in addition to the protein portion of 47%, the slime also contained 9.3% p r o t e i n - b o u n d hexose-like material, 20% m e t h y l pentose, and 2.8% sialic acid. No hexosamines were f o u n d w h e n the m e t h o d of Morgan and Elson, as m o d i f i e d by Winzler (31), was e m p l o y e d . It was n o t the purpose of this research to identify c o m p l e t e l y c o m p o n e n t s of the slime; however, an a t t e m p t was m a d e to answer the question of w h e t h e r this material is Journal of Dairy Science Vol. 67, No. 4, 1984

a glycoprotein or a polysaccharide. A m i n o acid analysis (Table 4) proved that protein is a c o n s t i t u e n t of the slime; however, its amino acid profile appears to be similar to that of dried d e p r o t e i n i z e d w h e y in which the culture was grown. This m a y suggest w h e y protein c o n t a m i n a t i o n of the dry sample. However, w h e y in this e x p e r i m e n t was " d e p r o t e i n i z e d " twice (refer to Materials and Methods), making c o n t a m i n a t i o n unlikely. This m e t h o d was considered to be adequate for isolation of slime p r o d u c e d by r o p y streptococci. Thus, even though a relatively large p o r t i o n of the m o l e c u l e remains to be identified and m o r e specific identification of protein and carbohydrate fractions is needed, f r o m present information, a glycoprotein-like c o m p o s i t i o n of this material is likely.

CONCLUSIONS

Of the four r o p y lactic streptococci, one (LLF) was a variant of S. lactis, previously n a m e d S. lactis longi (11). The o t h e r three strains (L416, 701, 705) were variants of S. cremoris (Table 1). We suggest these variants be named Streptococcus cremoris longi. S o m e degrading agent capable of hydrolyzing a glycoprotein and an unstable genetic factor

ROPY LACTIC STREPTOCOCCI TABLE 4. Amino acid composition of the dry slime produced by S. cremoris longi L416 and dry whey permeate (numbers indicate calculations of one sample). Amino acids

Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe Lys His Trp Arg Cys

Slime

Permeate

(mg/g)a

(%)b

(rag/g)

(%)

34.60 55.30 40.80 138.30 23.60 9.00 19.96 23.90 6.28 32.00 26.80 4.36 11.40 27.50 3.72 0 9.76 . . .c

7.4 11.8 8.7 29.5 5.1 1.0 4.3 5.1 1.4 6.9 5.7 .9 2.4 5.9 .8 0 2.1 . . . .

.96 8.2 .48 4.1 .40 3.4 3.02 25.7 .72 6.1 .22 1.9 .37 3.2 .52 4.4 .21 1.8 .39 3.3 1.42 12.1 .33 2.8 .23 2.0 1.70 14.5 .38 3.2 0 0 .38 3.2 . . . . .

aMilligrams of amino acid per gram of freeze-dried pure sample. bAmino acids composition of pure sample expressed as percent. CNot determined.

induces the loss o f slime. Thus, a decrease o f viscosity paralleled t h e n u m b e r o f culture transfers and length o f culture i n c u b a t i o n at g r o w t h t e m p e r a t u r e s (Tables 2, 3). C o n s t a n t viscosity should be a t t a i n a b l e if t h e s t o c k s are f r e q u e n t l y d e v e l o p e d f r o m r o p y colonies and if t h e p r o d u c t is s t o r e d in t h e cold (i.e., < 10°C) a f t e r m a x i m u m slime p r o d u c t i o n . Analysis o f the slime c o m p o s i t i o n s h o w e d t h a t the slime p r o d u c e d by S. cremoris longi L416 is m o s t likely a g l y c o p r o t e i n . It consists o f 47% p r o t e i n , 20% m e t h y l p e n t o s e , 9.3% p r o t e i n - b o u n d hexose-like material, and 2.8% sialic acid. REFERENCES

1 Amer, A. M., and A. M. Lammerding. I982. Health maintenance benefits of cultured dairy products. Nutr. Q. 6:26. 2 Davies, F. L., and M. J. Gasson. 1981. Reviews of the progress of dairy science: Genetics of lactic acid bacteria. J. Dairy Res. 48:363.

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