Free Proline Production by Strains of Propionibacteria1

Free Proline Production by Strains of Propionibacteria 1 T H O R L A N G S R U D 2, G. W. R E I N B O L D , and E. G. H A M M O N D Department of Food Technology Iowa State University Ames 50011

ABSTRACT

MATERIALS AND METHODS

Differences in the rate of proline production in sodium lactate broth by different species and strains of propionibacteria were great after 15 days, but after 35 days proline production showed little variation. Propionibacteria produced proline at 3 and 21 C, but strain differences were substantial. These variations were related to rates of growth and autolysis. The rate of proline production by propionibacteria was retarded at pH's and concentrations of sodium chloride in Swiss cheese, but eventually the amount produced reached that of control media. Cupric ion retarded growth and proline production in sodium lactate broth. In fortified milk, cupric ion did not affect growth but decreased proline production.

Media Used

Throughout the experiment sodium lactate broth (SLB) comprising 1% Trypticase (BBL), 1% yeast extract (Difco), 1.5% sodium lactate (60%), and .025% K2HPO4, in i liter of double distilled water was used. In studies of the influence of initial pH on proline production, the pH was adjusted to 6.8, 6.0, 5.5, and 5.2 with .1 N HCL; otherwise the pH was 6.8. The media used to study the effect of NaC1 on proline production contained I, 5, 10, or 15 g/liter of NaC1. For studies of the effect of copper, the SLB contained 8 ppm and 16 ppm Cu ++. In addition, reconstituted nonfat dry milk solids fortified with .1 g/liter sodium lactate, .1 g/liter Trypticase, and .1 g/liter yeast extract and containing the same amounts of Cu ++ were used. Cultures

INTRODUCTION

Proline is in greater amounts in Swiss cheese than in any other cheese (2) and has been associated with the sweet flavor (8, 19). In a peptide-containing medium, Propionibacterium sbermanii P-59 produced large amounts of proline, and enzymatic casein hydrolysates were especially efficient precursors (11). These studies were under conditions optimal for proline production and with only one strain of propionibacterium. This paper reports proline production under conditions related to cheesemaking with a number of strains of propionibacteria.

Received November 3, 1977. 1Journal Paper No. J-9015 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Project 2144. 2The Dairy Research Institutes, Box 36, The Agricultural University of Norway, N-1432 Vollebekk, Norway. 1978 J Dairy Sci 61:303-308

Frozen stock cultures were obtained from the culture collection of the Department of Food Technology, Iowa State University. The cultures were thawed at 37 C and streaked on sodium lactate agar plates which were incubated in a candle-oats jar for 5 days at 32 C (18). Single colonies were picked and transferred into tubes containing 10 ml of sterile SLB and incubated at 32 C for 48 h. The cultures were Gram-stained and checked for catalase activity and patterns of carbohydrate fermentation. All cultures were activated before use by transferring three times every 48 h. The organisms listed in Table 1 were tested to study proline-production capacity. For temperature studies, Propio~libacteria freudenreicbii P-15, Propionibacterium pentosaceum P-9, P. sbermanii P-l, P. sbermanii P-24 and Propionibacterium zeae P-35 were used. For the remaining experiments, P. sbermanii P-59 was the only strain used. Ten-milliliter portions of media dispensed in

303

304

LANGSRUD ET AL.

TABLE 1. Propionibacterium strains in the investigation.

Speciesa

Sourceb

ISU no.

Strain no.

P. freudenreicbii P. freudenreicbii P. freudenreicbii P, intermed ium P. interrnedium P. jensenii P, pentosaceum P, pentosaceum P, pentosa ceum P. pento sa ceum P. pentosa ceum P, pento sa ceum P. raffinosaceum P. raffinosaceum P rubrum P. rubrum P. rubrum P. rubrum P. sbermanii P, sbermanii P. sbermanii P. sbermanii P. sbermanii P. sbermanii P. tboenii P. tboenii P. tboenii P tboenii P. zeae P. zeae P. zeae P, zeae

C O F K F

P-19 P-89 P--104 P-74 P--I06

KP 1 5571

Original designation from source

ATCC 9614 PZ 99 ATCC 4964

J

P- 52

E. 5.1

B H J F K

P-9 P-42 P-50 P--58 P--79 P-90 P-25 P-107 P-IO P--17 P--105 P 108 P-1 P-11 P-24 P-59 P-83 P--98 P-4 P--15 P--20 P-53 P-35 P-46 P-54 P-86

129 10 E.7.1. ATCC 4875 PT 52 5578 J 17 ATCC 14073 R 9611 R 19 ATCC 4871 ATCC 14072 F 24

0

A F A A F F A A C B B F A A A J E J J L

P 31 C KP 2

ISU SAUER ATCC 9614 TH 25 TH 20 TtI 21 E.5,2. 1505 E.1.2. E.I.1. 11

P. sberrnanii P. freudenreicbii P. freudenreicbii P. zeae P, zeae P. p eterso nil P. arabinosum P. arabinosum P. arabinosum P. pentosaceum P. tboenii P. pentosaceum P. jensenii P. t e c bni c um P. rubrum P. rubrum P. rubrum P. intermedium P. freudenreicbii P. pe nt os ac e um P. sbermanii P. sbermanii P. sb ermanii P. sbermanii P. thoenii P, tboenii P. tboenii P, petersonii P. petersonii P. jensenii P. ]ensenii P. zeae

Min. growth temp. 6.8

15

10 6.8 6.8

6.8

aSpecies classified according to R. V. Ogden (14). bsources: A) Cornell University, Ithaca, NY; B) Iowa State University, Ames;C) Kraft Foods Co., Stockton, IL; E) K. W. Sahli, Station Federale D'lndustrie Laitiere Liebefeld-Bern, Switzerland; F) American Type Culture Collection, Rockville, MD; H) W. Kundrat, University of Munich, Munich, Germany; J) C. B. van Niel, Hopkins Marine Station, Pacific Grove, CA; K) Communicable Disease Laboratory, Atlanta, GA; L) Isolated from Gruyere cheese imported from France;O) Origin unknown.

18 x 125 m m s c r e w - c a p p e d test t u b e s were autoclaved at 121 C for 15 min and i n o c u l a t e d with 1 d r o p o f a 48-h culture o f p r o p i o n i b a c teria. The t u b e s were i n c u b a t e d at 32 C e x c e p t in t h e t e m p e r a t u r e studies in w h i c h t e m p e r a tures o f 15, 21, 27, 32, and 37 C were used. To s t u d y proline p r o d u c t i o n at 3 C, the p r o p i o n i bacteria were i n c u b a t e d at 32 C for 6 days and t h e n held at 3 C. Analyses

Cell n u m b e r s were d e t e r m i n e d by the p o u c h Journal of Dairy Science Vol. 61, No. 3, 1978

m e t h o d o f Hettinga et al. (7). Proline was d e t e r m i n e d by G o o d w i n ' s m e t h o d (4) with .2-ml samples o f SLB m e d i a and 1-ml samples o f milk media. RESULTS A N D DISCUSSION Strain Differences

The a m o u n t s o f proline p r o d u c e d b y differe n t species are in Fig. 1, and t h e average values o f proline p r o d u c e d f r o m t h e strains w i t h i n each species are in Table 2.

PROLINE PRODUCTION BY PROPIONIBACTERIA

305

to be related to cell numbers. The differences p r o b a b l y were caused by variations in the rate of autolysis (9) which affected release of intracellular proteases and peptidases.

800

700 600

Influence of Temperature on Proline Production

.o_ 500

•-o-

400

o-

,Q 3OO 200

100 Strains of Pr®i0niRtcteria

FIG. 1. Proline production in SLB by 32 strains of the genus Propionibacteriurn at 32 C for 15 days.

Differences in proline p r o d u c t i o n were great b e t w e e n species and b e t w e e n strains within species after 15 days. P. s b e r m a n i i p r o d u c e d the largest a m o u n t of proline after 15 days but with some variation of strain. A single strain of P. jerzsenii p r o d u c e d a b o u t the same a m o u n t of proline as the average P. s b e r m a n i i . P. i~lterm e d i u m p r o d u c e d no proline after 15 days. A f t e r incubation for 35 days, the differences in proline p r o d u c t i o n among the various species became m u c h smaller, and it was impossible to differentiate b e t w e e n species. The variation in proline p r o d u c t i o n after 15 days did not seem

Since propionibacteria can grow t h r o u g h o u t a considerable temperature range (15), proline p r o d u c t i o n was measured f r o m 15 to 37 C. The rate of proline p r o d u c t i o n was most rapid at 32 C as shown in Fig. 2, but even at 15 C, proline was p r o d u c e d by P. s b e r m a n i i P-59. Proline p r o d u c t i o n at the t e m p e r a t u r e in the warm r o o m (21 to 25 C) and in the cold r o o m (2 to 5 C) used in Swiss cheese m a n u f a c t u r e (16) is particularly significant; therefore, protine production was observed in six strains of propionibacteria in SLB at 3 and 21 C. The strains were selected for their ability to grow at low temperatures (6). The results for 21 C are in Fig. 3. The rate of proline p r o d u c t i o n was correlated with m i n i m u m growth t e m p e r a t u r e (Table 1). If proline is i m p o r t a n t for Swiss flavor, it

I000 32C

A t.o)

E

TABLE 2. Average amounts of free proline produced a f t e r 15 and 35 days by different species of propionibacteria.

500 O-

Time (days) Species

P. freudenreicbii P. intermedium P. jensenii P. pentosaceum P. raffinosaceum P. rubrum P. sberrnanii P. tboenii P. zeae

15

35

Proline (mg/liter) 607 895 0 660 724 896 581 785 201 777 223 770 715 857 431 829 457 865

I

15

I

I

30 45 Time (Days)

I

I

60

75

FIG. 2. Influence of different incubation temperatures on proline production from SLB by P. shermanii P-59. Journal of Dairy Science Vol. 61, No. 3, 1978

306

800

LANGSRUD ETAL.

800

}-24

F

3,c

tQa

/

cm E v 40C

p

-35

e

200

-

;5 FIG.

3.

~'o

¢5

Time(Days)

Proline

production by S L B a t 21 C.

]

loo

15

i

]20

strains

of

Propionibacterium f r o m

should increase at 3 C because most of the flavor develops in the cold room, and the flavor is commensurate with the extent of aging in normal fermentation (10). To test this, organisms were incubated at 32 C for 6 days to reach high cell numbers and to be in the decelerating phase of cell numbers before they were transferred to 3 C. The results are in Fig. 4. At 3 C proline continued to increase rapidly for all strains except P. p e n t o s a c e u m P-9 and P. zeae P-35. The most proline was produced by P. s b e r m a n i i P-59 and P-24 at 75 days, after which proline decreased. This probably was caused by degradation of proline. These data confirm the results of Hettinga and Reinbold (5) that propionibacteria that could grow at 3.8 and 6.8 C generally showed greater metabolic activity than those growing only at 10 and 15 C. Influence of Initial pH on Proline Production

The pH of Swiss cheese generally is about 5.2 when it is moved into the warm room. A rapid increase occurs in the warm room, and the pH will be about 5.6 when the cheeses are moved into the cold room (3). Different initial J o u r n a l o f D a i r y S c i e n c e Vol. 6 1 , N o . 3, 1 9 7 8

i

i

100

120

Time(Days) FIG.

six

p:35

i

50

4.

Proline p r o d u c t i o n b y six s t r a i n s o f Propionibacteriurn f r o m S L B a t 3 C. T h e i n o c u l a t e d media were incubated at 32 C for 6 days before they w e r e t r a n s f e r r e d to a 3-C i n c u b a t o r .

pH values may influence the proline-producing capacity of propionibacteria. The pH change and proline production by P. s b e r m a n i i P-59 in SLB with different initial ptt's are in Fig. 5. Differences in growth rates were nil. Proline production was slower at the lower pH's, but after 35 days the amounts produced were the same. The differences in rates probably were caused by variation in enzymatic activity with pH and in release of proteases and peptidases by autolysis. Influence of NaCl on Proline Production

Propionibacteria usually are sensitive to salt; therefore, Swiss cheese must be salted lightly if eyes are to develop normally (3). About 3% NaC1 was shown by Antila (1) to be necessary to reduce growth rate, but normally the salt concentration is about 1% in domestic Swiss cheese (16). The effect on pH and proline production of .1 to 1.5% NaC1 in SLB inoculated with P. s b e r m a n i i P-59 is in Fig. 6. Cell numbers were unaffected, but during the first 2 days increasing amounts of NaCl resulted in lower pH's. These differences persisted for 35 days. The

PROLINE PRODUCTION BY PROPIONIBACTERIA

3 07 8 poreCu++

pH 6.0

16 PPmCu++

I

-

i I~

11

J---~',.,

400

i

600 7

o_

I0

20

30

5

Time(Days) FIG. 5. Influence of initial pH of SLB on proline production and pH changes by P. s b e r m a n i i P-59. ( ) proline production, (--4 pH changes.

10

20

30

40

50

60

70

Time(Days)

FIG. 7. Influence of copper on proline production by P. s b e r m a n i i P-59 at 32 C in SLB.

higher percentages of NaC1 showed proline production, but after 35 days there was little difference among the salt percentages in protine production. Influence of Copper on Proline Production by P. s b e r m a n i i P-59

G e n e r a l l y , i m p o r t e d Swiss c h e e s e c o n t a i n s s o m e c o p p e r , a n d t h e a m o u n t varies w i t h t h e

• 1%

1.~.

600

60

"- I T U

.°

/_~°-z °~

4o0

~okA,TL.~'

country of origin. Propionibacteria were inhibited by copper. Copper retarded the formation of propionic acid but had little influence on proteolysis (12, 13). We found that addition of 8 ppm Cu ++, a fairly normal value, or 16 ppm Cu ++, a maximum value for cheese, caused slower growth and lower cell numbers. Proline production is shown in Fig. 7. Sixteen parts per million Cu ++ significantly decreased the rate of proline production, but after 75 days the

~o=

-

;

3;

~o°.cu++

,;

~

;o

,o

;0

6;

,o

Time (Days) Time(Days)

FIG. 6. Influence of NaCI percentage in SLB on proline production and pH changes by P. s b e r m a n i i P-59. ( - - ) proline production, (---) pH changes.

FIG. 8. Influence of copper on proline production by P. s b e r m a n i i P-59 at 32 C in reconstituted nonfat dry milk fortified with .1% sodium lactate, .1% Trypticase, and .1% yeast extract. Journal of Dairy Science Vol. 61, No. 3, 1978

308

LANGSRUD ET AL.

a m o u n t p r o d u c e d w a s a b o u t t h e s a m e as in t h e control. In t h e s a m e e x p e r i m e n t in f o r t i f i e d m i l k , 8 o r 16 p p m C u ++ h a d n o e f f e c t o n g r o w t h . P r o l i n e p r o d u c t i o n , as in Fig. 8, w a s less t h a n t h e c o n t r o l w h e n C u ++ w a s a d d e d . T h e e f f e c t o f Cu ++ o n g r o w t h o f P. sberrna,lii P - 5 9 w a s d i f f e r e n t in S L B a n d in m i l k . T h e r e a s o n p r o b a b l y is b e c a u s e in m i l k t h e C u ++ is a s s o c i a t e d w i t h t h e m i l k p r o t e i n s ( 1 7 ) . T h e d i f f e r e n c e in p r o l i n e p r o d u c t i o n in S L B c a u s e d b y C u ++ p r o b a b l y w a s t h e r e s u l t o f g r o w t h d i f f e r e n c e s . B u t t h e r e s u l t s in m i l k m a y indicate that some of the pathways of proline production partly are inhibited by copper.

ACKNOWLEDGMENT

To Kraftco Corp. for partial support of this project. REFERENCES

1 Antila, M. 1956. Der Aminosaiireabbau durch Propions[/urebakterein. Meijeritiet. Aikak. 18/19:1. 2 Antila, V., and M. Antila. 1968. The c o n t e n t of free amino acids in Finnish cheese. Milchwissenschaft 23: 597. 3 Foster, E. M., F. E. Nelson, M. L. Speck, R. N. Doetsch, and J. C. Olson. 1957. Dairy microbiology. Prentice-Hall, Inc., Englewood Cliffs, NJ. 4 Goodwin, J. F. 1972. S p e c t r o p h o t o m e t r y of proline in plasma and urine. Clin. Chem. 18:449. 5 Hettinga, D. H., and G. W. Reinbold. 1975. Split defect of Swiss cheese: II. Effect of low temperatures on the metabolic activity of Propionibacteriurn. J. Milk Food Technol. 38: 31. 6 Hettinga, D. H., G. W. Reinbold, and E. R. V e d a m u t h u . 1974. Split defect of Swiss cheese: I. Effect of strain of propionibacterium and wrapping material. J. Milk Food Technol. 37: 322.

Journal of Dairy Science Vol. 61, No. 3, 1978

7 Hettinga, D. H., E. R. V e d a m u t h u , and G. W. Reinbold. 1968. Pouch m e t h o d for isolating and e n u m e r a t i n g propionibacteria. J. Dairy Sci. 51:1707. 8 Hintz, P. C., W. L. Slatter, and W. J. Harper. 1956. A survey of various free amino and fatty acids in domestic Swiss cheese. J. Dairy Sci. 35:235. 9 Langsrud, T. 1974. Proline production by propionibacteria. Dissertation, Iowa State University. Xerox University Microfilms No. 75-3316. Ann Arbor, M1. 10 Langsrud, T., and G. Reinbold. 1973. Flavor development and microbiology of Swiss cheese. A review. II1. Ripening and flavor production. J. Milk Food Technol. 36:593. 11 Langsrud, T., G. W. Reinbold, and E. G. Hamm o n d . 1977. Proline production by Propionibacterium sbermanii P-59. J. Dairy Sci. 60:16. 12 Maurer, L., G. W. Reinhold, and E. G. H a m m o n d . 1975. Influence of copper on the characteristics of Swiss-type cheese. J. Dairy Sci. 58:645. 13 Maurer, L., G. W. Reinbold, and E. G. H a m m o n d . 1975. Effect of copper on microorganisms in the m a n u f a c t u r e of Swiss cheese. J. Dairy Sci. 58:•630. 14 Ogden, R. V. 1974. Classification and DNA base ratios of propionibacteria. Dissertation, Iowa State University. Xerox Microfilms No. 74-23,753. A n n Arbor, MI. 15 Park, H. S., G. W. Reinbold, E. G. H a m m o n d , and W. S. Clark, Jr. 1967. Growth of propionibacteria at low temperatures. J. Dairy Sci. 50: 589. 16 Reinbold, G. W. 1972. Swiss Cheese Varieties. Pfizer Cheese Monographs. Vol. IV. Chas. Pfizer, Inc., New York, NY. 17 Samuelsson, E. G. 1966. The copper c o n t e n t in milk and the distribution of copper to some various phases of milk. Milchwissenschaft 21 : 335. 18 V e d a m u t h u , E. R., and G. W. Reinbold. 1967. The use of candle-oats jar incubation for the enumeration, characterization, and t a x o n o m i c s t u d y of propionibacteria. Milchwissenschaft 22:428. 19 Virtanen, A. 1., and M. Krevla. 1948. On the significance of amino acids for the taste of Emmenthaler cheese. Meijeritiet. Aikak. 10:13.