Effect of Peptide-Rich Extracts on Acid Production by Oxytetracycline and Penicillin-Resistant Starters1

EFFECT

OF P E P T I D E - R I C H E X T R A C T S ON A C I D P R O D U C T I O N BY

O X Y T E T R A C Y C L I N E - AND P E N I C I L L I N - R E S I S T A N T STARTERS 1 H. E. KENNEDY 2 Department of Dairy Technology, Ohio State University, Columbus SU~5

Iv~ARY

The effect of peptide-rich extracts, including pancreas extract, yeast extract, liver fraction L, tryptone, tryptose, neopeptone, casein hydrolysate, milk protein hydrolysate, and peptonized milk, on the acid production of oxytetracycline-sensitive and -resistant counterparts of Streptococcus lactis (10D) in skimmilk was determined. Additions of 0.5% of each stimulant enabled the resistant culture to produce acid in 8 hr., in concentrations equal to or in excess of that of the sensitive culture. The additives did not enable the sensitive culture to produce acid in the presence of the antibiotic. The effect of pancreas extract on acid production by penicillin-sensitive and -resistant counterparts of five strains of S. lactis and two strains of Streptococcus cremoris was determined. Six of the seven penicillin-resistant cultures, supplemented with pancreas extract, produced acid in 8 hr. equal to or in excess of that developed by the parent strains. The pancreas extract did not enable the sensitive parent counterparts of these cultures to produce acid in the presence of low levels of penicillin. Likewise, the resistant cultures were unable to develop normal acid after supplementing with pancreas extract, if a level of penicillin was added in excess of the level tolerated by these cultures.

The use of antibiotic-resistant starter cultures to counteract the effect of antibiotic residues in milk generally has not proved successful, because such cultures do not produce acid at a rate or in quantities suitable for manufact u r i n g purposes (2, 4, 5, 8). The decreased acid production by antibiotic-resistant cultures may be a result of specific differences in metabolic activities reported to exist between resistant and sensitive counterparts of lactic acid bacteria (2, 7, 11, 12, 13). Several investigators have related slow acid production in milk to inability of the starter microorganisms to utilize protein (1, 3, 15). Addition to milk of numerous biological extracts has been reported to overcome this deficiency (1, 3, 6, 9, 15). The stimulatory action of certain of these materials has been attributed to their peptide content (1, 3, 10). Angelotti (2) reported that the addition of casein hydrolysate and yeast extract to milk improved acid production by the oxytetraeycline-resistant form of Streptococcus lavtis (10D). The purpose of the present study was to investigate the effect of certain peptide-rich extracts on antibiotic-resistant and -sensitive single strain cultures in milk with or without antibiotics added.

Received for publication December 7, 1960. 1Article No. 19:60 of the Department of Dairy Technology, The Ohio State University. Supported by a grant from the U. S. Public ttealth Service (National Institutes of ttealth). 2Present address: Johnson & Johnson, New Brunswick, N. J. 8Trade name---terramycin. 844

ACID PRODUCTION BY ANTIBIOTIC-RESISTANT

STARTERS

845

/~IATERIALS AND ICIETttODS

The cultures used were the oxytetracycline3-susceptible and -resistant counterparts of S. lactis (10D), and the penicillin-sensitive and -resistant counterparts of Streptococcus cremoris (Strains 806 and US3) and S. lactis (Strains 924, 10D, C10, F1K, and T13b). These cultures were studied in sterile skimmilk (10% reconstituted of nonfat dry milk) which was free of inhibitory substances (Matrix4). Daily transfers were made using 1% inoeulum, and incubation was for approximately 16 hr. at 30 ° C. Penicillin- and oxytetraeycline-resistant cultures were carried in the presence of 0.5 unit penicillin/ milliliter and 100 ~g. oxytetracycline/milliliter, respectively. The nutritional supplements studied included pancreas extract, liver fraction L, yeast extract, tryptone, tryptose, neopeptone, casein hydrolysate, milk protein hydrolysate, and peptonized milk. These nutrients were added as sterile aqueous solutions in concentrations of 0.1, 0.5, and 1.0% to the skimmilk medium. The antibiotics, oxytetracycline or penicillin, were added as aqueous solutions also. Constant volumes were maintained by the addition of small quantities of sterile distilled water. Inoculations were made using 5% of fresh 14- to 16-hr.-old cultures and incubation was at 30 ° C. Titratable acidity determinations were made immediately after inoculation (0 hr.) and after appropriate incubation intervals (4, 8, 12, and 24 hr.). The results were expressed as per cent developed acidity, which consisted of the difference between the original titratable acidity and the acidity at a given incubation time. RESULTS

Effect o/ additives on oxytetracycline-sensitive and -resistant counterparts of S. lactis (IOD). For the first phase of the study, the effect of pancreas extract on the rate and total acid production by the oxytetracycline-sensitive and -resistant counterparts of S. lactis (10D), was determined over a 24-hr. incubation period. The curves in Figure 1 illustrate that the sensitive and resistant cultures were stimulated by pancreas extract; however, the degree of relative stimulation was greater for the resistant form. Maximmn stimulation for the sensitive culture was obtained with 0.1% pancreas extract, whereas 0.5% gave highest acidity values for the resistant strain. Comparison of the curves which represent acid production by the resistant culture supplemented with 0.5% pancreas extract and by the sensitive control culture, reveals that the rate of acid production by the resistant culture was stimulated to such an extent that after 8 hr. of incubation it had developed acidity equal to that produced by the sensitive control, i.e., 0.47% developed acidity. This value is appreciably higher than that for the resistant control, which was 0.23%. The pancreas extract had a greater effect on the rate than on total acid production. For the second phase of the study, the effect of the other peptide-rich extracts on acid production by oxytetracycline-sensitive and -resistant S. Iactis (10D) within an 8-hr. incubation period was determined. The additives may 4 Galloway-West

Co., C h i c a g o .

846

~.

E. K E N N E D Y

0.7

Sensitive + 0.1% pancreas extract Sensitive control

~ 0.6

•

Sensitive + 0.5%

o.5

0.4

o

0.3

o ~z =

0.2

? n.1

4

,

i

i

i

8

12

16

20

,

24

Hours incubation

FIG. 1.

The effect of pancreas extract on acid production by oxytetracycline-resistant Streptococcus lactis ( I O D ) .

be grouped generally into three categories, i.e., water-soluble portions of glandular or yeast proteins, peptides obtained by digestion of casein or other proteins, and enzymatic digest of whole milk proteins. Generally, the lowest concentration (0.t%) effeeted somewhat less stimulation than the higher dosages, although little difference was observed between the results obtained with 0.5 and 1.0% of the additives. Table 1 contains data representative of average developed acidity values obtained with the supplements at the 0.5% concentration. These data reveal differences in acid production by the unsupplemented sensitive and resistant control cultures and the effectiveness of each of the stimulants on acid TABLE I Effect of 0.5% peptide-rieh extracts on acid production by oxytetracycline-sensitive and -resistant Streptococcus lactis (10D) P e r cent developed t i t r a t a b l e acidity a Sensitive Additive

Control

Glandular or yeast p r o t e i n s P a n c r e a s extract .41 Liver f r a c t i o n L .42 Yeast extract .42 Digested casein or other p r o t e i n s Tryptone .~0 Tryptose .41 Neopeptone .40 Casein hydrolysate .39 Digested whole milk p r o t e i n s Milk p r o t e i n hydrolysate .39 Peptonized milk .39 Average values for f o u r trials.

Resistant

Additive

P e r cent increase

Control

Additive

P e r cent increase

.54 .53 .54

32 28 29

.22 .23 .23

.48 .45 .4{}

129 96 100

.50 .53 .49 .51

25 29 23 31

.26 .26 .26 .23

.48 .4,2 .46 .46

85 62 73 100

.50 .46

28 18

.24 .24

.53 .40

121 67

A C I D I:)t~ODUCTION BY A N T I B I O T I C - R E S I S T A N T STAI%TEI~S

847

p r o d u c t i o n b y b o t h of these c u l t u r e c o u n t e r p a r t s . A c i d p r o d u c t i o n b y t h e res i s t a n t c u l t u r e s u p p l e m e n t e d w i t h 0.5% a d d i t i v e s r e a c h e d a level e q u a l to or i n excess of t h a t of t h e s e n s i t i v e controls. T h e r a n g e of p e r c e n t a g e i n c r e a s e over t h e c o n t r o l s b y t h e r e s i s t a n t c u l t u r e was f r o m 67 to 1 2 9 % , w h e r e a s t h e i a c r e a s e b y t h e s e n s i t i v e c u l t u r e was o n l y f r o m ] 8 to 3 2 % .

Effect of additives in the presence of oxytetracycline on acid production by oxytetracycline-sensitive and -resistant counterparts of S. lactis (IOD). T h e effect of t h e a d d i t i v e s on a c i d p r o d u c t i o n b y b o t h t h e s e n s i t i v e a n d r e s i s t a n t c u l t u r e s i n t h e p r e s e n c e of 5 ~g. o x y t e t r a c y c l i n e / m i l l i l i t e r was i n v e s t i g a t e d . F o r t h i s p u r p o s e , d e t e r m i n a t i o n s w e r e m a d e in m i l k s u p p l e m e n t e d w i t h e i t h e r 0.5% a d d i t i v e , 5 ~g. o x y t e t r a c y c l i n e / m i l l i l i t e r , or 0.5% a d d i t i v e p l u s 5 ~g. o x y t e t r a c y c l i n e / m i l l i l i t e r . T h e d a t a in T a b l e 2 i l l u s t r a t e t h a t a c i d p r o d u c t i o n TABLE 2 Effect of 0.5.% nutritional 'supplements in milk on acid production by oxytetracycline-sensitive and -resistant Streptococcus lactis (10D) in the presence of 5 ~g/ml oxytetraeyeline Per cent developed titratable acidity ~ Sensitive

Additive

Control

Glandular or yeast proteins l~ancreas extract .38 Liver fraction L .38 Yeast extract .38

Additive

O.T.b

l~esistant Additive + O.T.

Control

Additlve

O.T.

Additive + O.T.

.57 .51 .55

.02 .03 .03

.02 .05 .06

.20 .21 .21

.48 .43 .45

.20 .21 .21

.47 .46 .45

DigesCed casein or other proteins Tryptone .41 .51 Tryptose .41 .53 Neopeptone .41 .48 Casein hydrolysate .38 .53

.03 .03 .03 .03

.05 .05 .03 .06

.31 .31 .31 .21

.50 .46 .49 .46

.33 .33 .33 .21

.48 .46 .45 .44

Digested whole milk proteins Milk protein hydrolysate .41 .50 Peptonized milk .4I .46 Average values for two trials. b Oxytetracyeline (5 ~g/m]).

.03 .03

.05 .04

.31 .31

.62 .40

.33 .33

.53 .46

b y t h e sensitive c u l t u r e was i n h i b i t e d b y o x y t e t r a c y c l i n e , r e g a r d l e s s of t h e p r e s e n c e of t h e n u t r i t i o n a l a d d i t i v e s ; whereas, t h e a c i d i t y of t h e s u p p l e m e n t e d r e s i s t a n t c u l t u r e w a s i n c r e a s e d b e y o n d its c o n t r o l a n d to levels e q u a l to or in excess of t h e s e n s i t i v e controls. T h e r e s u l t s f o r a l l a d d i t i v e s m a y be r e p r e s e n t e d b y v a l u e s f o r p a n c r e a s e x t r a c t , w h i c h show t h a t 0.5% of this m a t e r i M i n c r e a s e d t h e a c i d i t y of t h e s e n s i t i v e c u l t u r e f r o m 0.38 to 0.57% ; however, u p o n a d d i t i o n of 5 ~g. o x y t e t r a e y c l i n e / m i l l i l i t e r , t h e d e v e l o p e d a c i d i t y was o n l y 0.02%. T h e resistant culture supplemented with pancreas extract had a developed acidity v a l u e of 0.48%, c o m p a r e d w i t h its c o n t r o l v a l u e of 0.20%. I n the p r e s e n c e of p a n c r e a s e x t r a c t a n d o x y t e t r a c y c l i n e , the r e s i s t a n t c u l t u r e p r o d u c e d 0.47% acidity.

848

H . E . KENNEDY

Effect of pancreas extract on penicillin-sensitive and -resistant counterparts of S. lactis and S. cremoris. T h e effect of 0.5% p a n c r e a s e x t r a c t p l u s 0.5 u n i t p e n i c i l l i n / m i l l i l i t e r m i l k on a c i d p r o d u c t i o n b y t h e p e n i c i l l i n - s e n s i t i v e a n d - r e s i s t a n t c o u n t e r p a r t s (8) of five s t r a i n s of S. lactis a n d two s t r a i n s of S. cremoris was d e t e r m i n e d . S i m i l a r l y , t h e effect of 0.5% p a n c r e a s e x t r a c t in t h e p r e s e n c e of 1.0 u n i t p e n i c i l l i n / m i l l i l i t e r m i l k on a c i d p r o d u c t i o u b y o n l y t h e r e s i s t a n t c o u n t e r p a r t of each c u l t u r e was s t u d i e d . One u n i t of p e n i c i l l i n / m i l l i l i t e r was a level twice as g r e a t as t h a t to w h i c h these c u l t u r e s w e r e a d a p t e d . D e v e l o p e d a c i d i t y v a l u e s , m e a s u r e d a f t e r 8 a n d 24 hr. of i n c u b a t i o n , a r e p r e s e n t e d i n T a b l e 3. T h e p a n c r e a s e x t r a c t d i d n o t c o u n t e r a c t t h e effect of p e n i TABLE 3 Effect of pancreas extract on acid production by penlcillin-sensitive and -resistant starter cultures Per cent developed titratable acidity " Sensitive

Cultures

Streptococc~lslactis

Hours Strain incuNo. bation

Resistant

Control

0.5~c p.e.b 0.5 U. pen.

Control

0.5% p.e. 0.5 U. pen.

0.5% p.e. 1.0 U. pen.

(924)

8 24

.41 .57

.10 .29

.19 .40

.43 .55

.23 .46

(10D)

8 24

.11 .36

.07 .37

.16 .44

.46 .53

.16 .44

(C10)

8 24

.38 .60

.25 .30

.25 .42

.46 .50

.22 .43

(FIK)

8 24

.18 .40

.07 .33

.28 .42

.50 .48

.19 .33

(T13b)

8 24

.16 .42

.24 .38

.27 .41

.48 .52

.42 .41

Streptococcus cremoris (806)

8 24

.45 .58

.12 .30

.15 .48

.48 .54

.15 .39

(US3)

8 24

.59 .58

.10 .28

.25 .43

.29 .45

.16 .33

Average values for two to three trials. b Pancreas extract (0.5%) and penicillin (0.5 unit or 1.0 unit/milliliter milk). cillin on t h e sensitive c u l t u r e s , b u t t h e r e s i s t a n t c u l t u r e s w e r e a p p r e c i a b l y s t i m u l a t e d b y p a n c r e a s e x t r a c t i n t h e p r e s e n c e of 0.5 u n i t p e n i c i l l i n / m i l l i l i t e r . F o r e x a m p l e , a f t e r 8 hr. of i n c u b a t i o n , the sensitive c u l t u r e , S. lactis ( C 1 0 ) , d e v e l o p e d 0.38% a c i d i t y ; however, t h i s c u l t u r e s u p p l e m e n t e d w i t h p a n c r e a s e x t r a c t a n d p e n i c i l l i n d e v e l o p e d o n l y 0.25%. T h e v a l u e s a f t e r 24 hr. of i n c u b a t i o n w e r e 0.60 a n d 0.30%, r e s p e c t i v e l y . T h e d e v e l o p e d a c i d i t y of t h e r e s i s t a n t S. lactis ( C 1 0 ) , f o r e x a m p l e , was o n l y 0.25% a f t e r 8 hr., b u t p a n c r e a s e x t r a c t s t i m u l a t e d t h e a c i d p r o d u c t i o n to a v a l u e of 0.46%. T h e r e s i s t a n t c u l t u r e s ( e x c e p t U S 3 ) , s u p p l e m e n t e d w i t h 0.5% p a n c r e a s e x t r a c t a n d 0.5 u n i t p e n i c i l l i n / m i l l i l i t e r , p r o d u c e d a c i d i t y e q u a l to o r in excess of t h a t p r o d u c e d b y t h e i r p a r e n t sensitive c o n t r o l s w i t h i n 8 hr. of i n c u b a t i o n .

ACID PRODUCTION BY ANTIBIOTIC-RESISTANT STARTERS

849

After 8 hr. the supplemented culture, S. lactis (T13b), in the presence of 1.0 unit penicillin/milliliter, produced 0.42% acidity compared to 0.27% for the control (Table 3). After 24 hr. there was no difference between the results obtained in supplemented and control milk. The remaining six cultures were inhibited by 1.0 unit penicillin, regardless of the addition of pancreas extract. Thus, in most cases partial penicillin resistance did not complement pancreas extract in overcoming the inhibitory effect of increased penicillin dosages. DISCUSSION

The results reported show that, by the addition of peptide-rich extracts, acid production by antibiotic resistant starter cultures may be stimulated to a level equal to that of their sensitive counterparts. Slow acid production by antibiotic-resistant lactic streptococci would appear to be due to an altered capacity of these bacteria to utilize the carbohydrates in milk. Indeed, the effect of antibiotic resistance on carbohydrate utilization by lactic streptococci has been well demonstrated (11). However, the necessity of a suitable nitrogen source for microorganisms to-conduct fermentative activities under normal conditions is generally recognized. The stimulatory properties of various extracts of plant and animal origin are attributed to their content of certain peptides, which are readily available for utilization by bacteria. Furthermore, it has been observed (14) that slowergrowing cultures responded to more peptides in various extracts than did faster-growing strains; however, all extracts contained certain common stimulatory factors. Presumably, the same situation exists for the antibiotic-resistant counterparts of sensitive cultures. Previous investigators (2, 3) have suggesteel-¢hat increased acid production by peptide supplements may be a result of enhanced enzyme formation. All stimulants employed in the present study were partially digested proteins and all of them had similar effects on acid production. These facts suggest that the reduced ability for acid production by oxytetracycline- or penicillin-resistant cultures may be associated with a change in ability to utilize protein, as indicated by a more specific requirement for available nitrogen. That is, the resistant cultures may have developed a dependence on specific nitrogen sources in order to synthesize the necessary materials for carbohydrate utilization. This view appears valid as the resistant forms of the cultures responded to stimulants which were inactive for the antibiotic-sensitive counterparts. ACKNOWLEDGlYiEN~T The author expresses appreciation to Dr. A. C. Findlay of Charles Pfizer and Company, who provided the antibiotics used ia these studies. REFERENCES

(1) A~DE~SO~, A. W., ~ ELLIKFA~, P. R. The Nutritional Requirements of Lactic Streptococci Isolated from Starter Cultures. II. A Stimulatory Factor Required for the l~apid Growth of Some Strains in l~econstituted :Nonfat Milk Solids. ft. Dairy Sci., 36: 608. 1953.

850

H.E.

KENNEDY

(2) ANG~L(rrT~, R. Studies on the Physiological Activities of Terramycin-Reslstant and -Susceptible Cultures of Cheese Ripening Strains of Streptovoccus lactis. Ph.D. thesis, Ohio State University. 1955. (3) GAaVIE, E. L, aND MABBI~'r, L. A. Acid Production in Milk by Starter Cultures--The Effect of Peptone and Other Stimulatory Substances. J. Dairy Research, 23: 305. 1956. (4) HARGROVt~, R. E., WALTEJ¢, H. E., MALI.:AI~IE:S, 5. P., JK., AND MASKEIJL, K. W. The Effect of Penicillin and Streptomycin on Swiss Cheese Starters. J. Dairy Sci., 33: 401. 1950. (5) KATZN~LS0N, t=I., AND H00J), E. G. Influence of Penicillin and Other Antibiotics on Lactic Streptococci in Starter Cultures Use(] in Cheddar Cheese Making. J. Dairy Set., 32: 96]. 1949. (6) K~-NmDY, H. E., AND SPEC~, M. L. Studies on Corn Steep Liquor in the Nutrition of Certain Lactic Acid Bacteria. J. Dairy Set., 38: 208. 1955. (7) MIKOLAJCIK, E. M. Physiology of Oxytetraeycline-Resistant and -Sensitive Streptococcus lactis. Ph.D. thesis, Ohio State University. 1959. (8) Rrc~Am)s, R. J., K~'CNm)Y, It. E., AND GOULD, I. A. Antibiotic Resistance and Acid Production Among Starter Cultures Belonging to the Genus Streptococc~s. J. Milk Food Technol. (In press.) (9) SAN~)INE, W. E., HANKnV, L., SPSCK, M. L., Ainu) AVRAI~, L. W. Observations on Bacterial Growth Stimulants Present in Pancreas Tissue. J. Dairy Set., 38: 597. 1955. (10) SAND]NIl, W. E., SPF~CK, M. L., ANg) AUaAND, L. W. Identification of Constituent Amino Acids in a Peptide Stimulatory for Lactic Aeid Bacteria. J. Dairy Set., 39: 1532. 1956. (11) SHAHANI,K. M. Carbohydrate and Pyruvate Metabolism of Oxytetraeyeline-Sensitive and Oxytetracycline-I~esistant Organisms. Antibiotics Ammal 1956-57, p. 523. Medical Encyclopedia, Inc. 1957. (12) SI~AHANI, K. IV[. Galactokinase and Ga]actowaldenase Enzyme Activity of Streptococc~ls lactis. J. Dairy Sci., 43: 852. 1960. (13) SHA~ANI, K. M., AND II.xl~PmR, W. J. Differences in the Acid Production and Phosphorus Metabolism of Oxytetracycline-Sensitive and -Resistant Organisms. Appl. Microbioh, 6: 9. 1958. (]4) SPECK, M. L., M c A ~ L Y , J. K., AND WILBUR, JEANNE D. Variability in Response of Lactic Streptococci to Stimulants in Extracts of Pancreas, Liver, and Yeast. J. Dairy Sci., 4 1 : 1 . 1958. (155 WmIGRT, L. D., ANn SKF~C~GS,H. R. The Growth Requirements of Certain Streptococci. J. Baeteriol., 48: 117. 1944.