Commensalism and Competition in Mixed Cultures of Lactobacillus bulgaricus and Streptococcus thermophil us 1

Transcription

1 337 J. Milk Food Techno!. Vol. 39. No. 5, Pages (May, 1976) Copyright 1976, nternational Association of Milk, Food, and Environmental Sanitarians Commensalism and Competition in Mixed Cultures of Lactobacillus bulgaricus and Streptococcus thermophil us 1 NANCY J. MOON and G. W. RENBOLD2 Department of Food Technology oa State University, Ames, oa 511 (Received for publication May 19, 1975) ABSTRACT Cultures of Streptococcus thermophilis and Lactobacillus bulgaricus produced more acid in mixed than in single strain culture. Groth of S. thermophilus in mixed culture as enhanced during the exponential phase and reached higher numbers in the stationary phase than hen gron alone. L. bulgaricus as inhibited in the exponential and stationary phases of groth in mixed culture. L. bulgaricus liberated Seit-filterable compounds during its groth that stimulated groth and acid production of S. themophilus. These compounds are believed to be responsible for the commensal response observed in mixed cultures. Because of its rapid groth, S. thermophilus as a better competitor than L. bulgaricus for limiting nutrients in the medium. This resulted in inhibition of the groth of L. bulgaricus. The competitive and commensal response as optimal at 37 C and at a ratio of numbers of lactobacilli to streptococci of 2:1 at inoculation. Manufacture of cultured dairy products depends at some point on controlled production of lactic acid. Generally, selected strains of organisms are added to milk at some stage of manufacture to bring about these changes. Single species seldom are sufficient, so mixtures of cultures possessing desirable capabilities are most often used. These organisms usually have complex nutritional requirements for groth and activity. When several species are combined for propagation in a medium ith finite groth nutrients, many different groth patterns or associations result. Thus, the controlled fermentation of milk using selected starters presents an unusual opportunity to study the interactions of the organisms. Effects of such associations have been identified in some dairy products. For example, the inhibitory action of nisin and diplococcin produced by some lactic streptococci as discovered in studies of delayed acid production in starters for the manufacture of Cheddar cheese (1 3). Strain dominance in mixed cultures of Streptococcus lactis and Streptococcus cremoris became evident in similar investigations (6,16). Little information is available, hoever, on the associative groth of Lactobacillus bulgaricus and Streptococcus thermophilus, the organisms used to manufacture yogurt and talian and Siss cheeses. Most reports of their associative 'Journal Paper 867 of' the oa Agriculture and Home Economics Experiment Station, Ames, loa 511. Project 'Present address: Vice President-Research and Development, Leprino Cheese Company, P.O. Box 84, Denver, Colorado 821. groth stress a symbiotic relationship (1,4,8,14,15) in hich groth and acid production of both organisms are stimulated. L. bulgaricus is believed to produce large quantities of amino acids that stimulate groth and acid production of S. thermophilus (1, 4, 14, 14). S. thermophi/us produced formic acid, hich similarly stimulated L. bulgaricus (8). These reports emphasied the stimulation of acid production by mixed cultures hich should result in shorter manufacturing times for products and economic savings. The purpose of this investigation as to describe the interactions of a single-strain pair of S. thermo phi/us and L. bulgaricus that resulted in stimulation of acid production because of commensalistic and competitive groth. Cultures MATERALS AND METHODS Single strains designated L. bulgaricus 1 and S. thermophilus A ere studied. These cultures ere obtained from the culture collection of the Department of Food Technology, oa State University. This pair as selected from a survey of pairs exhibiting stimulation of acid production as assessed by a modit1ed activity test of mixed cultures (N. J. Moon, M. S. Thesis, oa State University, 1974). Cultures ere maintained in 1 ml of sterile, reconstituted skimmilk. All trials ere conducted in 8 ml of skimmilk unless stated otherise. Enumeration of viable organisms and plating technique Lee's agar (9) as used for enumeration of S. themphilus in mixed and pure culture, but as modified to increase recovery efficiency by addition of.3o/ocarboxylmethyl cellulose,.1 o/o Teen 8, and.5 M phosphate buffer. S. thermophi1us colonies ere counted after a 3-h incubation at 32 C. At this time and temperature, L. bulgaricus ill not form colonies on this medium. Viable numbers of L. bulgaricus in mixed and pure culture ere determined by colony formation on LBS agar (BBL, Division of Bioquest, Cockeysville, MD). S. thermophilus did not form colonies on this agar. Pour plates ere used for enumeration according to standard procedures (2). Developed acidity Milk samples ithdran from the culture flask ere titrated to the phenolphthalein end point ith.1 N NaOH, and the percentage of developed acidity calculated as lactic acid. Stimulation or inhibition of developed acidity The acidity produced by mixed cultures as expressed as the percentage difference beteen the mixed-culture-developed acidity

2 338 MOON AND RENBOLD (MCDA) and the sum of the pure-culture-developed acidities (PCDA) from the equation, %difterence = [(MCDA - :L PCDA):L PCDA] x 1 f the percentage difference is negative, inhibition has occurred. A positive percentage difference ould indicate stimulation. This equation is reliable only if the total developed acidity is not greater than about.9%. At this acidity the streptococci are inhibited (7). L. hulgaricus is somehat more aciduric. Optimum ratio a,( organisms to produce stimulation o.f'acid production in mixed cultures The S. thennophilus inoculation level as held constant at.5%(v/v), hile the inoculation level of L. bulgaricus as varied from.2% to.oo/o, producing numerical ratios of lactobacilli to streptococci of.3:1 to 2: at inoculation. Pure culture controls ere alays included. To-tenths percent of L. bulgaricus corresponded to about 2.5 x 1 ~ organisms/ml and.5 o/o addition of S. thermophilus to 1.2 x [ 6 organisms/mi. The incubation temperature as 37 C and the percentage of stimulation or inhibition of developed acidity and viable numbers ere determined as noted. Temperature optimum for stimulation of developed acidity A linear temperature gradient for incubating cultures as produced by placing heating bars on one end of an aluminum block and circulating refrigerated ater through the opposite end. The 1.3-m long block had 18 parallel series of holes (22 x 11 mm) for placement of culture tubes (18 X 125 mm). The block as insulated on five sides by 1 em of expanded mica packing. The entire assembly as placed in a lucite box. Temperature fluctuations ere minimied (±.5 C) by fitting the ncite box ith a hinged lid that as kept closed during experiments. To facilitate rapid arm up of sample tubes 1 ml of ater as placed in each hole of the block. With this arrangement temperature equil,ibration time as about 5 min for a 1 C difference beteen sample tube and the block. For temperature optima studies, sterile milk tempered to 3 C as inoculated ith.5% (v/v) S. thermophilus and.2% (v /v) L. bulgaricus and 15 ml as dispensed into sterile culture tubes. This procedure as folloed to avoid errors in inoculating small volumes of milk ith reproducible inoculum sies. The tubes ere then placed in the prearmed aluminum block to obtain the desired temperatures 47, 42, 37, 32, and 21 C. Cell-free filtrate preparation Pure cultures ere incubated for 4 h at 37 C. At this time the milk had not coagulated. The cells ere removed by centrifugation at 12, x g for 15 min at 4 C. The supernatant fluid as adjusted to ph 4.5 ith sterile 1. N H to precipitate the casein. The precipitate formed after 3 min as removed by centrifugation at 12, x g for 15 min at 4 C. This precipitate also contained cells not removed in the first centrifugation. The supernatant fluid as adjusted to ph 6.8 ith 1. N NaOH. The slight precipitate that formed at this stage as removed by centrifugation at 12, x g for 15 min at 4 C. This precipitate obtained at ph 6.8 contained fe cells,< 1/g. The supernatant fluid as sterilied by passage through a preashed Seit filter. For heat stability tests, 15 ml of Seit filtrate as transferred aseptically to a sterile screcap culture tube and held in ater baths at 1 C for 1 min or 8 C for 1 min. or as autoclaved at 121 C for 15 min. The presence of active compounds as assessed only in the final Seit tiltrate and ph 6.8 precipitate. Other fractions ere not tested because they contained high numbers of cells. Filtrates ere tested by addition of!oo/o (v/v) to freshly inoculated pure cultures of the test organism. A 1% addition of.75% saline to a second culture served as a control. The ph 6.8 precipitate as tested for activity by addition to sterile groth media at l% (/v). The control contained no additions. The assay cultures ere incubated for 1 h, and tested every 2 h for an increase in cell numbers and developed acidity. RESULTS AND DSCUSSON Groth and stimulation of acid production. Preliminary studies ith these selected strains of L. > <1: (l.. > '1!1'1..9l.8.7l.6 o. sool.3 Oo2QO HOURS Figure 1. Developed acidity of mixed and pure cultures of L. bulgaricus and S. thermophilus. osed circle: Mixed culture. Open triangle: Expected value (.sum of developed acidity o.l both cultures). Open circle: S. thermophilus. osed triangle: L bulgaricus. bulgaricus and S. thermophilus indicated that stimulation of acid production occurred in mixed culture. With.5% S. thermophilus and.2% L. bulgaricus inocula, stimulation of the mixed culture as observed for the first lo h of incubation at 37 C (Fig. 1). Previous investigators have shon that stimulation of acid production in mixed culture is due to enhanced groth of streptococci. Our results further emphasie this effect (Fig. 2). Groth of S. thermophilus at about 4 h as increased by about SOo/o. This groth response of the streptococci can be termed commensalism. Later (4 to 8 h), viable numbers of S. thermophilus decreased in the mixture, and to a lesser extent in the control. The early sharp decline of S. thermophilus in the mixture is unexplained, but probably is not due to ph (4-h ph as 5.8). The sharp decline ins. thermophilus after 15 h may be due to ph (2-h ph as 3.8). Groth of L. bulgaricus in mixed culture as less vigorous than in the control. n mixed culture, the exponential period of groth as not maintained, and the stationary phase occurred sooner and ith feer numbers of cells present than in the pure culture. Groth of L. bulgaricus in the mixture as retarded throughout most of the groth cycle.

3 NTERACTONS AMONG LACTC BACTERA ~ 8. (/) ~ (/) <( ::: : 5 ::: LL 4 3 m 2 """ HOURS Figure 2. The groth of mixed and pure cultures of L. bulgaricus and S. thermophilus at 37 C. Open circle: S. thermophilus control. osed circle: S. thermophil us in mixed culture. Open triangle: L. bulgaricus control. osed triangle: L. bulgaricus in mixed culture. Competition and L. bulgaricus nhibition of L. bulgaricus throughout its incubation in mixed culture has not been previously reported. nhibition of an organism in mixed culture may theoretically be due to croding, suboptimal ph, competition for nutrients, or the presence of an inhibitory compound (1). Croding and ph differences ere unlikely causes for inhibition of L. bulgan cus in mixed culture because the control L. bulgaricus culture gre to greater cell densities and loer ph. No inhibitory compounds produced against L. bulgaricus by S. thermophilus seemed to be present because cell-free filtrates and ph 6.8 precipitates from S. thermophilus cultures did not inhibit groth or acid production of L. bulgaricus. t is possible that such a factor could be removed by coprecipitation or attachment to the Seit filter. To explore this possibility, e altered the ratio of cell numbers at inoculation. We reasoned that if S. thermophilus produced an inhibitory factor, inhibition of L. bulgaricus ould be maximal hen S. thermophilus predominated in the inoculum. Conversely, hen L. bulgaricus predominated in the inoculum, minimum inhibition ould be expected. Figure 3 shos that maximum inhibition occurred at a ratio of L. bulgaricus to S. thermophilus of 2:1 rather than hen S. thermophilus predominated (such as at ratios of 1:15). Minimum inhibition occurred either at very high or very lo ratios of L. bulgaricus to S RATO OF L. bulgaricus to S. thermophilus A AT NOCULATON Figure 3. Percent inhibition of the groth of lactobacilli in the exponential phase as a.function of the ratio of L. buigaricus to S. thermophilus at inoculation. thermophilus. Thus, the presence of an inhibitory factor against L. bulgaricus produced by S. thermophilus as very unlikely. As a final alternative, e considered the possibility of inhibition of L. bulgaricus by competition ith S. thermophilus for essential nutrients. Often, hen an inhibitory factor cannot be demonstrated, competition for essential nutrients restricts the groth of one species in mixed culture (12). n competitive environments here nutrient sources are limited, the faster-groing organism often has a greater advantage (3). At optimal conditions for stimulation of acid production, S. thermophilus gre much more rapidly than L. bulgaricus (Fig. 2). t may be that inhibition of L. bulgaricus as due to the competitive and rapid utiliation of some essential nutrient by the faster groing S. thermophilus. A competitive groth advantage for S. thermophilus also is suggested by the temperature optimum for stimulation, 37 C (Fig. 4). This temperature is closer to the groth optimum for S. thermophilus (4-45 C) than for L. bulgaricus (45-5 C) (5). The lack of stimulation at higher temperatures may be due to faster groth and better competitive ability of L. bulgaricus at these temperatures. No attempt as made to discover the identity of the proposed limiting nutrient. Because both of these organisms are knon to be fastidious, any one of a large number of compounds could be responsible.

4 34 MOON AND RENBOLD >- <( (L _j > >R , \ \ \.~~~~~~~----~.L------~~ TEMPERATURE (C) Figure 4. Developed acidity of mixed and pure cultures o( L. bulgaricus and S. thermophilus after 4-h incubation as a functi~n of temperature. osed circle: l.1ixed culture. Open circle: S. thermophilus. Open triangle: L. bulgaricus. osed triangle: expected value (sum of developed acidity ofl. bulgaricus and S. thermophilus). Commensalism and S. thermophilus Other orkers have indicated that L. bulgaricus produced groth factors for S. thermophilus. Our results further substantiate this claim. A 1% addition of cell-free Seit-filtrates of L. bulgaricus culture stimulated the acid production of S. thermophilus by 26% Table 1). This is somehat less than the 38o/o observed in mixed culture. This difference may be due to some dilution effect in the filtrate preparation. Additions of more than 1% ere not as stimulatory to S. thermophilus, probably because of depletion of nutrients essential for S. thermophilus by L. bu[garicus in the original pure culture. The stimulatory agents in the cell-free filtrate ere only partly heat stable, suggesting the involvement of more than one factor (Table 1). The identity of TABLE J. Stimulationa of developed acidity of 5. thermophilus by the additionb of heat treated Seit filtrates of 4-h L. culture Addition Seit-tiltrates Autoclaved Seit-tiltrates Seit-filtrates heated 8 C 1 min Seit-fiitrates heated 1 C 1 min % Stimulation developed acidity a Developed acidity determined after 4-h incubation and %stimulation determined by comparing ith the control containing no addition of filtrate. b Added at 1o/o (v/v). compounds as not determined, but they could be any of several ater-soluble, filterable, partly heat labile compounds knon to stimulate S. thermophilus and other lactic acid organisms (11). Stimulation of acid production in mixed cultures as temperature-dependent, having an optimum of 37 C (Fig. 4). This temperature ould presumably favor S. thermophilus as previously discussed. The optimum ratio of cell numbers at inoculum for stimulation of the groth of S. thermophilus as 2:1 ~- bulgaricus to S. thermophilus) (Fig. 5). S. thermophilus as maximally :=J ' L 14 f.- i 12 f- ; 1 ~/ 8~ 1/ <J; :r_ sf 3: 4~ (') 2l 4 2 RATO us to NOCU LAT! ~ Figure 5. Stimulation of the groth of S. thermophilus in the exponential phase as a function of the ratio of L. bulgaricus to S. thermophilus at inoculation. stimulated in the exponential phase of groth at this ratio. Presumably, the stimulating factors are produced in optimal amounts at this ratio and temperature. Commensalism and competition are to terms that have been applied to groth of these organisms in mixed culture. L. bulgaricus produces compounds that stimulate groth and acid production of S. thermophilus. The rapid groth appears to deplete the medium of some unknon compound essential for L. bulgaricus. The exact identity of the compounds involved in this instance is unknon but may be any of several knon groth factors for S. thermophilus and L. bulgaricus. nhibition of L. bulgaricus by S. thermophilus may be significant hen mixed cultures of these organisms are used for product manufacture. Reduced groth of L. bulgaricus could result in defective body characteristics in cheeses, and flavor problems in yogurt. These results illustrate the complexities of groth interactions of mixed cultures. t is likely, hoever, that

5 NTERACTONS AMONG LACTC BACTERA 341 similar interaction complexities exist and play a significant role in the population dynamics of natural environments and fermented oods. REFERENCES 1. Accolas, J.P., M. Veux, and J. Auclair Etude des interactions entre diverse bacteries lactiques thermophiles et mesophiles, en relation avec a fabrication des form ages a pate cuite. Lait 55-56: American Public health Association, nc Standard methods for the examination of dairy products. 13th ed. American Public Health Association nc., Washington, D.C. 3. Annear, D Mixed bacterial gro\\-th:. Studies on the mixed groth of a viridans streptococcus, a diptheroid and Staphyloccus a/bus. Aust. J. Exp. Bioi. Med. Sci. 29: Bautista. E., R. S. Dahiya, and M. L. Speck dentification of compounds causing symbiotic groth of Streptococcus thermophi/us and Lactobacillus bulgaricus in milk. J. Dairy Res. 33: Breed, R. S., E. G. D. Murray, and N. R. Smith Bergey's manual of determinative bacteriology, 7th ed. Wiliiams and Wilkins Co., Baltimore, Maryland. p Collins, E. B. 1%1. Domination among strains of lactic streptococci ith attention to antibiotic ;)roduction. Appl. Microbial. 9: Elliker, P. R Practical dairy bacteriology. McGra-Hill Book Co., Ne York. 8. Galesloot, T. E., F. Hassing, and H. A. Verenga Symbiosis in yogurt:. Stimulation of Lactobacillus bulgaricus by a factor produced by Streptococcus thermophilus. Neth. Milk Dairy J. 22: Lee. S. Y., E. R. Vedamuthu, and G. W. Reinbold An agar medium for the differential enumeration of yoghurt starter bacteria. J. Milk Food Techno!. 37: Meers, J. L Groth of bacteria in mixed cultures. CRC Crit. Rev. Microbial. 1: Nurmikko. V.. and E. Karha. 1%2. Nutritional requirements of lactic acid bacteria.. Vitamin and amino acid requirements of Streptococcus thermophilus strains. Ann. Acad. Sci. Fenn. A.. 114: Oberhofer. T. R., and W. C. Fraier.l961. Competition of Staphyloccus au reus ith other organisms. J, Milk Food Techno!. 24: Oxford, A. E Diplococcin, and anti-bacterial protein elaborated by certain milk streptococci. Biochem. J. 38: Pette, J. W., and H. Loekema Groeifactoren voor Sc. thermophilus. Neth. Milk Dairy J. 4: Pette, J. W.. and H. Loekema.l95. Yoghurt.. Symbiose en antibiose in mengcultures van Lb. bulgaricus en Sc. thermophilus. Neth. Milk Dairy J. 4: Reddy, M.S., E. R. Vedamuthu, C. H. Washam, and G. W. Reinbold Associative groth relationships in to strain mixtures of Streptococcus lactis and Streptococcus cremoris. J. Milk Food Techno!. 34: