(Northrup, 1912; Hastings and Catley, 1927; Slobodska- INFLUENCE OF A FILM YEAST, CANDIDA KRUSEI, ON

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1 INFLUENCE OF A FILM YEAST, CANDIDA KRUSEI, ON THE HEAT RESISTANCE OF CERTAIN LACTIC ACID BACTERIA GROWN IN SYMBIOSIS WITH IT', 2 H. J. PEPPLER AND W. C. FRAZIER Department of Agricultural Bacteriology, Univernity of Wisconsin Received for publication April 28, 1941 A comparison of the heat resistance of certain lactic acid bacteria grown in pure culture and in symbiosis with a filmforming yeast, Candida krusei, revealed that the bacteria nearest the yeast pellicle were more active after a severe heat treatment than were those farthest from the surface growth (Peppler and Frazier, 1941). No report on this result of symbiosis has been found in the literature. It was believed that better use might be made of this relationship in the preparation of cultures of thermoduric bacteria if more were known concerning the causes of the beneficial effect of Candida krusei. The influence of accessory growth substances, reduced acidity, reduced oxygen tension, and combinations of these factors was considered. Growth with acid-destroying yeasts is known to prolong markedly the viability of common dairy streptococci and lactobacilli (Northrup, 1912; Hastings and Catley, 1927; Slobodska- Zaykowska, 1926). The association of certain lactobacilli with film-forming yeasts is reported to increase their rate of acid production (Hastings and Catley, 1927; Lott, 1926; Severine and Paraschuk, cited by Palladina and Masjukewitsch, 1931). A new aspect of symbiosis, the stimulation of lipolysis by Mycotorula lipolytica during growth with lactic acid bacteria, was reported by Fouts (194). 1 This work was aided by a grant from the Wisconsin Alumni Research Foundation. 2 Published with the consent of the Director of the Agricultural Experiment Station. 181

2 182 H. J. PEPPLER AND W. C. FRAZIER Other acid-destroying and oxygen-consuming symbionts, notably Oospora lactis, also affect the longevity of certain lactic acid bacteria (Trioli-Petersson, 1899; Hastings and Catley, 1927; Linneboe and Hastings, 1936). The beneficial effects have been attributed to the reduction of acidity and oxygen tension (Northrup, 1912; Hastings and Catley, 1927), and the slight proteolytic action of film-forming yeasts during symbiosis (Hastings and Catley, 1927). EXPERIMENTAL Pure cultures of Lactobacillus helveticus, Strain 39aW, and Streptococcus thermophilus, strain Mc, were grown in reconstituted skimmilk; separate cultures were associated with Candida krusei, sometimes called a "mycoderm" (Graham, 1939), and the mixed cultures were designated as 39aW-my and Mc-my. Methods described by Peppler and Frazier (1941) were used in the preparation of cultures and in the determination of their activity at 37 C. after a heat treatment at 62 to 64. for 3 minutes. Heat resistance of bacteria in different parts of an associated culture The effect of Candida krusei on the heat resistance of S. thermophilus and L. helveticus was demonstrated with cultures held at 37 for 14 hours, and then stored at 2 for one week. After four successive transfers, subcultures from the top region, from the bottom region and from the mixture of top and bottom areas were made in reconstituted skimmilk and then heat-treated as indicated. Plate counts, with the medium of Kulp and White (1932), were prepared immediately before and after heating, and at hourly intervals until the eighth hour. The decreases in ph were recorded. The variations in heat resistance of S. thermophilus organisms from different parts of the associated culture are shown by curves of growth and decrease in ph in figure 1; thedesignation, -hour, refers to samples taken before the heat treatment. Bacteria nearest the surface growth of the aerobic film yeast were more active after the heat treatment than bacteria located farthest from it. The influence of Candida krusei extended far

3 HEAT RESISTANCE OF LACTIC ACID BACTERIA 183 below its surface pellicle, and the mixture of top andbottom zones showed an activity between those of separate subcultures from the top and bottom areas. a 4. I e Subculture of *- Top zone / &-& Bottom zone Mixed top and bottom S X / A ^~~~~~~~~~~~ Downloaded from O Hours after heating FIG. 1. Upper part shows growth curves of Streptococcus thermophilu obtained from the top or bottom zone, and their mixture, of a culture associated with Candida krusei and heat-treated at 64C. for 3 minutes; lower part shows acid increase of the same heat-treated subcultures at 37C. The effect of association on the heat resistance of L. helveticus from different regions of the culture is shown in figure 2. Although the influence of Candida krusei was less marked with L. helveticus than with S. thermophilus, similarities between on March 16, 219 by guest

4 184 H. J. PEPPLER AND W. C. FRAZIER different subcultures were indicated by the positions of the curves of growth and developed acidity. Just why cells of S. therrmophilus in intimate contact with cells of the film yeast received greater benefit than similarly located cells of L. helveticus is not evident. Observations of the rate and extent of acid destrucg. Subculture of * -*. Top zone 8. ^ ^ Bottom zone Mixed top 4 rn-rn ! '~ and bottom g;.4 -~~ p... rzi Hours after heating FIG. 2. Upper part shows growth curves of Lactobacillu8s helveticu8 obtained from the top or bottom zone, and their mixture, of a culture associated with Candida krusei and heat-treated at 62 for 3 minutes; lower part shows acid increase of the same heat-treated subcultures grown at 37C. tion during symbiosis between the film-forming yeast and these two bacterial species provided no explanation of the part played by the film yeast in benefiting S. thermophilus. It is probable that the level of lactic acid tolerance, which is relatively much lower for S. thermophilus than for L. helveticus, may be con-

5 HEAT RESISTANCE OF LACTIC ACID BACTERIA cerned; therefore, the same amount of reduction in acidity should be more helpful to the streptococci than to the lactobacilli. Subsequent experiments revealed that reduction in acidity alone could not account for the benefits of symbiosis. Effect of acidity on heat resistance of pure cultures of bacteria Pure cultures of S. thermophilus and L. helveticus were grown at 37 for 14 hours, adjusted to different acidities with NaOH, and then stored at 12 or 25 C. for one week. There were no marked differences in activity after heat treatment, as data of table 1 reveal. Under the less favorable conditions of storage at 25 C., adjustment of acidity to ph 5.7 to 6. appeared to be more helpful to S. thermophilus than to L. helveticus. Effect of Candida krusei culture filtrates on acid production by S. thermophilus following heat treatment Previous studies (Peppler and Frazier, 1941) had demonstrated the beneficial effect of small amounts of Neopeptone in the skimmilk culture medium on the heat resistance of S. thermophilus and L. helveticus. Since the improved culture medium resulted in the greater increase in activity of S. thermophilus after heating, and this organism also benefited most from the symbiosis with Candida krusei, the possible formation of accessory compounds by the film yeast during association was suggested, and the streptococcus was selected for study. Three portions of reconstituted skimmilk were acidified to ph 4.4 with lactic acid; one portion was inoculated heavily with a pure culture of S. thermophilus, the other two with Candida krusei. To one of the film-forming yeast cultures was added.1 per cent Neopeptone. After 1 days' incubation at room temperature, filtrates were prepared in the usual way, adjusted to ph 6.4, and sterilized by means of sintered glass filters. The filtrates were added. to the medium in which S. thermophilus was given the heat treatment, and their effect on theacidproduction of this organism after the heat treatment is shown in table 2. It is evident from the marked effect of all filtrates that Candida 185

6 186 H. J. PEPPLER AND W. C. FRAZIER TABLE 1 Effect of adjustment of acidity after incubation 14 hours at S7. followed by storage at 12' or 5. on the heat resistance of Streptococcus thermophilus and Lactobacillus helveticus INOCULATING CULTURE HEAT-TREATED SUBCULTURE Incubation Tempera- ph after ph after Platepcount Dro in ph time ture NaOH added storage heati afterheating after ht hour S. thermophilus conife hours "C. millions/ml. million/mi. 4.64* * L. helveticus * * Controls: no NaOH added. TABLE 2 Effect of different filtrates prepared from pure cultures of Candida krusei and Streptococcus thermophilus on acid production of Str. thermophilus following heat treatment at 64C. for 3 minutes DROP IN ph AFTER HEAT TREATMENT SOURCE OF FILTRATE hours hours hours hours hours S. thermophilus added to acid skimmilk.) Candida krusei grown in acid skimmilk Candida krusei grown in acid Neopeptone-skimmilk medium Control

7 HEAT RESISTANCE OF LACTIC ACID BACTERIA krusei cultures contained substances which stimulated S. thermophilus. Effect of neopeptone in culture medium during symbiosis on the heat resistance of S. thermophilus The stimulatory substances formed by Candida krusei in pure culture probably are formed also during growth with S. thermophilus. If Neopeptone were equally stimulatory, its addition to the culture medium would mask any effect of the film yeast. Therefore the associated culture, Mc-my, was carried in skimmilk TABLE 3 Effect of culture medium on the heat resistance of Streptococcus thermophilus after growth in symbiosis with Candidca krusei INOCULATING CULTURE BEAT-TREATED SUBCULTURE Kind of medium ph before Plateheating count after Plateheating count atrs Dr~in he ph millions/mi. miuions/ml. Skimmilk: Sample from top Sample from bottom Neopeptone-skimmilk: Sample from top Sample from bottom alone and in that containing.1 per cent Neopeptone. After numerous weekly transfers at 25 C., subcultures from the top area and from the bottom area of each culture were heated at 64 C. for 3 minutes and then incubated at 37 C. The comparison in table 3 shows that regardless of the medium employed, bacteria near the zone of film yeast growth were more active after heating than bacteria of the same culture farther from the surface pellicle. Neopeptone in the culture medium did not supersede the influence of Candida krusei, but rather enhanced the activities of both the film yeast and the bacteria. 187

8 188 H. J. PEPPLER AND W. C. FRAZIER Effect of air, hydrogen, Neopeptone and reduced acidity on heat resistance of S. thermophilus The rapid consumption of oxygen by Candida krusei resulting in lowered redox potentials may favor the facultative anaerobic h 8 ' Treatment of cultures after incubation at 37- * * NaOH, neopeptone added; stored under hydrogen & & NaOH added; stored under hydrogen *-* Neopeptone added - Nothing added : A Downloaded from m '.4 r ~~~~~~~~~~~.4. ~~~~ 9~~~~~~~~~~~~~~~~~~~~~~ p >4~~~ Hours after heating FIG. 3. Upper part shows growth curves at 37 of heat-treated subcultures (64 for 3 min.) of Streptococcus thermophilus made from cultures held at 37 for 14 hours and then treated with NaOH and Neopeptone, and stored under hydrogen for one week at 2C.; lower part shows increase in acidity of the same heat-treated subcultures. lactic acid bacteria grown in association with it. The cessation of fermentation and the death of Streptococcus lactis is reported on March 16, 219 by guest

9 HEAT RESISTANCE OF LACTIC ACID BACTERIA to be due partly to accumulation of acid, but mainly to oxygen (Rahn and Bigwood, 1938). Neutralization of mature cultures and storage under nitrogen prolonged the viability of S. lactis. In preliminary experiments it was shown that in litmus milk Candida krusei maintained a reduced condition at the surface of associated cultures, but that redox potentials of areas beneath the pellicle of the film yeast and at the bottom of the culture were not consistently different. If the maintenance of a reduced condition contributes to the vitality of bacteria, storage under hydrogen should provide a suitable environment. Pure cultures of S. thermophilus were stored under hydrogen, following incubation at 37 for 14 hours, and different combinations of reduced acidity, added Neopeptone, and displacement of air by hydrogen were tried. For storage under hydrogen, bottles were provided with special means for removing all the air above cultures and replacing it with commercially pure hydrogen under slight pressure. After incubation at 37 C., cultures were placed under hydrogen and held at 2 C. for one week, during which time the hydrogen was replaced every 48 hours. The effect of these different treatments on the heat resistance of S. thermophilus is shown in figure 3. A marked increase in heat resistance of the culture with reduced acidity, added Neopeptone, and replaced hydrogen was observed. Cultures, receiving these special treatments singly or in any combination of two, showed activities more like that of the untreated control. DISCUSSION The beneficial effect of Canrdida krusei suggests the use of this film yeast, or possibly other organisms with similar physiological properties, not only to enhance the longevity but also the heat resistance of acid-forming bacteria. Various microorganisms, such as certain film yeasts and molds or the acetic acid bacteria described by Ritter (1933) may be adapted to use in the preparation of cultures employed in industry, especially in certain phases of dairy manufacture. Since a large proportion of bacteria in associated cultures are benefited, special conditions of incubation may be recommended, 189

10 19 H. J. PEPPLER AND W. C. FRAZIER such as: growing associated cultures in thin layers with greater surface area, intermittent aeration, addition of carbonates, and use of temperatures favorable to the accessory symbiont. As an alternative certain artificial treatments replacing measurable changes caused by Candida krusei during symbiosis could be employed.to increase the resistance of Streptococcus thermophilus. Similar conditions could be applied to maintain the activity of thermoduric bacteria stored in pure culture. The high resistance of freshly-isolated organisms to physical and chemical agents (Hastings, 1923) may possibly be influenced by symbiosis in natural environments similar to the type observed in this study. SUMMARY 1. After symbiosis with Candida krusei, Streptococcus thermophilus and Lactobacillus helveticus from areas near the surface film were more active following a severe heat treatment than bacteria from the same culture but at lower levels. 2. The influence of the film yeast was shown to extend for some distance below its active growing region. 3. Filtrates of pure cultures of Candida krusei contained substances which stimulated acid production by S. thermophilus. 4. Mature bacterial cultures stored under conditions in which the apparent actions of the film yeast were simulated by a reduction of acidity, a lowering of oxygen tension, and the addition of Neopeptone, demonstrated that each of these treatments alone produced no consistent differences in activity but the combination of all three treatments markedly increased the heat resistance of S. thermophilus. REFERENCES FouTs, E. L. 194 Effect of lactic acid on the hydrolysis of fat in cream by pure cultures of lipolytic microbrganisms. J. Dairy Sci., 23, GRAHAM, V. C Studies on ffim-forming yeast and Mycoderma. Univ. Wisconsin. Doctor's thesis. HASTINGS, E. G Comparative resistance of bacteria from native habitats and from artificial cultures. J. Infectious Diseases, 33, HASTINGS, E. G., AND CATLEY, A. B A study of symbiosis in a Swiss cheese starter. Univ. Wisconsin. Research report. Unpublished.

11 HEAT RESISTANCE OF LACTIC ACID BACTERIA 191 KULP, W. L., AND WHITE, V A modified medium for plating Lactobacillus acidophilus. Science, 76, LINNEBOE, J. B., AND HASTINGS, E. G Ueber die symbiontischen Funktionen von Oidium lactis. Zehtr. Bakt. Parasitenk., II., 93, LoTT, W. A Biologie des Bacterium casei e (von Freudenreich) und des Mycoderma Th6ni. Milchw. Forsch., 3, NORTHRUP, Z The influence of certain acid-destroying yeasts upon lactic bacteria. Mich. Agr. Exp. Sta. Tech. Bull., 15, PALLADINA,. K., AND MA5JUKEWITSCH, W. A Ueber die Einwirkung verschiedener Hefearten auf das Wachstum und die physiologischen Eigenschaften der Milchsaurebakterien und Schimmelpilze. Festskrift til eare for S. Orla-Jensen. Odenske. pp PEPPLER, H. J., AND FRAZIER, W. C Factors affecting activity and heat resistance of Swiss cheese starter cultures. IV. Effect of variations in time and temperature of incubation and of storage on heat resistance. J. Dairy Sci., 24, RARN, O., AND BIGWOOD, F. M The effect of low temperatures on Streptococcus lactis. Arch. Microbiol., 1, 1-5. RITTER, W. 193 Oidium Arten. Milchw. Forsch., 11, RITTER, W Die beweglichen Kurzstibehen in Magenlab. Milchw. Forsch., 15, SLOBODSKA-ZAYKOWSKA, N Milchhefe auf der Milchsaurebakterien. Biochem. Z., 169, TROILI-PETERSSON, G Studien ueber saure Milch und Zahmilch. Z. Hyg. Infektionskrankh., 32, Downloaded from on March 16, 219 by guest