Interactions of Lactobacillus and Propionibacterium in Mixed Culture

Abstract

Four species of Lactobacillus (L. acidophilus, L. casei, L. delbrueckii and L. bulgaricus) were paired with four species of Propionibacterium (p. pentosaceum. P. shermanii, P. acidi· propionici and P. freudenreichit) and were grown in pure and mixed culture to assess associative interactions. Experiments were performed using a defined synthetic medium with glucose as an energy source and casamino acids as a nitrogen source. A differential respirometer (32°C) and Warburg flasks were used; growth, carbon dioxide, and acid production were measured. Fifteen of the 16 paired cultures had less growth, acid, and carbon dioxide produced than pure culture controls. In some pairs, both cultures were inhibited by mixed cultivation while in others only the Propionibacterium was inhibited. In several mixtures, the Lactobacillus culture was apparently unaffected by mixed culturing. In P. shermanii and L. acidophilus mixed culture, a beneficial synergistic effect on growth, carbon dioxide, and volatile and nonvolatile acid production was observed. Little lactate accumulated in the mixed culture medium and was apparently used as an energy source and resulted in increased carbon dioxide and acid production by the P. shermanii. Lactobacilli and propionibacteria are often found growing in mixed culture in food and other environments. For example in the manufacture of Swiss cheese, the action of both is necessary for proper flavor, body and texture development. The volatile and nonvolatile acid end-products of their metabolism contribute to the desirable characteristics of the final product (4). In the fermentation of kefir, a fermented milk product, propionibacteria are often added to ensure a secondary fermentation of lactate and production of vitamin B 12 , which enhances the nutritional value of the product (I). The interaction of these two microorganisms is important in other environments such as silage fermentations. Lactic acid bacteria are essential for proper pH reduction (I 1) and propionibacteria produce propionic acid later in the fermentation, which retards an undesirable secondary yeast fermentation after the silage is opened for feeding (8). The synergistic (beneficial) interaction of species of homofermentative Lactobacillus and Propionibacterium in mixed culture has not been widely studied but may have importance in various natural environments and in the commercial production of propionic acid by mixed fermentations. Since the nutritional requirements of each are complex, the possibility for several simultaneous metabolic interactions is evident and may depend substantially on the growth medium. The purpose of this investigation was to determine the associative growth patterns or growth dynamics of mixed cultures of four species from each of the two genera. The species were chosen to represent those occurring in foods and other natural environments. MATERIALS AND METHODS Cultures The Lactobacillus and Propionibacterium species used for this investigation are listed in Table l. Cultures were routinely maintained in a defined synthetic medium (6) modified to include 0.1 o/o (w /v} yeast extract and exclude 1 g sodium acetate/L. The medium contained glucose (5%, w/v) as an energy source and vitamin free casamino acids {5%, w/v} (Difco, Detroit, MI) as a nitrogen source. The medium contained 6 mMolar potassium phosphate (pH 6.8) to buffer pH changes. The synthetic medium was prepared from filter-sterilized (0.45-~.Lm membrane Millipore, New Bedford, MA} stock solutions. Before each experiment, the cultures were removed from storage at 4°C, transferred to synthetic media, incubated at 32°C for 24 h (three consecutive times to ensure activation). Mixed culture interactions Mixed and pure culture studies were performed in duplicate trials with the 16 possible paired combinations using a Gilson Single Valve Differential Respirometer (Gilson Medical Electronics, Inc., Middle· ton, WI) maintained at 32°C and shaking speed at about 100 RPM. An initial optical density of 0.1 (from an active 24-hold culture) for each pure culture (approximate viable population of 5 x 10 7 /ml) and an optical density of 0.2 (01. + 0.1) for the mixed culture was used. This level of inoculum was used to achieve a rapid growth rate and substrate conversions ·within the 12-h incubation period. A total volume of 5 ml of inoculated medium was dispensed into each sterile W arburg reaction vessel. Each of the two pure cultures and the mixed culture were prepared in quadruplicate in each trial and a single vessel containing media was used as a control. After 0, 3, 6, 9 and 12 h of incubation, samples were removed from the four replicate vessels for growth and metabolic end product analysis. Differences in results between replicate vessels and duplicate trials were less than lOo/o. Growth measurements Growth of the bacteria was determined by absorbance at 600 nm using a Bausch and Lomb Model 20 Spectrophotometer (Rochester, JOURNAL OF FOOD PROTECTION. VOL. 45. MARCH 1982