341 Journal of Food Protection, Vol. 56, No.4, Pages 34/-344 (April /994) Copyright©. International Association of Milk. Food and Environmental Sanitarians 5'L2{.esearcli ?{pte Competitive Inhibition of Propionibacterium acidipropionici by Mixed Culturing with Lactobacillus helveticus Downloaded from http://meridian.allenpress.com/jfp/article-pdf/57/4/341/1665023/0362-028x-57_4_341.pdf by guest on 29 September 2021 A. PEREZ CHAIA, A. M. STRASSER DE SAAD, A. PESCE DE RUIZ HOLGADO and G. OLIVER* /nstituto de Microbiolog{a, Facultad de Bioqu{mica. Qu{mica y Farmacia, Universidad Nacional de Tucumtin, and Centro de Referenda para Lactobacilos (CERELA), Chacabuco 145, 4000 San Miguel de Tucumtin, Argentina (Received March 15, 1993/Accepted October 30, 1994) ABSTRACT growth dynamics of mixed culture of one Lactobacillus strain and a strain of Propionibacterium isolated from a commercial Lactobacillus helveticus and Propionibacterium acidipropionici were grown in pure and mixed cultures in a complex medium to assess brand of Swiss-type cheese manufactured with selected milk and the associative interaction. The specific growth rates, substrate con- without starter. sumption coefficient, substrate utilization and product formation rates MATERIALS AND METHODS were determined in each case. Propionibacterium acidipropionici uti- lized glucose preferably when it grew in a medium containing a mixture Microorganisms and culture media of glucose and lactate. Its growth rate was higher on glucose than on The strains used in this study were Lactobacillus helveticus CRL lactate in pure culture. However, lactic acid was the substrate utilized 581 (Centro de Referencia para Lactobacilos - CERELA - collection) by propionibacteria in the associative growth. The fast pH reduction and Propionibacterium acidipropionici T, isolated from Swiss-type produced by the growth of lactobacilli and the slow lactate utilization cheese. by propionibacteria in mixed culture determined the inhibition of The culture medium used for maintenance and growth of these propionic acid bacteria in associative growth. microorganisms was the following: tryptone, 1.0%; yeast extract, 1.0%; dibasic potassium phosphate, 0.025%; manganese sulfate, 0.005%; Key Words: Propionibacterium acidipropionici, mixed culturing, cysteine, 0.05%; Tween 80, 0.05%; and glucose, 0.3%. The pH was Lactobacillus helveticus adjusted to 6.8 before autoclaving at 121°C for 20 min. In some cases, sodium lactate, 0.3%, or glucose, 0.15%, plus sodium lactate, 0.15%, were used as energy sources. The study of the interaction between lactobacilli and The cultures were kept in the media at 4°C. Before use they were propionibacteria have received little attention although they often incubated at 34°C for 12 h and activated by transfer to fresh medium grow in mixed culture in silage fermentations (13), fermented three times successively. dairy products (2,12) and commercial propionic acid production (1). Growth conditions The inocula were prepared from active cultures in logarithmic Lactobacilli growing on glucose produce lactic acid as the phase. Bacterial cells were harvested by centrifugation, washed in 0.1% main metabolic end product. Propionibacteria can utilize either peptone-water and diluted in fresh medium. glucose or lactic acid as a carbon and energy source and produces An initial optical density of 0.1 at 560 nm for each pure culture propionic acid, acetic acid and carbon dioxide (6,7). Lee et al. (9) (approximate viable population: 5 x 107/mifor lactobacilli and I x l()ll/ indicated that this organism preferentially uses lactic acid when ml for propionibacteria) and an optical density of 0.2 (0.1 + 0.1) for the both glucose and lactic acid are present in the medium Therefore, mixed cultures were used. These cultures were incubated at 34°C. when propionibacteria and lactobacilli grow in mixed culture, the Growth measurements former uses the lactic acid produced by lactobacilli instead of Growth of the bacteria was measured by plating serial competing for glucose (10). dilutions of each microorganism on the appropriate media (3). Parker and Moon (14) have determined that in mixed culture The number of lactobacilli was determined by using Lactobacillus one or both microorganisms may be affected by each other selective agar and incubating at 34°C for 7 days and LAPTglo (16) favorably or unfavorably. The study of growth dynamics of incubating aerobically at 34°C for 48 h. Sodium lactate agar (8) and Lactobacillus acidophilus and Propionibacteriwn shennanii has LAPTglO were used for the count of propionibacteria after incubation at shown synergistic or beneficial effects on mixed culturing (11). 34°C for 7 days in anaerobic jars with Anaerocult A (C02 atmosphere, On the other hand, in mixed culture of L helveticus and Merck). P. shennanii, an increase in their fermentative activities and a Substrates and metabolic end products lower cells yield of lactobacillus could be observed (15). The substrate and metabolic end products studied were deter- The purpose of this investigation was to determine the mined in the supernatant obtained by centrifugation of the media (5,000 342 CHAIA, STRASSER DE SAAD, HOLGADO AND OLIVER x g, 10 min; 4°C). Glucosewas detenninedwith an enzymaticglucose oxidase-peroxidasesystem (Wiener Lab., Argentina).D- and L-lactic - acids were determinedby enzymaticmethods using D- and L-LDH ! (4,5). 9,0 Propionicacid was measuredby gas liquidchromatographywith 1J - 'u a column packed with DEGS WT% 15 on 80-100mesh chromosorb .! u'" WAW. 6,8 j;j'" 'c u 0 -''" '~ Calcuwtion of the results c.. Rates of substrateutilization(VS in h'I), end product production 6,6 30,0 15,0 (Vp in h'I), and specific growth rate (11 in h'l) were obtained by calculating,respectively,the linear regressioncoefficientsof the loga- - rithmicvaluesof residualsubstrate,productsand CFU/mlaccordingto ~ 6,4 timeduring the exponentialgrowthphaseonly. Substrateconsumption E coefficients(as in llMolper cell)wereobtainedby calculatingthe linear J"- LL regressioncoefficientof thevaluesof residualsubstrateaccordingto the u 6,2 20,0 10,0 Cl1 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/57/4/341/1665023/0362-028x-57_4_341.pdf by guest on 29 September 2021 cell number per ml. .9 RESULTS AND DISCUSSION 6,0 The growth of P. acidipropionici T, on glucose and lactate was determined. 7,8 10,0 5,0 In pure culture on glucose (Fig. lA), it grows more rapidly than in pure culture on lactate (Fig. IB), although substrate utilization and propionic acid production grew more slowly on 7,6 glucose than on lactate. The higher growth on glucose was expected because more ATP is generated per mole of substrate during glucose metabolism. 2 3 4 5 6 7 8 9 10 time (hl The growth parameters and metabolic end products recov- Figure lB. Growth of P. acidipropioniciT, on lactate. Viable cell ered from pure cultures of P. acidipropionici T, are shown in number (ho), lactate utilization (.), propionic acid production ("). Table 1. The specific growth rate calculated from pure culture on glucose was higher than that determined on pure culture on indicated that P. acidipropionici has a more efficient growth on lactate. However, the rate of substrate utilization and propionic glucose than on lactate. acid production was slightly lower on glucose than on lactate. The growth of L helveticus CRL 581 in pure culture on The values obtained for substrate consumption coefficient glucose is shown in Fig. 2. The specific growth rate and the rate of glucose utilization of this microorganism were higher than those of propionibacteria on the same medium (Table 1). 0 =- ~ The growth of Lactobacillus helveticus CRL 581 and P. 0"- acidipropionici T, in mixed culture on glucose are shown in Fig. E 0"- 3. Lactobacillus grows more rapidly and reached a slightly higher 8.9 E E E population density in mixed culture than in pure culture. til 8,8 0'" ~u 1J In contrast, P. acidipropionici grew slowly and reached a 8,7 <!) u very low population density at the stationary phase in mixed '"u 8,6 l: culture. 15,0 ,Q a. The specific growth rates and the rate of glucose utilization 8,5 0 a: and propionic acid production from mixed 8,4 <i 5,0 culture are shown in Table 2. - 8,3 Propionibacterium acidipropionici exhibited a lower spe- E cific growth rate in mixed culture than in any pure cultures. This "- 6,2 10.0 4,0 J LL result showed that this microorganism was inhibited in the u 8,1 Cl1 associative growth but did not indicate whether glucose or lactic E 8,0 3,0 acid was the substrate utilized by this strain. 7,9 The growth of P. acidipropionici on a mixture of glucose and lactate was determined (Fig. 4). Only glucose was initially 7,8 5.0 2,0 utilized by this strain when the growth medium contained a 7,7 mixture of both substrates. After glucose was reduced to very low 7,6 1,0 levels, a significant shift in the substrate utilization was observed. 7,5 These results are in contrast to those reported by Lee et al. (9) for P. she171Ulnii. Thus, a competition for the substrate may be 3 4 5 6 7 8 9 10 time (hi expected for both microorganisms in mixed culture. Figure lAo Growth of P. acidipropionici TJ on glucose. Viable According to the biochemical pathway of substrate utiliza- cell number (ho), glucose utilization (0), propionic acid produc- tion, the amount of propionic acid produced and biomass formed tion ("). depend on the substrate used by propionibacteria Therefore, the COMPETITIVE INHIBITION OF PROPIONIBACTERIUM 343 TABLE I. Growth parameters and metabolic end products recovered from batch fermentations of pure cultures of L. helveticus CRL 581 and P. acidipropionici Tr Microorganisms Substrate Jl(h") G (h) a, (JlMol/cell) Vs (h") Vp(h") V/Jl P. acidipropionici Glucose 0.226 3.07 8.544 x 10,9 0.034 0.112 0.50 P.