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Journal of Food Protection, Vol. 56, No.4, Pages 34/-344 (April /994) Copyright©. International Association of Milk. Food and Environmental Sanitarians

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Competitive Inhibition of acidipropionici by Mixed Culturing with 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 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 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

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. acidipropionici Lactate 0.170 6.08 5.574 x IO-K 0.042 0.174 1.00 L helveticus Glucose 0.529 1.31 2.179 x IO-K 0.204 0.200

-• ~- "0"- = 9,0 - "-"0 E u .~ 'C l5 ....J .0'" l5" u u '" 'c ....J 0 '" '"u 15,0 30,0 'Q. 8,6 15,0 30p 'c 0 0 It 'Q. 0 n.e - 0 8,4 E - 5,0 III ::l"- ::> l1. U 'u 10,0 20,0 8,2 10,0 20,0 4,0 .0 .9 0'" '" 8,1 U ....J'" B,O 3,0 E "-::l l1. 5,0 10,0 u 7,8 5,0 10,0 2p .9'"

7,6 1,0

2 3 4 5 6 7 8 9 10 time (h I 2 3 4 5 6 7 8 9 10 time (h)

Figure 2. Growth of L. helveticus CRL 5810n glucose. Viable cell Figure 3. L. helveticus and P. acidipropionici grown in mixed number (0), glucose utilization (0), Zaftic acid production (.). culture on glucose. Viable cell number of P. acidipropionici (8), viable cell number of L. helveticus (0), glucose utilization (0), propionic acid production (A), lactic acid concentration (.).

TABLE 2. Growth parameters and metabolic end products recovered from batch fermentations of mixed cultures of L. helveticus CRL 581 and P. acidipropionici Tr

Microorganisms Substrate G (h)

P. acidipropionici 0.067 glucose 0.219(0) 0.92 L. helveticus 0.572 1.21 o Glucose utilization rate. b Propionic acid production rate. ratio V III identifies the substrate used by this strain in mixed tion of L helveticus was higher than the rate of lactate utilization p culture. This ratio calculated for mixed culture was similar to that of P. acidipropionici (Table 1). That may be the reason for the determined for pure culture on lactate alone (Tables 1 and 2). lactic acid accumulation and the pH reduction in mixed culture. This fact indicated that lactic acid was the substrate utilized by These results are in contrast to those reported by Liu and Moon propionibacteria in the associated growth. (11)for mixed culture of L acidophilus and P. she171Ul11ii.In their The low growth rate and the very low population density study, P. she171Ul11iiused lactate at a very high rate, lactic acid reached in mixed culture compared to pure culture of was not accumulated in the medium, and propionibacteria were propionibacteria on lactate might be caused by the fast pH stimulated by mixed culturing. reduction when Lactobacillus was present in the medium as The accumulation of lactic acid in the medium and the growth of propionibacteria is almost stopped when the medium inhibitory effect of pH on growth of propionibacterium have reaches pH values near to 5.0 (6,10). The pure cultures studies of previously been reported for the mixed culture of L. both microorganisms showed that the rate of lactic acid produc- helveticus and P. shermanii (15). 344 CHAlA, STRASSER DE SAAD, HOLGADO AND OLIVER

Lactobacillus casei in autoclave-sterilized whey. 1. Ind. Microbiol. 1:349- ~ ! 353. "-= 2. Cerna, 1. and H. Hrabova. 1977. Biologic enrichment of fermented "0 ="- "0 E milk beverages with vitamin BI2 and folic acid. Milchwissenschaft E i i 32:274-277. .. 3. C1arlc,W. S., A. R. Brazis, 1. L. Fowler, C. K. Johns and F. E. Nelson . Vl .. 0 • u :§ 1978. Standard plate count method. pp. 77-94. In E. M. Marth (ed.) ::> u - Standard methods for the examination of dairy products. 14th ed. E ~ -''" "- American Public Health Association, Washington, DC. => 10 20 lL.. 4. Gawehn, K. and H. U. Bergmeyer. 1974. D-(-) Lactate. pp. 1492-1493. U In H. U. Bergmeyer (ed.). Methods of enzymatic analysis. 2nd ed. .Q'" Vo!. III. Verlag Chemie, Weinheim, Germany. 5. Gutmann, 1. and A. H. Wahlefeld. 1974. L-(+)-Lactate. Determination 5 10 with lactate dehydrogenase and NAD. pp. 1464-1468. In H. U. Bergmeyer 7,80 (ed.). Methods of enzymatic analysis. 2nd ed. Vol. III. Verlag Chemie, Weinheim, Germany. 6. Hettinga, D. H. and G. W. Reinbold. 1972. The propionic acid bacteria: 3 5 9 12 15 18 21 24 time (hi A review. 1. Growth. 1. Milk Food Techno!. 35:295-301. 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 Figure 4. Growth of P. acidipropionici on a mixture of glucose 7. Hettinga, D. H. and G. W. Reinbold. 1972 . The propionic acid bacteria: 0.15% and lactate O.IS%. Viable cell number (.), glucose A review II. Metabolism. J. Milk Food Technol. 35:358-372. utilization (0), lactate utilization (.). 8. Hettinga, D. H., E. R. Vedamuthu and G. W. Reinbold. 1968. Pouch method for isolating and enumerating propionibacteria 1. Dairy Sci. The interaction of Lactobacillus and Propionibacterium 51:1707-1709. 9. Lee, E. H., A. G. Fredrickson and H. M. Tsuchiya. 1974. Diauxic growth has been described as exhibiting features typical of a of Propionibacterium shermanii. Appl. Microbiol. 28:831-835. commensal interaction (l0,11). 10. Lee, E. H., A. G. Fredrickson and H. M. Tsuchiya 1976. Dynamics of The results reviewed here show that the interaction in mixed culture of Lactobacillus plantarum and Propionibacterium mixed culture depends on the strains studied and on the shermanii. Biotechnol. Bioeng. 18:513-526. II. Liu, J. A. P. and N. 1. Moon. 1982. Commensalistic interaction between growth conditions. Our results also suggest that when Lactobacillus acidophilus and Propionibacterium shermanii. propionibacteria and lactobacilli grow in mixed culture in Appl. Environ. Microbiol. 44:715-722. food and other environments, the former would not com- 12. Mocquot, G. 1979. Review of the progress of dairy science: Swiss-type pete with lactobacilli and under only special conditions cheese. J. Dairy Res. 46: 133-160. 13. Moon, N. 1., L. O. Enly and E. M. Sudweeks. 1980. Aerobic deterioration (e.g., controlled pH) would contribute to the fermentation. of wheat, lucerne and maize silages prepared with Lactobacillus acidophilus and Candida spp. 1. Appl. Bacteriol. 49:75-87. ACKNOWLEDGMENTS 14. Parker, 1. A. and N. 1. Moon. 1982. Interaction of Lactobacillus and This work was partially supported by Grants from Consejo Nacional Propionibacterium in mixed culture J. Food. Prot. 45:326-330. de Investigaciones (CONICET) Argentina and Secretaria de Ciencia y 15. Perez Chaia, A., A. Pesce de Ruiz HolgOOo and G. Oliver. 1987. Tecnica de la la Universidad Nacional de Tucuman, Tucuman, Argentina. Interaction between Lactobacillus helveticus and Propionibacterium freudenreichii subsp. shermanii. MAN 5:325-331. REFERENCES 16. Raibaud, P., M. Caulet, 1. V. Galpin and G. Mocquot. 1961. Studies on the bacterial flora of the alimentary tract of pigs. II. Streptococci: 1. Bodie, E. A., N. Goodman and R. D. Schwartz. 1987. Production of Selective enumeration and differentiation of the dominant groups. Appl. propionic acid by mixed cultures of Propionibacterium shermanii and Bacteriol. 24:285-291.

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