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<I>Kluyveromyces Marxianus</I>

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<I>Kluyveromyces Marxianus</I> 189 Journal of Food Protection, Vol. 62, No. 2, 1999, Pages 189±193 Copyright Q, International Association of Milk, Food and Environmental Sanitarians A Differential Medium for the Isolation of Kluyveromyces marxianus and Kluyveromyces lactis from Dairy Products MARIA-JOSEÂ VALDERRAMA,* M. ISABEL DE SILOÂ NIZ, PILAR GONZALO, AND JOSEÂ M. PEINADO Departamento de MicrobiologõÂa, Facultad de BiologõÂa, Universidad Complutense de Madrid, 28040 Madrid, Spain MS 98-18: Received 26 January 1998/Accepted 10 August 1998 ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/2/189/1665745/0362-028x-62_2_189.pdf by guest on 28 September 2021 A selective and differential solid medium, called Kluyveromyces Differential Medium (KDM), is described for the isolation of Kluyveromyces marxianus and K. lactis from dairy products. Its discriminative potential is based on the detection of the enzyme b-galactosidase, in the absence of lactose. Of the more than 95 strains tested, including yeasts, bacteria, and ®lamentous fungus, only the strains of K. marxianus and K. lactis produced blue colonies on the medium due to the presence of X-Gal/ IPTG. The bacterial strains were not able to grow in KDM. On this basis, the medium was very satisfactory when testing naturally or experimentally contaminated dairy food products. When quality assessment tests were performed, optimal values of productivity (growth and color) and selectivity were obtained for K. marxianus and K. lactis. At present, microbial quality control in food industries MATERIALS AND METHODS is mainly focused on detecting pathogenic bacteria, while only total numbers of yeasts, together with ®lamentous fun- Strains. A total of 87 yeast strains have been used in this work (Table 1). They had been isolated in our laboratory from gi, are considered. Nevertheless, in recent years, economic spoiled food products or obtained from culture collections. Seven losses due to spoilage by yeasts have increased in European strains of bacteria and one ®lamentous fungus were also included. companies because of use of lower concentrations of pre- servatives, less severe preservation procedures, packaging Media. The new culture medium, called Kluyveromyces Dif- in modi®ed atmospheres, or new formulations that on oc- ferential Medium (KDM), consisted of a basal medium containing casion allow the growth of yeasts. the following: yeast extract (Difco Laboratories, Detroit, Mich.), When investigation of speci®c spoilage yeasts is re- 5 g/liter; proteose-peptone no. 3 (Difco), 3 g/liter; malt extract (Difco), 3 g/liter; glucose, 10 g/liter; and agar, 20 g/liter. After quired, traditional identi®cation methods are used after iso- autoclaving and cooling to 45 to 508C, adequate volumes of ®lter- lation of the yeasts, but these methods are laborious and sterilized solutions of the following substances were added: 5- time-consuming. bromo-4-chloro-3-indolyl-b-D-galactopyranoside (XGal) (Sigma- A number of selective culture media are currently Aldrich Quimica S.A., Barcelona, Spain), 0.08 mg/liter; isopropil- available for quantitative recovery of yeasts from foods. In b-D-thiogalactopyranoside (IPTG) (Sigma), 0.1 mg/liter; and general, they are acidi®ed or contain antimicrobial agents chloramphenicol (Sigma), 0.5 mg/liter. This medium was poured to inhibit bacterial growth. Certain substances, such as dich- into Petri dishes (15 ml/plate) and stored at 48C. The ®nal pH of 6 loran or rose bengal, are also added to inhibit radial growth the medium was 7.0 0.2. of microfungi (9, 19). As reference media, yeast morphology agar (YMA; Difco) was used for yeast and mold strains, and Trypticase soy agar Kluyveromyces marxianus and K. lactis are two of the (TSA; Panreac, Panreac Quimica S.A. Barcelona, Spain) for bac- most frequent spoilage yeasts of milk and dairy products teria. because of their capacity to assimilate or ferment lactose, as well as to hydrolyze fats or proteins present in milk (9, Inoculation. The media were inoculated with 10 ml of a sus- 10). In a previous work (8), we studied the enzymatic pro- pension in water (approximately 6 MacFarland) of an actively ®les of a number of spoilage yeasts in search of discrimi- growing culture of the strains. The drops were left to absorb on 8 native activities, and we found that K. marxianus was the the surface, and the plates were incubated at 28 C (yeasts and mold) or 378C (bacterial strains) for 7 days. Growth and color only species able to show b-galactosidase activity in the were checked daily. experimental conditions. Several chromogenic and ¯uoro- genic substrates were also screened for the detection of this In¯uence of incubation temperature. The strains of K. enzyme on solid media. On this basis, the aim of the present marxianus and K. lactis were inoculated on KDM plates as pre- work has been to develop a selective and differential culture viously described, and plates were incubated at 40 and 428C for medium for the isolation and rapid detection of Kluyvero- 7 days. Growth and color were recorded daily. As a growth con- myces in milk and dairy products. trol, the strains were inoculated in a similar manner on YMA plates, those being incubated in parallel at the same temperatures. * Author for correspondence. Tel: 34-1-3944964; Quality assessment of the culture medium. Twenty three E-mail: [email protected]. strains of K. marxianus and 9 strains of K. lactis, listed in Table 190 VALDERRAMA ET AL. J. Food Prot., Vol. 62, No. 2 TABLE 1. Growth of the studied strains on KDM TABLE 2. In¯uence of high incubation temperatures on the growth and color production of K. marxianus and K. lactis on No. of Organisms strains Growthb Colorc KDM Temperature (8C) Candida albicansa 2 1 2 C. guillermondii 1 1 2 40 42 C. lipolytica 1 1 2 C. parapsilosisa 2 1 2 Strains (no.) Growth Color Growth Color C. rugosa 1 1 2 K. marxianus (23) 15a 15 6 6 Debaryomyces hanseniia 7 1 2 K. lactis (9) 2 2 0 0 Issatchenkia orientalisa 4 1 2 a 1 1 Kluyveromyces lactis 8 a Results are expressed as number of strains that grew or produced 1 2 1 blue color at the tested temperatures. K. marxianusa 22 1 1 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/2/189/1665745/0362-028x-62_2_189.pdf by guest on 28 September 2021 1 1 2 K. thermotolerans 1 1 2 K. veronaea 1 1 2 TABLE 3. Productivity and selectivity of KDM assessed by the Pichia anomalaa 1 1 ecometric method and the modi®ed Miles-Misra methoda P. membranaefaciensa 1 1 2 Rhodotorula glutinis 1 1 2 Selec- R. minutaa 2 1 2 Strains RGi PR tivity R. mucilaginosaa 3 1 2 T Saccharomyces cerevisiaea 6 1 2 K. lactis CYC 1386 0.95 ND 1 Soporidiobolus salmonicolor 1 1 2 CYC 1009 0.93 ND 1 Torulaspora delbrueckii 8 1 2 CYC 1010 1 ND 1 Zygosaccharomyces bailiia 4 1 2 CYC 1361 1 ND 1 Z. bisporus 1 1 2 CECT 1362 0.81 1 1 Z. rouxiia 7 1 2 CECT 10.361 0.86 ND 1 Eurotium amstelodamii 1 1 2 CECT 10.366 1.18 ND 1 Bacillus cereus 1 2 2 CECT 10.356 1.06 ND 1 b Staphylococcus aureus 1 2 2 CECT 10.669 1 ND 1 Escherichia coli 1 2 2 K. marxianus CYC 1162T 1 1.4 1.1 Enterococcus faecalis 1 2 2 CYC 1059 1.25 0.6 1 Lactococcus lactis var. lactis 1 2 2 CYC 1165 1 1.1 1 Pseudomonas aeruginosa 1 2 2 CYC 1167 0.85 1 0.9 P. ¯uorescens 1 2 2 CYC 1182 1 0.9 1 CYC 1355 1 1 1 a Type strain included. CYC 1366 1 0.95 1 b 1 2 , growth; , absence of growth. CYC 1392 1 1.2 1 c 1 2 , blue color, , absence of blue color. CYC 1363 1 0.8 1 CYC 1364 1 0.9 1.1 CYC 1365 0.76 0.8 0.9 3, together with reference strains Zygosaccharomyces rouxii CYC 1394 1 1.4 1 NCYC 1152, Saccharomyces cerevisiae ATCC 7754, Eurotium CECT 10.315 1 1.4 1 amstelodamii ATCC 16018, and Bacillus cereus CECT 193 were CECT 10.257 1 1 1 used. Productivity and selectivity were tested by both the ecom- CECT 10.367 1.12 0.95 1 etric streaking procedure and the modi®ed Miles-Misra colony CECT 10.368 1 1 1 counting method (6, 14, 16), following the recommendations of CECT 10.369 1 1 1 the International Committee for Food Microbiology and Hygiene CECT 10.370 1 1.2 1 Working Party for Culture Media (ICFMH-WPCM) (6). Produc- CECT 10.371 1 1.1 1 tivity was expressed using two parameters: the relative growth CECT 10.374 1 1.4 1 index (RGi) (ecometric method) and productivity rate (PR) (mod- CECT 10.379 1 1 1 i®ed Miles-Misra method). CECT 10.668 1 0.9 1 CECT 10.649 1b 0.92b 1 Shelf life. In order to assess the shelf life of the medium, plates were stored at 48C for 1 month after preparation. They were S. cerevisiae ATCC 7754 1.1b 1b 1 checked weekly for performance (growth and color) using two Z. rouxii NCYC 1522 0.9b 0.8b 1 selected Kluyveromyces strains. Inoculation and incubation were B. cereus CECT 193 0 0 0 performed as previously described. a CYC, Complutensis Yeasts Collection; CECT, ColeccioÂn EspanÄ- Sample analysis. The performance of KDM as a differential ola de Cultivos Tipo; ATCC, American Type Culture Collection; medium for dairy products was analyzed on a total of 15 natural NCYC, National Collection of Yeast Cultures; T, Type strain; samples (Table 4).
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