The Bacterial Flora of Surface-Rlpened Cheeses with Special Regard to Coryneforms

Lait (1995) 75, 571-584 571 © Elsevier/INRA Original article The bacterial flora of surface-rlpened cheeses with special regard to coryneforms F Eliskases-Lechner, W Ginzinger Bundesanstalt für Alpenlëndiscne Milchwirtschaft, A-6200 Rotholz, Austria (Received 21 March 1995; accepted 20 June 1995) Summary - Randomly-selected Austrian bacterial surface-ripened cheeses were examined for changes in the microbiological composition of the smear. The bacterial counts of the Tilsit cheeses from 14 cheese plants and of 3 types of soft cheeses selected varied fram 1()4 ta 109 cfu/cm2 smear 3 d after manufacture and fram 108 ta 109 cfu/cm2 smear after a ripening period of 3 weeks. The flora tolerated a NaCI content of at least 80 g/kg in the plate count agar. A total of 386 isolates of coryneform bacteria were identified. The bacterial flora proved ta be of mixed population. However, Brevibacterium linens accounted for a large share of the flora, comprising 30% of the total bacterial count, Besides Bre- vibacterium linens, the other main types found ta be present in the heterogeneous flora were cream- coloured and yellow-pigmented coryneforms, which were predominantly identified as Arthrobacter globiformis and Brevibacterium ammoniagenes. The coryneforms isolated fram the cheeses 3 d after manufacture were more prateoly1ic than those isolated at later stages of ripening. Tilsit cheese / smear-ripened soft cheese / smear / coryneform bacteria / cheese ripening Résumé - Flore bactérienne de surface des fromages à pâte molle, en particulier les coryné- formes. L "évolution de la flore microbienne sur la croûte des fromages autrichiens emmorgés a été exa- minée. Des Tilsit provenant de 2 fromageries ainsi que 3 différents types de fromage à pâte molle fabriqués par la même fromagerie ont été étudiés pendant toute la période de raffinage, de même que des Tilsit provenant de 12fromageries à 3 différents stades d"affinage. Le nombre des germes aéro- bies mésophiles dans la morge était compris entre 1()4 et 109 ufc par cm2 de morge 3 j après la production et entre 1()B et 1()B ufc par cm2 après 3 sem d'etîineçe respectivement. La flore a toléré une teneur en NaCI de 80 glkg dans le milieu. Au total, 386 souches de bactéries corynéformes ont été iden- tifiées à raide de 52 tests biochimiques. La flore de ces morges se trouve être une population mixte. Brevibacterium linens, considéré comme dominant dans la morge, représentait 30% de la flore bac- térienne totale. Les autres espèces principales de cette flore hétérogène étaient de couleur jaune ou non pigmentées. Elles ont été identifiées en grande partie comme Arthrobacter globiformis et Brevi- bacterium ammoniagenes. Les bactéries corynéformes isolées de la morge des fromages de 3 j étaient plus protéolytiques que celles isolées plus tardivement. Tilsit / fromage à pâte molle / marge / bactérie corynéforme / affinage 572 F Eliskases-Lechner, W Ginzinger INTRODUCTION and the yeasts, are caused by contamination du ring the manufacturing and ripening pro- cesses. In a previous paper (Eliskases- On the surfaces of red smear cheeses, bacterial counts of between 109 and 1011 cfu/g Lechner and Ginzinger, 1995), we reported smear (1 OL 109 ctu/crn-' su rface) are on the yeast flora of su rface-ripened reached within the first 2 weeks of ripening cheeses. The aim of the present study was and remain constant until the time of con- the quantification and characterization of sumption (Accolas et al, 1978; Keller and the bacterial flora in the smear of these Puhan, 1985; Grand et al, 1992). The vari- cheeses. ations between different cheese varieties or cheese factories are low. Only during the initial period of ripening do differences occur MATERIALS AND METHODS with regard to the rapidity of the change from yeast dominance to bacterial dominance. Origin of cheese samples The importance of the coryneforms for the surface ripening of smear cheeses is The smears of Tilsit cheeses from 2 plants (A, often stated, but only a few studies have B) and 3 varieties of soft cheeses from 1 plant (C) were examined during the ripening period. In been made of the identity of these bacte- addition, Tilsit cheeses from 13 Austrian dairy ria. The following species were found on plants were examined at 3 stages of ripening, on the surfaces of different cheese varieties: the 3rd, 7th and 21st d alter manufacture. Arthrobacter aurescens, Arthrobacter cit- reus, Arthrobacter globiformis, Arthrobac- ter nicotianae, Arthrobacter protophormiae, Enumeration and isolation Arthrobacter uratoxydans, Arthrobacter vari- abilis, Arthrobacter spp, Brevibacterium 2 ammoniagenes, Brevibacterium erythro- The smear film of 100 cm of the cheese surface was scraped off with a sterilespatula,dilutedseri- genes, Brevibacterium helvolum, Brevibac- ally in Ringer solution and plated on plate count terium linens, Brevibacterium oxydans and agar + 30 g/kg NaCI, 80 g/kg NaCI and 150 g/kg Corynebacterium spp (Sei 1er,1986; Sauter, NaCI(PCA; Merck5463); it was then incubatedat 1986; Mayer, 1990; Piton and Fontanier, 30°C for 12 d (Seller, 1986) in daylight to make 1990; Piton-Malleret and Gorrieri, 1992). pigment production possible for those coryne- Among these species, the grey, the white forms which are colourless alter groW1hin dark- and the cream-coloured ones formed the ness (Mulder et al, 1966; EI-Erian, 1969). The yeast developmenton the plateswas suppressed main part (40%), the yellow strains 36% and by Pimaricin; before use 0.1 ml of a suspension the orange-pigmented strains only 22% (60 mg Pimaricin/3 ml H20) was plated on the (Sauter, 1986), in spite of using Brevibac- surface of the PCA (Sauter, 1986; Engel, 1993). terium linens cultures for each smearing. Three pigmented groups (orange, yellow, cream) This is why some commercial cultures for were counted separately. Ali counts are given use in smear-cheese ripening presently con- per cm2 smear surface; the use of 9 smear sur- tain different coryneforms besides Bre- face as a unit would result in values approxi- vibacterium linens such as Brevibacterium mately 100 x higher. Five colonies of each mor- phological type were selected randomly from the spp and Arthrobacterspp. PCA plates, purified and stored on plate count ln Austria it is common practice to use agar slants. Brevibacterium linens as the sole culture Yeast populations were detected on yeast organism. Ali other microorganisms found extract-glucose-chloramphenicol-agar (YGCA; on the cheese surfaces, both the bacteria Merck 1600) with 10 /lg/kg bromophenol blue Bacterial flora of Tilsit cheese 573 (Rapp, 1974) and incubated at 25°C for 5 d RESULTS (IOF,1990). Changes in bacterial counts Identification during ripening Three hundred eighly-six coryneforms were iden- Figures 1 and 2 show the increase in bac- tified using 52 biochemical characteristics terial counts on the surface of Tilsit cheeses described by Seiler et al (1980), Seiler (1983, from 2 plants (codes A, B) during ripening. 1986) and Valdés-Stauber and Sei 1er(1990, per- sonal information), including assimilation of sug- The bacterial counts of the Tilsit cheeses ars, ami no acids and acids, production of acid after removal from the brine were from carbon sources, nitrate reduction, NaCI tol- 104 ctu/cm'' in plant A and 106 ctu/crn'' in erance, KOH test (Greez and Oack, 1961), hydrol- plant B. lt must be considered that at this ysis of xanthine, starch, gelatine, casein and stage of ripening no "smear" had developed. catabolism of tyrosin. A microtitre tray technique Within 20 d, the maximum count, 109 and the computer program by Seiler and Braatz ctu/crnè, was reached and the count (1988) were used for the identification routine. remained at this level until the time of con- A screening of proteolytic activities on cal- sumption. The counts of the 80 g/kg-NaCI- cium-caseinate agar, with a 3-mm layer (Frazier and Rupp; Merck 5409) was made. The diameter resistant bacteria were equivalent ta the total of the clear zone around the colony was mea- number of cheese surface bacteria; accord- sured as the parameter of proteolytic activity. ingly, the bacterial flora can be character- 10 9 8 '"E o 7 ::::> u..-- Ü 6 0> 0 5 1---,/-/------------------1-- total count -4- 80 g/kg NaCI 4 -<>- 150 g/kg NaCI 3 0 10 20 30 40 50 60 70 8C ripening time (d) Fig 1. Oevelopment of the 80 g/kg-NaCI- and 150 g/kg-NaCI-tolerant bacteria compared with the total bacterial count of Tilsit cheese during the who le ripening period (plant A). Évolution de la flore bactérienne tolérant des teneurs de 80 g/kg et 150 g/kg NaCI comparée à la flore totale au cours de la maturation de Tilsit (fromagerie A). 574 F Eliskases-Lechner, W Ginzinger 10 9 8 '"E o 7 --:::> LL o 6 0> .2 5 1----1/----------------1---- total cou nt --<>- 80 g/kg NaCI 4 '------------------1-.--150 g/kg NaCI 3 0 10 20 30 40 50 6C ripening time (d) Fig 2. Development of the 80 g/kg-NaCI- and 150 g/kg-NaCI-tolerant bacteria compared with the total bacterial count of Tilsit cheese du ring the whole ripening period (plant B). Évolution de la flore bactérienne tolérant des teneurs de 80 g/kg et 150 g/kg NaCI comparée à la flore totale au cours de la maturation de Tilsit (fromagerie B). ized as 80 g/kg-NaCI tolerant. The 150 g/kg- By means of the results just given, 3 dif- NaCI-tolerant bacteria counts were approx- ferent times of ripening (3, 7 and 21 d after imately 1 log unit lower than the total cou nt. manufacture) were chosen to characterize The development of the total bacterial the smears of Tilsit cheeses from 14 cheese count of 3 different soft cheese varieties plants.

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