Lactobacillus in semi-hard cheese and their use as adjunct cultures

University dissertation from Department of Food Technology, Lund University, P.O. Box 124, SE-221 00 Lund. Telefax + 46 46 222 95 17

Abstract: The microflora of Herrgård cheese produced at three dairies was compared after three and six months maturation. The microflora of cheeses from two of the dairies was dominated by a spontaneous microflora of Lactobacillus after three months of ripening, while starter bacteria still dominated in cheese from the third dairy after six months. Isolates were typed by the PCR-based method Randomly Amplified Polymorphic DNA (RAPD). The Lactobacillus flora in cheese from different dairies consisted mainly of diverse RAPD-types, but of the 24 RAPD-types found, three were isolated from cheese made at two different dairies and two other RAPD-types were isolated from cheese from all three dairies. The RAPD-typing indicated a succession between different types of Non-Starter Lactic Acid Bacteria (NSLAB) in cheese from one of the dairies. The NSLAB in Danbo cheese was studied, with special emphasis on Lactobacillus, using six ripened cheeses of different ages and of different brands. The microflora in five cheeses was dominated by Lactobacillus, but the sixth cheese contained no Lactobacillus. Ten RAPD-types were found and 70 % of the isolates were of RAPD-types found in more than one cheese. The different RAPD-types of NSLAB were identified to species level by Temporal Temperature Gradient gel Electrophoresis (TTGE). Most NSLAB isolates were identified as Lactobacillus paracasei (76 %), but Lactobacillus plantarum, Lactobacillus curvatus and Lactobacillus rhamnosus were also found. 37 Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains of mostly human or cheese origin were classified by Restriction Endonuclease Analysis (REA) of the total chromosomal DNA and TTGE of PCR amplified 16s rDNA fragments. REA-typing singled out three major clusters, two composed of Lactobacillus paracasei and one of Lactobacillus rhamnosus strains. The groups obtained by TTGE corresponded generally with the REA-clusters. All Lactobacillus paracasei strains had one amplified 16s rDNA fragment incommon, but were separated into five subgroups due to the presence of one or two additional fragments amplified for some strains. The observed heterogeniety in the 16s rDNA of about half of the tested Lactobacillus paracasei strains might provide an explanation of the diversity within this species. A cheese model system, reproducible and comparable to cheese manufactured in full-scale in terms of moisture, salt content, pH and microbial ecology, was developed. Lactobacillus strains originating from cheese or human mucosa were tested as adjunct cultures in the cheese model system. The test-strains were mainly Lactobacillus paracasei and Lactobacillus rhamnosus strains. Adjuncts of Lactobacillus paracasei were re-isolated more frequently than adjuncts of Lactobacillus rhamnosus, but no correlation was found between re-isolation and the origin of the adjunct. The flavour development was generally strain dependent, but adjuncts of Lactobacillus paracasei were slightly more common in improving cheese flavour. Model cheese with a microflora dominated by an RAPD-type of Lactobacillus plantarum was associated with off-flavour and serine degradation. Two human-derived Lactobacillus paracasei strains were used as adjunct cultures in pilot scale cheese production. These strains improved cheese flavour and survived passage through the human gastro-intestinal tract when delivered in semi-hard cheese. The NSLAB did not share this characteristic.

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