Blowing In Cheese Research Articles

Spore-forming bacteria in the dairy chain

Spore-forming bacteria form the most diverse and most complex group of bacteria in terms of their elimination from the dairy chain, due to their ability to form highly resistant spores. As ubiquitous microorganisms, spore-formers can enter the product along the milk-processing continuum from different sources, and subsequently cause spoilage in various types of dairy products. The most important classes of spore-forming bacteria relevant to the dairy industry are Bacilli and Clostridia. Bacilli are responsible mainly for the spoilage and decreased shelf-life of fluid milk, while Clostridia cause late gas blowing in cheese. Spore-forming microorganisms contaminate raw milk primarily at the farm level, with potential for recontamination to occur at various points along the dairy production continuum. The most effective measure in reducing spore load at the farm level is adequate pre-milking teat preparation, while at the dairy plant level, bactofugation and microfiltration are applied. Understanding the ecology of spore-formers can improve application of systematic approaches for controlling the spoilage bacteria in dairy processing systems. Also, novel technologies, such as high-pressure processing, ultrasound treatment, irradiation etc., could provide the dairy industry with the powerful tools to eliminate these bacteria from the dairy chain.

Genomic approach to studying nutritional requirements of Clostridium tyrobutyricum and other Clostridia causing late blowing defects

Clostridium tyrobutyricum is the main microorganism responsible for the late blowing defect in hard and semi-hard cheeses, causing considerable economic losses to the cheese industry. Deeper knowledge of the metabolic requirements of this microorganism can lead to the development of more effective control approaches. In this work, the amino acids and B vitamins essential for sustaining the growth of C. tyrobutyricum were investigated using a genomic approach. As the first step, the genomes of four C. tyrobutyricum strains were analyzed for the presence of genes putatively involved in the biosynthesis of amino acids and B vitamins. Metabolic pathways could be reconstructed for all amino acids and B vitamins with the exception of biotin (vitamin B7) and folate (vitamin B9). The biotin pathway was missing the enzyme amino-7-oxononanoate synthase that catalyzes the condensation of pimeloyl-ACP and l-alanine to 8-amino-7-oxononanoate. In the folate pathway, the missing genes were those coding for para-aminobenzoate synthase and aminodeoxychorismate lyase enzymes. These enzymes are responsible for the conversion of chorismate into para-aminobenzoate (PABA). Two C. tyrobutyircum strains whose genome was analyzed in silico as well as other 10 strains isolated from cheese were tested in liquid media to confirm these observations. 11 strains showed growth in a defined liquid medium containing biotin and PABA after 6–8 days of incubation. No strain showed growth when only one or none of these compounds were added, confirming the observations obtained in silico. Furthermore, the genome analysis was extended to genomes of single strains of other Clostridium species potentially causing late blowing, namely Clostridium beijerinckii, Clostridium sporogenes and Clostridium butyricum. Only the biotin biosynthesis pathway was incomplete for C. butyricum and C. beijerincki. In contrast, C. sporogenes showed missing enzymes in biosynthesis pathways of several amino acids as well as biotin, folate, and cobalamin (vitamin B12). These observations agree with the results of growth experiments of these species in liquid media reported in the literature. The results of this study suggest that biotin and folate are potential targets for reducing late blowing in cheese and highlight the usefulness of genomic analysis for identifying essential nutrients in bacteria.

Multilocus variable-number of tandem repeat analysis (MLVA) for Clostridium tyrobutyricum strains isolated from cheese production environment

Clostridium tyrobutyricum is a gram-positive spore-forming anaerobe that is considered as the main causative agent for late blowing in cheese due to butyric acid fermentation. In this study, multilocus variable-number of tandem repeat (VNTR) analysis (MLVA) for C. tyrobutyricum was developed to identify the source of contamination by C. tyrobutyricum spores in the cheese production environment. For each contig constructed from the results of a whole genome draft sequence of C. tyrobutyricum JCM11008T based on next-generation sequencing, VNTR loci that were effective for typing were searched using the Tandem Repeat Finder program. Five VNTR loci were amplified by polymerase chain reaction (PCR) to determine their number of repeats by sequencing, and MLVA was conducted. 25 strains of C. tyrobutyricum isolated from the environment, raw milk, and silage were classified into 18 MLVA types (DI=0.963). Of the C. tyrobutyricum strains isolated from raw milk, natural cheese, and blown processed cheese, strains with identical MLVA type were detected, which suggested that these strains might have shifted from natural cheese to blown processed cheese. MLVA could be an effective tool for monitoring contamination of natural cheese with C. tyrobutyricum in the processed cheese production environment because of its high discriminability, thereby allowing the analyst to trace the source of contamination.

Detection of Clostridium Tyrobutyricum in Milk to Prevent Late Blowing in Cheese by Automated Ribosomal Intergenic Spacer Analysis

Clostridium tyrobutyricum has been identified as the main causal agent of the late blowing defect in cheese, with major effects on quality and commercial value. In this work, for the first time, we applied automated ribosomal intergenic spacer analysis (ARISA) approach to diagnose the presence of C. tyrobutyricum in raw milk before cheesemaking. A species-specific primer set was designed and used for this original application of the ARISA. Sensitivity of detection, reproducibility of the fluorescent PCR assay, and repeatability of the capillary electrophoretic analysis of amplicons were evaluated using DNA extracted from milk added with known amounts of C. tyrobutyricum genome copies, ranging from 3 × 10(6) to 3. Results indicated that the sensitivity of the technique permits to detect the bacterium in all the samples. The reproducibility, evaluated by analyzing 3 sets of serial dilutions, resulted satisfactory, with little deviation within PCR reactions amplifying the same starting amount of template (standard deviations ≤ 0.1, coefficients of variation ≤ 3%). The peaks' fluorescence displayed an evident correspondence with the number of genome copies contained in each dilution. The capillary electrophoretic analysis, tested by running a single PCR product per dilution point in 10 repeats, resulted efficient and highly repeatable, with excellent coefficients of variation ≤ 2% and standard deviations ≤ 0.1 in all the sample sets. This application of ARISA gives good estimates of the total C. tyrobutyricum DNA content allowing a specific, fine-scale resolution of this pollutant species in a complex system as milk. A further advantage linked to the automatization of the process.

Short communication: Jenny milk as an inhibitor of late blowing in cheese: A preliminary report

Late blowing on semihard and hard cheese may have an important economic effect on dairy production. Many studies have attempted to prevent this defect by physical treatment, the use of additives, and the use of bacteriocins. In this paper, we look at the effect of jenny milk as an inhibitor of blowing caused by clostridia and coliforms in ewe cheese making. Bulk ewe and jenny milk samples were collected in the morning by mechanical milking and were refrigerated at 4°C. On the collected samples, the count of somatic cells, coliforms, Clostridium butyricum, and Escherichia coli were determined. The bulk raw milk was divided in two 45-L vats: vat 1 was used as a control, whereas 0.5L of jenny milk was added to vat 2. Four semihard cheeses, weighing about 2kg each, were made from each vat. Cheese making was replicated twice. After a ripening period of 60 d, the count of coliforms and of C. butyricum was determined. In the treated group, a significant inhibition of coliform bacteria was observed. The addition of jenny milk in cheese making may prove to be a useful and innovative approach for the inhibition of spore-forming clostridia strains.

Control of late blowing in cheese by adding lacticin 3147-producing Lactococcus lactis IFPL 3593 to the starter

Bacteriocins produced by lactic acid bacteria offer potential as tools for ensuring food safety and quality. Lacticin 3147 is a two-peptide lantibiotic previously shown to inhibit a broad range of food spoilage and pathogenic bacteria. Lacticin 3147-producing Lactococcus lactis IFPL 3593 was investigated for its ability to inhibit the growth of clostridia as a means to prevent late blowing in cheese. L. lactis IFPL 3593 was shown to inhibit germination of clostridia spores and prevent late blowing in semi-hard cheeses, with a 5 log g −1 reduction in the numbers of spores when compared to control cheeses. Furthermore, this bacteriocin-producing strain demonstrated considerable potential as a biopreservative agent against heterofermentative lactobacilli and their associated blowing defects. The use of this strain to prevent late blowing in cheese thereby represents a promising alternative to the addition of lysozyme particularly given the increasing concerns regarding the potential allergenicity of this additive.

Quantitative Detection of Clostridium tyrobutyricum in Milk by Real-Time PCR

We developed a real-time PCR assay for the quantitative detection of Clostridium tyrobutyricum, which has been identified as the major causal agent of late blowing in cheese. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent in 40% of the reactions. The quantification was linear (R(2) > 0.9995) over a 5-log dynamic range, down to 10 genome equivalents, with a PCR efficiency of >0.946. With optimized detergent treatment and enzymatic pretreatment of the sample before centrifugation and nucleic acid extraction, the assay counted down to 300 C. tyrobutyricum spores, with a relative accuracy of 82.98 to 107.68, and detected as few as 25 spores in 25 ml of artificially contaminated raw or ultrahigh-temperature-treated whole milk.

The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process

A molecular biology method based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) was developed to detect Clostridium spp. in cheese samples suspected of late blowing. Strains of Clostridium spp. and different Lactic Acid Bacteria species, obtained from international collections, were used to determine the experimental conditions for the PCR amplification and DGGE differentiation. DNA extracted directly from cheeses with late blowing symptoms was subjected to PCR and DGGE analysis and traditional agar plating was performed for samples pasteurized and enriched overnight. Moreover, volatile fatty acids were determined for comparison purposes. The PCR-DGGE results were in agreement with the plating performed, and only samples presenting DGGE bands migrating at the same position as Clostridium spp. bands, showed the presence of Clostridium colonies on Reinforced Clostridial Medium plates. Butyric acid contents were high (>100 mg/kg) in the cases of positive DGGE results, underlining the suitability of the protocol for the study of cheese spoilage. The sensitivity of the method is estimated to be 10 4 CFU/g.

Identification of Clostridium tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification.

Butyric acid fermentation, the late-blowing defect in cheese, caused by the outgrowth of clostridial spores present in raw milk, can create considerable loss of product, especially in the production of semihard cheeses like Gouda cheese, but also in grana and Gruyère cheeses. To demonstrate the causative relationship between Clostridium tyrobutyricum and late blowing in cheese, many cheesemaking experiments were performed to provoke this defect by using spores from several strains of the major dairy-related clostridia. A method of PCR amplification of a part of the 16S rRNA gene in combination with hybridization with species-specific DNA probes was developed to allow the specific detection of clostridial sequences in DNAs extracted from cheeses. The sensitivity was increased by using nested PCR. Late blowing was provoked in experimental cheeses with 28 of the 32 C. tyrobutyricum strains tested, whereas experimental cheeses made with spores from C. beijerinckii, C. butyricum, and C. sporogenes showed no signs of butyric acid fermentation. In all experimental and commercial cheeses with obvious signs of late blowing, DNA from C. tyrobutyricum was detected; in some cheeses, signals for C. beijerinckii were also found. It was concluded that only C. tyrobutyricum strains are able to cause butyric acid fermentation in cheese.

Poster C3 Undesirable side-effects upon use of lactobacilli to prevent late blowing in cheese

Poster C3 Undesirable side-effects upon use of lactobacilli to prevent late blowing in cheese