Display omitted
•96 phages active against Clostridium tyrobutyricum were isolated and characterized.•Phages survived in cheese without changing starter, pH, DM or volatile compounds.•Cheese late ...blowing caused by C. tyrobutyricum was delayed 2 weeks by phage FA67.
Lytic bacteriophages (phages) offer a great potential as biocontrol agents for spoilage Clostridium tyrobutyricum, responsible for butyric acid fermentation in semi-hard and hard ripened cheeses, resulting in late gas blowing defect. With this aim, we have isolated, identified and characterized new lytic phages of C. tyrobutyricum, and have evaluated their efficacy to control cheese late blowing by adding them to manufacture milk. Silage, soil, milk and cheese from dairy farms were screened for anti-clostridial phages, obtaining 96 isolates active against C. tyrobutyricum. According to host range, source and plaque morphology, we obtained 20 phage profiles, 8 of them (represented by phages FA3, FA21, FA29, FA52, FA58, FA67, FA70 and FA88) showing a wider host range and high quality lysis, which were further characterized. Selected isolates showed a non-contractile tail, belonging to the Siphoviridae family, and were grouped into 3 restriction profiles. Viable phages were detected after storage in sodium-magnesium buffer (SM buffer), skim milk and acidified skim milk (pH 5) for 7 d at 4 °C, 12 °C and 37 °C, although a decline in infectivity was observed in some cases. Good phage survival was also detected during semi-hard cheese manufacture and ripening (60 d), and cheese lactococci counts, pH, dry matter values, and volatile compounds were not affected by phage addition. In semi-hard cheese, phage FA67 impaired the early germination of C. tyrobutyricum spores and caused a significant decrease in clostridial vegetative cells counts at 14 d of ripening, delaying by 2 weeks the consumption of lactic acid, formation of butyric acid and appearance of late blowing symptoms, compared to the spoilt control cheese without the phage. This is the first report on the application of phage to control C. tyrobutyricum in cheese.
Clostridium tyrobutyricum is described as the main causative agent of late blowing defect in cheese. Currently, there are no fast methods to detect this microorganism in raw milk, which would allow ...determining the use of milk for fresh or cured cheese. The technique commonly used is the Most Probable Number, which is laborious and non-specific. In this work, we present the optimization of a real-time PCR-based detection method for C. tyrobutyricum spores in raw milk samples. This novel approach extracts DNA in a semi-automatic system with magnetic beads. The applicability of the developed procedure has been tested in field milk samples from cow, ewe and goat (n = 202), allowing detection of low levels of butyric spores. Raw milk samples were also analyzed by microbiological culture in a selective medium for butyric bacteria, and positive colonies were identified by multiplex PCR and 16S rDNA sequencing. Apart from C. tyrobutyricum, other Clostridium spp. were identified, which should be considered for further development of detection methods.
•Method based on qPCR for C. tyrobutyricum spore detection in milk has been set up.•The limit of detection is 102 spores/mL and it can be performed in about 5 h.•The analyzed milk samples presented low levels of C. tyrobutyricum spores.
The impact of autochthonous and type-strains of Clostridium tyrobutyricum, Clostridium butyricum, Clostridium beijerinckii and Clostridium sporogenes on spoilage (late blowing defect, LBD), ...physico-chemical characteristics and volatile profile of cheese has been investigated. Five semi-hard cheeses were produced from ewe milk inoculated with 104 spores/mL of five Clostridium strains and ripened for 60 d. One cheese without clostridial spores served as control. C. tyrobutyricum CECT 4011 and INIA 68 resulted potent cheese spoilers, and caused the appearance of the earliest and greatest symptoms of LBD, affecting cheese pH and color, and leading to accumulation of volatile compounds like butyric, propionic and pentanoic acids and some aldehydes, alcohols and esters associated with cheese rancid and pungent off-odors. Cheeses contaminated with C. beijerinckii INIA 63 and C. sporogenes INIA 71 showed milder and late LBD symptoms, and a volatile profile characterized by higher levels of 2-butanone, 2,3-butanedione and 2-butanol than the rest of cheeses. Despite cheese inoculated with C. butyricum CECT 361 presented a slight blown-pack at the end of ripening, it showed physico-chemical characteristics and a volatile profile similar to control cheese. The first two axes of a principal component analysis (PCA) performed for the 21 significant volatile compounds out of 38, accounting for 91% of the variability between cheeses, separated cheeses made with C. tyrobutyricum CECT 4011 and INIA 68, with severe LBD symptoms, from the rest of cheeses, and also differentiated control cheese and cheese made with C. butyricum CECT 361, from cheeses with milder LBD symptoms made with C. beijerinckii INIA 63 and C. sporogenes INIA 71.
•Cheeses were made from milk deliberately contaminated with five Clostridium strains.•Cheeses with clostridia showed late blowing defect at different stages of ripening.•Clostridium tyrobutyricum were potent spoilers and markedly affected cheese volatile profile.•Clostridium beijerinckii and Clostridium sporogenes produced belated and milder cheese spoilage.•Clostridium butyricum strain action on cheese was weak and did not caused substantial spoilage.
Via butyric acid fermentation, clostridia – mainly Clostridium tyrobutyricum – are able to transform lactic acid into butyric acid, acetic acid and gas (H2 and CO2). The presence of clostridial ...endospores in milk may lead to severe quality defects in semi-hard and hard cheeses. As a consequence of butyric acid fermentation during ripening, cheeses tend to swell and develop undesired slits, irregular eyes and a rancid taste, thus resulting in high economic losses for producers. Several measures regarding stable, milking and feed hygiene have already been partly implemented to minimise the risk of raw milk contamination with clostridial endospores. Contamination, nevertheless, cannot be avoided completely. Moreover, some of the existing procedures to reduce the bacterial and endospore count in milk (e.g. bactofugation, addition of bacteriocins) are not always applicable or even prohibited for the production of certain cheese types. Therefore, cheese producers may benefit from integrating the determination of the initial count of clostridial endospores in milk into their quality control system of primary materials. This review discusses the role of butyric acid clostridia in the cheese processing environment and methods for the detection and enumeration of cheese-damaging clostridia in milk and cheese.
•Butyric acid producing bacteria may lead to major quality defects in hard cheese.•The respective contribution of different clostridial species is not fully understood yet.•The precise enumeration of the clostridial spore count in milk is a difficult task.•There is no standardised method for the quantification of clostridial spores in milk.
Late blowing is a type of microbiological spoilage that causes unwanted changes in the taste, odour and texture of cheese due to the metabolic activities of Clostridium contaminating raw milk. This ...study aimed to develop a multiplex quantitative real-time polymerase chain reaction (PCR) analysis method for the rapid and simultaneous detection of Clostridium butyricum, Clostridium sporogenes and Clostridium tyrobutyricum using specific primers and probes. The optimised method was determined to work with high sensitivity and specificity to quantify late-blowing Clostridium species in cheese samples. The lowest detectable gene copy number was 4.9 × 101, 7.8 × 101 and 8.5 × 101 for C. tyrobutyricum, C. butyricum and C. sporogenes, respectively. These results demonstrated that this method is a powerful tool for detecting and quantifying late-blowing agents in cheese. This is also the first multiplex qPCR study involving C. butyricum for detecting Clostridia, a late-blowing agent in cheese.
•Late-blowing is one of the most important problems of cheese producers.•The culture method does not provide quantitative results at species level.•This qPCR method was able to detect Clostridium species with high sensitivity.•This assay has low detection limit ̴ log10 1.0 CFU/mL.•This assay has the potential as a molecular tool for analyses cheese by rapidly.
This study investigated the stress factors that affect the content and the spatial distribution of Clostridium spores in Grana Padano (GP) wheels and in GP samples purchased from the market. The ...cheeses analyzed were produced adding lysozyme to prevent late blowing defects (LBD). The highest NaCl amount was detected in the external part (5.2 ± 0.6%) while a lower level was observed in the core (2.8 ± 0.3%). The clostridial community differed in cheeses with different ripening times along with pH and salt changes that occur slowly over time. The spore level in the core (0.24 ± 0.19 and 0.16 ± 0.14 MPN/g after 11 and 20 months of ripening) was significantly lower than in the peripheral zones (0.48 ± 0.26 and 0.45 ± 0.31 MPN/g). Moreover, in the wheels aged 11 months, the presence of C. tyrobutyricum was higher in the areas with the lowest salt content, while after 20 months of ripening when a homogeneous concentration of salt and pH within the wheel occurred, C. sporogenes was the prevalent species. Our results showed that in the GP wheels changes in the presence of clostridia responsible for LBD still occur until the salt content in the center of cheeses is higher than threshold NaCl needed to inhibit spore germination.
•Spore level in the core portion of the cheese was significantly lower.•Salt gradient within the cheese matrix affects the spatial distribution of Clostridum species.•C.tyrobutyricum and C.sporogenes are the most able to withstand adverse conditions.•C. tyrobutyricum content increases with the decrease of the NaCl concentration.•C. sporogenes was the prevalent species in internal and external areas.
Three strains of Lactobacillus were previously isolated from dairy products, Lactobacillus casei 26, Lactobacillus delbrueckii subsp. bulgaricus 76 and L. casei 95. Anti-costridial activities of ...these strains were evaluated against a collection of Clostridium isolates, belonging to species responsible for late blowing defect in cheese. The three strains of Lactobacillus inhibited the growth of all Clostridium isolates analyzed. The inhibitory effect of cell-free supernatant of each of these Lactobacillus strains combined with that of commercial strain L. casei BAL C, or with a solution of lysozyme, were compared by disc diffusion assay. Mixtures of cell-free Lactobacillus supernatants and lysozyme exhibited higher inhibitory activity than the supernatants and lysozyme solution separately (P < 0.05). Additionally, Lactobacillus strains were resistant to lysozyme concentrations usually used during cheese making process.
The genus
is a large and diverse group of species that can cause food spoilage, including late blowing defect (LBD) in cheese. In this study, we investigated the taxonomic status of strain FAM25158 ...isolated from Emmental cheese with LBD using a polyphasic taxonomic and comparative genomic approach. A 16S rRNA gene sequence phylogeny suggested affiliation to the
cluster, with
DSM 2637
being the closest related type strain (99.16% sequence similarity). Average Nucleotide Identity (ANI) analysis revealed that strain FAM25158 is at the species threshold with
, with ANI values ranging from 94.70 to 95.26%, while the digital DNA-DNA hybridization values were below the recommended threshold, suggesting that FAM25158 is significantly different from
at the genomic level. Moreover, comparative genomic analysis between FAM25158 and its four closest
relatives revealed a diversity of metabolic pathways, with FAM25158 differing from other
strains by the presence of genes such as
,
, and
, responsible for sucrose utilization, and the absence of many important functional genes associated with cold and osmolality adaptation, which was further supported by phenotypic analyses. Surprisingly, strain FAM25158 exhibited unique physiologic traits, such as an optimal growth temperature of 30°C, in contrast to its closest relatives,
species with an optimal growth temperature of 37°C. Additionally, the growth of FAM25158 was inhibited at NaCl concentrations higher than 0.5%, a remarkable observation considering its origin from cheese. While the results of this study provide novel information on the genetic content of strain FAM25158, the relationship between its genetic content and the observed phenotype remains a topic requiring further investigation.
Paucilactobacillus wasatchensis, a nonstarter lactic acid bacteria, can cause late gas production and splits and cracks in aging cheese when it metabolizes 6-carbon substrates, particularly ...galactose, to a 5-carbon sugar, resulting in the release of CO2. Previous studies have not explained late gas production in aging cheese when no galactose is present. Based on the genome sequence of Pa. wasatchensis WDC04, genes for potential metabolic pathways were mapped using knowledgebase predictive biology software. This metabolic modeling predicted Pa. wasatchensis WDC04 could metabolize gluconate. Gluconate contains 6 carbons, and Pa. wasatchensis WDC04 contains genes to convert it to 6-P-gluconate and then to ribulose-5-P by using 6-phosphogluconate dehydrogenase in a decarboxylating step, producing CO2 during its metabolism. The goal of this study was to determine if sodium gluconate, often added to cheese to reduce calcium lactate crystal formation, could be metabolized by Pa. wasatchensis WDC04, resulting in gas production. Carbohydrate-restricted DeMan, Rogosa, and Sharpe broth was mixed with varying ratios of ribose, sodium gluconate, or d-galactose (total added substrate content of 1% wt/vol). Oxyrase (Oxyrase Inc.; 1.8% vol/vol) was also used to mimic the anaerobic environment of cheese aging in selected tubes. Tubes were inoculated with a 4-d culture of Pa. wasatchensis WDCO4, and results were recorded over 8 d. When inoculated into carbohydrate-restricted DeMan, Rogosa, and Sharpe broth containing only sodium gluconate as the added substrate, Pa. wasatchensis WDC04 grew, confirming gluconate utilization. Of the 10 ratios used, Pa. wasatchensis WDC04 produced gas in 6 scenarios, with the most gas production resulting from the ratio of 100% sodium gluconate with no added ribose or galactose. It was confirmed that obligately heterofermentative nonstarter lactobacilli such as Pa. wasatchensis WDC04 can utilize sodium gluconate to produce CO2 gas. Addition of sodium gluconate to cheese thus becomes another risk factor for unwanted gas production and formation of slits and cracks.
The use of a sterilized product for washing cows' udders before milking may be useful to reduce or prevent Clostridium tyrobutyricum contamination, the main cause of the late-blowing defect in hard ...and semi-hard cheeses. The aim of this research was to evaluate the antibacterial efficacy of an experimental formula containing 15% condensed donkey milk (lysozyme content 825 mg/L). The antimicrobial activity of condensed milk was first evaluated in vitro, using the disk diffusion method, on the following microorganisms: Bacillus megaterium, Bacillus mojavensis, Clavibacter michiganensis, and Clostridium tyrobutyricum. These results were compared with the effects of 2 antibiotics, ampicillin (100 mg/mL) and kanamycin (50 mg/ mL), and a commercial pre-dipping formula. The results showed that the inhibitory activity of lysozyme from donkey milk on all the considered microorganisms was higher than that of the commercial product and similar to that of the 2 antibiotics. Next, the formula with lysozyme was compared with a commercial pre-dipping formula on 48 lactating cows (24 cows in each group). Skin tests were performed on teats before and after pre-dipping. Results showed that the formula with condensed milk significantly reduced the clostridial load detected on the skin of cows' teats before cleaning (−55.61% vs. −27.99%) and in the bulk milk of the experimental group compared with the control group with commercial product (−52.53% vs. −32.42%).
PDF
