Mechanisms of Clostridium tyrobutyricum removal through natural creaming of milk: A microscopy study

Abstract

Clostridium tyrobutyricum is the main spoilage agent of late blowing defect (LBD) in Grana Padano and Parmigiano-Reggiano cheeses; LBD is characterized by openings and holes and is sometimes accompanied by cracks and an undesirable flavor. Even a very few spores remaining in the cheese curd may cause LBD; thus, it is essential to eradicate them during milk natural creaming. By this process, most of the bacteria, somatic cells, and spores rise to the top of the milk, together with the fat globules, and are removed with the cream. Previous studies suggested that milk immunoglobulins mediate the interactions between fat globules and bacteria that occur upon creaming but no direct evidence for this has been found. Moreover, other physical chemical interactions could be involved; for example, physical entrapment of spores among globule clusters. To maximize the efficiency of the natural creaming step in removing Cl. tyrobutyricum, it is essential to understand the nature of spore-globule interactions. With this aim, raw milk was contaminated with spores of Cl. tyrobutyricum before going to creaming overnight at 8°C, after which spore and bacteria removal was >90%. The obtained cream was analyzed by light interference contrast and fluorescence microscopy and by transmission electron microscopy (TEM). Results showed that most of the vegetative cells and spores, which were stained with malachite green before addition to milk, adhered tightly to the surface of single fat globules, the membranes of which appeared heterogeneous when stained with the fluorescent dye DilC18(3)-DS. Using the same dye, we observed transient and persistent interactions among globules, with formation of clusters of different sizes and partial coalescence of adhering membranes. Transmission electron microscopy examination of replicates of freeze-fractured cream allowed us to observe tight adhesion of spores to fat globules. Ultrathin sections revealed that this adhesion is mediated by an amorphous, slightly electron-opaque material, sometimes granular in appearance. Bacteria also adhered to different fat globules, linking them together, which suggests that adhesion was strong enough to maintain a stable contact. Although we cannot exclude physical entrapment of bacteria among fat globule clusters, we show for the first time that most of the bacteria are adhered to fat globules by an electron-opaque material whose nature has yet to be determined. Immunoglobulins are certainly the best candidates for adhesion but other compounds may be involved.

Clostridium Tyrobutyricum Protect Intestinal Barrier Function from LPS-Induced Apoptosis via P38/JNK Signaling Pathway in IPEC-J2 Cells

“…C.t , a Gram-positive and strictly anaerobic bacterium, has received much attention from the dairy and chemical synthesis industries because of its efficiency in butyric acid production [19, 20]. However, the role of C.t in animal models or mammalian cells has not yet been reported.…”

New insight on crystal and spot development in hard and extra-hard cheeses: Association of spots with incomplete aggregation of curd granules

“…Based on previous cheese structure studies (Ong et al, 2010;Schrader, 2012;D'Incecco et al, 2015), various microscopy techniques and different dyes (light and fluorescence, confocal, confocal micro-Raman, and transmission electron microscopy) were used in combination with chemical data to achieve an unambiguous characterization of these particles in cheese. Our ultimate goal was to formulate a hypothesis on the origin of these structures as they appear in hard and extra-hard cheeses.…”

A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy

“…Due to their small size, Clostridium spores are often observed by conventional electron microscopy, either in scanning or transmission mode, that has a high resolution (D'Incecco et al, 2015(D'Incecco et al, , 2018Bassi et al, 2009;El Jaam et al, 2017;Trunet et al, 2017). A disadvantage of this technique is that it requires specific skills, since sample preparation procedures for specimen fixation have to be used This approach, however, involves dead vegetative cells and spores thus no in vivo studies can be performed.…”

“…incubated at 37°C in an anaerobic jar equipped with a anaerobic reagent kit (Anaerocult A,VWR, Leuven, Belgium). Spore suspension was obtained by inoculating 1% of a 24-h culture of C. tyrobutyricum culture in RCM broth, as previously described by D'Incecco et al (2015). The culture in the anaerobic jar was maintained at 37°C for 4 d and for a further 15 d at room temperature.…”