Abstract
A total of 130 strains of lactic acid bacteria (starter lactic acid bacteria -SLAB- and non-starter lactic acid bacteria -NSLAB-) were isolated from milk, whey and cheese. Anti-clostridial activity was detected in 56 isolates against Clostridium tyrobutyricum ATCC 25755. Seven strains with the highest inhibitory activity were selected to determine their inhibitory mechanisms. Anti-clostridial activity of strains Streptococcus macedonicus 23, S. macedonicus 24, Lactobacillus casei/paracasei 29 and Lactobacillus rhamnosus 104 was due to organic acids production. Inhibitory mechanisms of strains L. casei 26 and 95 were acid production, bacteriocin and hydrogen peroxide. While anti-clostridial activity of Lactobacillus delbrueckii subsp. bulgaricus 76 was due to acid and hydrogen peroxide production. Cell-free supernatants obtained from strains 26, 95 and 76 conserved their anti-clostridial activity after thermal treatments at 70 and 100 °C. Supernatants obtained from bacteriogenic strains 26 and 95 were analyzed by RP-HPLC. RP-HPLC fractions with anti-clostridial activity were analyzed by mass spectrometry (MALDI-TOF). A peptide with molecular weight of 1162.54 Da was detected in HPLC fraction of L. casei 95. No results were obtained for MALDI-TOF analysis of another HPLC fractions. Based on the thermostability and low molecular weight, L. casei 95 anti-clostridial peptide would belong to a class I or II bacteriocin according to Gram-positive bacteria antimicrobial peptides classification.
Highlights
Late blowing defect is one of the major causes of spoilage in semi-hard and hard ripened cheeses
Most isolates belonged to the Lactobacillus casei Group (66.1%) (L. casei, L. paracasei and L. rhamnosus), followed by Lactobacillus delbrueckii (7.1%), Streptococcus macedonicus (7.1%), Pediococcus pentosaceus (5.4%) and Enterococcus faecalis (5.4%)
Artisan cheese and whey samples analyzed in this work, 56 lactic acid bacteria (LAB) strains with inhibitory activity against C. tyrobutyricum ATCC 25755 were isolated
Summary
Late blowing defect is one of the major causes of spoilage in semi-hard and hard ripened cheeses. Clostridium metabolism of lactate includes the production of organic acids (butyric and acetic), carbon dioxide and hydrogen, resulting in irregular eyes, splits and cracks in the cheese, as well as undesirable aroma and flavors (Alvenäs, 2015; Calamari et al, 2018). Clostridium tyrobutyricum is considered the primary cause of late blowing in cheese, other clostridial species such as Clostridium sporogenes, Clostridium beijerinckii and Clostridium butyricum have been shown to significantly contribute to the appearance of this defect (Garde et al, 2013; Bermúdez et al, 2016). In the cheesemaking process many strategies have been proposed to prevent late blowing defect like addition of lysozyme and physical spores removal by bactofugation or microfiltration. The reduction in spore numbers achieved by bactofugation may be insufficient to prevent late blowing defect if spore load is very high. LAB are considered as natural preservatives due to the production of antimicrobial metabolites such as organic acids (lactic and acetic), hydrogen peroxide, acetaldehyde, diacetyl, acetoin, fatty acids, carbon dioxide, ethanol, Damino acids, reutericyclin, reuterin and bacteriocins (Özogul and Hamed, 2018; García-Cano et al, 2019)
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