The butyric acid fermentation, responsible for late blowing of cheese, is caused by the outgrowth in cheese of some species of Clostridium, resulting in texture and flavor defects and economical ...losses. The aim of this study was to evaluate the effectiveness of different antimicrobial compounds against vegetative cells and spores of C. tyrobutyricum, C. butyricum, C. beijerinckii and C. sporogenes strains isolated from cheeses with late blowing defect. Minimal inhibitory concentration (MIC) for reuterin, nisin, lysozyme and sodium nitrite were determined against Clostridium strains in milk and modified RCM (mRCM) after 7d exposure. Although the sensitivity of Clostridium to the tested antimicrobials was strain-dependent, C. sporogenes and C. beijerinckii generally had higher MIC values than the rest of Clostridium species. The majority of Clostridium strains were more resistant to antimicrobials in milk than in mRCM, and vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 0.51–32.5mM) and nisin (MIC values 0.05–12.5μg/ml) were able to inhibit the growth of vegetative cells and spores of all assayed Clostridium strains in milk and mRCM. Strains of C. tyrobutyricum exhibited the highest sensitivity to lysozyme (MIC values<0.20–400μg/ml) and sodium nitrite (MIC values 18.75–150μg/ml). These results suggest that reuterin and nisin, with a broad inhibitory activity spectrum against Clostridium spp. spores and vegetative cells, may be the best options to control Clostridium growth in dairy products and to prevent associated spoilage, such as late blowing defect of cheese. However, further studies in cheese would be necessary to validate this hypothesis.
•Clostridium sensitivity to antimicrobials was strain dependent and varied with the type of bacteria cell and growth media.•Reuterin and nisin, with the broadest anti-clostridial spectrum, inhibited all Clostridium strains.•Sodium nitrite and lysozyme had limited inhibitory effect against Clostridium strains.
In this study, reuterin-producing Lactobacillus reuteri INIA P572 was added to cheese as an adjunct culture together with 50 or 100 mM glycerol (required for reuterin production), with the aim of ...controlling Clostridium tyrobutyricum CECT 4011 growth and preventing the late blowing defect (LBD) of cheese caused by this strain. L. reuteri survived cheese manufacture and produced reuterin in situ, detected at 6 and 24 h. However, the produced reuterin was enough to inhibit the growth of Clostridium, showing undetectable spore counts from day 30 onward and, therefore, to prevent cheese LBD during ripening (60 d, 14 °C). The acidification of these cheeses was not affected, although from day 14 they showed significantly lower lactococci counts than cheese made only with the starter (control cheese). Cheeses with LBD showed lower levels of lactic acid than control cheese and the formation of propionic and butyric acids, but cheeses with reuterin showed the same organic acids profile than control cheese. The cheese made with L. reuteri and 100 mM glycerol showed a light pink colour, not observed in the cheese made with L. reuteri and 50 mM glycerol. These results demonstrated a potent anti-clostridial activity of reuterin produced in an actual food product like cheese, and proved to be a novel approach to prevent LBD of cheese.
•Clostridium tyrobutyricum CECT 4011 caused late blowing defect in cheese.•Reuterin-producing Lactobacillus reuteri was used as adjunct plus glycerol to control cheese blowing.•L. reuteri survived cheese manufacture and produced reuterin in situ.•Reuterin inhibited Clostridium growth and prevented cheese blowing.•Cheeses with L. reuteri and glycerol showed the same organic acid profile than control cheese.
In this study we evaluated the application of different high pressure (HP) treatments (200–500 MPa at 14 °C for 10 min) to industrial sized semi-hard cheeses on day 7, with the aim of controlling two ...Clostridium tyrobutyricum strains causing butyric acid fermentation and cheese late blowing defect (LBD). Clostridium metabolism and LBD appearance in cheeses were monitored by sensory (cheese swelling, cracks/splits, off-odours) and instrumental analyses (organic acids by HPLC and volatile compounds by SPME/GC–MS) after 60 days. Cheeses with clostridial spores HP-untreated and HP-treated at 200 MPa showed visible LBD symptoms, lower concentrations of lactic, citric and acetic acids, and higher levels of pyruvic, propionic and butyric acids and of 1-butanol, ethyl and methyl butanoate, and ethyl pentanoate than cheeses without spores. However, cheeses with clostridial spores and HP-treated at ≥ 300 MPa did not show LBD symptoms and their organic acids and volatile compounds profiles were comparable to those of their respective HP-treated control cheeses, despite HP treatments caused a low spore reduction. A decrease in C. tyrobutyricum spore counts was observed after curd pressing, which seems to indicate an early spore germination, suggesting that HP treatments ≥300 MPa were able to inactivate the emerged C. tyrobutyricum vegetative cells and, thereby, prevent LBD.
•High-pressure processing (HPP) offers new applications in cheese preservation.•Cheeses were made with spores of 2 potent Clostridium tyrobutyricum cheese spoilers.•HPP was used to control C. tyrobutyricum and late blowing defect (LBD).•HPP of 7-d-old cheeses at ≥ 300 MPa (14 °C, 10 min) prevented LBD at least for 60 d of ripening.
Clostridium tyrobutyricum is a bacteria of concern in the cheese industry, capable of surviving the manufacturing process and causing butyric acid fermentation and late blowing defect of cheese. In ...this work, we implement a method based on the cell wall-binding domain (CBD) of endolysin CTP1L, which detects C. tyrobutyricum, to monitor its evolution in cheeses challenged with clostridial spores and in the presence or absence of reuterin, an anti-clostridial agent. For this purpose, total bacteria were extracted from cheese samples and C. tyrobutyricum cells were specifically labelled with the CBD of CTP1L attached to green fluorescent protein (GFP), and detected by fluorescence microscopy. By using this GFP-CBD, germinated spores were visualized on day 1 in all cheeses inoculated with clostridial spores. Vegetative cells of C. tyrobutyricum, responsible for butyric acid fermentation, were detected in cheeses without reuterin from 30 d onwards, when LBD symptoms also became evident. The number of fluorescent Clostridium cells increased during ripening in the blowing cheeses. However, vegetative cells of C. tyrobutyricum were not detected in cheese containing the antimicrobial reuterin, which also did not show LBD throughout ripening. This simple and fast method provides a helpful tool to study the evolution of C. tyrobutyricum during cheese ripening.
•GFP-cell wall-binding domain (CBD) of endolysin CTP1L binds specifically to Clostridium.•Cheeses were made with C. tyrobutyricum spores and with or without reuterin.•Cheese bacteria were extracted and C. tyrobutyricum labelled with GFP-CBD-CTP1L.•Clostridial germinated spores and vegetative cells were detected in cheeses made with spores.•C. tyrobutyricum cells were not detected in cheeses with the antimicrobial reuterin.
Adjunct cultures originating from artisanal cheese environments may play an important role in recreating and developing traditional cheese flavours, thanks to their enzymatic activities, involved in ...different metabolic pathways that occur during cheese ripening. In this work, Ligilactobacillus salivarius SP36, a strain isolated from an old cheese seal, was added as an adjunct culture to the cheese’s raw milk, and its effect on the microbiological, physical–chemical and sensory characteristics of the cheese was studied. The use of L. salivarius SP36 in cheese manufacturing had no significant (p > 0.05) effect on the cheese microbiota, gross composition (fat percentage, protein, total solids, moisture and NaCl concentration), colour or texture of the cheese. However, L. salivarius SP36 increased (p < 0.01) the formation of 25 volatile compounds, including 10 esters, 1 aldehyde, 8 alcohols and 6 carboxylic acids. In addition, cheeses made with L. salivarius SP36 received higher scores (p < 0.01) for aroma intensity and quality than control cheeses. L. salivarius SP36 proved to be a good candidate as an adjunct culture for cheesemaking, since it improved the cheese flavour by making it more intense and recovering typical sensorial notes of traditional cheeses.
Streptococcus thermophilus INIA 463 became nisin-resistant after exposure in skim milk to subminimal inhibitory concentrations of nisin (1–3 IU/ml) for less than 2 h. Addition of 20 IU/ml caused a 4 ...log unit decrease in
S. thermophilus counts of a culture not exposed previously to nisin, whereas no decrease was observed in the culture exposed to nisin for 2 h. Transfer of immunity genes as responsible for nisin resistance was discarded. The presence of extracellular or intracellular specific nisin-degrading enzymes was not detected in the nisin-resistant variant of
S. thermophilus INIA 463. Nisin resistance was caused by the induction of a resistance mechanism. Transmission electron microscopy (TEM) revealed that the nisin-resistant variant of
S. thermophilus INIA 463 had a thickened cell wall compared to the wild strain. Resistance to nisin was lost after one transfer (4 h growth) in nisin-free skim milk.
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.
Raw goat milk collected at 11 Andalusian dairy plants was used to individually manufacture cheeses without starter culture addition. Microbial groups were monitored during ripening for 60 days. A ...total of 1442 lactic acid bacteria (LAB) isolates randomly chosen were identified by molecular methods. Counts of mesophilic LAB exceeded 9 log cfu g−1 on day 1 and decreased 0.5 log units during ripening whereas counts of lactobacilli and leuconostocs increased and those of enterococci remained constant. Thirty-three LAB species belonging to eight different genera were isolated, with higher biodiversity on day 1 (26 species) than on day 60 (20 species). Structural genes coding for nisin, lacticin 481, lactococcin B, six plantaricins and five enterocins were detected in LAB isolates. The information obtained on the technological properties of selected isolates may be useful in developing specific starters for goat milk cheeses, capable of maintaining their distinctive sensory characteristics.
The effect of the biopreservation system formed by Lactobacillus reuteri INIA P572, a reuterin-producing strain, and glycerol (required for reuterin production), on the volatile fraction, aroma and ...odour of industrial sized semi-hard ewe milk cheese (Castellano type) was investigated over a 3-month ripening period. The volatile compounds were extracted and analyzed by SPME-GC-MS and cheese odour and aroma profiles were studied by descriptive sensory analysis. Control cheese was made only with a mesophilic starter and experimental cheeses with L. reuteri were made with and without glycerol. The addition of L. reuteri INIA P572 to milk enhanced the formation of six volatile compounds. Despite the changes in the volatile compounds profile, the use of L. reuteri INIA P572 did not noticeably affect the sensory characteristics of cheese. On the other hand, the addition of L. reuteri INIA P572 coupled with 30mM glycerol enhanced the formation of twelve volatile compounds, but decreased the formation of five ones. The use of the biopreservation system did not affect overall odour and aroma quality of cheese although it resulted in a significant decrease of the odour intensity scores. In addition, this cheese received significant higher scores for “cheesy” aroma and significant lower scores for the aroma attributes “milky”, “caramel” and “yogurt-like”. The first two axes of a principal component analysis (PCA) performed for selected volatile compounds and sensory characteristics, accounting for 75% of the variability between cheeses, separated cheeses made with L. reuteri INIA P572 and glycerol from the rest of cheeses, and also differentiated control cheese from cheeses made with L. reuteri INIA P572 from day 60 onward. Our results showed that the reuterin-producing L. reuteri INIA P572 strain, when coupled with glycerol, may be a suitable biopreservation system to use in cheese without affecting odour and aroma quality.
•The effect of L. reuteri with and without glycerol on the volatile fraction of cheese was studied.•The addition of L. reuteri enhanced the formation of some volatile compounds.•L. reuteri did not noticeably affect the sensory characteristics of cheese.•The use of L. reuteri with glycerol modified the formation of several volatile compounds.•L. reuteri with glycerol did not affect odour and aroma quality but decreased odour intensity.
The volatile fraction of 30 Iberian dry-cured hams of different physicochemical characteristics and the effect of high pressure processing (HPP) at 600MPa on volatile compounds were investigated. ...According to the analysis of variance carried out on the levels of 122 volatile compounds, intramuscular fat content influenced the levels of 8 benzene compounds, 5 carboxylic acids, 2 ketones, 2 furanones, 1 alcohol, 1 aldehyde and 1 sulfur compound, salt concentration influenced the levels of 1 aldehyde and 1 ketone, salt-in-lean ratio had no effect on volatile compounds, and water activity influenced the levels of 3 sulfur compounds, 1 alcohol and 1 aldehyde. HPP-treated samples of Iberian ham had higher levels of 4 compounds and lower levels of 31 compounds than untreated samples. A higher influence of HPP treatment on volatile compounds than physicochemical characteristics was observed for Iberian ham. Therefore, HPP treatment conditions should be optimized in order to diminish its possible effect on Iberian ham odor and aroma characteristics.
•Iberian hams of different chemical composition were HPP-treated at 600MPa.•Intramuscular fat influenced the levels of 20 individual volatile compounds.•HPP treatment decreased the levels of 31 individual volatile compounds.•HPP treatment increased the levels of 2 sulfur compounds, 1 aldehyde and 1 alkane.
