The occurrence of late blowing defects in cheese produces negative effects on the quality and commercial value of the product. In this work, we verified whether the addition of raw jenny milk to bulk ...cow milk reduced the late blowing defects in semihard cheeses. During cheesemaking, different aliquots of jenny milk were poured into 2 groups of 4 vats, each containing a fixed amount of cow milk. A group of cheeses was created by deliberately contaminating the 4 vats with approximately 3 log10 cfu/mL milk of Clostridium tyrobutyricum CLST01. The other 4 vats, which were not contaminated, were used for a second group of cheeses. After 120 d of ripening, some physical, chemical, and microbiological parameters were evaluated on the obtained semihard cheeses. Differences in sensory properties among cheeses belonging to the uncontaminated group were evaluated by 80 regular consumers of cheese. Our results showed that the increasing addition of jenny milk to cow milk led to a reduction of pH and total bacterial count in both cheese groups, as well as C. tyrobutyricum spores that either grew naturally or artificially inoculated. We observed a progressive reduction of the occurrence of late blowing defects in cheese as consequence of the increasing addition of jenny milk during cheese making. Moreover, the addition of jenny milk did not affect the acceptability of the product, as consumers found no difference among cheeses concerning sensorial aspects. In conclusion, the important antimicrobial activity of lysozyme contained in jenny milk has been confirmed in the current research. It is recommend for use as a possible and viable alternative to egg lysozyme for controlling late blowing defects in cheese.
Summary
Clostridium tyrobutyricum causes swelling, cracks and off‐flavours of cheeses (late blowing defect, LBD) due to butyric acid fermentation. To control this spoilage bacterium, we investigated ...the use of nisinogenic Lactococcus lactis subsp. lactis INIA 415 as starter in cheeses contaminated with C. tyrobutyricum spores. Control cheese made with spores showed LBD after 14 days of ripening. However, in cheese made with the bacteriocin producer and spores, in which (unlike control cheese) bacteriocin activity was detected throughout ripening, LBD occurred after 21 days. At this stage, level of lactic acid was 1.22‐fold higher (P < 0.01) and concentrations of propionic and butyric acids were 2.15‐ and 2.32‐fold, respectively, lower (P < 0.01) in cheese made with the nisin producer than in control cheese, according to the less pronounced spoilage symptoms showed by the former cheese. The bacteriocin producer delayed the appearance of LBD, although it cannot arrest completely C. tyrobutyricum growth.
Late blowing defect is one of the major causes of microbial spoilage in semi‐hard and hard cheeses, resulting in texture and flavour defects, with an unfavourable economic impact on cheese production. In this study, the use of L. lactis subsp. lactis INIA 415 in cheese manufacture delayed late blowing defect appearance and diminished spoilage symptoms.
Late blowing, a microbiological spoilage in hard and semi-hard cheese caused by
Clostridium
spores in raw milk, results in high economic losses for cheese producers. This study compared the ...sensitivity of the newly developed multiplex qPCR method which employing novel oligonucleotide primers and fluorescent TaqMan probes, and the culture-based most probable number (MPN) method in detecting the late blowing agent Clostridium species in traditional Turkish cheese. A total of 50 naturally contaminated cheese samples obtained from producers were analysed by both methods.
Clostridium tyrobutyricum
was the most common species occurring in 74% of the cheese samples, followed by
C. butyricum
and
C. sporogenes
occurring in 50% and 16% of the samples, respectively. The results of the two methods were consistent in 42 out of the 50 (84%) cheese samples. Our results indicate that the multiplex qPCR method is more sensitive than the MPN method. The multiplex qPCR method provided a favourable alternative to traditional cultural methods. This alternative molecular method has great potential in the laboratory and in the field for the rapid detection of late blowing of cheese samples.
An obligatory heterofermentative lactic acid bacterium, Lactobacillus wasatchii sp. nov., isolated from gassy Cheddar cheese was studied for growth, gas formation, salt tolerance, and survival ...against pasteurization treatments at 63°C and 72°C. Initially, Lb. wasatchii was thought to use only ribose as a sugar source and we were interested in whether it could also utilize galactose. We conducted experiments to determine the rate and extent of growth and gas production in carbohydrate-restricted (CR) de Man, Rogosa, and Sharpe (MRS) medium under anaerobic conditions with various combinations of ribose and galactose at 12, 23, and 37°C, with 23°C being the optimum growth temperature of Lb. wasatchii among the 3 temperatures studied. When Lb. wasatchii was grown on ribose (0.1, 0.5, and 1%), maximum specific growth rates (µmax) within each temperature were similar. When galactose was the only sugar, compared with ribose, µmax was 2 to 4 times lower. At all temperatures, the highest final cell densities (optical density at 640nm) of Lb. wasatchii were achieved in CR-MRS plus 1% ribose, 0.5% ribose and 0.5% galactose, or 1% ribose and 1% galactose. Similar µmax values and final cell densities were achieved when 50% of the ribose in CR-MRS was substituted with galactose. Such enhanced utilization of galactose in the presence of ribose to support bacterial growth has not previously been reported. It appears that Lb. wasatchii co-metabolizes ribose and galactose, utilizing ribose for energy and galactose for other functions such as cell wall biosynthesis. Co-utilization of both sugars could be an adaptation mechanism of Lb. wasatchii to the cheese environment to efficiently ferment available sugars for maximizing metabolism and growth. As expected, gas formation by the heterofermenter was observed only when galactose was present in the medium. Growth experiments with MRS plus 1.5% ribose at pH 5.2 or 6.5 with 0, 1, 2, 3, 4, or 5% NaCl revealed that Lb. wasatchii is able to grow under salt and pH conditions typical of Cheddar cheese (4 to 5% salt-in-moisture, pH ~5.2). Finally, we found that Lb. wasatchii cannot survive low-temperature, long-time pasteurization but survives high-temperature, short-time (HTST) laboratory pasteurization, under which a 4.5 log reduction occurred. The ability of Lb. wasatchii to survive HTST pasteurization and grow under cheese ripening conditions implies that the presence of this nonstarter lactic acid bacterium can be a serious contributor to gas formation and textural defects in Cheddar cheese.
The suitability of the biopreservation system formed by reuterin-producing L. reuteri INIA P572 and glycerol (required for reuterin production) to prevent late blowing defect (LBD) was evaluated in ...industrial sized semi-hard ewe milk cheese contaminated with Clostridium tyrobutyricum INIA 68, a wild strain isolated from a LBD cheese. For this purpose, six batches of cheese were made (three with and three without clostridial spores): control cheeses with lactococci starter, cheeses with L. reuteri as adjunct, and cheeses with L. reuteri and 30 mM glycerol. Spores of C. tyrobutyricum INIA 68 germinated during pressing of cheese curd, causing butyric acid fermentation in cheese after 30 d of ripening. The addition of L. reuteri, without glycerol, enhanced the symptoms and the formation of volatile compounds associated with LBD. When glycerol was added to cheese milk contaminated with C. tyrobutyricum, L. reuteri was able to produce reuterin in cheese resulting in cheeses with a uniform cheese matrix and a volatile profile similar to cheese made with L. reuteri and glycerol (without spores). Accordingly, L. reuteri INIA P572 coupled with glycerol seems a novel biopreservation system to inhibit Clostridium growth and prevent LBD by means of in situ reuterin production.
•C. tyrobutyricum INIA 68 caused late blowing defect (LBD) in industrial size cheese.•Reuterin-producing L. reuteri was used as adjunct plus glycerol to control LBD.•L. reuteri coupled with glycerol produced reuterin in situ in the cheese paste.•Reuterin inhibited Clostridium growth and prevented LBD in cheese for at least 3 months.•Volatile profile of cheese with reuterin and Clostridium was not affected.
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.
Clostridium tyrobutyricum, a Gram-positive, anaerobic, spore-forming bacterium, is considered as one of the main causative agents for spoilage of hard and semihard cheeses. Growth of C. tyrobutyricum ...in cheese is critically influenced by ripening temperature and time, pH, salt and lactic acid concentration, moisture and fat content, and the presence of other microorganisms. Previous studies revealed high intraspecies diversity of C. tyrobutyricum strains and variable tolerance toward pH, temperatures, and salt concentrations. These findings indicate that strain-dependent characteristics may be relevant to assess the risk for cheese spoilage if clostridial contamination occurs. In this study, we aimed to compare the phenotypes of 12 C. tyrobutyricum strains which were selected from 157 strains on the basis of genotypic and proteotypic variability. The phenotypic analysis comprised the assessment of gas production and organic acid concentrations in an experimental cheese broth incubated at different temperatures (37, 20, and 14 °C). For all tested strains, delayed gas production at lower incubation temperatures and a strong correlation between gas production and the change in organic acid concentrations were observed. However, considering the time until gas production was visible at different incubation temperatures, a high degree of heterogeneity was found among the tested strains. In addition, variation among replicates of the same strain and differences due to different inoculum levels became evident. This study shows, that, among other factors, strain-specific germination and growth characteristics should be considered to evaluate the risk of cheese spoilage by C. tyrobutyricum.
A novel slow-growing, obligatory heterofermentative, nonstarter lactic acid bacterium (NSLAB), Lactobacillus wasatchensis WDC04, was studied for growth and gas production in Cheddar-style cheese made ...using Streptococcus thermophilus as the starter culture. Cheesemaking trials were conducted using S. thermophilus alone or in combination with Lb. wasatchensis deliberately added to cheese milk at a level of ~10(4) cfu/mL. Resulting cheeses were ripened at 6 or 12°C. At d 1, starter streptococcal numbers were similar in both cheeses (~10(9) cfu/g) and fast-growing NSLAB lactobacilli counts were below detectable levels (<10(2) cfu/g). As expected, Lactobacillus wasatchensis counts were 3×10(5) cfu/g in cheeses inoculated with this bacterium and below enumeration limits in the control cheese. Starter streptococci decreased over time at both storage temperatures but declined more rapidly at 12°C, especially in cheese also containing Lb. wasatchensis. Populations of fast-growing NSLAB and the slow-growing Lb. wasatchensis reached 5×10(7) and 2×10(8) cfu/g, respectively, after 16 wk of storage at 12°C. Growth of NSLAB coincided with a reduction in galactose concentration in the cheese from 0.6 to 0.1%. Levels of galactose at 6°C had similar decrease. Gas formation and textural defects were only observed in cheese with added Lb. wasatchensis ripened at 12°C. Use of S. thermophilus as starter culture resulted in galactose accumulation that Lb. wasatchensis can use to produce CO2, which contributes to late gas blowing in Cheddar-style cheeses, especially when the cheese is ripened at elevated temperature.
Lactococcal and enterococcal adjuncts with verified anticlostridial activity were successfully applied in the technology process of Dutch‐type low‐scalded cheese. The microbial status of experimental ...cheeses, physicochemical parameters and late blowing defects were evaluated for batches inoculated with Clostridium tyrobutyricum, and compared with noninoculated controls and the application of nisin. Two strains (CCDM 731 and CCDM 71) belonging to the species Lactococcus lactis subsp. lactis had significant anticlostridial effects which were comparable to nisin. Regardless of the noneffective enterococci adjunct, both lactococcal strains effectively supported the fermentation and ripening process in combination with the commercial starter itself or in combination with nisin.
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