Butyric Acid Generation by Clostridium tyrobutyricum from Low-Moisture Anhydrous Ammonia (LMAA) Pretreated Sweet Sorghum Bagasse.

Sweet sorghum bagasse (SSB) is an under-utilized feedstock for biochemical conversion to biofuels or high value chemicals. One such chemical that can be generated biochemically and applied to a wide array of industries from pharmaceuticals to the production of liquid transportation fuels is butyric acid. This work investigated cultivating the butyric acid producing strain Clostridium tyrobutyricum ATCC 25755 on low-moisture anhydrous ammonia (LMAA) pretreated SSB. Pretreated SSB hydrolysate was detoxified and supplemented with urea for shake flask batch fermentation to show that up to 11.4 g/L butyric acid could be produced with a selectivity of 87% compared to other organic acids. Bioreactor fermentation with pH control showed high biomass growth, but a similar output of 11.3 g/L butyric acid was achieved. However, the butyric acid productivity increased to 0.251 g/L∙hr with a butyric acid yield of 0.29 g/g sugar consumed. This butyric acid output represented an 83% theoretical yield. Further improvements in butyric acid titer and yield can be achieved by optimizing nutrient supplementation and incorporating fed-batch fermentation processing of pretreated SSB hydrolysate. Construction of ZGO:Sr NR- and ZGC@PDA NP-driven ratiometric aptasensor for CEA detection.

The effect of Clostridium tyrobutyricum Spo0A overexpression in the intestine of mice.

Clostridium tyrobutyricum shows probiotic properties and can affect the composition of gut microbiota and regulate the intestinal immune system. Compared with other probiotics, this spore-producing bacterium shows unparalleled advantages in commercial production. In addition to being resistant to extreme living environments for extended periods, its endophytic spores are implicated in inhibiting cancer cell growth. We speculated that C. tyrobutyricum spores can also promote gut health, which mean it can maintain intestinal homeostasis. To date, the beneficial effects of C. tyrobutyricum spores on gut health have not been reported. In this study, a Spo0A-overexpressing C. tyrobutyricum strain was developed to increase spore production, and its probiotic effects on the gut were assessed. Compared with the wild-type, the engineered strain showed significantly increased sporulation rates. Mice administered with the engineered strain exhibited enhanced intestinal villi and the villus height/crypt depth ratio, weight gain and improved Firmicutes/Bacteroidetes ratio to facilitate intestinal homeostasis. This study demonstrated for the first time that enhanced spore production in C. tyrobutyricum can improve intestinal homeostasis, which is advantageous for its commercial application in food and pharmaceutical industry.

Development of an in vivo fluorescence based gene expression reporter system for Clostridium tyrobutyricum.

C. tyrobutyricum, an acidogenic Clostridium, has aroused increasing interest due to its potential to produce biofuel efficiently. However, construction of recombinant C. tyrobutyricum for enhanced biofuel production has been impeded by the limited genetic engineering tools. In this study, a flavin mononucleotide (FMN)-dependent fluorescent protein Bs2-based gene expression reporter system was developed to monitor transformation and explore in vivo strength and regulation of various promoters in C. tyrobutyricum and C. acetobutylicum. Unlike green fluorescent protein (GFP) and its variants, Bs2 can emit green light without oxygen, which makes it extremely suitable for promoter screening and transformation confirmation in organisms grown anaerobically. The expression levels of bs2 under thiolase promoters from C. tyrobutyricum and C. acetobutylicum were measured and compared based on fluorescence intensities. The capacities of the two promoters in driving secondary alcohol dehydrogenase (adh) gene for isopropanol production in C. tyrobutyricum were distinguished, confirming that this reporter system is a convenient, effective and reliable tool for promoter strength assay and real time monitoring in C. tyrobutyricum, while demonstrating the feasibility of producing isopropanol in C. tyrobutyricum for the first time.

Abatement of the clostridial load in the teats of lactating cows with lysozyme derived from donkey milk.

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%).

Butanol and butyric acid production from Saccharina japonica by Clostridium acetobutylicum and Clostridium tyrobutyricum with adaptive evolution.

Optimal conditions of hyper thermal (HT) acid hydrolysis of the Saccharina japonica was determined to a seaweed slurry content of 12% (w/v) and 144 mM H2SO4 at 160 °C for 10 min. Enzymatic saccharification was carried out at 50 °C and 150 rpm for 48 h using the three enzymes at concentrations of 16 U/mL. Celluclast 1.5 L showed the lowest half-velocity constant (Km) of 0.168 g/L, indicating a higher affinity for S. japonica hydrolysate. Pretreatment yielded a maximum monosaccharide concentration of 36.2 g/L and 45.7% conversion from total fermentable monosaccharides of 79.2 g/L with 120 g dry weight/L S. japonica slurry. High cell densities of Clostridium acetobutylicum and Clostridium tyrobutyricum were obtained using the retarding agents KH2PO4 (50 mM) and NaHCO3 (200 mM). Adaptive evolution facilitated the efficient use of mixed monosaccharides. Therefore, adaptive evolution and retarding agents can enhance the overall butanol and butyric acid yields from S. japonica.

Use of fluorescent CTP1L endolysin cell wall-binding domain to study the evolution of Clostridium tyrobutyricum during cheese 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.

Effect of hot season on blood parameters, fecal fermentative parameters, and occurrence of Clostridium tyrobutyricum spores in feces of lactating dairy cows.

High temperature influences rumen and gut health, passage rate, and diet digestibility, with effects on fermentative processes. The main aim of the study was to investigate the effect of hot season on hindgut fermentation, the occurrence of Clostridium tyrobutyricum spores in bovine feces, and on their relationship with metabolic conditions in dairy cows producing milk used for Grana Padano cheese. The study was carried out on 7 dairy farms located in the Po Valley (Italy), involving 1,950 Italian Friesian dairy cows. The study was carried out from November 2013 till the end of July 2014. Temperature and relative humidity were recorded daily by weather stations. Constant management conditions were maintained during the experimental period. Feed and diet characteristics, metabolic conditions, and fecal characteristics were recorded in winter (from late November 2013 to the end of January 2014), spring (from April to May 2014), and summer (July 2014) season. In each season, blood samples were collected from 14 multiparous lactating dairy cows per herd to measure biochemical indices related to energy, protein, and mineral metabolism, as well as markers of inflammation and some enzyme activities. Fecal samples were also collected and measurements of moisture, pH and volatile fatty acids (VFA) were performed. The DNA extracted and purified from fecal samples was used to detect Clostridium tyrobutyricum spores in a quantitative real-time PCR assay. The daily mean temperature-humidity index was 40.7 ± 4.6 (range 25 to 55), 61.2 ± 3.7 (range 39 to 77), and 70.8 ± 3.2 (range 54 to 83) in winter, spring, and summer, respectively. Total VFA concentration in feces progressively decreased from winter to summer. The seasonal changes of acetate and propionate followed the same trend of total VFA; conversely, butyrate did not show any difference between seasons, and its molar proportion was greater in summer compared with winter. A greater occurrence of Cl. tyrobutyricum spores in summer compared with the other seasons was observed. The plasma concentrations of glucose, urea, albumin, Ca, Mg, Cl, Zn, and alkaline phosphatase activity were lower in summer compared with winter, whereas the opposite occurred for bilirubin and Na. Our results show that summer season, through direct and indirect effect of heat stress, affected fecal fermentative parameters and hindgut buffering capacity, and was responsible for the increasing occurrence of Cl. tyrobutyricum spores in feces.

Acetone-Butanol-Ethanol Production from Waste Seaweed Collected from Gwangalli Beach, Busan, Korea, Based on pH-Controlled and Sequential Fermentation Using Two Strains.

The optimal conditions for acetone-butanol-ethanol (ABE) production were evaluated using waste seaweed from Gwangalli Beach, Busan, Korea. The waste seaweed had a fiber and carbohydrate, content of 48.34%; these are the main resources for ABE production. The optimal conditions for obtaining monosaccharides based on hyper thermal (HT) acid hydrolysis of waste seaweed were slurry contents of 8%, sulfuric acid concentration of 138 mM, and treatment time of 10 min. Enzymatic saccharification was performed using 16 unit/mL Viscozyme L, which showed the highest affinity (Km = 1.81 g/L). After pretreatment, 34.0 g/L monosaccharides were obtained. ABE fermentation was performed with single and sequential fermentation of Clostridium acetobutylicum and Clostridium tyrobutyricum; this was controlled for pH. A maximum ABE concentration of 12.5 g/L with YABE 0.37 was achieved using sequential fermentation with C. tyrobutyricum and C. acetobutylicum. Efficient ABE production from waste seaweed performed using pH-controlled culture broth and sequential cell culture.

Development of a specific fluorescent phage endolysin for in situ detection of Clostridium species associated with cheese spoilage.

Late blowing defect (LBD) is a major cause of spoilage in cheeses, caused by the growth of Clostridium spp. in the cheese matrix. We investigated the application of CTP1L, a bacteriophage endolysin active against Clostridium tyrobutyricum, and its enzymatically active and cell wall-binding domains (EAD and CBD) attached to green fluorescent protein (GFP) to detect dairy-related Clostridium species by fluorescence microscopy. GFP-CTP1L and GFP-CBD demonstrated specificity for Clostridium spp. by labelling 15 and 17 of 20 Clostridium strains, respectively, but neither bound to other members of the cheese microbiota. However, GFP-EAD did not label any Clostridium strain tested. Unexpectedly, GFP-CTP1L and GFP-CBD were also able to bind to clostridial spores. In addition, GFP-CBD allowed us to visualize the vegetative cells of C. tyrobutyricum directly in the matrix of a LBD cheese. Site-directed mutants of GFP-CTP1L and GFP-CBD were made to examine the amino acids involved in binding and oligomer formation. Oligomerization was not essential for binding, but specific mutations in the CBD which affected oligomer formation also affected binding and lytic activity. We conclude that GFP-CTP1L and GFP-CBD could be good biomarkers for rapid detection of Clostridium spores in milk, so measures can be taken for the prevention of LBD in cheese, and also provide effective tools to study the development of Clostridium populations during cheese ripening.

Fermentative hydrogen production from Jerusalem artichoke by Clostridium tyrobutyricum expressing exo-inulinase gene.

Clostridium tyrobutyricum ATCC25755 has been reported as being able to produce significant quantities of hydrogen. In this study, the exo-inulinase encoding gene cloned from Paenibacillus polymyxa SC-2 was into the expression plasmid pSY6 and expressed in the cells of C. tyrobutyricum. The engineered C. tyrobutyricum strain efficiently fermented the inulin-type carbohydrates from Jerusalem artichoke, without any pretreatment being necessary for the production of hydrogen. A comparatively high hydrogen yield (3.7 mol/mol inulin-type sugar) was achieved after 96 h in a batch process with simultaneous saccharification and fermentation (SSF), with an overall volumetric productivity rate of 620 ± 60 mL/h/L when the initial total sugar concentration of the inulin extract was increased to 100 g/L. Synthesis of inulinase in the batch SSF culture was closely associated with strain growth until the end of the exponential phase, reaching a maximum activity of 28.4 ± 0.26 U/mL. The overall results show that the highly productive and abundant biomass crop Jerusalem artichoke can be a good substrate for hydrogen production, and that the application of batch SSF for its conversion has the potential to become a cost-effective process in the near future.

Industrial-scale application of Lactobacillus reuteri coupled with glycerol as a biopreservation system for inhibiting Clostridium tyrobutyricum in semi-hard ewe milk 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.

Application of high pressure processing for controlling Clostridium tyrobutyricum and late blowing defect on semi-hard 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.

The acquisition of Clostridium tyrobutyricum mutants with improved bioproduction under acidic conditions after two rounds of heavy-ion beam irradiation.

End-product inhibition is a key factor limiting the production of organic acid during fermentation. Two rounds of heavy-ion beam irradiation may be an inexpensive, indispensable and reliable approach to increase the production of butyric acid during industrial fermentation processes. However, studies of the application of heavy ion radiation for butyric acid fermentation engineering are lacking. In this study, a second (12)C(6+) heavy-ion irradiation-response curve is used to describe the effect of exposure to a given dose of heavy ions on mutant strains of Clostridium tyrobutyricum. Versatile statistical elements are introduced to characterize the mechanism and factors contributing to improved butyric acid production and enhanced acid tolerance in adapted mutant strains harvested from the fermentations. We characterized the physiological properties of the strains over a large pH value gradient, which revealed that the mutant strains obtained after a second round of radiation exposure were most resistant to harsh external pH values and were better able to tolerate external pH values between 4.5 and 5.0. A customized second round of heavy-ion beam irradiation may be invaluable in process engineering.

Genomic approach to studying nutritional requirements of Clostridium tyrobutyricum and other Clostridia causing late blowing defects.

Clostridium tyrobutyricum is the main microorganism responsible for the late blowing defect in hard and semi-hard cheeses, causing considerable economic losses to the cheese industry. Deeper knowledge of the metabolic requirements of this microorganism can lead to the development of more effective control approaches. In this work, the amino acids and B vitamins essential for sustaining the growth of C. tyrobutyricum were investigated using a genomic approach. As the first step, the genomes of four C. tyrobutyricum strains were analyzed for the presence of genes putatively involved in the biosynthesis of amino acids and B vitamins. Metabolic pathways could be reconstructed for all amino acids and B vitamins with the exception of biotin (vitamin B7) and folate (vitamin B9). The biotin pathway was missing the enzyme amino-7-oxononanoate synthase that catalyzes the condensation of pimeloyl-ACP and l-alanine to 8-amino-7-oxononanoate. In the folate pathway, the missing genes were those coding for para-aminobenzoate synthase and aminodeoxychorismate lyase enzymes. These enzymes are responsible for the conversion of chorismate into para-aminobenzoate (PABA). Two C. tyrobutyircum strains whose genome was analyzed in silico as well as other 10 strains isolated from cheese were tested in liquid media to confirm these observations. 11 strains showed growth in a defined liquid medium containing biotin and PABA after 6-8 days of incubation. No strain showed growth when only one or none of these compounds were added, confirming the observations obtained in silico. Furthermore, the genome analysis was extended to genomes of single strains of other Clostridium species potentially causing late blowing, namely Clostridium beijerinckii, Clostridium sporogenes and Clostridium butyricum. Only the biotin biosynthesis pathway was incomplete for C. butyricum and C. beijerincki. In contrast, C. sporogenes showed missing enzymes in biosynthesis pathways of several amino acids as well as biotin, folate, and cobalamin (vitamin B12). These observations agree with the results of growth experiments of these species in liquid media reported in the literature. The results of this study suggest that biotin and folate are potential targets for reducing late blowing in cheese and highlight the usefulness of genomic analysis for identifying essential nutrients in bacteria.

Butyric acid fermentation from pretreated and hydrolysed wheat straw by an adapted Clostridium tyrobutyricum strain.

Butyric acid is a valuable building-block for the production of chemicals and materials and nowadays it is produced exclusively from petroleum. The aim of this study was to develop a suitable and robust strain of Clostridium tyrobutyricum that produces butyric acid at a high yield and selectivity from lignocellulosic biomasses. Pretreated (by wet explosion) and enzymatically hydrolysed wheat straw (PHWS), rich in C6 and C5 sugars (71.6 and 55.4 g l(-1) of glucose and xylose respectively), was used as substrate. After one year of serial selections, an adapted strain of C. tyrobutyricum was developed. The adapted strain was able to grow in 80% (v v(-1) ) PHWS without addition of yeast extract compared with an initial tolerance to less than 10% PHWS and was able to ferment both glucose and xylose. It is noticeable that the adapted C. tyrobutyricum strain was characterized by a high yield and selectivity to butyric acid. Specifically, the butyric acid yield at 60-80% PHWS lie between 0.37 and 0.46 g g(-1) of sugar, while the selectivity for butyric acid was as high as 0.9-1.0 g g(-1) of acid. Moreover, the strain exhibited a robust response in regards to growth and product profile at pH 6 and 7.

Prevention of late blowing defect by reuterin produced in cheese by a Lactobacillus reuteri adjunct.

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.

The use of high pressure CO2 -facilitated pH swings to enhance in situ product recovery of butyric acid in a two-phase partitioning bioreactor.

Through the use of high partial pressures of CO2 (pCO2 ) to facilitate temporary pH reductions in two-phase partitioning bioreactors (TPPBs), improved pH dependent partitioning of butyric acid was observed which achieved in situ product recovery (ISPR), alleviating end-product inhibition (EPI) during the production of butyric acid by Clostridium tyrobutyricum (ATCC 25755). Through high pressure pCO2 studies, media buffering effects were shown to be substantially overcome at 60 bar pCO2 , resulting in effective extraction of the organic acid by the absorptive polymer Pebax® 2533, yielding a distribution coefficient (D) of 2.4 ± 0.1 after 1 h of contact at this pressure. Importantly, it was also found that C. tyrobutyricum cultures were able to withstand 60 bar pCO2 for 1 h with no decrease in growth ability when returned to atmospheric pressure in batch reactors after several extraction cycles. A fed-batch reactor with cyclic high pCO2 polymer extraction recovered 92 g of butyric acid to produce a total of 213 g compared to 121 g generated in a control reactor. This recovery reduced EPI in the TPPB, resulting in both higher productivity (0.65 vs. 0.33 g L(-1) h(-1) ) and yield (0.54 vs. 0.40). Fortuitously, it was also found that repeated high pCO2 -facilitated polymer extractions of butyric acid during batch growth of C. tyrobutyricum lessened the need for pH control, and reduced base requirements by approximately 50%. Thus, high pCO2 -mediated absorptive polymer extraction presents a novel method for improving process performance in butyric acid fermentation, and this technique could be applied to the bioproduction of other organic acids as well.

Potential application of aromatic plant extracts to prevent cheese blowing.

This study aimed to inhibit the growth of Escherichia coli and Clostridium tyrobutyricum, common bacteria responsible for early and late cheese blowing defects respectively, by using novel aqueous extracts obtained by dynamic solid-liquid extraction and essential oils obtained by solvent free microwave extraction from 12 aromatic plants. In terms of antibacterial activity, a total of 13 extracts inhibited one of the two bacteria, and only two essential oils, Lavandula angustifolia Mill. and Lavandula hybrida, inhibited both. Four aqueous extracts were capable of inhibiting C. tyrobutyricum, but none were effective against E. coli. After extracts' chemical composition identification, relationship between the identified compounds and their antibacterial activity were performed by partial least square regression models revealing that compounds such as 1,8 cineole, linalool, linalyl acetate, ß-phellandrene or verbene (present in essential oils), pinocarvone, pinocamphone or coumaric acid derivate (in aqueous extracts) were compounds highly correlated to the antibacterial activity.

Outcome of oral provocation test in egg-sensitive children receiving semi-fat hard cheese Grana Padano PDO (protected designation of origin) containing, or not, lysozyme.

PURPOSE: Lysozyme, obtained from egg white, is a potential food allergen used in the dairy industry to prevent late blowing of the loaf caused by the outgrowth of clostridial spores (Cl. butyricum and Cl. tyrobutyricum) during cheese aging. The aim of this study was to evaluate the possible correlation between egg protein allergy in pediatric age and sensitization to egg lysozyme, used for the preparation of Grana Padano cheese. METHODS: The tolerability of Grana Padano cheese has been evaluated in pediatric patients allergic to egg proteins through an oral provocation test with increasing amounts of cheese containing, or not, lysozyme at 12 and 24 months of aging. RESULTS: When lysozyme-sensitized children received 12-months aged and lysozyme-containing cheese, several immediate and late adverse reactions such as itching, abdominal pain, vomiting, nausea, dermatitis, rhinitis, bronchial asthma, urticaria, and angioedema were seen in 5 out of 21 subjects; only 1 out of 21 children showed an adverse reaction after challenge with 24-months-ripened lysozyme-containing cheese. CONCLUSIONS: There is a possible relationship between the severity of allergic reactions and the lysozyme-specific IgE level in blood. In particular vomiting, hypotension, and abdominal pain were present when IgE level was higher than 7 kU/L. A ripening time of 24 months may reduce allergy problems when lysozyme-containing cheese is given to sensitized subjects, probably due to the hydrolysis of antigenic epitopes during aging.