Immunological response in egg-sensitive adults challenged with cheese containing or not containing lysozyme.

OBJECTIVE: Lysozyme is an enzyme that hydrolyzes bacterial peptidoglicans. For this reason, it is used in cheese manufacturing in order to prevent a defect of long-ripened hard cheese called "late blowing" due to the outgrowth of spores of Clostridium tyrobutyricum and Clostridium butyricum. Moreover, germination of Listeria monocytogenes spores into vegetative cells is also sensitive to lysozyme. The enzyme can be an allergenic molecule, and for this reason there are concerns about its use in food industry. The immunological and clinical response of consumption of lysozyme-containing cheese has been evaluated in 25 egg-sensitive subjects with or without lysozyme sensitization. METHODS: A total of 25 egg-sensitive subjects were enrolled in this study. All the subjects were already treated for egg-sensitization and presented a positive skin prick test. All the subjects had a body mass index ≤ 25 kg/m(2) and were in the age range of 20-50 years. Each subject was studied twice and received randomly 30 g of Grana Padano (containing lysozyme) or TrentinGrana cheese (lysozyme-free) of two different aging periods: 16 or 24 months. A washout period of 1 week between each cheese intake was adopted. Blood samples were taken in fasting conditions and 1 hour after cheese intake and IgA, total IgE, and lysozyme-, ovomucoid-, and ovalbumin-specific IgE were measured. RESULTS: No adverse reactions were observed in both groups of patients after cheese samples were given. Lysozyme did not determine any variation of specific IgE compared with basal level. In lysozyme-sensitive patients a significant relationship between IgA and lysozyme-specific IgE was observed when lysozyme-containing cheese was given, confirming that lysozyme can pass the gut barrier. CONCLUSIONS: Neither adverse events nor immunological responses were observed after ingestion of cheese containing lysozyme. However, the immunological properties of peptides deriving from cheese protein hydrolysis need to be clarified, as does the effect of lysozyme on bacterial proteolytic activity.

Vaccination of lactating dairy cows for the prevention of aflatoxin B1 carry over in milk.

The potential of anaflatoxin B(1) (AnAFB(1)) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB(1)-KLH) in controlling the carry over of the aflatoxin B(1) (AFB(1)) metabolite aflatoxin M(1) (AFM(1)) in cow milk is reported. AFB(1) is the most carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB(1) is AFB(1) chemically modified as AFB(1)-1(O-carboxymethyl) oxime. In comparison to AFB(1), AnAFB(1) has proven to be non-toxic in vitro to human hepatocarcinoma cells and non mutagenic to Salmonella typhimurium strains. AnAFB(1)-KLH was used for immunization of cows proving to induce a long lasting titer of anti-AFB(1) IgG antibodies (Abs) which were cross reactive with AFB(1), AFG(1), and AFG(2). The elicited anti-AFB(1) Abs were able to hinder the secretion of AFM(1) into the milk of cows continuously fed with AFB(1). Vaccination of lactating animals with conjugated AnAFB(1) may represent a solution to the public hazard constituted by milk and cheese contaminated with AFs.

Conjugated linoleic acid (CLA) and fatty acid composition of milk, curd and Grana Padano cheese in conventional and organic farming systems.

CLA levels and fatty acid composition were measured to compare the fat composition in organic bulk milk, destined to the production of Grana Padano cheese, with those produced by conventional system. The curds and Grana Padano cheeses were also analysed to evaluate the effects of the production technology on the CLA content. All analysed organic samples were characterized by higher annual means of CLA, vaccenic acid (TVA) and linolenic acid (LNA) in comparison with conventional samples (with P<0.05). Nevertheless, no particular effect of the production technology was seen on the CLA content. The animal diet appears to be the factor which has the highest effect on the CLA concentration in milk and milk products and an organic diet based on fresh or dried forage, that is rich in CLA precursory fatty acids, may improve the yield of fatty acids with beneficial effects on health.

Absence of allergic reactions to egg white lysozyme additive in Grana Padano cheese.

OBJECTIVE: The negative effects on cheese quality of milk contaminated by spores of Clostridium butyricum and Cl.tyrobutyricum is prevented by the use of egg white lysozyme as additive. Since the presence of lysozyme in cheese could be possibly risky in allergic subjects, we aimed at investigating its absorption as well as serum IgE antibody titers after ingestion of Grana Padano cheese, an Italian DPO, long-ripened hard cheese, in white egg allergic subjects. METHODS: Cheese lysozyme was measured by HPLC. Ten healthy volunteers and 20 patients with hen egg hypersensitivity, RAST positive (binding > or = 3%) to lysozyme and/or ovomucoid and ovalbumin received 15, 30 and 60 g of cheese at distance of at least 2 weeks each. Serum lysozyme was measured by ELISA and specific IgE binding to lysozyme by the radioallergosorbent test (RAST). RESULTS: The concentration of lysozyme in cheese was 155 +/- 5 mg/kg. The area-under-the-curve of serum lysozyme after 15 g of cheese was 244.5 +/- 14.0 in controls and 330.2 +/- 9.9 in patients (p < 0.01). Similar results were obtained with 30 and 60 g of cheese. Only 3 patients (15%) showed positive IgE antibody responses to cheese (overall RAST mean 4.45 +/- 1.25 % vs. 4.24 +/- 1.02 % baseline, p = ns). CONCLUSIONS: The amount of lysozyme absorbed with cheese was globally very low, although it was significantly lower in healthy controls than in allergic patients, where it induced an increase of IgE RAST score in 15% of subjects, without any clinical reaction. Therefore, the use of lysozyme as additive in Grana Padano cheese, does not appear to be harmful in egg allergic subjects.

Protective effect of cyanidin 3-O-beta-D-glucoside on ochratoxin A-mediated damage in the rat.

The aim of the present study was to verify whether the oral administration of cyanidin 3-O-beta-D-glucoside (C3G) might counteract damage induced by chronic exposure (28 d) to ochratoxin A (OTA) in rats and if its effect may be mediated by haeme oxygenase-1 (HO-1). Forty male Sprague-Dawley rats, individually caged, were divided into four groups of ten animals. A control group received a commercial diet, group C3G received the control diet supplemented with C3G (1 g/kg feed), group OTA received the control diet supplemented with 200 parts per billion of OTA, and group OTA+C3G received the OTA group diet supplemented with C3G (1 g/kg feed). After 4 weeks of treatment animals were killed and the liver, kidneys and brain of each rat were collected and homogenised to evaluate non-proteic thiol groups (RSH), lipid hydroperoxide (LOOH) levels, HO-1 expression and DNA fragmentation. Rats of the OTA group showed a significant (P < 0.001) decrease in RSH content of kidney and liver and a significant (P < 0.001) increase of LOOH in all the examined tissues compared with the control group. In the OTA+C3G group both RSH content and LOOH levels were similar to those observed in the control group, demonstrating that C3G was able to counteract the effects of OTA. A significant (P < 0.001) induction of HO-1 was evident in kidney and liver of both OTA and C3G groups. DNA damage occurred in all the examined tissues of the OTA group, whereas C3G was able to prevent it. The present study confirmed that the effects of OTA are mediated by oxidative stress and demonstrated that C3G efficiently counteracted deleterious effects of OTA because of its antioxidant and HO-1-inducing properties.

Carnosic acid from rosemary extracts: a potential chemoprotective agent against aflatoxin B1. An in vitro study.

Since oxidative stress plays an important role in the toxicity mechanism of several mycotoxins such as aflatoxin B1 (AFB1), the use of natural or synthetic free radical scavengers could be a potential chemopreventive strategy. Carnosic acid (CA) is the major polyphenolic compound present in rosemary plants and it can also be found in sage leaves. Its free radical scavenging properties were tested with two chemical methods. It was found that it has good free radical scavenging capacity at pH 7.4. This study also found that a 24 h pre-treatment with 10, 20 and 30 microm CA led to a clear, dose-dependent protective effect on cell toxicity, reducing cell death induced by a 24 h exposure with 10 microm AFB1, respectively, by 16% (P < 0.05), 26% (P < 0.01) and 63% (P < 0.001). It was also found that a 24 h pre-treatment with 20 and 30 microm CA achieved a reduction of ROS levels, respectively, of 146% (P < 0.001) and 173% (P < 0.001) in HepG2 cells exposed to 10 microm AFB1 for 8 h. Moreover, in cells pre-incubated with 30 microm CA for 24 h the concentration of 8-OH-deoxyguanine decreased by 57% (P < 0.001) with respect to the cells exposed for 24 h to 10 microm AFB1 alone. The results obtained with the in vitro and chemical studies support the theory that AFB1 induced oxidative stress plays an important role in the cytotoxic mechanism of this mycotoxin. Furthermore these findings suggest a starting point for developing alimentary strategies in order to counteract the damage caused by AFB1 contamination in feed and food.

Assessing the transfer of genetically modified DNA from feed to animal tissues.

In Europe, public and scientific concerns about the environmental and food safety of GM (Genetically Modified) crops overshadow the potential benefits offered by crop biotechnology to improve food quality. One of the concerns regarding the use of GM food in human and animal nutrition is the effect that newly introduced sequences may have on the organism. In this paper, we assess the potential transfer of diet-derived DNA to animal tissues after consumption of GM plants. Blood, spleen, liver, kidney and muscle tissues from piglets fed for 35 days with diets containing either GM (MON810) or a conventional maize were investigated for the presence of plant DNA. Only fragments of specific maize genes (Zein, Sh-2) could be detected with different frequencies in all the examined tissues except muscle. A small fragment of the Cry1A(b) transgene was detected in blood, liver, spleen and kidney of the animals raised with the transgenic feed. The intact Cry1A(b) gene or its minimal functional unit were never detected. Statistical analysis of the results showed no difference in recovery of positives for the presence of plant DNA between animals raised with the transgenic feed and animals raised with the conventional feed, indicating that DNA transfer may occur independently from the source and the type of the gene. From the data obtained, we consider it unlikely that the occurrence of genetic transfer associated with GM plants is higher than that from conventional plants.

Evaluation of xenobiotics in human milk and ingestion by the newborn--an epidemiological survey in Lombardy (Northern Italy).

BACKGROUND: Many human milk benefits have been well documented; nevertheless the newborn potential risk to the xenobiotic exposition may be relevant and it requires a biological monitoring in general prevention. Concerning this problem, attention should be paid to mycotoxins and heavy metals. AIM OF THE STUDY: Assessing the presence of the xenobiotics aflatoxins, ochratoxin A, lead and cadmium in human milk, defining their level of contamination and evaluate the potential risk for the newborn derived from this xenobiotic ingestion. METHODS: A study has been carried out on lactating women randomly selected in seven hospitals in Lombardy (Northern Italy). Two hundred and forty-seven puerparae were recruited; 231 women participated in the study. Women's milk samples on the third or fourth day after delivery were tested to determine aflatoxins and ochratoxin A levels. Lead and cadmium were determined in 143 women because supplemental milk could be taken only from these women. RESULTS: Aflatoxin B1 (11.4 ng/l) and aflatoxin M1 (194 ng/l) were found only in one sample,while ochratoxin A was detected in 198 samples (85.7 %) at an average value of 6.01 +/- 8.31 ng/l. A total of 75.7% of samples were positive for lead; the cadmium situation was better with 87.4% of the sample with values below detection limits (2 microg/l). A high percentage of babies (71 %) are exposed to mycotoxin levels on day 6 greater than the TDI value of 0.2 ng/kg b.w. Lead and cadmium presence in human milk presented risk respectively for 8% and 0.7% of newborns on the fourth day; 9.5% and 1.4% on the sixth day. CONCLUSIONS: The study points out that mycotoxins and lead are present in maternal milk, and the data confirm the need to continue biological monitoring in general prevention.

Reduction of Carryover of Aflatoxin from Cow Feed to Milk by Addition of Activated Carbons.

According to a double-reversal experimental design on 12 late-lactation Friesian cows the effect of two activated carbons (ACs) (CAC1 and CAC2) and a hydrated sodium calcium aluminosilicate (HSCAS) on carryover of aflatoxin B1 (AFB1) from feed to aflatoxin M1 (AFM1) in milk was determined. Cows were fed a basal diet containing AFB1 naturally contaminated corn meal and copra, During week 1 cows were fed diets containing AFB1 alone (11.28 µg of AFB1/kg of feed); in week 2 the diets contained AFB1 plus 2.0% sorbent; and in week 3 the diets again contained AFB1 alone (13.43 µg of AFB1/kg of feed). ACs reduced the analytical content of AFB1 in the pelleted feed by from 40.6% to 73.6%, whereas reduction by HSCAS was 59.2%, The AFM1 concentrations in milk in weeks 1 and 3 were higher than that in week 2, Decreases in the AFM1 excreted in the milk by addition to feed of 2% of the sorbents ranged from 22% to 45%. CAC1 and HSCAS were significantly different from each other in reducing the AFM1concentration in milk (45.3% versus 32.5%); these reductions were significantly higher than that of CAC2 (22.0%). Carryover reduction by addition of CAC1 (50%) was significantly higher than that of HSCAS (36%). Addition of 2% CAC2 did not allow pelleting of feed because of the caking action of this carbon, The lower performance of CAC2 could be related to the unsuccessful pelleting. The addition of ACs did not influence feed intake, milk production, milk composition, or body weight. Our results suggest that ACs, high-affinity sorbents for AFB1 in vitro, are efficacious in reducing AFB1 carryover from cow feed to milk. Further in vivo investigations should establish lower amounts of ACs which can be efficacious.