Discrimination of different feed additives and poly-herbal formulations based on their untargeted phytochemical profiles.

INTRODUCTION: Feed additives represents a valid tool in animal nutrition to improve animal performance and livestock productivity under a sustainable perspective; however, there is a paucity of information about their comprehensive metabolomic and bioactive profiles. OBJECTIVE: In this study, we tested the ability of an untargeted metabolomics approach to discriminate nine commercial feed additives and unique blends of botanical extracts used in both ruminant and non-ruminant nutrition, according to their phytochemical profiles and different in vitro bioactive properties. METHODS: An ultra-high-performance liquid chromatography coupled with Orbitrap mass spectrometry and multivariate statistics were combined to search for potential markers, in order to better discriminate the different commercial samples. RESULTS: Several phytochemicals were identified, namely alkaloids, phenolics, organosulfurs, and terpenoids. The polyherbal formulation Zigbir was the best source of phytochemicals, accounting for a cumulative total content of phytochemicals equal to 3.03 mg Eq./g, being particularly abundant in terpenoids, stilbenes, phenolic acids, and small-molecular-weight phenolics. Multivariate statistics allowed to group the different products in 2 bioactive subclusters. The diterpenoid andrographolide recorded the highest abundance in Zigbir and Sangrovit. The most predictive biomarkers were: piperine, isoquercitrin, 6-methylthiohexyldesulfoglucosinolate, 6-methylumbelliferone, benzoic acid, (+)-(1R,2R)-1,2-diphenylethane-1,2-diol, and piperitenone. Flavonoids were highly correlated with both in vitro antioxidant and enzyme inhibition assays. CONCLUSIONS: Our findings provide new insights into the comprehensive phytochemical composition of commercial feed additives and blend of botanical extracts used for both ruminant and non-ruminant nutrition. A great importance of polyphenols in relation to the biological activities was detected.

A combined metabolomics and peptidomics approach to discriminate anomalous rind inclusion levels in Parmigiano Reggiano PDO grated hard cheese from different ripening stages.

Parmigiano Reggiano is a hard cheese with a Protected Designation of Origin (PDO) certification that also applies to the grated product. The percentage of rind in grated Parmigiano Reggiano is regulated by the PDO production Specification and must not exceed the limit of 18% (w/w). The present study evaluates the potential of an untargeted foodomics approach to detect anomalous inclusions of rind in grated Parmigiano Reggiano cheese. In particular, a combined metabolomics and peptidomics approach was used to detect potential markers of counterfeits (rind > 18%). In the framework of realistic food integrity purposes, non-Parmigiano Reggiano grated samples and different ripening times were also considered. Untargeted metabolomics allowed detecting 347 compounds, with a prevalence of amino acids and peptide derivatives, followed by fatty acyls and other compounds (such as lactones, ketones, and aldehydes) typically related to proteolysis and lipolysis events. Overall, the unsupervised multivariate statistics showed that the ripening time plays a hierarchically higher impact than rind inclusion in determining the main differences in the chemical profiles detected. Interestingly, supervised statistics highlighted distinctive markers for ripening time and rind inclusion, with only 16 common discriminant compounds being shared between the two conditions. The best markers of rind inclusion > 18% were 2-hydroxyadenine (VIP score = 1.937; AUC value = 0.83) and the amino acid derivatives argininic acid (VIP score = 1.462; AUC value = 0.75) and 5-hydroxyindole acetaldehyde (VIP score = 1.710; AUC value = 0.86). Interestingly, the medium-chain aldehyde 4-hydroperoxy-2-nonenal was a common marker of both ripening time and anomalous rind inclusion (>18%), likely arising from the lipid oxidation processes. Finally, among potential marker peptides of rind inclusion, the alpha-S1 casein proteolytic product (F)FVAPFPEVFGK(E) could be identified.

Screening of Regulated and Emerging Mycotoxins in Bulk Milk Samples by High-Resolution Mass Spectrometry.

In this work, a retrospective screening based on ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS) based on Orbitrap-Q-Exactive Focus™ was used to check the occurrence of regulated and emerging mycotoxins in bulk milk samples. Milk samples were collected from dairy farms in which corn silage was the main ingredient of the feeding system. The 45 bulk milk samples were previously analyzed for a detailed untargeted metabolomic profiling and classified into five clusters according to the corn silage contamination profile, namely: (1) low levels of Aspergillus- and Penicillium-mycotoxins; (2) low levels of fumonisins and other Fusarium-mycotoxins; (3) high levels of Aspergillus-mycotoxins; (4) high levels of non-regulated Fusarium-mycotoxins; (5) high levels of fumonisins and their metabolites. Multivariate statistics based on both unsupervised and supervised analyses were used to evaluate the significant fold-change variations of the main groups of mycotoxins detected when comparing milk samples from clusters 3, 4, and 5 (high contamination levels of the corn silages) with cluster 1 and 2 (low contamination levels of the corn silages). Overall, 14 compounds showed a significant prediction ability, with antibiotic Y (VIP score = 2.579), bikaverin (VIP score = 1.975) and fumonisin B2 (VIP score = 1.846) being the best markers. The k-means clustering combined with supervised statistics showed two discriminant groups of milk samples, thus revealing a hierarchically higher impact of the whole feeding system (rather than the only corn silages) together with other factors of variability on the final mycotoxin contamination profile. Among the discriminant metabolites we found some Fusarium mycotoxins, together with the tetrapeptide tentoxin (an Alternaria toxin), the α-zearalenol (a catabolite of zearalenone), mycophenolic acid and apicidin. These preliminary findings provide new insights into the potential role of UHPLC-HRMS to evaluate the contamination profile and the safety of raw milk to produce hard cheese.

Changes of Milk Metabolomic Profiles Resulting from a Mycotoxins-Contaminated Corn Silage Intake by Dairy Cows.

In this study, an untargeted metabolomics approach based on ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) was used for investigating changes in chemical profiles of cow milk considering diets based on mycotoxins-contaminated corn silages. For this purpose, 45 milk samples were classified into five clusters according to the corn silage contamination profile, namely (1) low levels of Aspergillus- and Penicillium-mycotoxins; (2) low levels of fumonisins and other Fusarium-mycotoxins; (3) high levels of Aspergillus-mycotoxins; (4) high levels of non-regulated Fusarium-mycotoxins; (5) high levels of fumonisins and their metabolites, and subsequently analyzed by UHPLC-HRMS followed by a multivariate statistical analysis (both unsupervised and supervised statistical approaches). Overall, the milk metabolomic profile highlighted potential correlations between the quality of contaminated corn silages (as part of the total mixed ration) and milk composition. Metabolomics allowed to identify 628 significant milk metabolites as affected by the five levels of corn silage contamination considered, with amino acids and peptides showing the highest metabolite set enrichment (134 compounds). Additionally, 78 metabolites were selected as the best discriminant of the prediction model built, possessing a variable importance in projection score >1.2. The average Log Fold-Change variations of the discriminant metabolites provided evidence that sphingolipids, together with purine and pyrimidine-derived metabolites were the most affected chemical classes. Also, metabolomics revealed a significant accumulation of oxidized glutathione in milk samples belonging to the silage cluster contaminated by emerging Aspergillus toxins, likely involved in the oxidative imbalance. These preliminary findings provide new insights into the potential role of milk metabolomics to provide chemical indicators of mycotoxins-contaminated corn silage feeding systems.

A Combined Metabolomic and Metagenomic Approach to Discriminate Raw Milk for the Production of Hard Cheese.

The chemical composition of milk can be significantly affected by different factors across the dairy supply chain, including primary production practices. Among the latter, the feeding system could drive the nutritional value and technological properties of milk and dairy products. Therefore, in this work, a combined foodomics approach based on both untargeted metabolomics and metagenomics was used to shed light onto the impact of feeding systems (i.e., hay vs. a mixed ration based on hay and fresh forage) on the chemical profile of raw milk for the production of hard cheese. In particular, ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) was used to investigate the chemical profile of raw milk (n = 46) collected from dairy herds located in the Po River Valley (Italy) and considering different feeding systems. Overall, a total of 3320 molecular features were putatively annotated across samples, corresponding to 734 unique compound structures, with significant differences (p < 0.05) between the two feeding regimens under investigation. Additionally, supervised multivariate statistics following metabolomics-based analysis allowed us to clearly discriminate raw milk samples according to the feeding systems, also extrapolating the most discriminant metabolites. Interestingly, 10 compounds were able to strongly explain the differences as imposed by the addition of forage in the cows' diet, being mainly glycerophospholipids (i.e., lysophosphatidylethanolamines, lysophosphatidylcholines, and phosphatidylcholines), followed by 5-(3',4'-Dihydroxyphenyl)-gamma-valerolactone-4'-O-glucuronide, 5a-androstan-3a,17b-diol disulfuric acid, and N-stearoyl glycine. The markers identified included both feed-derived (such as phenolic metabolites) and animal-derived compounds (such as lipids and derivatives). Finally, although characterized by a lower prediction ability, the metagenomic profile was found to be significantly correlated to some milk metabolites, with Staphylococcaceae, Pseudomonadaceae, and Dermabacteraceae establishing a higher number of significant correlations with the discriminant metabolites. Therefore, taken together, our preliminary results provide a comprehensive foodomic picture of raw milk samples from different feeding regimens, thus supporting further ad hoc studies investigating the metabolomic and metagenomic changes of milk in all processing conditions.

Milk metabolomics based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry to discriminate different cows feeding regimens.

The feeding system represents one of the main factors driving raw milk composition, thus determining differences in nutritional value and technological properties. In this preliminary study, untargeted metabolomics with ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) coupled with both unsupervised and supervised multivariate statistics was used to investigate the chemical profile of bulk milk collected from dairy cows (n = 103) following different feeding regimens, being corn silage (MS-FS, n = 51), hay (H-FS, n = 35) and a mixed ration consisted in fresh forage and hay (MR-FS, n = 17). Overall, a total of 1686 metabolites was identified by means of UHPLC-QTOF, with significant differences (p < 0.05) between the three feeding regimens under investigation. The metabolites detected mainly belonged to lipids (mainly glycerophospholipids and triglycerides), followed by oligopeptides, steroid derivatives, and secondary metabolites (such as phenolic compounds and terpenoids). Interestingly, multivariate statistics applied to the metabolomics data revealed intriguing differences in the discriminant markers detected. The markers identified included both feed-derived (such as phenolic metabolites) but also animal-derived compounds (such as fatty acids). Therefore, our results provide comprehensive insights into the metabolomics profile of different bulk milk samples, suggesting also an indirect influence of feeding regimens on its chemical signature.

Untargeted metabolomics reveals differences in chemical fingerprints between PDO and non-PDO Grana Padano cheeses.

The purpose of this preliminary study was to discriminate the chemical fingerprints of Protected Designation of Origin (PDO) Grana Padano cheeses from non-PDO "Grana-type" cheeses by means of an untargeted metabolomic approach based on ultra-high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC/QTOF-MS). Hierarchical cluster analysis and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) allowed discriminating PDO vs. non-PDO cheeses. Lipids (fatty acids and their derivatives, phospholipids and monoacylglycerols), amino acids and oligopeptides, together with plant-derived compounds were the markers having the highest discrimination potential. It can be postulated that Grana Padano value chain, as strictly defined in the PDO production specification rules, can drive the biochemical processes involved in cheese making and ripening in a distinct manner, thus leaving a defined chemical signature on the final product. These preliminary findings provide the basis for further authenticity studies, aiming to protect the designation of origin of PDO Grana Padano cheese by applying a comprehensive foodomics-based approach.

Impact of boiling on free and bound phenolic profile and antioxidant activity of commercial gluten-free pasta.

Cooking by boiling dry pasta could have varying degrees of influence on nutritional and functional components. In the present study, its effect on total phenolic content and antioxidant capacity, as well as on the comprehensive profile of free and bound phenolics, was investigated in six commercial gluten-free (GF) pasta products. Overall, the heat treatment caused a significant reduction (P<0.01) of the total phenolic content as well as FRAP reducing power and ORAC radical scavenging, with significant differences among the pasta samples considered. The highest values were recorded in free phenolic fraction remaining in black rice (41mggallic acid equivalents100g-1 and 25mmolTrolox Equivalents100g-1) and quinoa (24mggallic acid equivalents100g-1 and 14mmolTrolox Equivalents100g-1) cooked GF pasta. Significant correlations (P<0.01) could be found between total phenolics and both the antioxidant capacity assays performed. UHPLC-ESI/QTOF-MS mass profiling allowed confirming the spectrophotometric results, while identifying the amount of free and bound fractions. Among phenolic classes, lignans exhibited the highest decrease during the cooking process, followed by stilbenes and flavonoids. However, phenolic acids and other phenolics showed the highest stability. Furthermore, cooking by boiling strongly lowered the bound-to-free ratio of phenolic compounds, by an averaged factor ranging from 14-folds for flavonoids to 5-folds for other classes of phenolics.

Phenolic profile and fermentation patterns of different commercial gluten-free pasta during in vitro large intestine fermentation.

The fate of phenolic compounds, along with short-chain fatty acids (SCFAs) production kinetics, was evaluated on six different commercial gluten-free (GF) pasta samples varying in ingredient compositions, focussing on the in vitro faecal fermentation after the gastrointestinal digestion. A general reduction of both total phenolics and reducing power was observed in all samples, together with a substantial change in phenolic profile over 24h of faecal fermentation, with differences among GF pasta samples. Flavonoids, hydroxycinnamics and lignans degraded over time, with a concurrent increase in low-molecular-weight phenolic acids (hydroxybenzoic acids), alkylphenols, hydroxybenzoketones and tyrosols. Interestingly, discriminant analysis also identified several alkyl derivatives of resorcinol as markers of the changes in phenolic profile during in vitro fermentation. Furthermore, degradation pathways of phenolics by intestinal microbiota have been proposed. Considering the total SCFAs and butyrate production during the in vitro fermentation, different fermentation kinetics were observed among GF pasta post-hydrolysis residues.

Evaluation of phenolic profile and antioxidant capacity in gluten-free flours.

The characterization of phenolic fingerprints in common gluten-free flours is still scarce. Total phenolic and anthocyanin contents, antioxidant capacity and the entire phenolic profile were investigated for extracts from chickpea, sorghum, quinoa, black rice, lentil, amaranth, brown rice, oat and white rice flours, using soft wheat flour as a comparison. The highest phenolic content was found in black rice, followed by quinoa extracts (147.9 and 87.2mg gallic acid equivalents 100g-1, respectively). Consistently, antioxidant capacity was highest in black rice and quinoa flours (34mmol trolox equivalents 100g-1), while anthocyanins were highest in black rice flour. Data showed a high correlation between phenolic content and both reducing and scavenging activities, with Pearson's coefficient of 0.90 and 0.91, respectively. Although the entire phenolic profile was diverse and differed among flours, these represent a valuable source of health-promoting compounds, mainly belonging to flavonoids, phenolic acids and lignans.

Corn silage replacement with barley silage in dairy cows' diet does not change milk quality, cheese quality and yield.

BACKGROUND: Considering that water availability for agricultural needs is being restricted, an alternative to corn in animal nutrition should be explored in the Po Valley. The present study aimed to evaluate the effects of either a partial (Trial I) or a total (Trial II) corn silage substitution with barley silage in dairy cows' diet on milk yield and composition, its coagulation properties, cheese yield and the sensorial profile of 16-month-aged Grana Padano cheese. RESULTS: A partial or a total substitution of corn silage with barley silage had no effect on milk yield. Milk fat content in Trial I and milk urea content in both trials were higher with barley silage based diets than in corn silage based diets. No effects were observed concerning the lactodinamographic profile for milk aptitude to cheese-making, cheese yield and its organoleptic traits between feed treatments in Trials I and II. In both trials, hardness, friability and solubility scores were generally lower than reference values, whereas deformability, elasticity and stickiness scores were generally higher than reference values. CONCLUSION: A partial or a total substitution of corn silage with barley silage in diets for dairy cows did not induce any negative effects on animal performance, nor on milk-quality traits, cheese quality and yield. © 2016 Society of Chemical Industry.

New assessment based on the use of principal factor analysis to investigate corn silage quality from nutritional traits, fermentation end products and mycotoxins.

BACKGROUND: A survey on 68 dairy farms was carried out to evaluate the ensiling procedures adopted to store corn silage. Samples from core, lateral and apical zones of the feed-out face of silos were analysed. A principal factor analysis (PFA) was carried out on the entire database (196 silage samples and 36 variables) and 11 principal factor components (PCs) were retained and interpreted. RESULTS: Ensiling procedures influenced the area exposed to risk of air penetration. Cores had higher dry matter, starch and lactic acid content or lower pH, fibre, propionate and butyrate concentrations than peripheral samples (P < 0.05). The highest (P < 0.05) mycophenolic acid and roquefortina C concentrations were detected in lateral samples. Chemical and digestibility variables loaded on two PCs; four PCs were characterized by end-products associated with clostridia, heterolactic, homolactic and aerobic fermentations; two PCs were associated with mycotoxins, whereas three PCs explained ensiling procedures. CONCLUSION: The main quality traits of corn silages differed throughout the entire silo face. Minimization of the area exposed to risk of air penetration represents the best strategy to preserve the nutritional value and safety of corn silages. PFA allowed a clusterization of original variables into 11 PCs, appearing able to discriminate well and poorly preserved corn silages.

Use of principal factor analysis to generate a corn silage fermentative quality index to rank well- or poorly preserved forages.

BACKGROUND: To investigate corn silage fermentative quality, a principal factor analysis was carried out on a database consisting of 196 corn silages sampled from the core, lateral and apical parts of silo feed-out face and characterised by 36 variables. Eleven principal factor components (PCs) were retained and interpreted. Two PCs were related to chemical and digestibility variables; four PCs were characterised by end-products associated with clostridia, heterolactic, homolactic or aerobic fermentations; two PCs were associated with mycotoxins produced by Penicillium roqueforti or by Aspergillus fumigatus and Fusarium spp., while three PCs explained ensiling procedures adopted to store corn silages. RESULTS: Lower (P < 0.05) yeast or mould counts and greater (P < 0.05) aerobic stability were measured in core than in peripheral samples. Excluding PCs related to ensiling procedures, other PCs were able to predict microbiological counts, aerobic stability or biogenic amine content as verified by multiple linear regression analysis. Based on these results, several corn silage quality index calculations were computed by using a summative equation approach in which different PCs as well as diverse relative weights multiplying each PCs were combined. To compute definitive index calculation, only PCs explaining clostridia, heterolactic and homolactic fermentations were used with relative weights of 30%, 50% and 20%. CONCLUSIONS: The new proposed fermentative quality index was highly correlated to parameters related to corn silage fermentative quality, such as microbiological counts, aerobic stability or biogenic amines and it properly discriminated well- and poorly preserved forages.

Cooking quality and starch digestibility of gluten free pasta using new bean flour.

The use of rice/leguminous blend may be nutritionally convenient in gluten free product manufacturing. Gluten free spaghetti was prepared with rice flour and different concentrations of bean flour (included at levels of 0%, 20% and 40%, w/w) derived from a new developed white-seeded low phytic acid and lectin free (ws+lpa+lf) bean cultivar. Protein, ash and dietary fibre contents increased linearly (P<0.05) while total starch decreased quadratically (P<0.05) with the inclusion of ws+lpa+lf bean flour. The colour of spaghetti was influenced (P<0.05) by ws+lpa+lf bean inclusion. With respect to 0% spaghetti, the inclusion of ws+lpa+lf bean increased linearly (P<0.05) the optimal cooking time and the water absorption capacity, without affecting cooking loss and texture properties. The ws+lpa+lf bean inclusion increases quadratically (P<0.05) the resistant starch content, while decreasing quadratically (P<0.05) the in vitro glycemic index. The partial replacement of rice flour with bean flour can favourably be used in gluten free spaghetti formulation.

Vaccination of heifers with anaflatoxin improves the reduction of aflatoxin b1 carry over in milk of lactating dairy cows.

It was previously reported that injection of anaflatoxin B1 (AnAFB1) conjugated to keyhole limpet hemocyanin (KLH), together with Freund's adjuvant, was effective in inducing in cows a long lasting titer of anti-aflatoxin B1 (AFB1) antibodies (Abs), cross-reacting with other aflatoxins, which were able to hinder, proportionally to their titer, the secretion of aflatoxin M1 (AFM1) into the milk of cows continuously fed with AFB1. According to anti-AFB1 Ab titer, 50% of the vaccinated cows were recognized as high responder animals. In an attempt to prepare a more effective formulation for vaccination of cows, it was compared the immunogenicity, in Holstein Friesian heifers, of AnAFB1 covalently conjugated to KLH or to recombinant diphtheria toxin (CRM197) molecules, and injected together with various adjuvants. This study demonstrated that injection of AnAFB1 conjugated to KLH and mixed with complete (priming) and incomplete Freund's adjuvant (boosters), as in the previous schedule of immunization, was the most effective regimen for inducing Ab responses against AFB1, although pre-calving administration could increase the effectiveness of vaccination, resulting in 100% high responder animals. After one booster dose at the beginning of the milk production cycle, anti-AFB1 Ab titers were comparable to those recorded at the end of the immunization schedule, and proved to be effective in reducing significantly AFB1 carry over, as AFM1, from feed to milk. Pre-calving vaccination of dairy heifers with conjugated AnAFB1, adjuvated with complete and incomplete Freund's adjuvant, may represent the most effective tool for preventing the public health hazard constituted by milk and cheese contaminated with aflatoxins.

Food for Healthy Living and Active Ageing.

The link between diet and health has been recognized since the Grecian period; as Hippocrates said, "Let food be your medicine and medicine be your food". Although the primary goals of diet are meeting nutritional requirements and providing energy, there is increasing awareness that a correct and balanced diet may prevent the insurgence of diet-related pathologies and/or improve well-being and life expectancy, also reflecting on the ageing process. Research on the interaction among nutrients, gut microbiota and host metabolism is presently unravelling the molecular mechanisms underlying the positive and negative effects of traditional diets on health and ageing, providing useful information for the design of innovative foods targeting specific needs and segments of the population. The food supply chain plays a key role in ensuring quality and safety through both comprehensive quality management and inspection systems and a focused innovation process mainly devoted to the creation of functional foods. However, innovation and scientific development pose a problem of information asymmetry towards final consumers; thus, regulatory aspects and private and public communication strategies must be efficiently developed.

Effect of water-saving irrigation regime on whole-plant yield and nutritive value of maize hybrids.

BACKGROUND: The effect of a water-saving irrigation regime on yield, chemical composition, rumen in situ dry matter disappearance (DMD) and neutral detergent fiber disappearance (NDFD), along with 7 h in vitro starch degradability (7 h IVSD), in maize hybrids selected for whole-plant silage making was investigated. A plot experiment was conducted in a continental climate location and four commercial maize hybrids (FAO class 700) were used in a completely randomized design with a factorial arrangement of irrigation treatments (fully irrigated (FI) and water-saving regime (WS)) and four replicates/treatment. The total amount of irrigation water was 494 mm in FI plots and 367 mm in WS plots, the latter achieved by skipping irrigations at vegetative growth stage, silking and blistering. RESULTS: Whole-plant yield, chemical composition, DMD, NDFD and 7 h IVSD slightly differed among hybrids and were not influenced by irrigation treatments. Plant dry matter content was lower in FI than WS plots (320 vs. 341 g kg⁻¹) , respectively; P < 0.05). Differences among hybrids were recorded for starch and acid detergent fiber contents (P < 0.05). CONCLUSIONS: The lack of differences on yield and nutritive value in tested maize hybrids grown under different water supply suggests the water-saving regime could be suitable for an optimal use of available water in maize management.

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.