Lactic acid bacteria bioprotection applied to the malting process. Part I: Strain characterization and identification of antifungal compounds

Lactic acid bacteria (LAB) are of interest to cereal and beverage industry due to their contribution to the microbial safety and quality of fermented food/beverages. The main objectives of this study were to optimize the production of an antifungal cell-free-supernatant (cfs), based on lactic acid bacteria, using a wort-base substrate; and the subsequent identification of its active acid-base antifungal compounds. Two antifungal strains (Lactobacillus amylovorus DSM19280 and Lactobacillus reuteri R29) and a negative control strain (L. amylovorus DSM20552) were used. Barley based malt extract (wort) was produced and used as a fermentation substrate. Worts fermented with LAB were characterized in detail (total cell counts, pH, and total titratable acids (TTA) over time (0–120 h). A microtiter plate and an overlay agar based assay were used to evaluate the antifungal activity of the LAB cfs. Sugars, organic acids of the fermented and non-fermented wort (control) were analysed by HPLC. LAB antifungal metabolites were quantified using a QuEChERS method and an HPLC-UV/PDA system. Results show that wort produced from barley malt is a suitable substrate for LAB and dependent on the species and the fermentation time. The type of antifungal compounds can vary significantly. L. amylovorus DSM19280 cfs inhibited Fusarium culmorum spores at levels of 10⁴ spores.mL⁻¹, for 7 days, whereas L. reuteri R29 cfs inhibited up to 10⁵ spores.mL⁻¹ for the same time. Thirteen acid-base antifungal compounds were identified in L. reuteri R29 cfs and twelve in L. amylovorus DSM19280. Among them, phenyllactic, OH-phenylactic and benzoic acids were present at a significant concentration. This study demonstrates that the LAB wort-base cfs exhibits potent antifungal activity and is the ideal substrate for applications in a wide range of cereal products.     

Biopreservation potential of lactic acid bacteria from Andean fermented food of vegetal origin

Microbial fermentations have long represented a way of natural biopreservation of raw materials, which frequently originated new food products. Among them, traditionally fermented products still manufactured by native populations all around the world are source of lactic acid bacteria (LAB) strains with high biotechnological potential. LAB are food grade microorganisms and therefore a good alternative to chemicals to be applied in food preservation. A total of 130 LAB isolates recovered from “chicha” and “tocosh”, traditional fermented Andean products of vegetal origin, were screened for antimicrobial activities against spoiler fungi Meyerozyma guilliermondii CECT 1021 (synonym Pichia guilliermondii), Penicillium roqueforti CECT 2905^NT, Aspergillus oryzae CECT 2094^NT and Aspergillus niger CECT 2807 as well as against foodborne pathogens Escherichia coli O157:H7 CECT 5947, Listeria innocua CECT 910^T and Salmonella enterica subsp. enterica serovar Typhi CECT 4138. LAB isolates represented nine species and four genera that exhibited a general inhibition of food pathogens and were also active against A. oryzae and M. guilliermondii while a poor inhibition of A. niger and P. roqueforti was produced. Antifungal activity of cell free supernatants (CFS) from seven selected strains grown in MRS was confirmed against toxigenic fungi Aspergillus parasiticus CECT 2681, Penicillium expansum CECT 2278 and Fusarium verticilloides CECT 2987 and also on the three foodborne bacteria included in the study. Phenyllactic and 3,5-Di-O-caffeoylquinic acids were identified as the predominant bioactive compounds in CFS by QuEChERS extraction with LC-MS-LIT detection approach. Four out of seven strains free of antibiotic resistances involving L. plantarum M5MA1 and M9MM1 from chicha and L. fermentum T3M3 and Lc. mesenteroides T1M3 from tocosh showed high potential to be used as biopreservatives in food applications.     

Identification of antifungal peptides produced by Lactobacillus plantarum IS10 grown in the MRS broth

The use of secondary metabolites of lactic acid bacteria for preservation of foods is increasingly gaining interest to the food industry to replace synthetic preservatives. In this study, the cell free supernatant containing peptides obtained from Lactobacillus plantarum IS10 was fractionated by size exclusion chromatography using sephadex G-25, and tested against Aspergillus flavus MD3, Penicillium roqueforti MD4 and Eurotium rubrum MD5. Among the fractions, fraction number 10 showed 60% antifungal activity at a concentration of 0.02 mg peptide/mL. Four novel peptides out of twenty peptides obtained from fraction 10 were identified and determined by de novo sequencing. Peptide FPSHTGMSVPPP with a net charge +1, hydrophobicity ratio 58% and molecular weight of 1253 was further studied. The selected peptide showed a good activity at a concentration of 5 mg/mL against selected fungi and poor activity at low concentrations. This work indicates that L. plantarum IS10 has the capability of producing peptides which are affective against spoilage fungi.     

Biodiversity,dynamics and antimicrobial activity of lactic acid bacteria involved in the fermentation of maize flour for doklu production in Côte d'Ivoire

Doklu is a maize-based spontaneously fermented dough produced and consumed in parts of West Africa, particularly in Côte d'Ivoire. The characterization of the microbial ecosystem of doklu was carried out using a polyphasic approach. First, culture-dependent methods were used for bacterial enumeration and the phenotypic and molecular identification of 250 lactic acid bacteria (LAB) isolates. Then, culture-independent methods, including PCR-TTGE (V3 region of the 16S rRNA gene), provided a fingerprinting of bacterial DNA directly extracted from doklu. Bio preservative abilities were also tested and strains producing antimicrobial compounds were genotyped using PFGE. During maize dough fermentation, LAB became dominant and their load increased from 4.2 ± 0.2 log CFU/g to 9.0 ± 0.7 log CFU/g only after 48 h. Culture-dependent methods highlighted the presence of five LAB groups with the species Lactobacillus plantarum (28%), Lactobacillus fermentum (41.6%), Pediococcus acidilactici (6.8%), Pediococcus pentosaceus (18%) et Weissella cibaria (5.6%), succeeding during the fermentation. Lb. fermentum being practically the only species present at the end of fermentation, is with Lb. plantarum, the predominant species of fermenting dough. Culture-independent analysis underlined the undoubted role of Lb. fermentum, actively involved in the dough fermentation. These Lb. fermentum species, with a diversity of strains also showed important antimicrobial activity, due to production of bacteriocins. Being able to produce antimicrobial compounds, Lb. fermentum species may act as both bio protective culture as well as fermenting agent in cereal products and could be exploited to create functional starter cultures.     

Minerals in vitro bioaccessibility and changes in textural and structural characteristics of uncooked pre-germinated brown rice influenced by ultra-high pressure

Ultra-high pressure (UHP) is considered as an effective processing method to enhance micronutrients utilization efficiency in germinated brown rice (GBR), but there is little information available for the effects of UHP treatments on mineral bioaccessibility and related structural characteristics. Therefore, this work examined the changes induced by UHP in the in vitro bioaccessibility of selected minerals and structural and textural properties at pressures, as well as their potential relationship. UHP treatments were applied at 100, 300 and 500 MPa for 10 min, and 0.1 MPa as control. Proximate analysis showed that free fatty acids (FFA) and moisture in GBR greatly increased after UHP treatment. Three representative minerals with different levels were chosen to assess the bioaccessibility, including phosphorus (P), magnesium (Mg) and manganese (Mn). The results demonstrated that the minerals bioaccessibility, measured by percentage solubility, were the highest when at 300 MPa whereas it decreased at 500 MPa. Correspondingly, microstructure imaging by scanning electron microscope (SEM) showed that the gelatinization of starch granule occurred at 300 MPa and it became more obvious when increased to 500 MPa. Starch gelatinization significantly lowered the hardness of GBR grains, simultaneously accompanied with the decrease of cohesiveness, gumminess and resilience through texture profile analysis (TPA). Multivariate analysis by principal component analysis and canonical correspondence analysis indicated a highly intra-associated (p < 0.01) TPA parameters and a close correlation between the bioaccessibility and hardness, chewiness and springiness. These results provide information on minerals bioaccessibility and structural properties of uncooked GBR after UHP treatment, which could lay the foundation for further elucidating the correlation between structure and micronutrients bioaccessibility.     

Effect of sulfur dioxide and ethanol concentration on fungal profile and ochratoxin a production by Aspergillus carbonarius during wine making

Fungal profiles and ochratoxin A (OTA) accumulation during wine making were investigated using five different wine grape cultivars, Cabernet Sauvignon, Pinot Noir, Merlot, Syrah and Petit Verdot and the intrinsic influences caused by sulfur dioxide, ethanol and combine effect of ethanol and reducing sugar were analyzed using Cabernet Sauvignon and inoculation of Aspergillus carbonarius. Aspergillus spp. and Penicillium spp. were found as the major fungi in all winemaking processes and were highly correlated with OTA accumulation in wine. Most fungi died and OTA production decreased after 48 h of alcoholic fermentation, being consistent with the period when ethanol accumulation increased. The addition of SO₂ significantly inhibited the growth and OTA production of A. carbonarius with complete inhibition at 500 mg/L. When the ethanol concentration in the must increased to the range of 2–4%, growth and OTA production of A. carbonarius were significantly inhibited. Reducing sugar concentration had no significant effect on the growth and OTA production of A. carbonarius within the levels changing during the winemaking. Therefore, the increase of ethanol concentration played an important role in causing the decrease of fungal contamination and OTA accumulation during winemaking.     

Combination of nisin and ε-polylysine with chitosan coating inhibits the white blush of fresh-cut carrots

Effects of the combination of nisin and ε-polylysine with chitosan coating on quality maintenance and white blush inhibition were investigated in fresh-cut carrots. Fresh-cut carrots were treated with 1% lactic acid solution (v/v), 1% chitosan solution (w/v), or 1% chitosan solution containing 64 μg/mL nisin and 250 μg/mL ε-polylysine (LA + CH + Nisin + ε-PL). The samples were packed in polyethylene plastic bags and stored at 4 °C for 9 days. Changes in sensory attributes, physicochemical indices, respiration rate, microbiological counts and white blush were measured. Results showed that LA + CH + Nisin + ε-PL significantly (P < 0.05) inhibited respiration rate, decline of ascorbic acid and growth of microorganism (yeast and mold, total viable counts, total coliforms counts, Staphylococcus aureus and Pseudomonas spp.), and increased total phenol content and phenylalanine ammonialyse (PAL) activity compared with the control after 9-day storage. It was also strongly effective in inhibiting the white blush of fresh-cut carrots. Furthermore, LA + CH + Nisin + ε-PL significantly (P < 0.05) reduced the lignin synthesis in fresh-cut carrots by inhibiting the cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL) activity, as well as Dc4CL and DcC4H gene expression. Our results may provide some basis for the use of the combination of nisin and ε-polylysine with chitosan coating as an alternative preservation method for fresh-cut carrots.     

Starter culture fermentation of Chinese sauerkraut: Growth,acidification and metabolic analyses

Chinese sauerkraut is a kind of traditional and typical fermented food. Four lactic acid bacteria (LAB) strains, Leuconostoc mesenteroides NCU1426, Lactococcus lactis NCU1315, Lactobacillus plantarum NCU1121 and Lactobacillus casei NCU1222 isolated from Chinese sauerkraut, were used in single starter cultures. Microbiological changes and pH values were monitored during fermentation. Metabolic substrates and products during the fermentation were examined using high performance liquid chromatography (HPLC) technology. Results have shown that Leu. mesenteroides and Lc. lactis grew faster, produced lactic acid earlier and were poorly acid-resistant, whereas Lb. plantarum and Lb. casei produced much more lactic acid throughout fermentation and showed better acid-tolerance. Two Lactococcus had outstanding performance in sucrose utilization while the other two Lactobacillus were likely to use glucose and fructose during fermentation. Unexpectedly, Leu. mesenteroides and Lc. lactis showed weak citric acid metabolism in fermentation. All the four LAB strains were able to utilize malic acid in early fermentation. In conclusion, these LAB strains have shown notable differences in growth and fermentative properties during starter culture fermentation of Chinese sauerkraut, probably resulting from LAB fermentative function and a mixture of complex substrates.     

Whole soybean as probiotic lactic acid bacteria carrier food in solid-state fermentation

For the purpose of preparing lactic acid bacteria (LAB) carrier food, the solid-state fermentation of whole soybean was performed using Bifidobacterium animalis 937, Lactobacillus casei Zhang and Lactobacillus plantarum P-8 mixed with Bacillus subtilis natto, respectively. The physicochemical properties, the amino nitrogen content and peptide molecular weight distribution of the fermented whole soybean products were examined during this process. After 48 h of fermentation, the viable counts of the three samples were 1.41 × 10⁸ CFU/g (B. animalis 937), 1.74 × 10¹⁰ CFU/g (L. casei Zhang) and 2.19 × 10¹⁰ CFU/g (L. plantarum P-8), with the pH declined rapidly from 6.32 to 5.78, 5.60 and 5.44 at the early stage of the fermentation and increased to 6.71, 6.47 and 6.60 at the later stage of the fermentation. The fermentation caused a sharply increase in the content of the free amino nitrogen from 99.7 μmol/g to 301.9 μmol/g, 390.1 μmol/g and 529.1 μmol/g in the solid fermented soybean products, due to the multiplication of microorganism and the effect of enzyme system. Furthermore, the levels of soybean peptide with molecular weight less than 1000 Da increased 30.7%, 71.2% and 81.3% relative to that of the control group at 48 h. The result of the present work implied that whole soybean fermented by LAB can provide the different probiotics for the host, and there is potential to develope nutritious fermented soybean products.     

Occurrence and molecular identification of anisakid nematodes isolated from Pacific cod (Gadus macrocephalus) caught off Korea

Anisakids nematodes from Pacific cod (Gadus macrocephalus) in Korea were investigated and their molecular identification was conducted, to assess the epidemiological role of Pacific cod in human anisakidosis in Korea. Totally 238 Pacific cod were caught from 5 different areas around Korean peninsula. Fish were dissected and carefully examined for collecting nematodes. PCR-RFLP and the subsequent sequencing were conducted for molecular identification of those nematodes. A high prevalence of infection (193/238, 81.1%) in Pacific cod was observed, and 1694 nematodes were collected. 79.1% (1340/1694) of the nematodes were found freely in the body cavity of Pacific cod, and the rest of them (20.9%, 354/1694) were in the digestive tract or attached to other organs. PCR-RFLP analysis using HinfI and RsaI restriction enzymes revealed 3 different banding patterns corresponding to Anisakis pegreffii, Hysterothylacium aduncum and hybrid genotype (Anisakis simplex × A. pegreffii), respectively. Of 1694 nematodes, 1280 (75.6%) were identified as A. pegreffii and 406 (24.0%) were H. aduncum. A. pegreffii occupied 84.0% (1125/1340) of the nematodes in the body cavity and 40.0% (132/330) of them in the digestive tract, but no nematodes were found in Pacific cod muscles.     

In vitro antifungal activity of lactic acid bacteria against mycotoxigenic fungi and their application in loaf bread shelf life improvement

Food spoilage caused by mycotoxigenic fungi represents an important food safety problem. Lactic acid bacteria (LAB) are used as starter cultures in a larger number of food products. In this study, 16 strains of LAB were cultivated in MRS broth under anaerobiosis. Then, cell free supernatants were obtained by centrifugation and their antifungal activity against Aspergillus parasiticus and Penicillium expansum was tested using the disc-diffusion method. Furthermore, the LABs that showed in vitro antifungal activity were used in bread fermentation with yeast in order to study fungal growth inhibition and aflatoxin (AF) reduction in processed bread previously inoculated with A. parasiticus. The compounds present in the fermented medium of six LAB strains induced inhibition of P. expansum growth, whereas five probiotic strains produced antifungal compounds against A. parasiticus. The analysis by liquid chromatography coupled to mass spectrometry in tandem showed a reduction of the AF content in bread samples fermented with yeast and LABs. The reduction of AFs ranged from 84.1 to 99.9%. Moreover, bread sample studies showed a shelf life increase of about 3–4 days.     

Effect of nutmeg (Myristica fragrans) essential oil on the oxidative and microbial stability of cooked sausage during refrigerated storage

This paper examines the oxidative and microbial stability of cooked sausages, produced with the addition of 10 ppm (NO1) and 20 ppm (NO2) nutmeg (Myristica fragrans) essential oil. Instrumental parameters of color (CIE L*, CIE a* and CIE b*), Thiobarbituric acid-reactive substance (TBARS) values, microbial profile and sensory properties of aroma have been determined on the 1st, 30th, 45th, and 60th day of storage. Addition of the nutmeg essential oil had no effect on the color of cooked sausages. At the end of the storage, NO2 sausages had the best oxidative and microbial stability. TBARS values of NO1 and NO2 sausages were 1.21 mg MDA/kg and 0.95 mg MDA/kg, respectively, and were significantly lower (P < 0.05) compared to control (1.53 mg MDA/kg). Total number of aerobic mesophilic bacteria was lowest in NO2 sausages (78.3 cfu/g) and highest in control (185 cfu/g). After 45 and 60 days of storage, sensory properties of aroma of NO1 (4.21; 3.92) and NO2 sausages (4.39, 4.28) were better compared to those in control (4.07, 3.25). Hence, the addition of nutmeg essential oil in amount of 20 ppm can be successfully applied in order to extend the shelf life of cooked sausages.     

Phenolic fingerprint allows discriminating processed tomato products and tracing different processing sites

Tomatoes are important in human nutrition, as they are a source of carotenoids and phenolic compounds. However, transformation processes may alter the nutritional value of foods, decreasing the concentration of health-promoting compounds. This work aimed to explore the effects of industrial transformation on processed tomatoes (crushed pulp, puree and paste), as well as the effect of the different pre-processing technologies, rather than different manufacturing sites, in producing tomato paste. Results demonstrate that phenolics profiling can distinguish between different processed products as well as different paste pre-treatments (namely cold, warm and hot break), even though the latter underwent a final thermal treatment at >100 °C. Analogously, the different processing sites could be discriminated thanks to their characteristic phenolic fingerprint. The greatest differences identified were between conjugated forms of flavonoids, phenylpropanoids and lignans. The latter were the most labile phenolics, followed by flavonoids and then phenylpropanoids. Results provide evidence for the potential of phenolic fingerprint to support traceability of transformation processes and to investigate their effect on the nutritional value of processed tomatoes.     

Determination of heavy metals in selected black sea fish species

Heavy metals can be accumulated by marine organisms thought a variety of pathways, including respiration, adsorption and ingestion. The levels of heavy metals are known to increase drastically in marine environment through mainly anthropogenic activities. Fish are good indicators for the long term monitoring of metal accumulation in the marine environment. The aim of this study was to determine the levels of Cd, As, Hg, Pb, Zn and Cu in edible part and gill of seven most consumed Bulgarian fish species collected from north-east coast of Black Sea. These fish species are sprat (Sprattus sprattus sulinus), Mediterranean horse mackerel (Trachurus mediterraneus ponticus), Black sea gobies (Neogobius melanostromus), shad (Alosa pontica), Atlantic bonito (Sarda sarda), bluefish (Pomatomus saltatrix) and grey mullet (Mugil cephalus). The fish samples were collected during 2010. The analytical determination of As, Cd, Pb, Zn and Cu were performed by using flame and graphite furnace atomic absorption spectrometry after microwave digestion procedure. The total mercury determination was determined using a direct mercury analyzer (DMA-80). The metal concentration of analyzed elements was highest in the gill for all fish species. The maximum metal concentration was measured for Cu (1.40 mg kg⁻¹ w.w), Zn (11 mg kg⁻¹ w.w) and Pb (0.08 mg kg⁻¹ w.w) in muscle tissues of shad and sprat. The edible part of horse mackerel has the maximum value for Hg (0.12 mg kg⁻¹ w.w) while Atlantic bonito predominantly accumulates As (1.10 mg kg⁻¹ w.w). The analytical results obtained from this study were compared within acceptable limits for human consumption set by various health institutions.     

Histamine reduction by Maillard reaction with glucose

Histamine, well known as a toxic biogenic amine, is found in a variety of foods. Reducing its concentration and toxicity is desirable. In this study, the glucose/histamine Maillard reaction was proposed as a novel tool for histamine control. Effects of temperature, heating time, initial pH value, NaCl concentration, initial histamine concentration and initial glucose concentration on percentage removal of histamine in the glucose/histamine Maillard reaction model were investigated. The results showed that histamine reduction was affected by these variables, and could be almost eliminated under appropriate conditions. Fluorescence intensity and ultraviolet–visible spectroscopy analyses were used to characterize the glucose/histamine Maillard reaction. Cytotoxicity assay revealed that the glucose/histamine Maillard reaction significantly reduced the toxicity of histamine (P < 0.05). Furthermore, histamine concentrations in canned tuna samples were significantly reduced by thermal treatment with glucose (P < 0.05). This study demonstrates that the glucose/histamine Maillard reaction is a promising method for histamine control.     

Determination of melamine in milk using surface plasma effect of aggregated Au@SiO2 nanoparticles by SERS technique

A simple and rapid method to detect melamine in liquid milk by Surface-enhanced Raman Scattering (SERS) technique was presented. The pretreatment procedure of milk samples only contains hydrochloric acid treatment and twice centrifugation. In order to reduce the distortion about the Raman signals originating from charge transfer and electronic tunnelling effect, SiO₂ shell-isolated gold nanoparticles (Au@SiO₂ NPs) instead of Au NPs were employed to enhance signal intensity. Aggregation occurs when the Au@SiO₂ NPs colloid is mixed with the melamine solution or the treated milk containing melamine. Different from aggregated Au NPs, these aggregated Au@SiO₂ NPs on Cu substrate can undistortedly enhance the Raman signals of melamine using surface plasma effect. Quantitative analysis was tried and the results showed a good linearity (R² ≈ 0.99) when the melamine concentration was between 0.5 mgL⁻¹ and 5 mgL⁻¹. The detection sensitivity satisfies the requirement of the Codex Alimentarius Commission and this method can be practically used for melamine detection in milk.     

Isolation of lactic acid bacteria with antifungal activity against the common cheese spoilage mould Penicillium commune and their potential as biopreservatives in cheese

Moulds are the most common cheese spoilage organisms which can lead to economic loss as well as raising public health concerns due to the production of mycotoxins. In this study, 897 lactic acid bacteria (LAB) isolated from different herbs, fruits and vegetables were screened for their antifungal activity in an agar plate overlay assay. Thirty-six isolates had weak activity, 11 had moderate activity and 12 were confirmed as having strong activity. The strong antifungal isolates were obtained from a range of different sources but were all identified by 16S rDNA sequencing as being Lactobacillus plantarum. The antifungal spectra for these 12 isolates were determined against eight other moulds commonly associated with cheese spoilage and all isolates were found to possess inhibition against Penicillium solitum, Aspergillus versicolor and Cladosporium herbarum, but not against Penicillium roqueforti, Penicillium glabrum, Mucor circinelloides, Geotrichum candidum or Byssochlamys nivea. The absence of sodium acetate from MRS agar resulted in no inhibition of Penicilium commune, suggesting the synergistic effect of acetic acid with the antifungal LAB, similarly to that previously reported. To determine their potential as biopreservatives in cheese, LAB isolates were inoculated into cottage cheese prior to the addition of P. commune. All Lb. plantarum isolates were found to prevent the visible growth of P. commune on cottage cheese by between 14 and >25 days longer than cottage cheese that contained either no added LAB or LAB that did not have antifungal activity (Lactococcus lactis, Weissella soli, Leuconostoc inhae and Leuconostoc mesenteroides isolates). The results of this study shows that LAB isolated from various herbs, fruits and vegetables possess antifungal activity and have potential for use as biopreservatives in cheese.     

Antimicrobial effects of cinnamon bark oil on microbial composition and quality of grass carp (Ctenopharyngodon idellus) fillets during chilled storage

In this study, 11 essential oils were initially screened for antimicrobial activity against Aeromonas veronii, Acinetobacter johnsonii, Shewanella putrefaciens, and Pseudomonas jessenii. Cinnamon bark oil exhibited the highest antimicrobial activity and was used as a natural preservative in grass carp fillets stored at 4 ± 1 °C. Sensory assessment, total volatile basic nitrogen (TVB-N), color, biogenic amines, adenosine triphosphate (ATP)-related compounds, K-value, and presence of microbiota were analyzed during storage. Cinnamon bark oil treatment was found to be effective in enhancing organoleptic quality, inhibiting microbial growth, and delaying the increase of TVB-N, putrescine, cadaverine and K-value. Based on sensory analysis, shelf-life of grass carp fillets was 8 days for the control and 12 days for the treatment group. According to high-throughput sequencing, Aeromonas and Pseudomonas were the predominant microbiota in fresh grass carp. As storage time progressed, the microbial composition of both control and treatment samples became less diverse. Pseudomonas followed by Aeromonas were dominant in spoiled control samples. However, there were significant differences in microbial composition between control and treatment samples at the end of storage. Cinnamon bark oil treatment inhibited the growth of Aeromonas and Shewanella, and Pseudomonas was the only predominant microbiota found in spoiled treatment samples.     

Detection of selected corticosteroids and anabolic steroids in calf milk replacers by liquid chromatography–electrospray ionisation – Tandem mass spectrometry

The use of corticosteroids and anabolic steroids in food producing animals is regulated or banned in the European Union (EU). However, their use as growth promoters cannot be excluded. Milk replacers, considered by EU legislation as feeds, may be a good way of administration of these compounds. In order to improve the control of growth promoter utilization in animal husbandry and preventing possible consequences to animal welfare, we developed a method for multiresidue analysis of prednisolone, prednisone, dexamethasone, cortisone, cortisol, 17α- and 17β-boldenone and their precursor androstadienedione (ADD), testosterone, epitestosterone, 17α- and 17β-nandrolone, and trenbolone in powdered milk for calves. All analytes were extracted, after a common deproteinization and defatting sample pre-treatment, by a unique immunoaffinity column and analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS) in both positive and negative electrospray ionization (ESI) modes. The method was validated according to the criteria of the Commission Decision 2002/657/CE. The analytical limits were from 0.39 to 0.73 ng mL⁻¹ for the decision limit (CCα) and 0.46–0.99 ng mL⁻¹ for detection capability (CCβ). The analysis of 50 samples of milk replacers for calves, always revealed the presence of cortisol and cortisone (average concentrations 2.56 and 1.06 ng mL⁻¹, respectively), frequently testosterone and epitestosterone (1.24 and 0.63 ng mL⁻¹, respectively), occasionally β-nandrolone (0.82 ng mL⁻¹) and prednisolone (0.41 ng mL⁻¹). The other anabolic steroids were never found.