Screening for Antifungal Indigenous Lactobacilli Strains Isolated from Local Fermented Milk for Developing Bioprotective Fermentates and Coatings Based on Acid Whey Protein Concentrate for Fresh Cheese Quality Maintenance.

The demand for healthy foods without artificial food additives is constantly increasing. Hence, natural food preservation methods using bioprotective cultures could be an alternative to chemical preservatives. Thus, the main purpose of this work was to screen the indigenous lactobacilli isolated from fermented cow milk for their safety and antifungal activity to select the safe strain with the strongest fungicidal properties for the development of bioprotective acid whey protein concentrate (AWPC) based fermentates and their coatings intended for fresh cheese quality maintenance. Therefore, 12 lactobacilli strains were isolated and identified from raw fermented cow milk as protective cultures. The safety of the stains was determined by applying antibiotic susceptibility, haemolytic and enzymatic evaluation. Only one strain, Lacticaseibacillus paracasei A11, met all safety requirements and demonstrated a broad spectrum of antifungal activity in vitro. The strain was cultivated in AWPC for 48 h and grew well (biomass yield 8 log10 cfu mL-1). L. paracasei A11 AWPC fermentate was used as a vehicle for protective culture in the development of pectin-AWPC-based edible coating. Both the fermentate and coating were tested for their antimicrobial properties on fresh acid-curd cheese. Coating with L. paracasei A11 strain reduced yeast and mould counts by 1.0-1.5 log10 cfu mL-1 (p ≤ 0.001) during cheese storage (14 days), simultaneously preserving its flavour and prolonging the shelf life for six days.

Zearalenone and Its Metabolites in Blood Serum, Urine, and Milk of Dairy Cows.

After oral contamination, zearalenone (ZEN) is rapidly absorbed in organisms and can be detected in biological fluids. In this study, we investigated the metabolites of ZEN in the biological fluids of cows (blood, urine, milk). The study was divided into three stages: preparation (the first stage), investigation (the second stage), and final stage (the third stage). Samples of biological fluids were taken 7, 21, and 35 days after the beginning of the study. At the first stage and at the second stage, the cows were fed a total mixed ration (TMR) with naturally contaminated mycotoxin-zearalenone (500 ± 75 µg/kg). In the third stage, the cows were fed a TMR without mycotoxins. This study established that at the second stage, the alpha-zearalenol concentrations in the serum increased by 92% and the beta-zearalanol in the urine decreased by 48% compared to the first stage (p < 0.05). The beta-zearalenol and zearalanone concentrations in the urine were higher compared to that of the alpha-zearalenol. The zearalenone concentration in the milk at the second stage was 35% higher than at the first stage (p < 0.05). A significant negative correlation (r = −0.540) was determined between the beta-zearalenol and beta-zearalanol concentrations in the urine and the positive significant correlation (r = 0.826) between the beta-zearalanol and alpha-zearalenol concentrations in the serum (p < 0.05). During the study, it was determined that feeding cows for two weeks with a TMR without mycotoxins can reduce concentrations of alpha-zearalanol, beta-zearalenol, and beta-zearalanol in the biological fluids and can reduce the concentrations of ZEN in the milk, but does not reduce the concentration of zearalanone.

Effects of Ethanol Extracts of Origanum vulgare and Thymus vulgaris on the Mycotoxin Concentrations and the Hygienic Quality of Maize (Zea mays L.) Silage.

The aim of this study was to assess the usefulness of oregano (Origanum vulgare L.) and thyme (Thymus vulgaris L.) extracts to decrease mycotoxin contents and improve the hygienic quality of maize (Zea mays L.) silage. Under laboratory conditions, maize silage samples were fermented with oregano (OE), thyme (TE), oregano and thyme mixture (MIX), and two commercial inoculants. After 90 days of fermentation, silos were opened and silage samples were taken for evaluation of mycotoxin concentrations and for hygienic quality analysis: assessment of fermentation parameters, the content of biogenic amines, and microbiological status. It was determined that the mycotoxin concentrations decreased differentially: ochratoxin A concentration was below the detection limit after treatment with the TE and MIX extracts, the lowest zearalenone and deoxynivalenol concentrations were achieved with the OE extract treatment, T-2 toxin concentration was significantly lower after treatment with the TE extract, and HT-2 toxin concentration was lower after treatment with the MIX extract. The lowest content of biogenic amines in maize silage was established with the MIX extract. Concerning the silage hygienic quality, the best results of fermentation parameters and microbiological status were also achieved with the MIX extract. The present study indicated that oregano and thyme herbal plant extracts can be used to decrease mycotoxin concentrations and improve the hygienic quality of maize silage.

Prevalence of Mycotoxins and Endotoxins in Total Mixed Rations and Different Types of Ensiled Forages for Dairy Cows in Lithuania.

In this study, 119 samples of total mixed rations and different types of ensiled forage (maize and grass silage, and haylage) collected in 2019-2020 from dairy farms in Lithuania were analyzed to evaluate the quantitative occurrence of mycotoxins and endotoxins. Samples were analyzed using high-performance liquid chromatography (HPLC) with a fluorescent (FLD) and an ultraviolet detector (UV) of mycotoxins and a detection assay based on the ELISA technology for endotoxins. The study included toxins regulated within the European Union (aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON) and T-2 toxin) and nonregulated toxins (endotoxins). Mycotoxin analysis showed that 49.58% of the samples out of 119 were positive for AFB1, 52.11% for ZEA and DON, 55.47% for T-2 toxin and 84.04% for endotoxins. In the contaminated samples, the highest mean values of AFB1 and T-2 toxin were determined in the grass silage samples, while ZEA and DON-were determined in the maize silage samples. Maize silage samples had the highest ZEA and DON concentrations, exceeding the EU maximum permissible concentration limits. In the haylage samples, AFB1 mycotoxin exceeded the maximum concentration limits. The highest mean value of endotoxins was determined in the total mixed rations samples. This is the first study to provide information about the concentrations of mycotoxins and endotoxins in total mixed rations and different types of ensiled forages for dairy cows in Lithuania.

Origanum vulgare and Thymus vulgaris Extract Usability to Improve Silage Hygienic Quality and Reduce Mycotoxin Concentrations.

Silage is one of the main feed components of ruminants in the world. Which can consist about 50 - 80% part of the ration of dairy cows during the winter. The aim of this study was to evaluate oregano (Origanum vulgare L.) and thyme (Thymus vulgaris L.) aqueous and ethanol extracts usability to improve the hygienic quality of perennial ryegrass, red clover and blue alfalfa silage samples and estimate their effect to mycotoxins concentrations. During the experiment under laboratory conditions 63 silage samples (21 perennial ryegrasses, 21 blue alfalfa, 21 red clover) has been fermented with inserted oregano and thyme aqueous and ethanol extracts and two commercial inoculants with mesophilic lactic acid bacteria. After 96 days of fermentation in silage samples were established fermentation parameters, microbiological status and mycotoxins concentrations. It was determined that the best results for perennial ryegrass and red clover silage samples hygienic quality was achieved with inserted oregano aqueous and thyme ethanol extracts. While in blue alfalfa samples, the best results of silage hygienic indicators were determined with inserted oregano aqueous and ethanol extracts. Aflatoxin B1 (AFB1), deoxynivalenol (DON), zearalenone (ZEA) and T-2 toxin concentrations in perennial ryegrass, red clover and blue alfalfa silage samples were best reduced with inserted aqueous and ethanol extracts of oregano and thyme. The present study shows that oregano (Origanum vulgare L.) and thyme (Thymus vulgaris L.) aqueous and ethanol extracts can be used to improve the silage hygienic quality, reduce mycotoxins concentrations and thus ensure the wellness of cattle.

Influence of microbial and chemical contaminants on the yield and quality of ethanol from wheat grains.

BACKGROUND: Higher alcohols and esters have a negative impact on ethanol quality; therefore, to analyze the influence of mycotoxins and pesticides on higher alcohols and esters formation during ethanol production from contaminated cereal grains is of outstanding importance. RESULTS: In this study, the yield of ethanol and the composition of volatile by-products (acetaldehyde, methyl acetate, ethyl acetate, methanol, propanol, isobutanol, amyl and isoamyl alcohols) after fermentation of wheat grains artificially contaminated with Fusarium sporotrichioides and pesticides (triasulfuron and sulfosulfuron) were analyzed. Wheat grains contamination with triasulfuron significantly reduced ethanol yield and increased isobutanol and ethyl acetate content. Moreover, wheat grains infection with F. sporotrichioides significantly increased methyl acetate, ethyl acetate, isobutanol, and amyl and isoamyl alcohols content. Significant differences between pesticides and wheat variety on volatile compounds formation in ethanol has been estimated. CONCLUSION: The results showed that grains contaminated with F. sporotrichioides and pesticides significantly influenced the process of alcoholic fermentation that leads to variation of by-products in ethanol, including higher alcohols, esters, and aldehydes. © 2018 Society of Chemical Industry.

Application of hydrolases and probiotic Pediococcus acidilactici BaltBio01 strain for cereal by-products conversion to bioproduct for food/feed.

The aim of this study was to apply the enzymatic treatment and fermentation by Pediococcus acidilactici BaltBio01 strain for industrial cereal by-products conversion to food/feed bioproducts with high amount of probiotic lactic acid bacteria (LAB). LAB propagated in potato media and spray-dried remained viable during 12 months (7.0 log10 cfu/g) of storage and was used as a starter for cereal by-products fermentation. The changes of microbial profile, biogenic amines (BAs), mycotoxins, lactic acid (L+/D-), lignans and alkylresorcinols (ARs) contents in fermented cereal by-product were analysed. Cereal by-products enzymatic hydrolysis before fermentation allows to obtain a higher count of LAB during fermentation. Fermentation with P. acidilactici reduce mycotoxins content in fermented cereal by-products. According to our results, P. acidilactici multiplied in potato juice could be used for cereal by-products fermentation, as a potential source to produce safer food/feed bioproduct with high amount of probiotic LAB for industrial production.

Stable isotope ratio method for the characterisation of the poultry house environment.

Stable isotope analysis was applied to describe the poultry house environment. The poultry house indoor environment was selected for this study due to the relevant health problems in animals and their caretakers. Air quality parameters including temperature, relative humidity, airflow rate, NH3, CO2 and total suspended particles, as well as mean levels of total airborne bacteria and fungi count, were measured. Carbon isotope ratios (13C/12C) were obtained in size-segregated aerosol particles. The carbon (13C/12C) and nitrogen (15N/14N) isotope ratios were measured in feed, litter, scrapings from the ventilation system, feathers and eggs. Additionally, the distribution of δ13C and δ15N values in different tissues of the chicken was examined. The airborne bacteria and fungi extracted from the air filters collected from poultry farms were grown in the laboratory in media with known isotope values and measured for stable isotope ratios. Analysis of isotope fractionation between microorganisms and their media indicated the applicability of stable isotope analysis in bulk samples for the identification of source material. The analysed examples imply that stable isotope analysis can be used to examine the indoor environment along with its biology and ecology, and serve as an informative bioanalytical tool.

Antimicrobial activity of two essential oils.

The aim of the study was to evaluate the antimicrobial activity of essential oils in vitro for possible application to reduce the content of microorganisms in the air of animal houses. The essential oils of Cymbopogon citrarus L. and Malaleuca alternifolia L. were screened against bacteria Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and yeast Candida albicans. The minimal inhibitory concentration of the active essential oils was tested using broth dilution assay. The essential oils concentrations ranged from 0.1-50.0%. The combined effects of essential oils were tested for Malaleuca alternifolia L. and Cymbopogon citrarus L. concentrations ranged from 0.005-50.0%. The oils showed a wide spectrum of antibacterial activity. Concentrations of 0.1-0.5% of Cymbopogon citrarus L. and Malaleuca alternifolia L. reduced total microorganisms count of Proteus mirabilis and Candida albicans. High antibacterial activity was also revealed for Cymbopogon citrarus L. with bactericidal concentrations of 0.8% for Escherichia coli, 5.0% for Enterococcus faecium, 5.0% for Pseudomonas aeruginosa and 8.0% for Staphylococcus aureus. Bactericidal concentrations of Malaleuca alternifolia L. were 5.0% for Pseudomonas aeruginosa and Enterococcus faecium, and 8.0% for Staphylococcus aureus. The essential oils of Cymbopogon citrarus and Malaleuca alternifolia may be a promising alternative of air disinfection in animal houses.

Mycological and mycotoxicological evaluation of grain.

Grain storage conditions affect its quality. In Lithuania, different types of farms have various harvesting, processing and storing conditions. Grain samples were tested from agricultural granaries of 3 different types with different grain storage conditions in Lithuania. During March-April in 2001 the investigation on mycological and mycotoxicological state of stored grain from different types of agricultural granaries were performed. Wheat (Triticum aestivum L.) samples (n = 33) were tested from small, medium and large granaries. Barley (Hordeum distichon L.) (n = 22) was tested from small and medium granaries. Considering this issue, 31 species of micromycetes ascribed to 8 genera were isolated and identified. The results obtained indicate that highest levels of micromycetes contamination are found in small granaries with good storing and drying equipment. Micromycetes of some species belonging to the Penicillium Link, Aspergillus Mich. Ex Fr., Fusarium Link, and other genera, are able to produce secondary metabolites - mycotoxins of various compositions that are toxic to plants, animals, and humans. The levels of mycotoxins zearalenone and ochratoxin A were established. The highest concentration of zearalenone and ochratoxin A were found in grains from small farm granaries.