Meat Starter Culture Reduces Aspergillus parasiticus Production of Aflatoxins on Meat-Based and Salami Model Media.

There is great concern about the risk posed by the consumption of food contaminated with aflatoxins (AF), produced mostly by Aspergillus strains, that can also be found in dry-fermented meat products (DFMPs). The aim of this study was to investigate the inhibitory effect of meat starter culture (SC), frequently used for fermentation in the meat industry, on A. parasiticus growth and the production of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), and sterigmatocystin (STE) on different meat-based (CMA) and salami model (SM-G) media. Incubation was carried out under optimal conditions for fungal growth and under typical conditions for ripening of DFMPs for 21 days. Reversed-phase UPLC-MS/MS analysis was performed to determine mycotoxin production. SC reduced A. parasiticus growth more on CMA than on SM-G media. AFB1 formation was inhibited on both types of SC-containing media, although SC generally had a stronger inhibitory effect on AFB1 production on CMA than on SM-G. AFB1 and AFB2 were produced on CMA, while AFB1 dominated in SM-G, AFG1, and AFG2 were not detected in any media. The results show that SC inhibited AFB1 formation of A. parasiticus on SM-G media after 21 days of incubation under typical conditions for the production of DFMPs. These results indicate the necessity to investigate AF on natural matrices in an environment that is as similar as possible to real conditions in the production of DFMPs.

Contribution of Insoluble Bound Antioxidants from Germinated Seeds of Wheat and Spelt to the Nutritional Value of White Bread.

This research aimed to study the dynamic changes in phenolic antioxidants between the germ and the rest of the germinated seed of wheat and spelt and to evaluate the whole grain flour from germinated seeds as a functional supplement. Longer germination resulted in higher TPC, DPPH, and ABTS values when considering the entire germinated seed, while the optimal germination time was not consistent when considering the germ and the remaining germinated seed separately. While in the germinated seed (without germ) the majority of TPC was determined to be bound phenolics (up to 92%), the extractable form dominated in the germ (up to 69%). The most abundant phenolic antioxidants in germinated wheat and spelt seeds, trans-ferulic acid, cis-ferulic acid, and p-coumaric acid, increased significantly with germination. Only breads with 5% germinated spelt or wheat flour were suitable for the production of a food product, showing higher extractable TPC, antioxidant activity, individual phenolic acids, and improved specific volume, and were preferred because of their appearance, aroma, and color. The PCA biplot showed that the addition of 15% and 30% germinated flours had the greatest positive impact on phenolic properties, while breads with the addition of 5% germinated flour had the greatest positive impact on specific volume and color.

Identification and Quantification of Selected Benzoxazinoids and Phenolics in Germinated Spelt (Triticum spelta).

In this study, we investigated the effects of germination on the secondary metabolite composition in spelt grains. Germination significantly increased the content of various metabolites in free and bound forms. Benzoxazinoids were the most important compounds in the free fraction of the 96 h germinated grains (MBOA content as the predominant compound was 277.61 ± 15.29 µg/g DW). The majority of phenolic acids were present in the bound fraction, with trans-ferulic acid as the main component, reaching 753.27 ± 95.87 µg/g DW. The often neglected cis-isomers of phenolic acids accounted for about 20% of the total phenolic acids. High levels of apigenin di-C-glycosides were found in spelt grains, and the schaftoside content was most affected by germination, increasing threefold. The accumulation of secondary metabolites significantly increased the antioxidant activity of germinated spelt. According to the results of this study, the content of most bioactive compounds was highest in spelt grains after 96 h of germination. These data suggest that germinated spelt could potentially be valuable for the production of functional foods.

The Occurrence of Five Unregulated Mycotoxins Most Important for Traditional Dry-Cured Meat Products.

This study investigated the occurrence of 5 unregulated mycotoxins in a total of 250 traditional dry-cured meat products sampled in 2020 and 2021 in five Croatian regions (eastern, northern, central, western, and southern). Aflatoxin B1 (AFB1), ochratoxin A (OTA), sterigmatocystin (STC), citrinin (CIT), and cyclopiazonic acid (CPA) concentrations were related to the geographical region of the product's origin and to local weather. The results revealed the contamination of 27% of samples, namely, STC in 4% of samples in concentrations of up to 3.93 µg/kg, OTA in 10% of samples in concentrations of up to 4.81 µg/kg, and CPA in 13% of samples in concentrations of up to 335.5 µg/kg. No AFB1 or CIT contamination was seen. Although no statistically significant differences in concentrations of individual mycotoxins across the production regions were found, differences in mycotoxin occurrence were revealed. The eastern and western regions, with moderate climate, delivered the largest number of contaminated samples, while the southern region, often compared with subtropics, delivered the smallest, so that the determined mycotoxins were probably mainly produced by the Penicillium rather than the Aspergillus species. Due to the interaction of various factors that may affect mycotoxin biosynthesis during production, the detected concentrations cannot be related solely to the weather.

Trans fatty acids in frequently consumed products from Serbian and Slovenian market.

OBJECTIVES: Industrially produced trans fatty acid (iTFA) have adverse health effects and thus their consumption should be limited. The aim of this study was to determine and compare the iTFA content in frequently consumed food products by young adults from the Serbian and Slovenian market with supposedly elevated iTFA content in 2015. At the time of this study, there was no recommended limit of iTFA in both countries, and reduction of iTFA in foods was on voluntary basis. METHODS: We determined iTFA content in food products, 19 from the Serbian and 22 from the Slovenian market, blinded and analysed in the same analytical run. Contents of fatty acids (FA) methyl esters were analysed by capillary gas chromatography with a flame ionisation detector. Heptadecanoic acid was used as internal standard. Individual FA along with TFA were expressed as percentages of total measured FA. The amount of each FA in the sample was then calculated from the response factor and the transformation factor of the FA from the FA methyl ester content. RESULTS: Elaidic acid (C18:1t) was found as the most abundant TFA in analysed products, ranging from 0.52 g/100 g of total FA in chocolate candy up to 60.4 g/100 g in a salami from Serbian market. In Slovenian products, the values for elaidic acid were lower, 0.04-3.95 g/100 g of total FA, except in one type of wafers (24.3 g/100 g). CONCLUSIONS: The majority of analysed products from the Serbian and three from Slovenian market exceeded the recommended WHO and EU limit of 2% iTFA of total fat in foods. Samples of frequently consumed salami, wafers, tea biscuits, and snacks were identified as products with potentially higher burden of iTFA in diets of young adults in Serbia.

Production of Aflatoxin B1 by Aspergillus parasiticus Grown on a Novel Meat-Based Media.

The aim of the present study was to develop meat-based media with compositions similar to those of dry-fermented meat products and to evaluate their use in studying the growth of Aspergillus parasiticus and the kinetics of aflatoxin B1 (AFB1) production. In our previous experiments, we found that the strain A. parasiticus ZMJ7 produced a high amount of AFB1. Cooked meat agar (CMA2) was used as a novel complex meat-based medium with four variations: CMA2G (CMA2 supplemented with 1% glucose), CMA2YE (CMA2 supplemented with 0.2% yeast extract), and CMA2GYE (CMA2 supplemented with 1% glucose and 0.2% yeast extract). Media were inoculated with an A. parasiticus spore suspension (105 spores/mL) and incubated at 25 °C for up to 15 days. The A. parasiticus lag phase lasted less than 1 day, irrespective of the growth medium, with the exception of control medium CMA1 (cooked meat agar) as an already known meat-based medium. The highest mean colony growth rate was observed on CMA2 and CMA2G. Reversed-phase UPLC-MS/MS analysis was performed to determine the AFB1 concentration in combination with solid phase extraction (SPE). The highest AFB1 concentration in meat-based media was detected in CMA2GYE after 15 days of incubation (13,502 ± 2367 ng/mL media). The results showed that for studying AFB1 production in dry-fermented meat products, novel suitable media such as CMA2-based media are required. This finding could represent a potential concern with regard to the production of dry-fermented meat products.

Accumulation and Transformation of Biogenic Amines and Gamma-Aminobutyric Acid (GABA) in Chickpea Sourdough.

In general, sourdough fermentation leads to an improvement in the technological, nutritional, and sensory properties of bakery products. The use of non-conventional flours with a specific autochthonous microbiota may lead to the formation of secondary metabolites, which may even have undesirable physiological and toxicological effects. Chickpea flours from different suppliers have been used to produce sourdoughs by spontaneous and inoculated fermentations. The content of nutritionally undesirable biogenic amines (BA) and beneficial gamma-aminobutyric acid (GABA) was determined by chromatography. Fenugreek sprouts, which are a rich source of amine oxidases, were used to reduce the BA content in the sourdoughs. Spontaneous fermentation resulted in a high accumulation of cadaverine, putrescine, and tyramine for certain flours. The use of commercial starter cultures was not effective in reducing the accumulation of BA in all sourdoughs. The addition of fenugreek sprouts to the suspension of sourdough with pH raised to 6.5 resulted in a significant reduction in BA contents. Enzymatic oxidation was less efficient during kneading. Baking resulted in only a partial degradation of BA and GABA in the crust and not in the crumb. Therefore, it could be suggested to give more importance to the control of sourdough fermentation with regard to the formation of nutritionally undesirable BA and to exploit the possibilities of their degradation.

Germinated Buckwheat: Effects of Dehulling on Phenolics Profile and Antioxidant Activity of Buckwheat Seeds.

The aim was to investigate the effects of the cold dehulling of buckwheat seeds on their germination, total phenolic content (TPC), antioxidant activity (AA) and phenolics composition. Cold dehulling had no negative effects on germination rate and resulted in faster rootlet growth compared to hulled seeds. Although the dehulling of the seeds significantly decreased TPC and AA, the germination of dehulled seeds resulted in 1.8-fold and 1.9-fold higher TPC and AA compared to hulled seeds. Liquid chromatography coupled to mass spectrometry identified several phenolic compounds in free and bound forms. Rutin was the major compound in hulled seeds (98 µg/g dry weight), orientin and vitexin in 96-h germinated dehulled seeds (2205, 1869 µg/g dry weight, respectively). During germination, the increases in the major phenolic compounds were around two orders of magnitude, which were greater than the increases for TPC and AA. As well as orientin and vitexin, high levels of other phenolic compounds were detected for dehulled germinated seeds (e.g., isoorientin, rutin; 1402, 967 µg/g dry weight, respectively). These data show that dehulled germinated seeds of buckwheat have great potential for use in functional foods as a dietary source of phenolic compounds with health benefits.

Adaptation Response Mechanisms of Staphylococcus epidermidis Strains Exposed to Increasing Concentrations of Didecyldimethylammonium Chloride.

Staphylococcus epidermidis is a commensal inhabitant of human skin and mucosa, and a common nosocomial pathogen in immunocompromised patients. S. epidermidis strains were isolated from places with precisely defined and controlled air quality and regular cleaning and disinfection regimes-cleanrooms. These strains were adapted to increasing concentrations of the quaternary ammonium disinfectant didecyldimethylammonium chloride (DDAC). Compared to nonadapted strains, these strains became adapted to up to 180-fold higher concentrations of DDAC, as seen by their increased minimal inhibitory concentrations. Examination of the stability of adaptation showed that three strains became permanently adapted to DDAC and named as resistant strains, and four strains were temporarily adapted to DDAC and named as strains with higher tolerance to DDAC. Some adapted strains showed cross-resistance to benzalkonium chloride and/or antibiotics. The adaptation response mechanisms of these DDAC-adapted strains were also investigated. The majority of adapted strains showed modifications to cell size and fatty acid composition. Some of the adapted strains showed changes in biofilm formation and overexpression of efflux pumps. Three adapted strains also showed altered growth rates. In this first report of adaptation of S. epidermidis strains to DDAC, the fatty acid profiling showed that the majority of strains had reduced ratio of saturated to unsaturated fatty acids and decreased content of straight-chain fatty acids, at the expense of the anteiso-branched fatty acids. We can conclude that S. epidermidis strains can adapt or become resistant to DDAC. We have revealed several adaptive response mechanisms that can be targeted for control and inhibition of S. epidermidis in cleanrooms and other clean processing environments.

A Kinetic Approach in the Evaluation of Radical-Scavenging Efficiency of Sinapic Acid and Its Derivatives.

A kinetic approach was used to determine the radical scavenging activities of sinapic acid and its derivatives: sinapine, 4-vinylsyringol, syringic acid, syringaldehyde, and ethyl, propyl and butyl sinapate. The responses were expressed as rates of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙) scavenging (RS), superoxide radical (O2˙-) scavenging (RFF), and ß-carotene bleaching in the emulsion system (RB). For RS and RB, the esters of sinapic acid showed the highest responses while, for RFF, this was seen for syringic acid. The effectiveness of the selected compounds for scavenging these free radicals was also determined at a fixed endpoint. The early response parameters were demonstrated to be good discriminators in assessing differences for antioxidants with comparable fixed endpoint activity. The primary feature that ranks the kinetic data and the endpoint determinations is interpreted in terms of the mechanisms of the reactions involved in each of the assays conducted.

LC-MS analysis of phenolic compounds and antioxidant activity of buckwheat at different stages of malting.

The impact of malting on the profile of the phenolic compounds and the antioxidant properties of two buckwheat varieties was investigated. The highest relative increases in phenolic compounds were observed for isoorientin, orientin, and isovitexin, which are consequently major inducible phenolic compounds during malting. Only a minor relative increase was observed for the most abundant phenolic compound, rutin. The radical-scavenging activity of buckwheat seeds was evaluated using ABTS and DPPH assays. A considerable increase in total phenolic compounds and higher antioxidant activity were observed after 64h of germination, whereas kilning resulted in decreased total phenolic compounds and antioxidant activity. Higher antioxidant activities for extracts were found for buffered solvents than for pure methanol and water. Changes in the composition of the phenolic compounds and increased antioxidant content were confirmed by several methods, indicating that buckwheat malt can be used as a food rich in antioxidants.

Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering.

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering.

Oxidative stability of n-3-enriched chicken patties under different package-atmosphere conditions.

The oxidation processes were studied in chicken patties, enriched with n-3 fatty acids, after 8days of storage at 4°C, under different aerobic conditions, and following heat treatment. Significant effects were seen on lipid and cholesterol oxidation and the sensory qualities for whole flaxseed addition in the chicken feed (i.e., n-3 fatty acid enrichment), and for the different package-atmosphere conditions. For the raw chicken patties, n-3 enrichment increased the colour L(∗) values while, after the heat treatment, there were higher thiobarbituric acid-reactive substances (TBARs) and cholesterol oxidation products (COPs), and the rancidity was more pronounced. In comparison with the low O2 (<0.5%) package-atmosphere condition, O2 enrichment (80%) increased the instrumentally measured colour values, TBARs, total and individual COPs, and the rancidity became pronounced. The most suitable package-atmosphere condition of these raw n-3-enriched chicken patties is a very low O2 atmosphere, with or without an O2 scavenger.

Effects of industrial and home-made spread processing on bilberry phenolics.

Bilberries processed into spreads represent an important source of anthocyanins if these remain rich in the final product. The effects of thermal processing were studied with non-ground and ground bilberries processed into spreads according to industrial and home-made procedures. Samples were analysed by LC-DAD-MS/MS and LC-MS. The spreads had 28-60% less total phenolics, 4-62% less anthocyanins, and 1-fold to 2-fold more phenolic acids and total flavonols than the bilberries, but approximately equal flavanols. The home-made spread from ground bilberries had ca. 26% higher antioxidant activity. Delphinidin 3-glucoside and cyanidin 3-glucoside were taken through the two spread procedures, with their degradation to gallic acid (38-57%), protocatechuic acid (1-2%) and 2,4,6-trihydroxybenzaldehyde determined. The amounts of gallic and protocatechuic acids did not reflect well for anthocyanin degradation. The industrial spread procedure with non-ground bilberries is a more suitable procedure to maintain the final content of anthocyanins.

Insight into the molecular mechanisms of propolis activity using a subcellular proteomic approach.

The effects of a fractionated 70% ethanolic extract of propolis were analyzed at the subproteome level by two-dimensional electrophoresis. Differential detergent fractionation was used to fractionate proteins from the yeast Saccharomyces cerevisiae according to their subcellular localization. Thus, four subcellular proteomes were obtained: cytosolic, membrane/organelle, nuclear, and cytoskeletal. Yeast treatment resulted in changes in the levels of proteins involved in carbohydrate and energy metabolism, antioxidant defense, actin filament dynamics, folding of proteins, and others. On the basis of this information, we can obtain better insights into the processes that are carried out in cells exposed to propolis extract.

Phenolic content of strawberry spreads during processing and storage.

This study provides a comprehensive and systematic evaluation of phenolics in strawberry spreads processed according to different industrial procedures and stored under several storage conditions for up to 19 weeks. Total phenolics were determined spectrophotometrically, and individual phenolics were determined by combined liquid chromatography and mass spectrometry: six anthocyanins, four phenolic acids, two flavonols, one flavanol, and one flavone. During storage, the phenolics were modified. The total anthocyanins, vanillic acid, kaempferol, and luteolin decreased, while salicylic and gallic acids increased. Total phenolics, cyanidin 3-(6″-succinyl-glucoside) (here observed for the first time), protocatechuic acid, quercetin, and catechin remained stable. The best phenolic retention was observed in spreads stored at 4 °C. Therefore, the proposed storage process (use of a cold chain) indicates good retention of phenolics in strawberry spreads, which maintain high nutritional and sensorial quality.

Identification of the chelocardin biosynthetic gene cluster from Amycolatopsis sulphurea: a platform for producing novel tetracycline antibiotics.

Tetracyclines (TCs) are medically important antibiotics from the polyketide family of natural products. Chelocardin (CHD), produced by Amycolatopsis sulphurea, is a broad-spectrum tetracyclic antibiotic with potent bacteriolytic activity against a number of Gram-positive and Gram-negative multi-resistant pathogens. CHD has an unknown mode of action that is different from TCs. It has some structural features that define it as 'atypical' and, notably, is active against tetracycline-resistant pathogens. Identification and characterization of the chelocardin biosynthetic gene cluster from A. sulphurea revealed 18 putative open reading frames including a type II polyketide synthase. Compared to typical TCs, the chd cluster contains a number of features that relate to its classification as 'atypical': an additional gene for a putative two-component cyclase/aromatase that may be responsible for the different aromatization pattern, a gene for a putative aminotransferase for C-4 with the opposite stereochemistry to TCs and a gene for a putative C-9 methylase that is a unique feature of this biosynthetic cluster within the TCs. Collectively, these enzymes deliver a molecule with different aromatization of ring C that results in an unusual planar structure of the TC backbone. This is a likely contributor to its different mode of action. In addition CHD biosynthesis is primed with acetate, unlike the TCs, which are primed with malonamate, and offers a biosynthetic engineering platform that represents a unique opportunity for efficient generation of novel tetracyclic backbones using combinatorial biosynthesis.

Fractionation of phenolic compounds extracted from propolis and their activity in the yeast Saccharomyces cerevisiae.

We have here investigated the activities of Slovenian propolis extracts in the yeast Saccharomyces cerevisiae, and identified the phenolic compounds that appear to contribute to these activities. We correlated changes in intracellular oxidation and cellular metabolic energy in these yeasts with the individual fractions of the propolis extracts obtained following solid-phase extraction. The most effective fraction was further investigated according to its phenolic compounds.

FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis.

BACKGROUND: FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses. RESULTS: Three putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively. CONCLUSIONS: Our results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of S. tsukubaensis for improving the yield of fermentative processes for production of FK506.

Chemical properties and antioxidant and antimicrobial activities of Slovenian propolis.

The chemical composition as well as the antioxidant and antimicrobial activities of two EtOH extracts of propolis (PEEs) from Slovenia were determined. EtOH was used as extracting solvent at 70 and 96%, providing the extracts PEE70 and PEE96, respectively. The extraction with 70% EtOH was more efficient than that with 96% EtOH, as the PEE70 was richer in total phenolic compounds than the PEE96. The Slovenian propolis was characterized by different phenolic acids and flavonoids. The PEE96 was slightly richer in three specific compounds, i.e., caffeic acid, ferulic acid, and luteolin, while all other substances detected showed higher contents in the PEE70. The PEE70 showed a stronger reducing power and ability to scavenge free radicals and metal ions than the PEE96. Both PEEs were in the main more effective against Gram-positive bacteria than against fungi and Gram-negative bacteria like Salmonella and Escherichia coli, with the exception of Campylobacter. The PEE96 decreased the intracellular oxidation in Saccharomyces cerevisiae in a dose-dependent manner. The antimicrobial activities and antioxidant properties were related to the total phenolic contents. The two PEEs have the potential for use as natural antimicrobial and antioxidant additives in foods.