Mycotoxins and pyrrolizidine alkaloids in herbal dietary supplements.

The market demand for herbal dietary supplements is rapidly growing and such products are becoming more common and accessible to consumers. However, the knowledge about their safety remains incomplete. Herbal dietary supplements are one of the food groups that can contribute significantly to human health concerns arising from chronic exposure to pyrrolizidine alkaloids and mycotoxins. This study aimed to simultaneously determine 79 natural contaminants, including mycotoxins, as well as pyrrolizidine and tropane alkaloids in herbal dietary supplements in one analytical run. Exposure assessment and human health risks were assessed for all compounds included in this study. The total concentration of naturally occurring contaminants in herbal dietary supplements reached 5.3 mg kg-1 and the most frequently detected mycotoxins were tentoxin and alternariol monomethyl ether. The latter was detected with the highest frequency, reaching concentrations up to 2.5 mg kg-1. The obtained results indicate a potential risk to public health related to herbal dietary supplement consumption.

An Occurrence Study of Mycotoxins in Plant-Based Beverages Using Liquid Chromatography-Mass Spectrometry.

Mycotoxins are toxic mold metabolites that can adversely affect human and animal health. More than 400 mycotoxins have been identified so far. Cereals and nuts are the predominant mycotoxin-contaminated foodstuffs. Plant-based drinks produced from cereals, nuts, and legumes have grown in popularity. The mycotoxins accumulated in these crops may transfer to these beverages. A liquid chromatography-tandem mass spectrometry method was developed and optimized for the assessment of 22 mycotoxins in commercially available plant-based drinks in Latvia and Lithuania. A total of 64% of the seventy-two analyzed beverages were positive for one to sixteen mycotoxins, with deoxynivalenol, beauvericin, and enniatins A, B, B1, T-2, and HT-2 toxins detected most frequently. The European Commission has not yet set guidelines for the maximum mycotoxin concentrations in plant-based beverages, nor has the European Food Safety Authority conducted a risk assessment. Therefore, acute exposure studies were provided for the Latvian population based on the assumed replacement of dairy milk with plant-based beverages to ascertain the safety of plant-based milk substitutes. Based on the observed levels of mycotoxin prevalence and contamination levels and assumed exposure, it can be concluded that tested plant-based beverages may be relatively safe. However, exposure to emerging mycotoxins should be considered.

The Effect of Ustilago maydis and Delayed Harvesting on A- and B-Type Trichothecene Concentrations in Maize Grain.

The aim of this study was to investigate whether, in the context of a higher incidence of Ustilago maydis and Fusarium spp. at optimal and delayed harvest times, a higher incidence of mycotoxin contamination in maize grains could be expected. The field experiment was carried out at the Lithuanian Research Centre for Agriculture and Forestry experimental fields over three consecutive years (2020-2022). Two maize hybrids (Duxxbury and Lapriora) with different FAO numbers were used. The experimental design in the field was a randomized complete block design. Harvesting took place at three different times: first at physiological maturity, and then 10 (±2) and 20 (±2) days after the first harvest. Each hybrid had four repetitions at different harvest times. The U. maydis infection was only detected in 2021 and after the first harvest cobs were further divided into four different groups with four repetitions: healthy cobs, cobs visually infected with Fusarium spp., cobs visually infected with common smut, and cobs visually infected with both pathogens. No U. maydis-damaged maize cobs were found in 2020 and 2022. The levels of Fusarium microscopic fungi in maize grains were also from 4 to 16 times higher in 2021 than in 2020 and 2022. Harvest delays in 2020 led to a significant deoxynivalenol concentration increase in the Duxxbury hybrid and an HT-2 concentration increase in the Lapriora hybrid. In 2021, deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, and HT-2 concentrations significantly rose in both hybrids, but the T-2 concentration significantly increased only in the Lapriora hybrid. Deoxynivalenol concentrations were, respectively, 110 and 14.6 times higher than in cobs only infected with Fusarium spp. or U. maydis. Concentrations of 15-acetyl-deoxynivalenol were, respectively, 60, 67, and 43 times higher than in asymptomatic cobs and cobs only infected with Fusarium spp. or U. maydis. Cobs contaminated with both pathogens also had higher concentrations of 3-acetyl-deoxynivalenol. T-2 and HT-2 were detected in maize grains harvested from cobs infected only with Fusarium spp. The presence of U. maydis and Fusarium fungi in maize cobs, along with harvest delays, led to significant increases in mycotoxin concentrations, highlighting the importance of timely harvesting and pathogen management to mitigate mycotoxin contamination in maize grains.

Submerged and Solid-State Fermentation of Spirulina with Lactic Acid Bacteria Strains: Antimicrobial Properties and the Formation of Bioactive Compounds of Protein Origin.

The aim of this study was to investigate the changes in bioactive compounds (L-glutamic acid (L-Glu), gamma-aminobutyric acid (GABA) and biogenic amines (BAs)) during the submerged (SMF) and solid-state (SSF) fermentation of Spirulina with lactobacilli strains (Lacticaseibacillus paracasei No. 244; Levilactobacillus brevis No. 173; Leuconostoc mesenteroides No. 225; Liquorilactobacillus uvarum No. 245). The antimicrobial properties of the untreated and fermented Spirulina against a variety of pathogenic and opportunistic strains were tested. The highest concentrations of L-Glu (3841 mg/kg) and GABA (2396 mg/kg) were found after 48 h of SSF with No. 173 and No. 244 strains, respectively. The LAB strain used for biotreatment and the process conditions, as well as the interaction of these factors, had statistically significant effects on the GABA concentration in Spirulina (p ≤ 0.001, p = 0.019 and p = 0.011, respectively). In all cases, the SSF of Spirulina had a higher total BA content than SMF. Most of the fermented Spirulina showed exceptional antimicrobial activity against Staphylococcus aureus but not against the other pathogenic bacteria. The ratios of BA/GABA and BA/L-Glu ranged from 0.5 to 62 and from 0.31 to 10.7, respectively. The GABA content was correlated with putrescine, cadaverine, histamine, tyramine, spermidine and spermine contents. The L-glutamic acid concentration showed positive moderate correlations with tryptamine, putrescine, spermidine and spermine. To summarize, while high concentrations of desirable compounds are formed during fermentation, the formation of non-desirable compounds (BAs) must also be considered due to the similar mechanism of their synthesis as well as the possibility of obtaining high concentrations in the end products.

Bio-Converted Spirulina for Nutraceutical Chewing Candy Formulations Rich in L-Glutamic and Gamma-Aminobutyric Acids.

This study aimed at evaluating changes of microalgae Spirulina during its fermentation with Lactiplantibacillus plantarum No. 122 strain, and further at incorporating Spirulina bio-converted for nutraceuticals rich in L-glutamic (L-Glu) and gamma-aminobutyric acids (GABA) into sucrose-free chewing candy (gummy) preparations. Fermented Spirulina had higher b* (yellowness) coordinates than untreated (non-fermented), and fermentation duration (24 and 48 h) had a statistically significant effect on colour coordinates. The highest contents of L-glutamic and gamma-aminobutyric acids (4062 and 228.6 mg/kg, respectively) were found in 24 and 48 h-fermented Spirulina, respectively. Fermentation increased the content of saturated fatty acids and omega-3 in Spirulina, while monounsaturated fatty acids and omega-6 were reduced. The addition of fermented Spirulina (FSp) significantly affected hardness, decreased lightness and yellowness, and increased the greenness of chewing candies. All chewing candy samples (with xylitol) prepared with 3 and 5 g of FSp and 0.2 µL of Citrus paradise essential oil received the highest scores for overall acceptability, and the highest intensity (0.052) of emotion "happy" was elicited by the sample group containing xylitol, agar, ascorbic acid, 3 g of FSp, and 0.1 µL of Mentha spicata essential oil. As an outcome of this research, one may conclude that fermented Spirulina has significant potential as an innovative ingredient in the production of healthier sucrose-free nutraceutical chewing candies.

Changes in Spirulina's Physical and Chemical Properties during Submerged and Solid-State Lacto-Fermentation.

The aim of this study was to select a lactic acid bacteria (LAB) strain for bio-conversion of Spirulina, a cyanobacteria ("blue-green algae"), into an ingredient with a high concentration of gamma-aminobutyric acid (GABA) for human and animal nutrition. For this purpose, ten different LAB strains and two different fermentation conditions (SMF (submerged) and SSF (solid state fermentation)) were tested. In addition, the concentrations of fatty acids (FA) and biogenic amines (BA) in Spirulina samples were evaluated. It was established that Spirulina is a suitable substrate for fermentation, and the lowest pH value (4.10) was obtained in the 48 h SSF with Levilactobacillus brevis. The main FA in Spirulina were methyl palmitate, methyl linoleate and gamma-linolenic acid methyl ester. Fermentation conditions were a key factor toward glutamic acid concentration in Spirulina, and the highest concentration of GABA (2395.9 mg/kg) was found in 48 h SSF with Lacticaseibacillus paracasei samples. However, a significant correlation was found between BA and GABA concentrations, and the main BA in fermented Spirulina samples were putrescine and spermidine. Finally, the samples in which the highest GABA concentrations were found also displayed the highest content of BA. For this reason, not only the concentration of functional compounds in the end-product must be controlled, but also non-desirable substances, because both of these compounds are produced through similar metabolic pathways of the decarboxylation of amino acids.

Toxigenicity of F. graminearum Residing on Host Plants Alternative to Wheat as Influenced by Environmental Conditions.

Fusarium graminearum is an important pathogen that causes Fusarium head blight (FHB) in several cereal crops worldwide. The potential of this pathogen to contaminate cereals with trichothecene mycotoxins presents a health risk for both humans and animals. This study aimed to evaluate the potential of different trichothecene genotypes of F. graminearum isolated from an alternative host plant to produce mycotoxins under different spring wheat grain incubation conditions. Fourteen F. graminearum strains were isolated from seven alternative host plants and identified as 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) genotypes. These strains were cultivated on spring wheat grains at 25 °C and 29 °C for 5 weeks. The mycotoxins produced were analysed with a high-performance liquid chromatograph (HPLC) coupled to a Thermo Scientific TSQ Quantiva MS/MS detector. The obtained results showed that the F. graminearum strains from alternative host plants could produce nivalenol (NIV), deoxynivalenol (DON), fusarenon-X (FUS-X), 3-ADON, deoxynivalenol-3-ß-d-glucoside (D3G), 15-ADON, and zearalenone (ZEA). F. graminearum strains produced DON and ZEA under both temperatures, with the mean concentrations varying from 363 to 112,379 µg kg-1 and from 1452 to 44,816 µg kg-1, respectively. Our results indicated the possible role of dicotyledonous plants, including weeds, as a reservoir of inoculum sources of F. graminearum-induced Fusarium head blight, associated with the risk of mycotoxin contamination in spring wheat.

Nanoflow liquid chromatography mass spectrometry method for quantitative analysis and target ion screening of pyrrolizidine alkaloids in honey, tea, herbal tinctures, and milk.

A method for the determination of pyrrolizidine alkaloids in tea, honey, herbal tinctures, and milk samples was developed by employing nano-LC-MS with high-resolution Orbitrap mass spectrometry. Quantitation was performed using the available analytical standards, and a MS2 target ion screening approach was developed using fragment ions that were specific for pyrrolizidine alkaloids under collision-induced dissociation. Proof of concept was delivered for the screening approach, proposing that the C6H8N+ fragment ion is a highly selective fragment ion for the detection of potential pyrrolizidine alkaloids. The elaborated quantitation was applied for the occurrence study of pyrrolizidine alkaloids in food products available on the Latvian market, including samples of tea (n = 15), honey (n = 40), herbal tinctures (n = 15), and milk (n = 10). The median LOQ over all analytes was 0.33 µg kg-1 in honey, 3.6 µg kg-1 in tea, 3.3 µg kg-1 in herbal tinctures, and 0.32 µg kg-1 in milk. The herbal tinctures samples and milk samples did not contain pyrrolizidine alkaloids above LOQ values. Analytes were detected in 33% of honey and 47% of tea samples. Most common were echimidine, intermedine, and enchinatine N-oxide. Pyrrolizidine alkaloids in tea samples were mainly N-oxides, with the highest total concentration being 215 µg kg-1 among the samples, exceeding the maximum limit of 200 µg kg-1 set by Commission Regulation (EU) 2020/2040. In honey samples, lycopsamine-type alkaloids were detected most frequently, with the highest total concentration equal to 74 µg kg-1. Advantages of the developed nano-LC-MS methods included increased sensitivity in comparison with conventional flow LC-MS, low solvent consumption typical with nano-LC and the novel use of a selective common target ion for detection and discovery of potential pyrrolizidine alkaloids using high resolution mass spectrometry.

Application of Wastewater-Based Epidemiology for Tracking Human Exposure to Deoxynivalenol and Enniatins.

Wastewater-based epidemiology (WBE) is a promising biomonitoring approach with the potential to provide direct information on human intake and exposure to food contaminants and environmental chemicals. The aim of this study was to apply WBE while employing the normalization method for exploring human exposure to selected mycotoxins according to population biomarker 5-hydroxyindoleacetic acid (5-HIAA). This type of normalization technique has been previously used to detect various other compounds. However, to the best of our knowledge, this is the first study tracking human exposure to mycotoxins. A sensitive analytical methodology was developed to achieve reliable quantification of deoxynivalenol, enniatins, and beauvericin in wastewater (WW) samples. The applicability of the method was evaluated by testing 29 WW samples collected at WW treatment plants in Latvia. With frequency of detection greater than 86%, enniatins B, B1, A, and A1 were revealed in WW samples. The estimated total daily intake for enniatins was in the range of 1.8-27.6 µg/day per person. Free deoxynivalenol (DON) was determined in all analysed WW samples. Based on the average 5-HIAA excretion level and the determined 5-HIAA content in the samples, the intake of DON by the human population of Riga was estimated at 325 ng/kg b.w. day.