A two-tiered system of analysis to tackle rice fraud: The Indian Basmati study.

Demand for high quality Basmati rice has increased significantly in the last decade. This commodity is highly vulnerable to fraud, especially in the post COVID-19 era. A unique two-tiered analytical system comprised of rapid on-site screening of samples using handheld portable Near-infrared NIR and laboratory confirmatory technique using a Head space gas chromatography mass spectrometry (HS-GC-MS) strategy for untargeted analysis was developed. Chemometric models built using NIR data correctly predicted nearly 100% of Pusa 1121 and Taraori, two high value types of Basmati, from potential adulterants. Furthermore, rice VOC profile fingerprints showed very good classification (R2 >0.9, Q2 > 0.9, Accuracy > 0.99) for these high quality Basmati varieties from potential adulterant varieties with aldehydes identified as key VOC marker compounds. Using a two-tiered system of a rapid method for on-site screening of many samples alongside a laboratory-based confirmatory method can classify Basmati rice varieties, protecting the supply chain from fraud.

Food Fingerprinting: Using a Two-Tiered approach to Monitor and Mitigate Food Fraud in Rice.

BACKGROUND: Rice is an important staple food that is consumed around the world. Like many foods, the price of rice varies considerably, from very inexpensive for a low-quality product to premium pricing for highly prized varieties from specific locations. Therefore, like other foods it is vulnerable to economically motivated adulteration through substitution or misrepresentation of inferior-quality rice for more expensive varieties. OBJECTIVE: In this article we describe results of a research project focused on addressing potential food fraud issues related to rice supplies in China, India, Vietnam, and Ghana. Rice fraud manifests differently in each country; therefore, tailored solutions were required. METHOD: Here we describe a two-tiered testing regime of rapid screening using portable Near Infrared technology supported by second tier testing using mass spectrometry-based analysis of suspicious samples. RESULTS: Portable Near Infrared spectroscopy models and laboratory-based Gas Chromatography-Mass Spectrometry methods were developed to differentiate between: high-value Basmati rice varieties and their potential adulterants; six Geographic Indicated protected rice varieties from specific regions within China; various qualities of rice in Ghana and Vietnam; and locally produced and imported rice in Ghana. Furthermore, an Inductively Coupled Plasma-Mass Spectrometry method was developed to support the Chinese rice varieties methods as well as a Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry method for quality differentiation in Vietnam. CONCLUSIONS/HIGHLIGHTS: This two-tier approach can provide a substantially increased level of testing through rapid screening outside of the laboratory with the reassurance of corroborating mass spectrometry-based laboratory analysis to support decision making.

A Human Relevant Defined Mixture of Persistent Organic Pollutants (POPs) Affects In Vitro Secretion of Glucagon-Like Peptide 1 (GLP-1), but Does Not Affect Translocation of Its Receptor.

Environmental exposure to persistent organic pollutants (POPs) has been suggested as a contributing factor for the increased rate of type 2 diabetes and obesity. A complex mixture of 29 POPs (Total mixture), based on human blood concentrations, was used to expose a glucagon-like peptide 1 (GLP-1) secreting enteroendocrine cell line (pGIP/neo: STC-1) in vitro for 3 and 24 h. Significant increases of GLP-1 occurred when cells were exposed to the Total mixture at ×500 blood levels. Six sub-mixtures representing chlorinated (Cl), brominated (Br), and perfluorinated chemicals (PFAA), and their combinations (Cl + Br, Cl + PFAA, Br + PFAA) were also tested at ×500. Secretion levels seen for these remained lower than the Total mixture, and the Br mixture had no effect. After 24 h, increased secretion was seen with all mixtures at ×1 blood levels. Cytotoxicity was present for ×100 and ×500 blood levels. When tested in a GLP-1 receptor translocation assay (U2OS-GLP1R-EGFP), neither agonistic nor antagonist effects on receptor internalization were seen for any of the mixtures. We conclude individual classes of POPs, alone or in combination, can affect GLP-1 secretion and may contribute as a molecular mechanism linking environmental toxicants and diabetes.

Improvements in single-use bioreactor film material composition leads to robust and reliable Chinese hamster ovary cell performance.

Single-use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO-K1, and CHO-DP12, cultured in media conditioned in an older single-use bioreactor (SUB) film (F-1) and a newer generation film (F-2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F-1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F-2 film. Proteomic profiling of CHO cells cultured in the F-1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC-MS revealed a reduction in the abundance of the analyzed leachates from F-2 films when compared to F-1 films including bDtBPP, potentially explaining improved CHO cell growth in F-2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess.

In vitro bioassay investigations of suspected obesogen monosodium glutamate at the level of nuclear receptor binding and steroidogenesis.

Monosodium glutamate (MSG) is a commonly used flavour enhancer in households, catering and food production. Recently it has been highlighted as a suspected dietary obesogen in epidemiological studies indicating a link between MSG consumption and weight gain. Additionally, animal studies have shown that MSG exposure has profound effects on sex steroid hormone levels and receptors; which have an important role in energy metabolism. However, the exact mechanism by which MSG exerts its effects has yet to be elucidated. Reporter gene assays (RGAs) and the H295R steroidogenesis assay have been used to investigate the endocrine disrupting potential of MSG. Receptor (ant)agonism was not observed in the MMV-Luc (oestrogen responsive) or TM-Luc (progestagen responsive) cell lines following exposure to MSG. Also, no effects on hormone production were observed. However, MSG exhibited an antagonist response in the androgen and progestagen responsive TARM-Luc cell line, with a dose dependent reduction in androgen response of 33%, 36.9% and 50.6% (in comparison to the solvent control) at 50, 250 and 500 µg/ml MSG, respectively (P ≤ 0.05; P ≤ 0.05; P ≤ 0.001). No cytotoxicity or pre-lethal cytotoxicity was observed at the concentrations tested. These findings demonstrate one potential pathway whereby MSG may act as a dietary obesogen.

The endocrine disrupting potential of monosodium glutamate (MSG) on secretion of the glucagon-like peptide-1 (GLP-1) gut hormone and GLP-1 receptor interaction.

Monosodium glutamate (MSG) is a suspected obesogen with epidemiological evidence positively correlating consumption to increased body mass index and higher prevalence of metabolic syndrome. ELISA and high content analysis (HCA) were employed to examine the disruptive effects of MSG on the secretion of enteroendocrine hormone glucagon-like peptide-1 (GLP-1) and GLP-1 receptor (GLP-1R), respectively. Following 3h MSG exposure of the enteroendocrine pGIP/neo: STC-1 cell line model (500µg/ml) significantly increased GLP-1 secretion (1.8 fold; P≤0.001), however, 72h exposure (500µg/ml) caused a 1.8 fold decline (P≤0.05). Also, 3h MSG exposure (0.5-500µg/ml) did not induce any cytotoxicity (including multiple pre-lethal markers) but 72h exposure at 250-500µg/ml, decreased cell number (11.8-26.7%; P≤0.05), increased nuclear area (23.9-29.8%; P≤0.001) and decreased mitochondrial membrane potential (13-21.6%; P≤0.05). At 500µg/ml, MSG increased mitochondrial mass by 16.3% (P≤0.01). MSG did not agonise or antagonise internalisation of the GLP-1R expressed recombinantly in U2OS cells, following GLP-1 stimulation. In conclusion, 72h exposure of an enteroendocrine cell line at dietary levels of MSG, results in pre-lethal cytotoxicity and decline in GLP-1 secretion. These adverse events may play a role in the pathogenesis of obesity as outlined in the obesogen hypothesis by impairing GLP-1 secretion, related satiety responses and glucose-stimulated insulin release.

An in vitro investigation on the cytotoxic and nuclear receptor transcriptional activity of the mycotoxins fumonisin B1 and beauvericin.

Fumonisin B1 (FB1) and beauvericin (BEA) are secondary metabolites of filamentous fungi, which under appropriate temperature and humidity conditions may develop on various foods and feeds. To date few studies have been performed to evaluate the toxicological and endocrine disrupting effects of FB1 and BEA. The present study makes use of various in vitro bioassays including; oestrogen, androgen, progestagen and glucocorticoid reporter gene assays (RGAs) for the study of nuclear receptor transcriptional activity, the thiazolyl blue tetrazolium bromide (MTT) assay to monitor cytotoxicity and high content analysis (HCA) for the detection of pre-lethal toxicity in the RGA and Caco-2 human colon adenocarcinoma cells. At the receptor level, 0.001-10µM BEA or FB1 did not induce any agonist responses in the RGAs. However at non-cytotoxic concentrations, an antagonistic effect was exhibited by FB1 on the androgen nuclear receptor transcriptional activity at 10µM and BEA on the progestagen and glucocorticoid receptors at 1µM. MTT analysis showed no decrease in cell viability at any concentration of FB1, whereas BEA showed a significant decrease in viability at 10µM. HCA analysis confirmed that the reduction in the progestagen receptor transcriptional activity at 1µM BEA was not due to pre-lethal toxicity. In addition, BEA (10µM) induced significant toxicity in both the TM-Luc (progestagen responsive) and Caco-2 cells.

In vitro bioassay investigations of the endocrine disrupting potential of steviol glycosides and their metabolite steviol, components of the natural sweetener Stevia.

The food industry is moving towards the use of natural sweeteners such as those produced by Stevia rebaudiana due to the number of health and safety concerns surrounding artificial sweeteners. Despite the fact that these sweeteners are natural; they cannot be assumed safe. Steviol glycosides have a steroidal structure and therefore may have the potential to act as an endocrine disruptor in the body. Reporter gene assays (RGAs), H295R steroidogenesis assay and Ca(2+) fluorimetry based assays using human sperm cells have been used to assess the endocrine disrupting potential of two steviol glycosides: stevioside and rebaudioside A, and their metabolite steviol. A decrease in transcriptional activity of the progestagen receptor was seen following treatment with 25,000 ng/ml steviol in the presence of progesterone (157 ng/ml) resulting in a 31% decrease in progestagen response (p=<0.01). At the level of steroidogenesis, the metabolite steviol (500-25,000 ng/ml) increased progesterone production significantly by 2.3 fold when exposed to 10,000 ng/ml (p=<0.05) and 5 fold when exposed to 25,000 ng/ml (p=<0.001). Additionally, steviol was found to induce an agonistic response on CatSper, a progesterone receptor of sperm, causing a rapid influx of Ca(2+). The response was fully inhibited using a specific CatSper inhibitor. These findings highlight the potential for steviol to act as a potential endocrine disruptor.