Alkalihalobacillus clausii PA21 transcriptome profiling and functional analysis revealed the metabolic pathway involved in glycoalkaloids degradation.

Glycoalkaloids (GAs), including α-solanine and α-chaconine, are secondary metabolites found in potato, which are toxic to higher animals. In a previous study, Alkalihalobacillus clausii PA21 showed the capacity to degrade GAs. Herein, the transcriptome response of PA21 to α-solanine or α-chaconine was evaluated. In total, 3170 and 2783 differential expressed genes (DEGs) were found in α-solanine- and α-chaconine-treated groups, respectively, with most DEGs upregulated. Moreover, GAs activated transmembrane transport, carbohydrate metabolism, transcription, quorum sensing, and bacterial chemotaxis in PA21 to withstand GA-induced stress and promote GAs degradation. Furthermore, qRT-PCR analysis confirmed the upregulation of degrading enzymes and components involved in GA degradation in PA21. In addition, the GAs-degrading enzymes were heterologous expressed, purified, and incubated with GAs to analyze the degradation products. The results showed that α-solanine was degraded to ß1-solanine, ß2-solanine, γ-solanine, and solanidine by ß-glucosidase, α-rhamnosidase, and ß-galactosidase. Meanwhile, α-chaconine was degraded to ß1-chaconine, ß2-chaconine, γ-chaconine, and solanidine by ß-glucosidase and α-rhamnosidase. Overall, the molecular mechanism underlying GAs degradation by PA21 was revealed by RNAseq combined with protein expression and function studies, thus providing the basis for the development of engineered bacteria that can efficiently degrade GAs to promote their application in the control of GAs in potatoes.

The application of an aqueous two-phase system combined with ultrasonic cell disruption extraction and HPLC in the simultaneous separation and analysis of solanine and Solanum nigrum polysaccharide from Solanum nigrum unripe fruit.

An aqueous two-phase system was used in conjunction with ultrasonic cell disruption to extract and separate solanine (mainly solasonine and solamargine) and Solanum nigrum polysaccharide from Solanum nigrum unripe fruit. The optimized conditions of the present study were determined by a single-factor experiment and a multifactor experiment. The concentration of ethanol was set at 60% and the duration of the ultrasonic cell disruption extraction was 50 min. In the ethanol-K2CO3 aqueous two-phase separation system, the concentration of ethanol was 36%, the concentration of K2CO3 was 0.21 mg·mL-1, and the temperature was 15 °C. The solasonine and solamargine were determined by high-performance liquid chromatography, and the Solanum nigrum polysaccharide was determined by an ultraviolet-visible spectrophotometer in accordance with the phenol-sulfuric acid method. xUnder optimized conditions, the average extraction efficiencies of solasonine, solamargine and Solanum nigrum polysaccharide were 95.86%, 95.95% and 96.95%, respectively, and the average separation efficiencies of solasonine, solamargine and Solanum nigrum polysaccharide were 2.07 mg·g-1, 2.05 mg·g-1 and 8.15 mg·g-1, respectively.

Organic-inorganic nanoparticles molecularly imprinted photoelectrochemical sensor for α-solanine based on p-type polymer dots and n-CdS heterojunction.

In this study, a molecularly imprinted polymer photoelectrochemcal (MIP-PEC) sensor based on semiconducting organic polymer dots (Pdots) and inorganic CdS quantum dots (QDs) has been established for the determination of α-Solanine. Specifically, p-type Pdots (p-Pdots) and n-type CdS QDs (n-CdS) were utilized to form organic-inorganic nanoparticles p-n heterojunction to enhance signal response, and their specific energy levels (VB/CB or HOMO/LUMO) were calculated for photoelectrochemical (PEC) bioanalysis application. At the same time, the combination of molecular imprinting technology and photoelectrochemistry overcomes the defeats of photoelectrochemistry which is the absence of selectivity, offers a new MIP-PEC sensor with high sensitivity and excellent selectivity based heterojunction enhanced strategy. In short, this study proposes the semiconducting organic-inorganic nanoparticles p-n heterojunction for molecularly imprinted polymer photoelectrochemcal bioanalysis application, and the MIP-PEC sensor was successfully fabricated based on these materials and methods. In the phosphate buffer solution (PBS), it was clearly observed that the photocurrent has a significant change between elution in acetic acid-ethanol mixture and incubation in template molecular solution because of the faster electron transfer speed, this phenomenon fully showed that the MIP-PEC sensor can specifically detect the target. Thus, the work typically offers a linear range from 0.01 to 1000 ng mL-1 with a detection limit of 6.5 pg mL-1 for α-Solanine. Furthermore, the fabricated MIP-PEC sensor will confirm the actual application.

Impact of light-exposure on the metabolite balance of transgenic potato tubers with modified glycoalkaloid biosynthesis.

Metabolite profiling (liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC-MS)) was used to assess the impact of light on the composition of transgenic potato (Solanum tuberosum L. cv. Desirée) with reduced glycoalkaloid content via the down-regulation of the SGT1 gene. Transgenic tubers exhibited an almost complete knock-out of α-solanine production and light had little impact on its accumulation. Levels of α-chaconine increased significantly in the peel of both the control and transgenic lines when exposed to light, particularly in the transgenic line. Major differences in metabolite profiles existed between outer and inner tuber tissues, and between light and dark-treated tubers. Many of the light-induced changes are explicable in terms of pathways known to be affected by stress responses. The impact of transgenesis on profiles was much less than that of tissue type or light and most differences were explicable in terms of the modification to the glycoalkaloid pathway.

Analysis of metabolic changes in plant pathosystems by imprint imaging DESI-MS.

The response of plants to microbial pathogens is based on the production of secondary metabolites. The complexity of plant-pathogen interactions makes their understanding a challenging task for metabolomic studies requiring powerful analytical approaches. In this paper, the ability of ambient mass spectrometry to provide a snapshot of plant metabolic response to pathogen invasion was tested. The fluctuations of glycoalkaloids present in sprouted potatoes infected by the phytopathogen Pythium ultimum were monitored by imprint imaging desorption electrospray ionization mass spectrometry (DESI-MS). After 8 d from the inoculation, a decrease of the relative abundance of potato glycoalkaloids α-solanine (m/z 706) and α-chaconine (m/z 722) was observed, whereas the relative intensity of solanidine (m/z 398), solasodenone (m/z 412), solanaviol (m/z 430), solasodiene (m/z 396), solaspiralidine (m/z 428), γ-solanine/γ-chaconine (m/z 560) , ß-solanine (m/z 706), and ß-chaconine (m/z 722) increased. The progression of the disease, expressed by the development of brown necrotic lesions on the potato, led to the further decrease of all the glycoalkaloid metabolites. Therefore, the applicability of imprint imaging DESI-MS in studying the plant metabolic changes in a simple pathosystem was demonstrated with minimal sample preparation.

Glycoalkaloid and calystegine levels in table potato cultivars subjected to wounding, light, and heat treatments.

Potato tubers naturally contain a number of defense substances, some of which are of major concern for food safety. Among these substances are the glycoalkaloids and calystegines. We have here analyzed levels of glycoalkaloids (α-chaconine and α-solanine) and calystegines (A3, B2, and B4) in potato tubers subjected to mechanical wounding, light exposure, or elevated temperature: stress treatments that are known or anticipated to induce glycoalkaloid levels. Basal glycoalkaloid levels in tubers varied between potato cultivars. Wounding and light exposure, but not heat, increased tuber glycoalkaloid levels, and the relative response differed among the cultivars. Also, calystegine levels varied between cultivars, with calystegine B4 showing the most marked variation. However, the total calystegine level was not affected by wounding or light exposure. The results demonstrate a strong variation among potato cultivars with regard to postharvest glycoalkaloid increases, and they suggest that the biosynthesis of glycoalkaloids and calystegines occurs independently of each other.

Simple and reliable methods for the determination of three steroidal glycosides in the eight species of Solanum by reversed-phase HPLC coupled with diode array detection.

INTRODUCTION: Solanum species are important ingredients of many traditional Indian medicines and thus the quality control of their herbal formulations is of paramount concern. OBJECTIVE: To establish a simple and effective high-performance liquid chromatographic (HPLC) method to evaluate the quality of Solanum species and their herbal formulations. METHODOLOGY: A rapid, simple, sensitive, robust and reproducible HPLC method was developed for the determination of three steroidal glycosides (SG); indioside D, solamargine and α-solanine in eight species of the genus Solanum. The analytes were separated on a monolithic performance RP-18e column (100 mm × 4.6 mm i.d.) using a gradient elution of acetonitile-water containing 0.1% trifluoroacetic acid (TFA) as the mobile phase with a flow rate 0.4 mL/min and UV detection at λ 210 nm. RESULTS: The method was linear over the range 3-15 µg/mL (r > 9994). Accuracy, precision and repeatability were all within the required limits. The mean recoveries measured at the three concentrations were higher than 98.8% with RSD < 2% for the targets. CONCLUSION: The established method is simple and can be used as a tool for quality control of plant material or herbal formulation containing SG.

Preparative separation of glycoalkaloids α-solanine and α-chaconine by centrifugal partition chromatography.

The main glycoalkaloids of a commercial potato cultivar, α-chaconine and α-solanine, were extracted from sprouts of Solanum tuberosum cv. Pompadour by a mixture of MeOH/H(2)O/CH(3)COOH (400/100/50, v/v/v). In these conditions, 2.8±0.62g of crude extract were obtained from 50g of fresh sprouts and the total glycoalkaloid content was determined by analytical HPLC at 216.5mg/100g. α-Chaconine and α-solanine were separated in a preparative scale using centrifugal partition chromatography (CPC). In a solvent system composed of a mixture of ethyl acetate/butanol/water (15/35/50, v/v/v), α-chaconine (54mg) and α-solanine (15mg) were successfully isolated from the crude extract in one step of purification. The purity of isolated compounds was determined to be higher than 92% by HPLC analysis.

Polyphenol and glycoalkaloid contents in potato cultivars grown in Luxembourg.

The polyphenol (phenolic acids, flavanols and flavonols) and glycoalkaloid (α-chaconine and α-solanine) contents of potato tubers grown in Luxembourg were analyzed by UPLC-DAD and HPLC-MS/MS separately in peel (approx. 2mm), outer (approx. 1cm) and inner flesh. Polyphenol contents decreased from the peel via the outer to the inner flesh and differed among the cultivars. The cultivars Vitelotte and Luminella had the highest polyphenol contents (5202 and 572 µg/g dry weight (DW) in the outer flesh), whereas Charlotte and Bintje had the lowest contents (19.5 and 48.0 µg/g DW). Chlorogenic acid and its isomers (neo- and cryptochlorogenic acid) were the major polyphenols. Glycoalkaloid contents were highest in the peel and lowest in the inner flesh, values in the flesh were below guideline limits in all cultivars. In conclusion, potatoes contribute to the daily intake of polyphenols and their consumption, thereby, may have positive effects on health.

Compositional and toxicological analysis of a GM potato line with reduced α-solanine content--a 90-day feeding study in the Syrian Golden hamster.

Steroidal glycoalkaloids (GAs) are toxins, produced by plants of the Solanaceae family. The potato plant (Solanum tuberosum L.) and its tubers predominantly contain the two GAs α-chaconine and α-solanine. These compounds are believed to act in synergy, and the degree of toxicity may therefore depend on their ratio in the potato. To determine the influence of α-solanine: α-chaconine ratio in potatoes on toxicity, a GM potato line (SGT 9-2) with reduced α-solanine content, and the parental control line (Desirée wild-type) having a traditional α-solanine: α-chaconine ratio were (1) studied for compositional similarity by analysing for a range of potato constituents, and (2) used in a 90-day feeding trial with the Syrian Golden hamster to study differential toxicity. The animal feeding study used diets with up to 60% freeze-dried potato powder from either line. Whilst data indicated some compositional differences between the GM line and its wildtype control these did not raise concerns related to nutritional value or safety. Results of the feeding trials showed a low number of significant differences between potato lines with different α-solanine: α-chaconine ratio but none were considered to raise safety concerns with regard to human (or animal) consumption.

Glycoalkaloids (α-chaconine and α-solanine) contents of selected Pakistani potato cultivars and their dietary intake assessment.

Glycoalkaloids (α-solanine and α-chaconine) are naturally occurring toxic compounds in potato tuber (Solanum tuberosum L.) that cause acute intoxication in humans after their consumption. Present research was conducted to evaluate α-chaconine, α-solanine, and total glycoalkaloids (TGAs) contents in the peel and flesh portions by high-performance liquid chromatography method in selected Pakistani potato cultivars. The α-solanine content varies 45.98 ± 1.63 to 2742.60 ± 92.97 mg/100 g of dry weight (DW) in peel and from 4.01 ± 0.14 to 2466.56 ± 87.21 mg/100 g of DW in flesh. Similarly, α-chaconine content varied from 4.42 ± 0.16 to 6818.40 ± 211.07 mg/100 g of DW in potato peel and from 3.94 ± 0.14 to 475.33 ± 16.81 mg/100 g DW in flesh portion. The TGA concentration varied from 177.20 ± 6.26 to 5449.90 ± 192.68 mg/100 g of DW in peel and from 3.08 ± 0.11 to 14.69 ± 0.52 mg/100 g of DW in flesh portion of all the potato cultivars tested. All the potato cultivars contained lower concentration of TGA than the limits recommended as safe, except 2 cultivars, that is FD8-3 (2539.18 ± 89.77 mg/100 g of DW) and Cardinal (506.16 ± 17.90 mg/kg). The dietary intake assessment of potato cultivars revealed that Cardinal, FD 35-36, FD 8-3, and FD 3-9 contained higher amount of TGA in whole potato, although FD 8-3 only possessed higher content of TGA (154.93 ± 7.75) in its flesh portion rendering it unfit for human consumption. Practical Application: This paper was based on the research conducted on toxic compounds present in all possible potato cultivars in Pakistan. Actually, we quantify the toxic compounds (glycoalkaloids) of potato cultivars through HPLC and their dietary assessment. This paper revealed safety assessment and their application in food industries especially potato processing.

Naturally occurring glycoalkaloids in potatoes aggravate intestinal inflammation in two mouse models of inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) may be initiated following disruption of the intestinal epithelial barrier. This disruption, in turn, permits luminal antigens unfettered access to the mucosal immune system and leads to an uncontrolled inflammatory response. Glycoalkaloids, which are found in potatoes, disrupt cholesterol-containing membranes such as those of the intestinal epithelium. Glycoalkaloid ingestion through potatoes may play a role in the initiation and/or perpetuation of IBD. AIM: To determine if commercial and high glycoalkaloids containing fried potato skins aggravate intestinal inflammation using two different animal models of IBD. METHODS: Fried potato skins from commercial potatoes containing low/medium glycoalkaloid levels and high glycoalkaloids potatoes were fed for 20 days to interleukin 10 gene-deficient mice and dextran sodium sulfate-induced colitic mice. Intestinal permeability, mucosal cytokine and myeloperoxidase levels and body weight were determined to assess intestinal injury. RESULTS: Deep frying potato skins markedly increased glycoalkaloid content. Interleukin 10 gene-deficient mice fed fried commercial potato skins with medium glycoalkaloid content exhibited significantly elevated levels of ileal IFN-γ relative to controls. Mice in the dextran sodium sulfate colitis model that were fed the same strain of potatoes demonstrated significantly elevated levels of pro-inflammatory cytokines IFN-γ, TNF-α, and IL-17 in the colon in addition to an enhanced colonic permeability. Inflammatory response was intensified when the mice were fed potatoes with higher glycoalkaloid contents. CONCLUSIONS: Our results demonstrate that consumption of potato skins containing glycoalkaloids can significantly aggravate intestinal inflammation in predisposed individuals.

Composition of phenolic compounds and glycoalkaloids alpha-solanine and alpha-chaconine during commercial potato processing.

The influence of a commercial production process for dehydrated potato flakes on the content of free phenolic compounds, total phenolics, and glycoalkaloids in potatoes during the subsequent processing steps was determined. Processing byproducts, such as potato peel (steam peeling), mashed potato residues, and side streams (blanching and cooking waters), have also been investigated. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify caffeic acid, gallic acid, ferulic acid, p-coumaric acid, p-hydoxybenzoic acid, protocatechuic acid, vanillic acid, catechin, and three isomers of caffeoylquinic acid: chlorogenic, neochlorogenic and cryptochlorogenic acid. Determination of the glycoalkaloids alpha-solanine and alpha-chaconine was performed by using a high-performance thin-layer chromatography (HPTLC) method. The deliverables reveal that processing potatoes to potato flakes remarkably diminishes the content of the analyzed compounds, mainly due to peeling and leaching. The influence of thermal exposure is less significant. About 43% of the initial phenolic acids and 10% of the glycoalkaloids remain after processing. The results of the total phenolic content assay by Folin-Ciocalteu reagent are proportional to the content of phenolic compounds determined by HPLC. Steam peeling has a higher influence on glycoalkaloid losses compared to that on phenolics. The highest amounts of phenolic compounds and glycoalkaloids were found in peeling byproduct. During processing, the amount of chlorogenic acid decreased, whereas the concentration of neochlorogenic acid increased due to isomerization. The impact of the results on potato processing technology is discussed.

Glycoalkaloids as biomarkers for recognition of cultivated, wild, and somatic hybrids of potato.

Cultivated and wild potato species synthesize a wide variety of steroidal glycoalkaloids (GAs). During breeding programs, species genomes are often put together through either sexual or somatic hybridization. Therefore, the determination of the GA composition of hybrids is very important in that it may affect either human consumption, or resistance to pathogen and pests. Here, we report the results of GA analysis performed on wild Solanum bulbocastanum, haploids of cultivated potato S. tuberosum and their interspecific somatic hybrids. GAs were extracted from tubers and analyzed by HPLC. HPLC Profile of S. tuberosum haploids showed, as expected, the presence of alpha-solanine and alpha-chaconine. The profile of S. bulbocastanum extract showed lack of alpha-solanine and alpha-chaconine, and the presence of four GAs. The GA pattern of the somatic hybrids was the sum of their parents' profile. This represents a noteworthy tool for their unequivocal recognition. Interestingly, two hybrids produced not only GAs of both parents but also new compounds to be further investigated. This provided evidence that somatic hybridization induced the synthesis of new metabolites. The nature of the probable unidentified GAs associated to S. bulbocastanum and its somatic hybrids was ascertained by chemical degradation and spectroscopic analysis of their aglycones and sugar moieties. Our results suggest their close relation with GAs of both wild and cultivated potato species.

Probabilistic modelling of exposure doses and implications for health risk characterization: glycoalkaloids from potatoes.

Potatoes are a source of glycoalkaloids (GAs) represented primarily by alpha-solanine and alpha-chaconine (about 95%). Content of GAs in tubers is usually 10-100 mg/kg and maximum levels do not exceed 200 mg/kg. GAs can be hazardous for human health. Poisoning involve gastrointestinal ailments and neurological symptoms. A single intake of >1-3 mg/kg b.w. is considered a critical effect dose (CED). Probabilistic modelling of acute and chronic (usual) exposure to GAs was performed in the Czech Republic, Sweden and The Netherlands. National databases on individual consumption of foods, data on concentration of GAs in tubers (439 Czech and Swedish results) and processing factors were used for modelling. Results concluded that potatoes currently available at the European market may lead to acute intakes >1 mg GAs/kg b.w./day for upper tail of the intake distribution (0.01% of population) in all three countries. 50 mg GAs/kg raw unpeeled tubers ensures that at least 99.99% of the population does not exceed the CED. Estimated chronic (usual) intake in participating countries was 0.25, 0.29 and 0.56 mg/kg b.w./day (97.5% upper confidence limit). It remains unclear if the incidence of GAs poisoning is underreported or if assumptions are the worst case for extremely sensitive persons.

Fate of toxic potato glycoalkaloids in a potato field.

The toxic glycoalkaloids, alpha-solanine and alpha-chaconine, are present in all parts of the potato plant and are possibly transferred to the terrestrial environment. The amounts of glycoalkaloids in plant, soil, and groundwater were followed in a potato field to investigate their distribution and fate during the season. The amount of glycoalkaloids in the plants was up to 25 kg/ha during maturity and decreased to below 0.63 kg/ha during plant senescence. The glycoalkaloids were detected in the upper soil (up to 0.6 kg/ha); this amount accounted only for a minor fraction of the amount present in the plants. Maximum glycoalkaloid concentration of 2.8 mg/kg dry weight soil was detected in September. Dissipation during winter appeared to be slow because glycoalkaloids were still present in the soil in March. No traces of glycoalkaloids were detected in the groundwater (detection limit 0.2 microg/L). From these results, the leaching potential of the glycoalkaloids is evaluated to be small.

Acute toxicity of high doses of the glycoalkaloids, alpha-solanine and alpha-chaconine, in the Syrian Golden hamster.

Sprouted, stressed, or spoiled potato tubers have reportedly led to human acute intoxication, coma, and death when consumed in high amounts. These effects have been attributed to glycoalkaloids (GAs), primarily alpha-solanine and alpha-chaconine, naturally present in all potatoes. The level of GAs in potato tubers has previously been shown to increase substantially as a result of improper handling and postharvest storage. A short-term study was performed to investigate the dose-response profile of alpha-solanine and alpha-chaconine alone or in combination, administered daily by oral gavage to Syrian Golden hamsters. Daily doses of 100 mg of alpha-solanine [kg body weight (BW)] (-1) induced death in two of four hamsters within 4 days, when administered by gavage to female Syrian hamsters. Doses of 100 mg of alpha-chaconine alone or alpha-solanine and alpha-chaconine combined in a ratio of 1:2.5, in doses of 75 or 100 mg (kg BW) (-1), induced death in one of four hamsters within the same period. Animals dosed with alpha-solanine alone or in combination with alpha-chaconine suffered from fluid-filled and dilated small intestines. The GA administration had no effect on acetyl cholinesterase (AChE) or butyryl cholinesterase (BuChE) activity in plasma or brain. Liquid chromatography-mass spectrometry-based metabolomics showed that there was a specific accumulation of alpha-chaconine in the liver tissues. In addition, metabolomics gave direct evidence of glycolytic metabolism of the GA with the beta 1, beta 2, and gamma-GAs detected in the urine and, to a lesser extent, the feces. Doses from 75 mg (kg BW) (-1) of alpha-chaconine, alpha-solanine, or the two compounds combined were potentially lethal within 4-5 days in the Syrian Golden hamster. However, the cause of death in these studies could not be established. No synergistic effects of alpha-solanine combined with alpha-chaconine were evident.

A major QTL and an SSR marker associated with glycoalkaloid content in potato tubers from Solanum tuberosum x S. sparsipilum located on chromosome I.

New potato (Solanum tuberosum) varieties are required to contain low levels of the toxic glycoalkaloids and a potential approach to obtain this is through marker-assisted selection (MAS). Before applying MAS it is necessary to map quantitative trait loci (QTLs) for glycoalkaloid content in potato tubers and identify markers that link tightly to this trait. In this study, tubers of a dihaploid BC(1) population, originating from a cross between 90-HAF-01 (S. tuberosum(1)) and 90-HAG-15 (S. tuberosum(2) x S. sparsipilum), were evaluated for content of alpha-solanine and alpha-chaconine (total glycoalkaloid, TGA) after field trials. In addition, tubers were assayed for TGA content after exposure to light. A detailed analysis of segregation patterns indicated that a major QTL is responsible for the TGA content in tubers of this potato population. One highly significant QTL was mapped to chromosome I of the HAG and the HAF parent. Quantitative trait loci for glycoalkaloid production in foliage of different Solanum species have previously been mapped to this chromosome. In the present research, QTLs for alpha-solanine and alpha-chaconine content were mapped to the same location as for TGA content. Similar results were observed for tubers exposed to light. The simple sequence repeat marker STM5136 was closely linked to the identified QTL.

Potato glycoalkaloids in soil-optimising liquid chromatography-time-of-flight mass spectrometry for quantitative studies.

Potato glycoalkaloids are produced in high amounts in potato fields during the growth season and losses to soil potentially impact shallow groundwater and via tiles to fresh water ecosystems. A quantitative liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) method for determination and quantification of potato glycoalkaloids and their metabolites in aqueous soil extracts was developed. The LC-ESI-TOF-MS method had linearities up to 2000microg/L for alpha-solanine and alpha-chaconine and up to 760microg/L for solanidine. No matrix effect was observed, and the detection limits found were in the range 2.2-4.7microg/L. The method enabled quantification of the potato glycoalkaloids in environmental samples.