Steviol Glycosides Research Articles

Physicochemical and microbiological properties and moisture adsorption isotherms characteristics of commercial steviol glycoside, rebaudioside A

Physicochemical and microbiological properties and moisture adsorption isotherms characteristics of commercial steviol glycoside, rebaudioside A

Impact of Stevia rebaudiana Culturing in Liquid Medium: Elevation of Yield and Biomass, Mitigation of Steviol Glycosides

An efficient micropropagation system is developed by culturing nodal segments containing the axillary shoot buds on liquid Murashige and Skoog (MS) medium. Direct shoot and root formation are highly effective in both solid and liquid MS media without any plant growth regulators (PGRs). Interestingly, a significant difference in yield is obtained between solid and liquid cultures. It is revealed that a relatively higher amount of plant biomass is obtained after culturing for 4 weeks in a liquid MS medium. However, the shoots produced on solid MS medium produce a remarkable decline in all physiological parameters. On contrary, the bioactive steviol glycosides (rebaudioside-A and stevioside) content is higher in shoots grown in solid MS on a comparative basis, which could be compensated by higher yield. After hardening off, all the regenerants are effectively grown in the field with a negligible loss (<1 %), and steviol glycosides spectra is again obtained by conducting high-performance liquid chromatography (HPLC) analysis after 10 weeks of the plantation. This method has great potential to be applied on large scale in bioreactors.

Safety of the proposed amendment of the specifications for enzymatically produced steviol glycosides (E 960c): Rebaudioside D produced via enzymatic bioconversion of purified stevia leaf extract.

The EFSA Panel on Food Additives and Flavourings (FAF Panel) provides a scientific opinion on the safety of a proposed amendment of the specifications of enzymatically produced steviol glycosides (E 960c) with respect to the inclusion of rebaudioside D produced via enzyme‐catalysed bioconversion of purified stevia leaf extract. Rebaudioside D (95% on dry basis) is produced via enzymatic bioconversion of purified stevia leaf extract using uridine diphosphate (UDP)‐glucosyltransferase (UGT) and sucrose synthase enzymes produced by the genetically modified yeast K. phaffii UGT‐A, that facilitates the transfer of glucose to purified stevia leaf extract via glycosidic bonds. The same enzymes from K. phaffii UGT‐A may be used in the manufacturing process of the food additive, rebaudioside M produced via enzyme modification of steviol glycosides from stevia (E 960c(i)). The Panel considered that separate specifications would be needed for this food additive produced via the manufacturing process described in the current application, aligned with those already established for E 960c(i). The Panel concluded that there is no toxicological concern for Rebaudioside D produced via enzymatic bioconversion of purified stevia leaf extract using UDP‐glucosyltransferase and sucrose synthase produced by a genetically modified strain of the yeast K. phaffii. However, based on the available data, the Panel could not exclude the possibility that some residual amount of DNA coding for the kanamycin resistance gene could remain in the final product. Should this gene propagate in microbiota due to the presence of recombinant DNA in the final product, this would be of concern. Therefore, the Panel concluded that the safety of Rebaudioside D produced via this enzymatic bioconversion was not sufficiently demonstrated with the available data given that the absence of recombinant DNA was not shown.

Improving Thermostability and Catalytic Activity of Glycosyltransferase From Panax ginseng by Semi-Rational Design for Rebaudioside D Synthesis.

As a natural sweetener and sucrose substitute, the biosynthesis and application of steviol glycosides containing the component rebaudioside D have attracted worldwide attention. Here, a glycosyltransferase PgUGT from Panax ginseng was first reported for the biosynthesis of rebaudioside D. With the three-dimensional structures built by homology modeling and deep-learning–based modeling, PgUGT was semi-rationally designed by FireProt. After detecting 16 site-directed variants, eight of them were combined in a mutant Mut8 with both improved enzyme activity and thermostability. The enzyme activity of Mut8 was 3.2-fold higher than that of the wild type, with an increased optimum reaction temperature from 35 to 40°C. The activity of this mutant remained over 93% when incubated at 35°C for 2 h, which was 2.42 times higher than that of the wild type. Meanwhile, when the enzymes were incubated at 40°C, where the wild type was completely inactivated after 1 h, the residual activity of Mut8 retained 59.0% after 2 h. This study would provide a novel glycosyltransferase with great potential for the industrial production of rebaudioside D and other steviol glycosides.

Maltitol based sugar-free chocolates may not promote dental caries: An open-label clinical study

Background: Sugar substitutes like maltitol are reported to be noncariogenic ingredients and may not lead to the development of dental caries (DC), in contrast to sugars and other fermentable carbohydrates. The current study aimed to investigate the effects of maltitol-based sugar-free chocolates on factors associated with DC. Methods: This was an open-label study involving healthy human volunteers. A total of 15 healthy human subjects were screened and enrolled in the study after obtaining their signed informed consent. The subjects were administered the test product, maltitol-based sugar-free chocolate, which contains no added sugar (sucrose). Sucrose was completely replaced with the sweeteners maltitol (minimum 45%) and steviol glycoside (0.05%). Salivary pH was measured using a digital pH meter at baseline and 0 h, 1 h, 2 h, and 4 h after test product consumption. The saliva samples were collected for measurement of Streptococcus mutans colonies at baseline and at 0 h and 4 h after test product consumption. The data were subjected to two-sided tests, at a 5% level of significance using Statistical Analysis System (SAS) 9.4 software (SAS Institute Inc., NC Cary). Results: A statistically significant increase in the salivary pH level from the baseline at 0 h measurement (p = 0.0181) and a reduction in S. mutans formation from baseline to 0 h and 4 h, respectively (p = 0.0001) were noted. Conclusion: Maltitol-based sugar-free chocolates were found to be safe for consumption and were devoid of cariogenic potential.

Development and Validation of the Chinese Version Non-Nutritive Sweetener Food Frequency Questionnaire with Urinary Biomarker in Children and Adolescents.

The purpose of this study was to develop a validated food frequency questionnaire (FFQ) to evaluate the intake of non-nutritive sweeteners (NNSs) in child and adolescent Asian populations. Intensive and overall market research was performed to create the applicable NNS-FFQ with 13 food categories and 305 items. Six intense sweeteners, including acesulfame potassium, aspartame, sucralose, glycyrrhizin, steviol glycosides and sorbitol, were investigated. The validity and reproducibility of the NNS-FFQ were evaluated. The validity was further assessed by examining the consistency of reported NNS intake compared with urinary biomarkers using Cohen's κ analysis. This work was considered to be relevant in Asian societies. One hundred and two children and adolescents recruited from several clinics were invited to participate in this study. High content validity indices and high content validity ratio levels were revealed for each sweetener and food category. Reproducibility among subjects was satisfactory. Significant moderate correlations between estimated steviol glycoside/sucralose consumption and sensitive urinary biomarker levels were demonstrated (κ values were 0.59 and 0.45 for steviol glycosides and sucralose, respectively), indicating that the NNS-FFQ can be used to assess an individual's NNS intake. The dietary intense sweetener consumption pattern evaluated in this measurement was similar to those observed in other Asian countries but differed from those observed in Western populations with respect to types and amounts of NNSs. This validated NNS-FFQ can be an applicable and useful tool to evaluate NNS intake in future epidemiological and clinical studies.

Culturing Important Plants for Sweet Secondary Products under Consideration of Environmentally Friendly Aspects

Some sweet tasting plant secondary metabolites are non-caloric or low nutritive compounds that have traditional use in food formulations. This mini-review focuses on conventional and advanced cultivation regimes of plants that accumulate sweet tasting or sweet taste modulating secondary metabolites of potential economic importance, in particular mogrosides (Siraitia grosvenorii), phyllodulcin (Hydrangea macrophylla), glycyrrhizin (Glycyrrhiza glabra), steviol glycosides (Stevia grosvenorii), and rubusoside (Rubus suavissimus). Consequential obstacles during the cultivation of Hydrangea macrophylla cultivars outside their natural habitat in a protected cultivation environment are addressed. Culturing at non-habitat locations facilitates short transportation routes of plant material for processing, which can be a key to an economically and environmentally compatible usage. The biosynthetic pathways, as far as known, are shortly mentioned. The proved or hypothetical degradation pathways of the compounds to minimalize environmental contamination are another focal point.

The Effects of Stevia Consumption on Gut Bacteria: Friend or Foe?

Stevia, a zero-calorie sugar substitute, is recognized as safe by the Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA). In vitro and in vivo studies showed that stevia has antiglycemic action and antioxidant effects in adipose tissue and the vascular wall, reduces blood pressure levels and hepatic steatosis, stabilizes the atherosclerotic plaque, and ameliorates liver and kidney damage. The metabolism of steviol glycosides is dependent upon gut microbiota, which breaks down glycosides into steviol that can be absorbed by the host. In this review, we elucidated the effects of stevia’s consumption on the host’s gut microbiota. Due to the lack of randomized clinical trials in humans, we included in vitro using certain microbial strains and in vivo in laboratory animal studies. Results indicated that stevia consumption has a potential benefit on the microbiome’s alpha diversity. Alterations in the colonic microenvironment may depend on the amount and frequency of stevia intake, as well as on the simultaneous consumption of other dietary components. The anti-inflammatory properties of stevioside were confirmed in vitro by decreasing TNF-α, IL-1β, IL-6 synthesis and inhibiting of NF-κB transcription factor, and in vivo by inhibiting NF-κB and MAPK in laboratory animals.

Daminozide enhances the vigor and steviol glycoside yield of stevia (Stevia rebaudiana Bert.) propagated in temporary immersion bioreactors

Stevia (Stevia rebaudiana Bertoni) produces steviol glycosides (SG), a group of diterpenoid secondary metabolites widely used as natural sweeteners. SGs share the same precursor with gibberellins (GAs), ent-kaurenoic acid. In this study, we attempted to increase the SG content in stevia propagated in Temporary Immersion Bioreactors (TIBs) by blocking ent-kaurenoic acid entry to the GA synthesis pathway using Daminozide (0, 10, 20 ppm), a known GA inhibitor. The maximum increase in biomass weight and SG content (up to 40% for stevioside and rebaudioside A) was observed following 10 ppm Daminozide treatment. The treatment also increased transcript accumulation for genes involved in SG biosynthesis, such as SrKA13H, SrUGT85C2, and SrUGT76G1, indicating that the SG pathway became more active due to GA pathway inhibition. Furthermore, GA inhibition following inhibitor exposure was also indicated by the upregulated expression of a GA biosynthesis gene (GA3ox) due to feedback regulation, and the downregulated expression of a GA catabolism gene (GA2ox2) as a result of feed-forward regulation.

A Hybrid RSM-ANN-GA Approach on Optimization of Ultrasound-Assisted Extraction Conditions for Bioactive Component-Rich Stevia rebaudiana (Bertoni) Leaves Extract.

Stevia rebaudiana (Bertoni) leaves consist of dietetically important diterpene steviol glycosides (SGs): stevioside (ST) and rebaudioside-A (Reb-A). ST and Reb-A are key sweetening compounds exhibiting a sweetening potential of 100 to 300 times more intense than that of table sucrose. Ultrasound-assisted extraction (UAE) of SGs was optimized by effective process optimization techniques, such as response surface methodology (RSM) and artificial neural network (ANN) modeling coupled with genetic algorithm (GA) as a function of ethanol concentration (X1: 0–100%), sonication time (X2: 10–54 min), and leaf–solvent ratio (X3: 0.148–0.313 g·mL−1). The maximum target responses were obtained at optimum UAE conditions of 75% (X1), 43 min (X2), and 0.28 g·mL−1 (X3). ANN-GA as a potential alternative indicated superiority to RSM. UAE as a green technology proved superior to conventional maceration extraction (CME) with reduced resource consumption. Moreover, UAE resulted in a higher total extract yield (TEY) and SGs including Reb-A and ST yields as compared to those that were obtained by CME with a marked reduction in resource consumption and CO2 emission. The findings of the present study evidenced the significance of UAE as an ecofriendly extraction method for extracting SGs, and UAE scale-up could be employed for effectiveness on an industrial scale. These findings evidenced that the UAE is a high-efficiency extraction method with an improved statistical approach.

Modification on the length of glucosyl chain in glucosyl steviol glycosides and its effect on product taste quality

Modification on the length of glucosyl chain in glucosyl steviol glycosides and its effect on product taste quality

GlucoMedix®, an extract of Stevia rebaudiana and Uncaria tomentosa, reduces hyperglycemia, hyperlipidemia, and hypertension in rat models without toxicity: a treatment for metabolic syndrome

BackgroundThe objective of this in vivo study is to evaluate in five rat models the pharmacologic effects and toxicity of a commercial hydro-alcoholic extract, GlucoMedix®, derived from Stevia rebaudiana and the pentacyclic chemotype of Uncaria Tomentosa (Willd.) DC, for use as a treatment for metabolic syndrome. The extract contains phytochemicals of Stevia (e.g., steviol glycosides) and Uncaria (e.g., pentacyclic oxindole alkaloids, but lacks tetracyclic oxindole alkaloids).MethodsThe pharmacologic assessments in three rat models include reductions in chemically induced hyperglycemia, hyperlipidemia (cholesterol and triglycerides), and hypertension, all of which are comorbidities of metabolic syndrome. Acute toxicity and 28-day subacute toxicity were assessed in rat models at doses higher than those used in the efficacy models.ResultsThe acute oral toxicity was evaluated in Holtzman rats and the extract did not produce acute toxic effects or lethality, with the LD50 > 5000 mg/kg (extract wet weight). Furthermore, subacute oral toxicity was evaluated in rats for 28 days at daily doses as high as 2000 mg/kg without toxicity or abnormal clinical chemistry or hematological effects. Daily oral doses of 250 - 1000 mg/kg were used to evaluate the treatment effects in hyperglycemic (alloxan-induced and glibenclamide-controlled), hyperlipidemic (cholesterol-induced and atorvastatin-controlled), and hypertensive (L-NAME-induced and enalapril-controlled) rat models. Alloxan-induced hyperglycemia was reduced in a dose-dependent manner within 28 days or less. Cholesterol-induced hyperlipidemic rats exhibited dose-dependent reductions in cholesterol and triglycerides at 21 days. Furthermore, GlucoMedix® produced a dose-dependent decrease in systolic and diastolic arterial blood pressure in L-NAME-induced hypertensive rats at 28 days.ConclusionsThe five in vivo rat models revealed that the all-natural phytotherapy GlucoMedix® is a safe and effective treatment for hyperglycemia, hyperlipidemia, and hypertension. This extract is expected to affect multiple comorbidities of metabolic syndrome, without any acute or subacute oral toxicity in humans. Although multiple prescription drugs are well known for the treatment of individual comorbidities of metabolic syndrome, no drug monotherapy concurrently treats all three comorbidities.

A UV/Vis spectrophotometric methodology for quality control of stevia-based extracts in the food industry

This study established a methodology for quality control of stevia extracts in food industry where one, or a mixture of major stevia glycosides, rebausioside A and stevioside, are present. The methodology relies on pre-treatment to eliminate glucose that may be already present, prior to acid hydrolysis of stevia glycosides toward isosteviol plus glucose and ensuing released glucose quantification using the glucose oxidase/peroxidase coupled assay. Subsequent isosteviol isolation and concentration assessment using UV spectroscopy, permits the quantification of an average number of glucose molecules released per a generated molecule of isosteviol. The latter increases the assay specificity, allowing confirmatory assessment of stevia glycoside identity and purity. Favorable technical characteristics, satisfying international specifications for method validation, were determined for each individual assay (glycoside quantification by acid hydrolysis and isosteviol quantification) for rebaudioside A and stevioside: Standard curves were linear (R2 ≥ 0.993 [R2 ≥ 0.984 for the glucose/isosteviol quantification assay]) over a concentration range from approximately 10−1 mg/mL for the acid assay and 3.0 mg/mL for the glucose/isosteviol assay up to at least 10 mg/mL glycoside. The CV% on all quality control levels was ≤6.33% for repeatability and ≤12.03% for intermediate precision. Bias was ≤±5.00% in all cases. Upon comparison with the gold standard LC/UV method for rebA quantification, we demonstrated a very good agreement and acceptable bias (slope: 1.02, y-intercept: −0.73, R2: 0.994). Acid hydrolysis was tested successfully on commercial products, stevia extracts and commodity sweeteners, containing either of the two major glycosides. We demonstrated that the proposed methodology was able to distinguish between commercial samples containing pure rebaudioside A or stevioside alone and samples containing additional steviol glycosides in combination. When verifying glycoside content in binary mixtures of rebaudioside A and stevioside, bias was sufficiently low (<10%). The proposed methodology requires minimal equipment and expertise and is applicable to a significant percentage (approximately 50%) of all commercial stevia extracts and sweeteners. Our findings should provide a useful tool for stevia quality control, thus allowing an increase in the frequency and extent of testing, even in minimally equipped settings, while containing cost.

Directional bioconversion and optimization of stevioside into rubusoside by Lelliottia sp. LST-1.

The present study aimed to specifically transform stevioside (ST) into rubusoside (RS) through bioconversion with high efficiency, seeking to endow steviol glycosides (SGs) with subtle flavours for commercial acceptability. An endophytic bacterium named Lelliottia LST-1 was screened and confirmed to specifically convert ST into RS, reaching a conversion rate of 75.4% after response surface optimization. Phylogenetic analysis combined with complete genome sequencing demonstrated that LST-1 was also presumed to be a new species. To further explore the principle and process of biological transformation, the potential beta-glucosidases GH3-1, GH3-2, GH3-3 and GH3-4 were expressed, purified and reacted with SGs. High-performance liquid chromatography revealed that all enzymes hydrolysed ST and generated RS, but substrate specificity analysis indicated that GH3-2had the highest substrate specificity towards STs and the highest enzyme activity. The potential β-glucosidase GH3-2 in Lelliottia sp. LST-1 was found to specifically and efficiently convert ST to RS. The efficient biotransformation of ST into RS will be beneficial to its large-scale production and extensive application in the food and pharmaceutical industries.

The Potential of Cold Plasma and Electromagnetic Field as Stimulators of Natural Sweeteners Biosynthesis in Stevia rebaudiana Bertoni.

Stevioside (Stev) and rebaudioside A (RebA) are the most abundant steviol glycosides (SGs) responsible for the sweetness of Stevia rabaudiana Bertoni. As compared to Stev, RebA has a higher sweetening potency, better taste and therefore is the most preferred component of the stevia leaf extracts. The aim of this study was to determine the effect of pre-sowing seed treatment with abiotic stressors cold plasma (CP) and electromagnetic field (EMF) on the amount and ratio of RebA and Stev in the leaves of stevia. Additionally, the effect on total phenolic content, flavonoid content and antioxidant activity was investigated. Seeds were treated 5 and 7 min with cold plasma (CP5 and CP7 groups) and 10 min with electromagnetic field (EMF10 group) six days before sowing. The germination tests in vitro demonstrated that all treatments slightly increased germination rate and percentage. HPLC analysis revealed that CP and EMF had strong stimulating effect on SGs accumulation. All treatments increased RebA concentration approximately 1.6-fold; however, the ratio of RebA/Stev decreased from 8.5 in the control to 1.9, 2.5 and 1.1 in CP5, CP7 and EMF10 groups respectively, since the concentration of Stev increased more than RebA, 7.1, 4.6 and 11.0-fold, respectively, compared to control. However, treatments had opposite effect on total phenolic content, flavonoid content, and antioxidant activity. We have demonstrated for the first time that short time pre-sowing treatment of stevia seeds with CP and EMF can be a powerful tool for the enhancement of biosynthesis of RebA and Stev, however it can have negative impact on the content of other secondary metabolites.

Metabolic Circuits in Sap Extracts Reflect the Effects of a Microbial Biostimulant on Maize Metabolism under Drought Conditions.

The use of microbial biostimulants in the agricultural sector is increasingly gaining momentum and drawing scientific attention to decode the molecular interactions between the biostimulants and plants. Although these biostimulants have been shown to improve plant health and development, the underlying molecular phenomenology remains enigmatic. Thus, this study is a metabolomics work to unravel metabolic circuits in sap extracts from maize plants treated with a microbial biostimulant, under normal and drought conditions. The biostimulant, which was a consortium of different Bacilli strains, was applied at the planting stage, followed by drought stress application. The maize sap extracts were collected at 5 weeks after emergence, and the extracted metabolites were analyzed on liquid chromatography-mass spectrometry platforms. The acquired data were mined using chemometrics and bioinformatics tools. The results showed that under both well-watered and drought stress conditions, the application of the biostimulant led to differential changes in the profiles of amino acids, hormones, TCA intermediates, phenolics, steviol glycosides and oxylipins. These metabolic changes spanned several biological pathways and involved a high correlation of the biochemical as well as structural metabolic relationships that coordinate the maize metabolism. The hypothetical model, postulated from this study, describes metabolic events induced by the microbial biostimulant for growth promotion and enhanced defences. Such understanding of biostimulant-induced changes in maize sap pinpoints to the biochemistry and molecular mechanisms that govern the biostimulant–plant interactions, which contribute to ongoing efforts to generate actionable knowledge of the molecular and physiological mechanisms that define modes of action of biostimulants.

Yield response, accumulation of bioactive ingredient and ion uptake of Stevia rebaudiana to different soil-moisture and nitrogen levels

The importance of stevia (Stevia rebaudiana) is increasing rapidly as a potential industrial crop throughout the world. Soil-moisture and nitrogen (N) are the key components for agricultural productivity. However, the information on the effects of availability of soil moisture and N dose on the growth of stevia, N dynamics in the plant and soil, and NUE is limited. Thus, a field study involving three soil-water regimes (irrigation schedule at 20–75 kPa) and five different levels of N (0–140 kg ha−1) was conducted. Results showed that the dry leaf and total steviol glycosides (SGs) yield of stevia was found maximum with the irrigation at 50 kPa. At harvest, the plants irrigated at 50 kPa produced 26.9–32.5% higher dry matter over limited application of water (75 kPa). The N at 105 kg ha−1 registered maximum leaf yield (2.59 and 2.37 t ha−1) and N uptake (83.23 and 74.73 kg ha−1). The higher dose of N (140 kg ha−1) did not enhance dry leaf and total SGs yield compared with N at 105 kg ha−1. The low apparent N-recovery and agronomic N-efficiency at higher doses of N indicate higher accumulations of N into the environment. The soil N-balance sheet indicates that the maximum N has been harvested with the higher N under water limiting conditions. Thus, it can be concluded that the irrigation at 50 kPa in combination with N at 105 kg ha−1 could improve leaf and total SGs yield, N uptake, and NUE of stevia without deteriorating soil health.

Safety evaluation of glucosylated steviol glycosides as a food additive in different food categories.

The EFSA Panel on Food Additive and Flavourings (FAF) assessed the safety of glucosylated steviol glycosides proposed for use as a new food additive in different food categories. Glucosylated steviol glycosides consist of a mixture of glucosylated steviol glycosides, containing 1–20 additional glucose units bound to the parent steviol glycosides. Glucosylated steviol glycosides consist of not less than 95% (on dry, dextrin‐free, basis) of total steviol glycosides, comprised of glucosylated and parent steviol glycosides. Glucosylated steviol glycosides are produced via enzymatic bioconversion using cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19), derived from a non‐genetically modified strain of Anoxybacillus caldiproteolyticus, that catalyses the transfer of glucose from starch to steviol glycosides mixtures isolated from the dried leaves of Stevia Rebaudiana. The Panel considered that the metabolism of glucosylated steviol glycosides is sufficiently similar to the already authorised steviol glycosides, and thus, the toxicological data previously assessed by the ANS Panel for steviol glycosides (E 960) were considered to support their safety as food additive. The existing acceptable daily intake (ADI) for steviol glycosides (E 960) of 4 mg/kg body weight (bw) per day expressed as steviol can also be applied to glucosylated steviol glycosides. The Panel concluded that there is no safety concern for the use of glucosylated steviol glycosides as a new food additive at the proposed use and use levels. The Panel recommended some modifications to the specifications proposed by the applicant for glucosylated steviol glycosides with respect to the assay, the definition of the proposed new food additive and the proposed maximum limits for arsenic.

Arbuscular mycorrhizal symbiosis as sustainable alternative in the Stevia rebaudiana Bertoni production

L Objective: Stevia rebaudiana Bertoni is a crop that can meet the demand for natural sweeteners; however, this demand requires a sustainable production, as a result of the inclusion of its steviol glycosides or active ingredients in the food and pharmaceutical industries. Design/methodology/approach: Modern agriculture implies the integrated use of valid natural resources, such as the arbuscular mycorrhizal fungi (AMF). These microorganisms establish a symbiotic relationship with, at least, 80% of the plants, to which they provide multiple benefits. They can directly and indirectly improve crop productivity, through nutrient (particularly, phosphorus) translocation of the soil solution. Results: As a sustainable alternative for the production of S. rebaudiana, they improve its nutritional state, resulting in a higher biomass production and glycoside concentration —fundamental yield parameters. Additionally, they promote resistance to biotic and abiotic stress factors and improve soil quality. Limitations/implications: It is worth mentioning that this fungi-plant mutualism is approximately 400 million years-old; however, it has only aroused interest during the last few years. Findings/conclusions: Although arbuscular mycorrhizal fungi (AMF) are an exploitable resource, their communities are threatened by biotic factors —such as the interaction with other microorganisms— and abiotic factors —which involve bad agricultural practices.

Changes in nonnutritive sweetener intake in a cohort of preschoolers after the implementation of Chile's Law of Food Labelling and Advertising.

The first phase of Chile's Law of Food Labelling and Advertising showed important declines in the sugar content of packaged foods, but it is unknown whether the law led to an increase in nonnutritive sweetener (NNS) intake, particularly among preschool children. Estimate the changes in preschoolers' NNS intake after the first phase of the Chilean law. We used 24-h dietary recalls collected in 2016 (pre-law) and 2017 (post-law) from a cohort of preschoolers (n=875). The primary caretaker was the respondent of the recalls. Information on NNS was obtained from nutrition facts panels collected annually and linked to dietary data. We used logistic regression to estimate the changes in the proportion of preschoolers who consume NNS and two-part models to estimate the changes in mean intake. We determined the percentage of children that surpassed the acceptable daily intake (ADI) of each NNS using the National Cancer Institute method. The proportion of consumers of at least one NNS increased from 77.9% to 92.0% (p-value < 0.01). The mean intake increased for sucralose, aspartame, acesulfame-K and steviol glycosides (+20.3, +15.1, +6.1 and +3.3mg/day, respectively). In addition, NNS dietary sources changed for sucralose and steviol glycosides, becoming industrialized juices and dairy beverages more relevant while tabletop NNS became less relevant. None of the children surpassed the ADI. NNS intake increased in preschoolers after the first phase of a national policy that promoted sugar reformulation.