Do high-protein diets have the potential to reduce gut barrier function in a sex-dependent manner?

PURPOSE: Impaired gut barrier function is associated with systemic inflammation and many chronic diseases. Undigested dietary proteins are fermented in the colon by the gut microbiota which produces nitrogenous metabolites shown to reduce barrier function in vitro. With growing evidence of sex-based differences in gut microbiotas, we determined whether there were sex by dietary protein interactions which could differentially impact barrier function via microbiota modification. METHODS: Fermentation systems were inoculated with faeces from healthy males (n = 5) and females (n = 5) and supplemented with 0.9 g of non-hydrolysed proteins sourced from whey, fish, milk, soya, egg, pea, or mycoprotein. Microbial populations were quantified using fluorescence in situ hybridisation with flow cytometry. Metabolite concentrations were analysed using gas chromatography, solid phase microextraction coupled with gas chromatography-mass spectrometry and ELISA. RESULTS: Increased protein availability resulted in increased proteolytic Bacteroides spp (p < 0.01) and Clostridium coccoides (p < 0.01), along with increased phenol (p < 0.01), p-cresol (p < 0.01), indole (p = 0.018) and ammonia (p < 0.01), varying by protein type. Counts of Clostridium cluster IX (p = 0.03) and concentration of p-cresol (p = 0.025) increased in males, while females produced more ammonia (p = 0.02), irrespective of protein type. Further, we observed significant sex-protein interactions affecting bacterial populations and metabolites (p < 0.005). CONCLUSIONS: Our findings suggest that protein fermentation by the gut microbiota in vitro is influenced by both protein source and the donor's sex. Should these results be confirmed through human studies, they could have major implications for developing dietary recommendations tailored by sex to prevent chronic illnesses.

Reduced free asparagine in wheat grain resulting from a natural deletion of TaASN-B2: investigating and exploiting diversity in the asparagine synthetase gene family to improve wheat quality.

BACKGROUND: Understanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine concentrations, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties. RESULTS: Natural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine concentrations in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high concentrations as a result of sulphur deficiency. CONCLUSIONS: Selecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine concentrations in commercial wheat grain.

Residue dynamics of a contact and a systemic fungicide in pollen, nectar, and other plant matrices of courgette (Cucurbita pepo L.).

Pollen and nectar can be contaminated with a range of pesticides, including insecticides, fungicides, and herbicides. Since these matrices are important food sources for pollinators and other beneficial insects, their contamination can represent a key route of exposure. However, limited knowledge exists with respect to pesticide residue levels and their dynamics in these matrices for many crops and active ingredients (AIs). We used controlled glasshouse studies to investigate the residue dynamics of a systemic (cyprodinil) and a contact (fludioxonil) fungicide in the floral matrices and other plant parts of courgette/zucchini (Cucurbita pepo L.). We aimed to better understand the processes behind residue accumulation and decline in pollen and nectar. Each AI was applied to plants, either by spraying whole plants or by targeted spraying onto leaves only. Samples of pollen, nectar, anthers, flowers, and leaves were taken on the day of application and each subsequent morning for up to 13 days and analysed for residues using LC-MS/MS. Significant differences in residue levels and dynamics were found between AIs and floral matrices. The present study allowed for the identification of potential routes by which residues translocate between tissues and to link those to the physicochemical properties of each AI, which may facilitate the prediction of residue levels in pollen and nectar. Residues of the contact AI declined more quickly than those of the systemic AI in pollen and nectar. Our results further suggest that the risk of oral exposure for pollinators may be considerably reduced by using contact AIs during the green bud stage of plants, but application of systemic compounds could still result in a low, but continuous long-term exposure for pollinators with limited decline.

Effects of different thawing methods on physical and physicochemical properties of frozen dough and quality of corresponding steamed bread.

The thawing method is critical for the final quality of products based on the frozen dough. The effects of ultrasound thawing, proofer thawing, refrigerator thawing, water bath thawing, ambient thawing, and microwave thawing on the rheology, texture, water distribution, fermentation characteristics, and microstructure of frozen dough and the properties of steamed bread were investigated. The results indicated that the ultrasound thawing dough had better physicochemical properties than other doughs. It was found that ultrasound thawing restrained the water migration of dough, improved its rheological properties and fermentation capacity. The total gas volume value of the ultrasound thawing dough was reduced by 21.35% compared with that of unfrozen dough. The ultrasound thawing dough displayed a thoroughly uniform starch-gluten network, and an enhanced the specific volume and internal structure of the steamed bread. In conclusion, ultrasound thawing effectively mitigated the degradation of the frozen dough and enhanced the quality of steamed bread.

Reducing Dietary Acrylamide Exposure from Wheat Products through Crop Management and Imaging.

The nutritional safety of wheat-based food products is compromised by the presence of the processing contaminant acrylamide. Reduction of the key acrylamide precursor, free (soluble, non-protein) asparagine, in wheat grain can be achieved through crop management strategies, but such strategies have not been fully developed. We ran two field trials with 12 soft (biscuit) wheat varieties and different nitrogen, sulfur, potassium, and phosphorus fertilizer combinations. Our results indicated that a nitrogen-to-sulfur ratio of 10:1 kg/ha was sufficient to prevent large increases in free asparagine, whereas withholding potassium or phosphorus alone did not cause increases in free asparagine when sulfur was applied. Multispectral measurements of plants in the field were able to predict the free asparagine content of grain with an accuracy of 71%, while a combination of multispectral, fluorescence, and morphological measurements of seeds could distinguish high free asparagine grain from low free asparagine grain with an accuracy of 86%. The acrylamide content of biscuits correlated strongly with free asparagine content and with color measurements, indicating that agronomic strategies to decrease free asparagine would be effective and that quality control checks based on product color could eliminate high acrylamide biscuit products.

Genetic control of grain amino acid composition in a UK soft wheat mapping population.

Wheat (Triticum aestivum L.) is a major source of nutrients for populations across the globe, but the amino acid composition of wheat grain does not provide optimal nutrition. The nutritional value of wheat grain is limited by low concentrations of lysine (the most limiting essential amino acid) and high concentrations of free asparagine (precursor to the processing contaminant acrylamide). There are currently few available solutions for asparagine reduction and lysine biofortification through breeding. In this study, we investigated the genetic architecture controlling grain free amino acid composition and its relationship to other traits in a Robigus × Claire doubled haploid population. Multivariate analysis of amino acids and other traits showed that the two groups are largely independent of one another, with the largest effect on amino acids being from the environment. Linkage analysis of the population allowed identification of quantitative trait loci (QTL) controlling free amino acids and other traits, and this was compared against genomic prediction methods. Following identification of a QTL controlling free lysine content, wheat pangenome resources facilitated analysis of candidate genes in this region of the genome. These findings can be used to select appropriate strategies for lysine biofortification and free asparagine reduction in wheat breeding programs.

The acrylamide problem: a plant and agronomic science issue.

Acrylamide, a chemical that is probably carcinogenic in humans and has neurological and reproductive effects, forms from free asparagine and reducing sugars during high-temperature cooking and processing of common foods. Potato and cereal products are major contributors to dietary exposure to acrylamide and while the food industry reacted rapidly to the discovery of acrylamide in some of the most popular foods, the issue remains a difficult one for many sectors. Efforts to reduce acrylamide formation would be greatly facilitated by the development of crop varieties with lower concentrations of free asparagine and/or reducing sugars, and of best agronomic practice to ensure that concentrations are kept as low as possible. This review describes how acrylamide is formed, the factors affecting free asparagine and sugar concentrations in crop plants, and the sometimes complex relationship between precursor concentration and acrylamide-forming potential. It covers some of the strategies being used to reduce free asparagine and sugar concentrations through genetic modification and other genetic techniques, such as the identification of quantitative trait loci. The link between acrylamide formation, flavour, and colour is discussed, as well as the difficulty of balancing the unknown risk of exposure to acrylamide in the levels that are present in foods with the well-established health benefits of some of the foods concerned.

Understanding the Relationships between Free Asparagine in Grain and Other Traits to Breed Low-Asparagine Wheat.

Since the discovery of acrylamide in food, and the identification of free asparagine as the key determinant of acrylamide concentration in wheat products, our understanding of how grain asparagine content is regulated has improved greatly. However, the targeted reduction in grain asparagine content has not been widely implemented in breeding programmes so far. Here we summarise how free asparagine concentration relates to other quality and agronomic traits and show that these relationships are unlikely to pose major issues for the breeding of low-asparagine wheat. We also outline the strategies that are possible for the breeding of low-asparagine wheat, using both natural and induced variation.

Comparison of the sensory properties of fragrant and non-fragrant rice (Oryza sativa), focusing on the role of the popcorn-like aroma compound 2-acetyl-1-pyrroline.

2-Acetyl-1-pyrroline (2-AP) has been widely reported as a key contributor to the popcorn-like aroma of fragrant rice (Oryza sativa). To gain a greater understanding of its contribution to the aroma in both fragrant and non-fragrant rice, sensory profiling was conducted with a trained panel to examine the sensory properties of six boiled rice samples, three fragrant and three non-fragrant varieties. The intensity of the popcorn note as an orthonasal odour, a retronasal flavour and as an after-effect was significantly higher in fragrant rice than in non-fragrant rice. However, panellists could not differentiate these popcorn attributes between the three different fragrant rice varieties. 2-AP was extracted from the boiled rice samples by headspace solid-phase microextraction and quantified by gas chromatography-mass spectrometry. 2-AP was below the limits of quantitation in non-fragrant varieties; however, gas chromatography-olfactometry of samples indicated the presence of 2-AP in both raw fragrant and non-fragrant rice varieties.

Effect of sugar reduction on flavour release and sensory perception in an orange juice soft drink model.

To examine the effect of sugar reduction on the sensory perception of sweetened beverages, an orange juice soft drink model flavoured with seven characteristic compounds (hexanal, decanal, linalool, ethyl butanoate, α-pinene, ß-myrcene and (Z)-3-hexen-1-ol) was developed. Five samples were prepared with relevant sugar contents (5.2, 8.2, 9.7, 11.2 and 14.2 °Brix). Using retronasal quantitative descriptive analysis (QDA), nine attributes were found to differ significantly (p < 0.05) with sugar content. When the samples were evaluated orthonasally, only the attribute "overripe orange" significantly decreased (p < 0.05) with reduction of sugar content. Headspace solid-phase microextraction with gas chromatography-mass spectrometry showed that as sugar concentration decreased, the headspace concentration of six of the volatile compounds decreased, whilst ethyl butanoate remained constant. Principal component analysis revealed that the total release of the flavour compounds was highly correlated with the perceived intensity of the orthonasal attribute "overripe orange".

Chemical characterisation of potential pheromones from the shoulder gland of the Northern yellow-shouldered-bat, Sturnira parvidens (Phyllostomidae: Stenodermatinae).

Bats of the genus Sturnira (Family Phyllostomidae) are characterised by shoulder glands that are more developed in reproductively mature adult males. The glands produce a waxy secretion that accumulates on the fur around the gland, dyeing the fur a dark colour and giving off a pungent odour. These shoulder glands are thought to play a role in their reproductive behaviour. Using gas chromatography-mass spectrometry, we analysed solvent extracts of fur surrounding the shoulder gland in the northern-shouldered bat, Sturnira parvidens to (i) characterise the chemical composition of shoulder gland secretions for the first time, and (ii) look for differences in chemical composition among and between adult males, sub-adult/juvenile males and adult females. Fur solvent extracts were analysed as liquids and also further extracted using headspace solid-phase microextraction to identify volatile components in the odour itself. Odour fingerprint analysis using non-metric multidimensional scaling plots and multivariate analysis revealed clear and significant differences (P < 0.001) between adult males vs both juvenile males and adult females. The chemical components of the shoulder gland secretion included terpenes and phenolics, together with alcohols and esters, most likely derived from the frugivorous diet of the bat. Many of the compounds identified were found exclusively or in elevated quantities among adult (reproductive) males compared with adult females and non-reproductive (juvenile) males. This strongly suggests a specific role in male-female attraction although a function in male-male competition and/or species recognition is also possible.

A review of the factors that influence pesticide residues in pollen and nectar: Future research requirements for optimising the estimation of pollinator exposure.

In recent years, the impact of Plant Protection Products (PPPs) on insect pollinator decline has stimulated significant amounts of research, as well as political and public interest. PPP residues have been found in various bee-related matrices, resulting in governmental bodies worldwide releasing guidance documents on methods for the assessment of the overall risk of PPPs to different bee species. An essential part of these risk assessments are PPP residues found in pollen and nectar, as they represent a key route of exposure. However, PPP residue values in these matrices exhibit large variations and are not available for many PPPs and crop species combinations, which results in inaccurate estimations and uncertainties in risk evaluation. Additionally, residue studies on pollen and nectar are expensive and practically challenging. An extrapolation between different cropping scenarios and PPPs is not yet justified, as the behaviour of PPPs in pollen and nectar is poorly understood. Therefore, this review aims to contribute to a better knowledge and understanding of the fate of PPP residues in pollen and nectar and to outline knowledge gaps and future research needs. The literature suggests that four primary factors, the crop type, the application method, the physicochemical properties of a compound and the environmental conditions have the greatest influence on PPP residues in pollen and nectar. However, these factors consist of many sub-factors and initial effects may be disguised by different sampling methodologies, impeding their exact characterisation. Moreover, knowledge about these factors is ambiguous and restricted to a few compounds and plant species. We propose that future research should concentrate on identifying relationships and common features amongst various PPP applications and crops, as well as an overall quantification of the described parameters; in order to enable a reliable estimation of PPP residues in pollen, nectar and other bee matrices.

Long-term exposure to sensory feed additives during the gestational and postnatal periods affects sows' colostrum and milk sensory profiles, piglets' growth, and feed intake.

This study investigated the effect of feed supplementation in sows and/or their progeny with 2 sensory feed additives (FA1: limonene and cinnamaldehyde; FA2: menthol, carvone, and anethole) on sows' feed intake, body weight, fat deposition, and colostrum/milk composition, as well as piglets' feed intake growth and feed efficiency from birth to slaughter at postnatal day 160 (PND160). During the last third of gestation and the whole of lactation, sows were subjected to a control diet (C) or the same diet containing FA1 or FA2 at 0.1% of complete feed content. Colostrum/milk samples were taken at days 1, 14, and 28 of lactation for gas chromatography-mass spectrometry analyses. After weaning, the progeny was subjected to a control diet (C) or experimental diets with a sweetener (0.015%) but no other additive (S), or to diets with a sweetener and the additive FA1 (FA1S) or FA2 (FA2S). There was no effect of dietary treatment on sows' feed intake, body weight, or adiposity (P > 0.15 for all), but the sensory characteristics of their colostrum/milk were modified by the diet and diet*time interaction. Limonene concentrations were higher in FA1 samples from PND1 to PND28, whereas carvone and anethole concentrations were higher in FA2 samples from PND1 to PND28. The concentration of these 3 compounds increased with time in the respective groups where they were mostly detected. Menthol concentrations were higher in FA2 samples at PND14 and PND28, but there was no time effect. Overall, cinnamaldehyde was always below the detection range. Piglets born from FA1 and FA2 sows had higher body weight (P = 0.034 at PND160), average daily gain (ADG; P = 0.036 for PND0-160), and average daily feed intake (ADFI; P = 0.006 for PND28-160) than piglets born from C sows. Overall, piglets that were never exposed to FA or only after weaning had lower ADG (P = 0.030 for PND0-160) and ADFI (P = 0.016 for PND28-160) than piglets that were exposed to FA only via the maternal diet, the condition combining both pre- and post-natal exposure being intermediary. In conclusion, FA1 and FA2 provided to gestating and lactating sows increased the progeny's feed intake and growth, suggesting nutritional programming and/or sensory conditioning during the perinatal period. Addition of FA only in the progeny's diet was not beneficial.

Changes in free amino acids and sugars in potatoes due to sulfate fertilization and the effect on acrylamide formation.

To examine how sulfur deprivation may affect acrylamide formation in cooked potatoes, three varieties of potato were grown under conditions of either severe sulfur deprivation or an adequate supply of sulfur. In all three varieties sulfur deprivation led to a decrease in acrylamide formation, even though the levels of sugars, which are acrylamide precursors, were higher in tubers of the sulfur-deprived plants. In one variety the concentration of free asparagine, the other precursor for acrylamide, was also higher. There was a very close correlation between the concentration of asparagine in the tubers expressed as a proportion of the total free amino acid pool and the formation of acrylamide upon cooking, whereas sugars were poorly correlated with acrylamide. In potatoes, where concentrations of sugars are usually limiting, competition between asparagine and other amino acids participating in the Maillard reaction may be a key determinant of the amount of acrylamide that is formed during processing.

Evaluation of 2-acetyl-1-pyrroline in foods, with an emphasis on rice flavour.

The popcorn-like aroma compound 2-acetyl-1-pyrroline (2-AP) is a key contributor to the desirable aroma of fragrant rice and is also important in the aroma of other foods, such as pandan leaf, popcorn and Mediterranean sausage. It can be formed enzymatically in the rice grain as it grows and is also formed, as part of the Maillard reaction, when rice is heated. This review examines the formation of 2-AP in rice and other foods, particularly its formation during cooking, focusing on the importance of the Maillard reaction between reducing sugar breakdown products and 1-pyrroline derived from the amino acids proline and ornithine. The synthesis of 2-AP is discussed alongside the attempts that have been made to stabilise this relatively unstable compound. The analysis of 2-AP by instrumental techniques, particularly gas chromatography-mass spectrometry and gas chromatography-olfactometry, alongside the use of sensory studies, is also discussed.

Acrylamide-forming potential of potatoes grown at different locations, and the ratio of free asparagine to reducing sugars at which free asparagine becomes a limiting factor for acrylamide formation.

Acrylamide is produced from free asparagine and reducing sugars during high-temperature cooking and food processing, and potato products are major contributors to dietary acrylamide intake. The present study analysed twenty varieties of potatoes grown at two sites (Doncaster and Woburn) in the United Kingdom to assess the effect of location of cultivation on acrylamide-forming potential. Analysis of variance revealed a full site by variety nested within type (French fry, boiling and crisping) by storage interaction for acrylamide (p<0.003, F-test), reducing sugars and total sugars (p<0.001, F-test). There was much greater free asparagine in potatoes grown at the Doncaster site compared with the Woburn site. Modelling of the relationship between the ratio of free asparagine to reducing sugars and the levels of acrylamide identified a value of 2.257±0.149 as the tipping point in the ratio below which free asparagine concentration could affect acrylamide formation.

Effect of citric acid and glycine addition on acrylamide and flavor in a potato model system.

Acrylamide levels in cooked/processed food can be reduced by treatment with citric acid or glycine. In a potato model system cooked at 180 degrees C for 10-60 min, these treatments affected the volatile profiles. Strecker aldehydes and alkylpyrazines, key flavor compounds of cooked potato, were monitored. Citric acid limited the generation of volatiles, particularly the alkylpyrazines. Glycine increased the total volatile yield by promoting the formation of certain alkylpyrazines, namely, 2,3-dimethylpyrazine, trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, tetramethylpyrazine, and 2,5-diethyl-3-methylpyrazine. However, the formation of other pyrazines and Strecker aldehydes was suppressed. It was proposed that the opposing effects of these treatments on total volatile yield may be used to best advantage by employing a combined treatment at lower concentrations, especially as both treatments were found to have an additive effect in reducing acrylamide. This would minimize the impact on flavor but still achieve the desired reduction in acrylamide levels.

Formation of high levels of acrylamide during the processing of flour derived from sulfate-deprived wheat.

When wheat was grown under conditions of severe sulfate depletion, dramatic increases in the concentration of free asparagine were found in the grain of up to 30 times as compared to samples receiving the normal levels of sulfate fertilizer. The effect was observed both in plants grown in pots, where the levels of nutrients were carefully controlled, and in plants grown in field trials on soil with poor levels of natural nutrients where sulfate fertilizer was applied at levels from 0 to 40 kg sulfur/Ha. Many of the other free amino acids were present at higher levels in the sulfate-deprived wheat, but the levels of free glutamine showed increases similar to those observed for asparagine. In baked cereal products, asparagine is the precursor of the suspect carcinogen acrylamide, and when flours from the sulfate-deprived wheat were heated at 160 degrees C for 20 min, levels of acrylamide between 2600 and 5200 microg/kg were found as compared to 600-900 microg/kg in wheat grown with normal levels of sulfate fertilization.

The use of asparaginase to reduce acrylamide levels in cooked food.

Strategies proposed for reducing the formation of the suspected carcinogen acrylamide in cooked foods often rely on a reduction in the extent of the Maillard reaction, in which acrylamide is formed from the reaction between asparagine and reducing sugars. However, the Maillard reaction also provides desirable sensory attributes of cooked foods. Mitigation procedures that modify the Maillard reaction may negatively affect flavour and colour. The use of asparaginase to convert asparagine to aspartic acid may provide a means to reduce acrylamide formation, while maintaining sensory quality. This review collates research on the use of enzymes, asparaginase in particular, to mitigate acrylamide formation. Asparaginase is a powerful tool for the food industry and it is likely that its use will increase. However, the potential adverse effects of asparaginase treatment on sensory properties of cooked foods and the need to achieve sufficient enzyme-substrate contact remain areas for future research.

Measurement of acrylamide and its precursors in potato, wheat, and rye model systems.

The relationship between acrylamide and its precursors, namely, free asparagine and reducing sugars, was studied in cakes made from potato flake, wholemeal wheat, and wholemeal rye, cooked at 180 degrees C, from 5 to 60 min. Between 5 and 20 min, major losses of asparagine, water, and total reducing sugars were accompanied by large increases in acrylamide, which maximized in all three products between 25 and 30 min, followed by a slow linear reduction. Acrylamide formation did not occur to a large degree until the moisture contents of the cakes fell below 5%. Linear relationships were observed for acrylamide formation with the residual levels of asparagine and reducing sugars for all three food materials.