Stability of individual anthocyanins from black carrots stored in light and darkness - Impact of acylation.

Black carrot anthocyanins have gained increasing attention as natural coloring agent, owing to their higher stability than anthocyanins from berries. The stability has been attributed to their higher degree of acylation. This study investigated the impact of acylation on the stability of individual anthocyanins during storage in light and darkness. We hypothesized that the acylated anthocyanins would be more stable than the non-acylated ones. The major five anthocyanins were fractioned by semi-preparative HPLC and stored at pH 4.5 in light and darkness to investigate how acylation affected the stability. The stability was evaluated by absorption spectroscopy and mass spectrometry (MS). Two of the anthocyanins were non-acylated; 3-xylosyl(glucosyl)galactoside and cyanidin 3-xylosylgalactoside, and three were acylated; cyanidin 3-xylosyl(sinapolyglucosyl)galacto-side, cyanidin 3-xylosyl(feruloylglu-cosyl)galactoside, and cyanidin 3-xylosyl(coumaroyl-glucosyl)galactoside. Both methods (spectroscopy and MS) showed a clear effect of acylation when stored in light, but surprisingly the two non-acylated anthocyanins, showed higher stability than the three acylated ones.

In vitro post-ovulatory oocyte ageing in grass carp Ctenopharyngodon idella affects H4K12 acetylation pattern and histone acetyltransferase activity.

Delayed fertilization leads to the ageing of post-ovulatory oocytes and reduces the developmental competence of arising embryos. Little information is available about the molecular processes during fish oocyte ageing. The current study investigated the functional consequences of oocyte ageing in grass carp Ctenopharyngodon idella embryos. In addition, the dynamics of selected post-transcriptionally modified histones (acetylation of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16) were analyzed during oocyte ageing. Ovulated oocytes were aged in vitro for 4 h in the laboratory incubator at 20 °C and studied for selected post-translational modification of histones. In addition, histone acetyltransferase activity was investigated as an important regulator of histone acetylation modification. The results indicated a significant decrease in oocyte fertilizing ability through 1 h of post-ovulatory ageing, and a complete loss of egg fertilizing abilities was detected at 4-h aged oocytes. Furthermore, post-ovulatory oocyte ageing for 1 and 4 h led to decreased levels of H4K12 acetylation. The activity of histone acetyltransferases increased significantly after ageing of the oocytes for 30 h in vitro. This modification may partly contribute to explaining the failures of egg viability and embryo development in the offspring from the aged oocytes. The results are the first to report histone modifications as a crucial epigenetic regulator during oocyte ageing in fish and might also benefit other vertebrates.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Influence of food matrix delivery system on the bioavailability of vitamin D3: A randomized crossover trial in postmenopausal women.

OBJECTIVES: Vitamin D insufficiency (blood 25-hydroxyvitamin D <50 nmol/L) is a global health problem. Vitamin D food fortification might be a solution, but knowledge is sparse on which food matrices yield the highest bioavailability. The aim of this study was to investigate the influence of different food matrices including complex formations with whey proteins on the human bioavailability of vitamin D. METHODS: In this randomized, multiple crossover trial, we enrolled 30 postmenopausal women 60 to 80 y of age with vitamin D insufficiency. We measured changes in serum concentrations of vitamin D3 (D3) postprandially for 24 h in response to the intake of 500 mL of different food matrices with 200 µg D3 added compared with a control (500 mL of water). Foods included apple juice with whey protein isolate (WPI), apple juice, semi-skim milk, and water (with D3). The food matrices were provided in a randomized order with ≥10-d washout period between them. On each intervention day, blood samples were collected at 0, 2, 4, 6, 8, 10, 12 and 24 h. RESULTS: D3 with WPI in juice did not enhance area under the curve (AUC) of serum D3 compared with juice without WPI (370 nmol ×  24 h/L; 95% confidence interval [CI], 321-419 versus 357 nmol ×  24 h/L; 95% CI, 308-406 nmol ×  24 h/L; P = 0.65). However, compared with juice, the AUC was significantly higher in response to the intake of D3 in milk (452 nmol ×  24 h/L; 95% CI, 402-502 nmol ×  24 h/L) and water with D3 (479 nmol ×  24 h/L; 95% CI, 430-527 nmol ×  24 h/L; P < 0.05). No difference was observed between milk and water (P = 0.34). CONCLUSIONS: The bioavailability of D3 was superior in milk and water compared with juice, regardless of whether WPI was added.

Administration of whey protein complexed vitamin D3 to vitamin D3-deficient growing Sprague-Dawley rats.

Vitamin D deficiency is a global health issue with consequences for bone health. Complexation of vitamin D3 with specific whey proteins might increase the bioavailability and enhance the effect of dietary supplementation on health outcomes. The current rat study was set up to investigate if complexation of vitamin D3 with whey protein isolate (WPI) or ß-lactoglobulin (B-LG) increases bioavailability of the vitamin and how it impacts markers of bone turnover and bone structure. For 8 weeks, growing male Sprague Dawley rats (n = 48) were fed a vitamin D-deficient diet and during the final 4 weeks gavage dosing of vitamin D3 either alone (VitD) or complexed with WPI (VitD + WPI) or ß-LG (VitD + B-LG) was administered. A placebo treatment (placebo) was also included. After sacrifice, samples of bone were collected and analyzed using biomechanical testing and µCT scanning. The concentrations of vitamin D3, vitamin D3 metabolites and bone markers (P1NP and CTX) were measured in serum. The results showed that VitD + B-LG appeared to induce lower levels of 25-hydroxy vitamin D3 in serum compared to VitD alone. Markers of bone turnover were generally higher in the VitD group compared to placebo and the VitD + WPI and VitD + B-LG treatments. No effects of treatments on bone strength or bone microstructure were detected. In conclusion, whey protein complexation of vitamin D3 supplements appeared to have no beneficial effects on circulating vitamin D3 metabolites but this did not impose changes in bone strength or trabecular bone microstructure.

Biorefinery of Green Biomass─How to Extract and Evaluate High Quality Leaf Protein for Food?

There is a growing need for protein for both feed and food in order to meet future demands. It is imperative to explore and utilize novel protein sources such as protein from leafy plant material, which contains high amounts of the enzyme ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCo). Leafy crops such as grasses and legumes can in humid climate produce high protein yields in a sustainable way when compared with many traditional seed protein crops. Despite this, very little RuBisCo is utilized for foods because proteins in the leaf material has a low accessibility to monogastrics. In order to utilize the leaf protein for food purposes, the protein needs to be extracted from the fiber rich leaf matrix. This conversion of green biomass to valuable products has been labeled green biorefinery. The green biorefinery may be tailored to produce different products, but in this Review, the focus is on production of food-grade protein. The existing knowledge on the extraction, purification, and concentration of protein from green biomass is reviewed. Additionally, the quality and potential application of the leaf protein in food products and side streams from the green biorefinery will be discussed along with possible uses of side streams from the protein production.

Thermal degradation of metabolites in urine using multiple isotope-labelled internal standards for off-line GC metabolomics - effects of injector and oven temperatures.

Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). An optimized derivatization protocol has been successfully applied using multiple isotope labelled analytical internal standards of selected deuterated and 13C selected compounds, covering a range of different groups of metabolites for non-automated GC metabolomics (off-line). Moreover, the study was also realized in a pooled urine sample, following metabolic profiling. A study of thermal degradation of metabolites due to GC inlet and oven programs (fast, slow) was performed, where the results indicated that both GC oven programs (fast and slow) negatively affected the thermal stability of the metabolites, while the fast-ramp GC program also suppressed MS signals. However, the use of multiple internal standards can overcome this drawback. The application of extended temperature ramp GC program presented identical behaviour on metabolite stability and better chromatographic separation combined with much lower signal suppression, compared to a short temperature ramp program. No effects were observed for organic acids, fatty acids, sugars and sugar alcohols, while significant differences were observed for amino acids. GC metabolomics is a strong tool that can facilitate analysis, but special attention is required for sampling handling and heating, before and during the GC analysis. The use and application of multiple multi-group internal standards is highly recommended.

Histone Acetylation Dynamics during In Vivo and In Vitro Oocyte Aging in Common Carp Cyprinus carpio.

Aging is the most critical factor that influences the quality of post-ovulatory oocytes. Age-related molecular pathways remain poorly understood in fish oocytes. In this study, we examined the effect of oocyte aging on specific histone acetylation in common carp Cyprinus carpio. The capacity to progress to the larval stage in oocytes that were aged for 28 h in vivo and in vitro was evaluated. Global histone modifications and specific histone acetylation (H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac) were investigated during oocyte aging. Furthermore, the activity of histone acetyltransferase (HAT) was assessed in fresh and aged oocytes. Global histone modifications did not exhibit significant alterations during 8 h of oocyte aging. Among the selected modifications, H4K12ac increased significantly at 28 h post-stripping (HPS). Although not significantly different, HAT activity exhibited an upward trend during oocyte aging. Results of our current study indicate that aging of common carp oocytes for 12 h results in complete loss of egg viability rates without any consequence in global and specific histone modifications. However, aging oocytes for 28 h led to increased H4K12ac. Thus, histone acetylation modification as a crucial epigenetic mediator may be associated with age-related defects, particularly in oocytes of a more advanced age.

Correction: Milk protein complexation enhances post prandial vitamin D3 absorption in rats.

Correction for 'Milk protein complexation enhances post prandial vitamin D3 absorption in rats' by Ida Emilie I. Lindahl et al., Food Funct., 2020, 11, 4953-4959, DOI: 10.1039/D0FO01062F.

Milk protein complexation enhances post prandial vitamin D3 absorption in rats.

This study investigated the effect of complexation with whey and casein protein, respectively, on post prandial absorption of vitamin D3. For this purpose, Sprague-Dawley rats (n = 78) were administered 840 IU vitamin D3 dissolved in ethanol and either (i) complexed with whey protein isolate (protein : vitamin ration 2 : 1), (ii) complexed with caseinate (protein : vitamin ration 2 : 1), or (iii) provided in a water solution. Serum concentrations of vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were measured before and 2, 3, 4, 5, 6, 7, 8, and 10 hours after administration of vitamin D3. Significant effects of complexation on serum concentrations of vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were demonstrated. Complexation with whey protein isolate resulted in the fastest and highest absorption of vitamin D3 while an effect of complexation with caseinate was evident but more modest and non-significant. In conclusion, the study demonstrates that complexation with milk proteins is an efficient strategy to enhance bioaccessibility of vitamin D3.

Simultaneous Determination of L- and D-Amino Acids in Proteins: A Sensitive Method Using Hydrolysis in Deuterated Acid and Liquid Chromatography-Tandem Mass Spectrometry Analysis.

Determination of the L- and D-amino acid composition in proteins is important for monitoring process-induced racemization, and thereby protein quality loss, in food and feed. Such analysis has so far been challenging due to the need for sample hydrolysis, which generates racemization, thereby leading to an overestimation of D-amino acids. Here, validation of an LC-MS/MS-based method for the simultaneous determination of L- and D-amino acids in complex biological matrixes, like food and feed, was performed in combination with deuterated HCl hydrolysis. This approach eliminated a racemization-induced bias in the L- and D-amino acid ratios. The LC-MS/MS method was applied for the analysis of 18 free amino acids, with a quantification limit of either 12.5 or 62 ng/mL, except for D-phenylalanine, for which quantification was impaired by background interference from the derivatization agent. For hydrolyzed samples, the composition of 10 L- and D-amino acids pairs could be determined in protein. The average relative standard deviation was 5.5% and 6.1%, depending on the type of hydrolysis tubes. The method was applied on a green protein isolate (lucerne), which contained an average of 0.3% D-amino acids. In conclusion, this method allows for an unbiased analysis of L- and D-amino acid ratios in complex protein samples, such as food and feed.

Mechanism behind the degradation of aqueous norbixin upon storage in light and dark environment.

Buffered aqueous solutions of norbixin were stored in light and dark, and analyzed using mass spectrometry. Compounds with both higher and lower masses than norbixin were detected, suggesting the formation of oxidation products and oxidative cleavage products of norbixin. The norbixin oxidation products included compounds containing several oxidations. The amounts of oxidation products of norbixin increased during storage in both light and dark, but in light, the development accelerated. Scavengers of superoxide radical anion (superoxide dismutase), hydrogen peroxide (catalase), hydroxyl radicals (mannitol) and singlet oxygen (sodium azide) and carbon-centered radicals (DMPO) were tested to determine if any of the reactive species were involved in the degradation of norbixin. Of these, only DMPO decreased the bleaching of norbixin indicating the involvement of carbon-centered radicals. Multiple oxidations of norbixin might be a result of a radical chain reaction involving peroxyl and carbon-centered radicals even though not detectable with electron spin resonance.

Incorporation of bixin in aqueous media: Self-formulation with sorbitol ester of norbixin.

We have previously reported how the natural food colorant, bixin, was enzymatically modified by appending sorbitol to the bixin scaffold. The resulted product, sorbitol ester of norbixin (SEN) was expected to be more hydrophilic. The present study aimed to investigate the physical behaviour of SEN in aqueous media. The property of SEN was studied together with non-reacted bixin as separation of the two compounds was unsuccessful. The SEN molecules behaved as a bolaamphiphile in aqueous media, underwent self-association and develop a hydrophilic aggregate. SEN-aggregates could uptake the non-reacted bixin molecules inside its hydrophobic moiety and dispersed it in aqueous media. Aggregation of SEN molecules with incorporated bixin resulted in a hypsochromic shift of the absorption spectra indicting H-aggregation. Dynamic light scattering showed the formation of aggregates with an average hydrodynamic radius 38 ±â€¯2 nm. The dispersibility of the aggregates was affected by pH and the ionic strength of the media.

Hydrophilization of bixin by lipase-catalyzed transesterification with sorbitol.

Bixin is one of the most used yellow-orange food colorants in the food industry. The polyene chain of bixin makes it highly hydrophobic and less suitable for water-based food formulations. Lipase-catalyzed reactions of bixin with sorbitol were studied to synthesize a new derivative of bixin with potential hydrophilic properties. Interestingly, we show that the lipase-catalyzed reaction of bixin leads to a transesterification reaction and formation of a transesterified product, sorbitol ester of norbixin (SEN). The reaction efficiency was optimized with various immobilized lipases at different water activity levels in the organic solvent, 2-methyl-2-butanol. Among the examined lipases, immobilized Candida antarctica lipase B (Novozyme 435) provided the highest reaction yield at a water activity close to zero. Tetrahydrofuran (THF) was used as co-solvent to improve bixin solubility. The optimization of the reaction conditions with 20% THF lead to a total reaction yield of 50% of SEN.

An in vivo characterization of colostrum protein uptake in porcine gut during early lactation.

Understanding the bioactive roles of colostrum proteins has gained much attention, and in particular, their potential use in human and veterinary medicine has been extensively studied. However, studies of bioactivity have mainly been conducted in vitro, but it has not yet been well characterized at the individual protein level which colostrum components are internalized by the intestinal tissue of the neonate. The aim of this study was to characterize the in vivo processing of porcine colostrum in the gastrointestinal tract, and describe which of the potential bioactive proteins can be observed in the small intestinal tissue, and therefore may be functionally important. Using 2D-LC-MS/MS analysis we mapped the proteins in porcine colostrum. The colostrum proteins were then traced in the stomach content, as well as in the small intestinal tissue of 5 piglets suckled for 24h. For comparison, we also mapped the proteins present in the intestinal tissue of a newborn piglet that had not received colostrum. This analysis allowed us to identify the colostrum proteins that are internalized and retained in the tissue from the small intestine, indicating their functional importance. Our studies have shown that in early lactation, some colostrum proteins are protected against proteolytic degradation in the stomach. Furthermore, colostrum proteins with immuno-protective, antimicrobial or other bioactive functions are more prone to uptake in the small intestine than the caseins and beta-lactoglobulin, which are amongst the most abundant in colostrum.

Farm animal proteomics--a review.

In agricultural sciences as in all other areas of life science, the implementation of proteomics and other post-genomic tools is an important step towards more detailed understanding of the complex biological systems that control physiology and pathology of living beings. Farm animals are raised in large-scale operations, with the aim to obtain animal products for human consumption. Hence, understanding the biological traits that impact yield and quality of these products is the specific aim of much biological experimentation. However, most of the data gathered from experiments on e.g. swine and cattle are relevant not only for farm animal sciences, but also for adding to our understanding of complex biological mechanisms of health and disease in humans. The aim of this review is to present an overview of the specific topics of interest within farm animal proteomics, and to highlight some of the areas where synergy between classic model organism proteomics and farm animal proteomics is rapidly emerging. Focus will be on introducing the special biological traits that play an important role in food production, and on how proteomics may help optimize farm animal production.

Advances in porcine genomics and proteomics--a toolbox for developing the pig as a model organism for molecular biomedical research.

Our current knowledge of human biology is often based on studying a wide range of animal species. In particular, for understanding human diseases, the development of adequate animal models is of immediate importance. Although genetic strains and transgenic animal model organisms like fruit fly (Drosophila), zebrafish and rodents are highly informative about the function of single genes and proteins, these organisms do not always closely reflect human biology, and alternative animal models are thus in great demand. The pig is a non-primate mammal that closely resembles man in anatomy, physiology and genetics. Pigs, although not easily kept for laboratory research, are, however, readily available for biomedical research through the large scale industrial production of pigs produced for human consumption. Recent research has facilitated the biological experimentation with pigs, and helped develop the pig into a novel model organism for biomedical research. This toolbox includes the near completion of the pig genome, catalogues of genes and genetic variation in pigs, extensive characterization of pig proteomes and transcriptomes, as well as the development of transgenic disease models. The aim of this review is to highlight the current progress of these ongoing areas of research, which are mandatory for successful development of biomedical pig models that are in demand for understanding human biology in health and disease.

Quantitative milk proteomics--host responses to lipopolysaccharide-mediated inflammation of bovine mammary gland.

Intramammary infusion of lipopolysaccharide (LPS) in cows induces udder inflammation that partly simulates mastitis caused by infection with Gram-negative bacteria. We have used this animal model to characterize the quantitiative response in the milk proteome during the time course before and immediately after the LPS challenge. Milk samples from three healthy cows collected 3 h before the LPS challenge were compared with milk samples collected 4 and 7 h after the LPS challenge, making it possible to describe the inflammatory response of individual cows. Quantitative protein profiles were obtained for 80 milk proteins, of which 49 profiles changed significantly for the three cows during LPS challenge. New information obtained in this study includes the quantified increase of apolipoproteins and other anti-inflammatory proteins in milk, which are important for the cow's ability to balance the immune response, and the upregulation of both complement C3 and C4 indicates that more than one complement pathway could be activated during LPS-induced mastitis. In the future, this analytical approach may provide valuable information about the differences in the ability of individual cows to resist and recover from mastitis.

Effects of bacterial colonization on the porcine intestinal proteome.

The gastrointestinal tract harbors a complex community of bacteria, of which many may be beneficial. Studies of germ-free animal models have shown that the gastrointestinal microbiota not only assists in making nutrients available for the host but also contributes to intestinal health and development. We studied small intestinal protein expression patterns in gnotobiotic pigs maintained germ-free, or monoassociated with either Lactobacillus fermentum or non-pathogenic Escherichia coli. A common reference design in combination with labeling with stable isobaric tags allowed the individual comparison of 12 animals. Our results showed that bacterial colonization differentially affected mechanisms such as proteolysis, epithelial proliferation, and lipid metabolism, which is in good agreement with previous studies of other germ-free animal models. We have also found that E. coli has a profound effect on actin remodeling and intestinal proliferation, which may be related to stimulated migration and turnover of enterocytes. Regulations related to L. fermentum colonization involved individual markers for immunoregulatory mechanisms.

Proteome profiles of mucosal immunoglobulin uptake in inflamed porcine gut.

Acquisition of passive immunity by endocytosis of intact immunoglobulins (Ig) from colostrum is critical for prevention of intestinal and systemic diseases in neonatal mammals. We compared proteome patterns of healthy and inflamed gut tissues from pre-term piglets to investigate the effect of inflammation on acquisition of passive immunity. A clear difference in the two-dimensional gel electrophoresis protein patterns between healthy and inflamed intestinal tissues was observed, suggesting that inflamed tissues failed to absorb and transfer Ig from colostrum to epithelial cells. We have mapped and identified the Ig proteins that are taken up by healthy intestinal tissues, and found that isoforms of the IgA and IgG heavy chain and Ig kappa and lambda light chains were internalized. Our results indicate that colostrum protein uptake in the porcine gut is a selective process that is obstructed in inflamed pre-term gut.