This study focused on identifying amylase-trypsin inhibitors (ATIs) in seven Norwegian-cultivated wheat varieties, including common wheat and ancestral species, and identifying potentially harmful opioid peptides within the ex vivo digesta of these wheats. LC-MS/MS analysis of tryptic peptides from ATI fractions revealed that the common wheat variety B & oslash;rsum exhibited the highest diversity of ATIs (n = 24), while they were less represented in tetraploid emmer (n = 11). Hexaploid wheat Bastian showed low diversity and relative abundance of ATIs. Nevertheless, digestion of Mirakel and Bastian by human gastrointestinal juices released the highest number of opioid-containing peptides, representing both gluten exorphins and gliadorphin. In conclusion, emmer had the lowest levels of ATIs, while einkorn and spelt released the fewest opioid-containing peptides after ex vivo digestion. These results point to the potential lower harmful effects of ancestral wheat compared to common hexaploid wheat varieties for wheat-sensitive individuals.

Six cattle breeds native to Norway, have for almost half a century been at risk of extinction. Due to their small population sizes, they have hardly been improved by breeding for many decades. Still, the endangered breeds represent a source of genetic diversity with special milk qualities compared to the modern breed, Norwegian red (NRF). This study reports for the first time a detailed overview of their milk composition. Milk from seven native breeds, in total 200 individuals, were included in the study. Rare genetic variants of αs1- and αs2-casein, and β-casein A1 and κ-casein B were more prevalent in milk form the endangered breeds compared to NRF. Moreover, milk from these six breeds showed better renneting properties and lower incidences of non-coagulating milk, compared to the NRF milk, which showed better acid coagulation properties. This study shows the potential for native breeds in small-scale production of high-quality rennet cheeses.

Safety is the most important criteria of any substance or microorganism applied in the food industry. The whole-genome sequencing (WGS) of an indigenous dairy isolate LL16 confirmed it to be Lactococcus lactis subsp. lactis with genome size 2,589,406 bp, 35.4% GC content, 246 subsystems, and 1 plasmid (repUS4). The Nextera XT library preparation kit was used to generate the DNA libraries, and the sequencing was carried out on an Illumina MiSeq platform. In silico analysis of L. lactis LL16 strain revealed non-pathogenicity and the absence of genes involved in transferable antimicrobial resistances, virulence, and formation of biogenic amines. One region in the L. lactis LL16 genome was identified as type III polyketide synthases (T3PKS) to produce putative bacteriocins lactococcin B, and enterolysin A. The probiotic and functional potential of L. lactis LL16 was investigated by the presence of genes involved in adhesion and colonization of the host's intestines and tolerance to acid and bile, production of enzymes, amino acids, and B-group vitamins. Genes encoding the production of neurotransmitters serotonin and gamma-aminobutyric acid (GABA) were detected; however, L. lactis LL16 was able to produce only GABA during milk fermentation. These findings demonstrate a variety of positive features that support the use of L. lactis LL16 in the dairy sector as a functional strain with probiotic and GABA-producing properties.

We investigated the digestive peptide profiles of pea and faba bean proteins to evaluate possible bioactive peptides (BAPs), and to test immune-modulating properties of selected peptides using in vitro cell model. More than 400 unique peptides were released upon gastrointestinal digestion of pea and faba bean proteins and identified by high-resolution mass spectrometry (HPLC-ESI-MS/MS). Among these, 24 peptides were potentially bioactive as predicted by in silico analysis. Also, up to 275 recognized BAPs and eight known allergens were identified within the peptide sequences of the digests. Four selected peptides significantly decreased the IL-8 response in Caco-2 cells, with a specific pea peptide, DKPWWPK, reducing the IL-8 response up to 40 % during IL-1β induction. Combined with other nutritional benefits, the immune-modulatory properties of legume proteins point to a regular introduction of legumes in the diet, as they represent a still scarcely exploited food category, especially in the Nordic countries.

Aflatoxin M1 was determined in 240 milk samples collected from Hulla and Dale districts, Ethiopia, by Enzyme linked immunosorbent assays (ELISA) detection. It has been found that 96.7% of the respondents did not have any awareness about aflatoxin. Laboratory analysis showed that AFM1 was detected in 77.91% of the total raw milk samples collected within a range of 0.00–0.038 and with an average of 0.0068 µg/L. Average AFM1 in raw milk during dry season (0.008 ± 0.0093 µg/L) was significantly higher (P < .05) than wet season (0.0049 ± 0.0041 µg/L). The level AFM1 detected in raw milk under the current study is lower than the limits set by EU and USA and hence can be considered as safe milk to consume. But the lower level of awareness about aflatoxin in the farmers showed that there is risk of aflatoxin contamination, so awareness creation and aflatoxin control measured should be implemented.

Several factors influence the composition of milk. Among these, genetic variation within and between cattle breeds influences milk protein composition, protein heterogeneity, and their posttranslational modifications. Such variations may further influence technological properties, which are of importance for the utilization of milk into dairy products. Furthermore, these potential variations may also facilitate the production of differentiated products (e.g., related to specific breeds or specific genetic variants). The objective of this study was to investigate the genetic variation and relative protein composition of the major proteins in milk from 6 native Norwegian dairy breeds representing heterogeneity in geographical origin, using the modern Norwegian breed, Norwegian Red, as reference. In total, milk samples from 144 individual cows were collected and subjected to liquid chromatography-electrospray ionization/mass spectrometry-based proteomics for identification of genetic and posttranslational modification isoforms of the 4 caseins (αS1-CN, αS2-CN, β-CN, κ-CN) and the 2 most abundant whey proteins (α-lactalbumin and β-lactoglobulin). Relative quantification of these proteins and their major isoforms, including phosphorylations of αS1-CN and glycosylation of κ-CN, were determined based on UV absorbance. The presence and frequency of genetic variants of the breeds were found to be very diverse and it was possible to identify rare variants of the CN, which, to our knowledge, have not been identified in these breeds before. Thus, αS1-CN variant D was identified in low frequency in 3 of the 6 native Norwegian breeds. In general, αS1-CN was found to be quite diverse between the native breeds, and the even less frequent A and C variants were furthermore detected in 1 and 5 of the native breeds, respectively. The αS1-CN variant C was also identified in samples from the Norwegian Red cattle. The variant E of κ-CN was identified in 2 of the native Norwegian breeds. Another interesting finding was the identification of αS2-CN variant D, which was found in relatively high frequencies in the native breeds. Diversity in more common protein genetic variants were furthermore observed in the protein profiles of the native breeds compared with milk from the high-yielding Norwegian Reds, probably reflecting the more diverse genetic background between the native breeds.

The demand for sustainably produced proteins is increasing with the world population and is prompting a dietary shift toward plant sourced proteins. Vegetable proteins have lower digestibility and biological value compared to animal derived counterparts. We explored sprouting of chickpea seeds as a strategy for improving digestibility. Protein evolution associated with by the sprouting process was assessed by proteomics. The sprouting induced breakdown of seed storage proteins and doubled the release of free alpha-amino nitrogen in sprouted chickpea flour. During sprouting, several enzymes involved in plant development were newly expressed. An ex vivo model of gastroduodenal and jejunal digestion was applied to assess the bioaccessibility of the protein digests. Proteins from chickpea sprouts showed a greater susceptibility to digestion with a 10% increase in alpha amino nitrogen. Peptides with potential immunoreactivity or bioactivity were catalogued in both digested chickpea sprouts and seeds using an in-silico approach. Peptides belonging to the non-specific transfer proteins, which are allergens in pulses, and peptides belonging to an IgE-binding hemagglutinin protein could only be identified in the digested chickpea sprouts. The observation collected paved the way to immune-based evaluations to assess the effect of germination on the allergenic potential.

In vitro digestion studies often use animal digestive enzyme extracts as substitutes of human gastric and pancreatic secretions. Pancreatin from porcine origin is thus commonly used to provide relevant pancreatic enzymes such as proteases, amylase and lipase. Rabbit gastric extracts (RGE) have been recently introduced to provide gastric lipase in addition to pepsin. Before preparing simulated gastric and pancreatic extracts with targeted enzyme activities as described in in vitro digestion protocols, it is important to determine the activities of enzyme preparations using validated methods. The purpose of this inter-laboratory study within the INFOGEST network was to test the repeatability and reproducibility of lipase assays using the pH-stat technique for measuring the activities of gastric and pancreatic lipases from various sources. Twenty-one laboratories having different pH-stat devices received the same protocol with identical batches of RGE and two pancreatin sources. Lipase assays were performed using tributyrin as a substrate and three different amounts (50, 100 and 200 µg) of each enzyme preparation. The repeatability results within individual laboratories were satisfactory with coefficients of variation (CVs) ranging from 4 to 8% regardless of the enzyme amount tested. However, the inter-laboratory variability was high (CV > 15%) compared to existing standards for bioanalytical assays. We identified and weighted the contributions to inter-laboratory variability of several parameters associated with the various pH-stat equipment used in this study (e.g. reaction vessel volume and shape, stirring mode and rate, burette volume for the automated delivery of sodium hydroxide). Based on this, we established recommendations for improving the reproducibility of lipase assays using the pH-stat technique. Defining accurate and complete recommendations on how to correctly quantify activity levels of enzyme preparations is a gateway to promising comparison of in vitro data obtained from different laboratories following the same in vitro digestion protocol.

This study was conducted in the southern region of Ethiopia. The objectives were to assess the occurrence and level of aflatoxin B1 contamination in dairy cow feeds. The analytical technique used was Enzyme-linked immunosorbent assay(ELISA). Two-way ANOVA was used to analyze +the season and location interaction effect. Laboratory analysis showed that 81.2% of the feed samples had a detectable level of AFB1 within the range of 0.067-29.69 µg/kg and with an average of 7.91±5.83 µg/kg. From the positive samples, 52.5 and 4.58% of the feed sample were above EU (5 µg/kg) and USA (20 µg/kg) limits for dairy cattle feed respectively. Average AFB1 concentration during the dry season (8.74± 5.6 µg/kg) is significantly higher (P<0.05) than wet season (7.02± 5.86 µg/kg). This result implies that sustainable good practices should be maintained for all feed harvesting, storage, and feeding practices by feed producers and dairy farmers regarding aflatoxin contamination.

The study was investigated to develop a fermented probiotic beverage using whey from Chhana (milk curds) production and pineapple juice. Four variants of drinks were prepared by blending whey and pineapple juice and coded as A (0:100), B (15:85), C (25:75) and D (35:65), and each blends have 8% sugar. The whey and pineapple juice blends were amended based on sensory and physico-chemical evaluation. The probiotic starter culture used was Lactobacillus acidophilus LA-5 (ATCC 4356). The blend of 25% whey and 75% pineapple juice was fermented for 5 h at 37 °C presented the maximum overall acceptability scores from sensorial analysis. The blends shelf-life was investigated in response to sensory evaluation, acidity, phase separation, and probiotic cell viability. The results revealed that the probiotic whey-pineapple beverage could be preserved for 42–56 days at 4 °C with satisfactory acceptability and shelf-life. The viable probiotic cell counts in the beverage after 42 and 56 days were 4.92 Log10 CFU mL−1 and 4.20 Log10 CFU mL−1, respectively. Survivability of the probiotic culture in the beverage during in vitro gastrointestinal digestion was more than 80% during the preservation period of 56 days. This study showed that a probiotic beverage of pineapple juice and whey, a by-product from Chhana production, may have good functional and nutritional qualities for human consumption.

Milk oligosaccharides are of high interest due to their bioactive properties. This study is the first to characterise milk oligosaccharides from native North European cattle breeds, as represented by 80 milk samples collected from eight native breeds originated from Norway (Norwegian Doela cattle and Norwegian Telemark cattle), Sweden (Swedish Mountain cattle), Denmark (Danish Red anno 1970), Iceland (Icelandic cattle), Lithuania (native Lithuanian Black and White) and Finland (Western Finncattle and Eastern Finncattle). Using high-performance liquid-chromatography chip/quadrupole time-of-flight mass-spectrometry, 18 unique monosaccharide compositions and a multitude of isomers were identified. No N-glycolylneuraminic acid was identified among these breeds. Western Finncattle milk was most abundant in neutral, acidic and fucosylated oligosaccharides. Further, Eastern Finncattle milk was significantly higher in acidic oligosaccharides and Icelandic cattle milk significantly higher in fucosylated oligosaccharides, compared to the mean. This study highlights specific native breeds of particular interest for future exploitation of milk oligosaccharides and breeding strategies.

Little is known about the extent of variation and activity of naturally occurring milk glycosidases and their potential to degrade milk glycans. A multi-omics approach was used to investigate the relationship between glycosidases and important bioactive compounds such as free oligosaccharides and O-linked glycans in bovine milk. Using 4-methylumbelliferone (4-MU) assays activities of eight indigenous glycosidases were determined, and by mass spectrometry and 1H NMR spectroscopy various substrates and metabolite products were quantified in a subset of milk samples from eight native North European cattle breeds. The results showed a clear variation in glycosidase activities among the native breeds. Interestingly, negative correlations between some glycosidases including β-galactosidase, N-acetyl-β-d-glucosaminidase, certain oligosaccharide isomers as well as O-linked glycans of κ-casein were revealed. Further, a positive correlation was found for free fucose content and α-fucosidase activity (r = 0.37, p-value < 0.001) indicating cleavage of fucosylated glycans in milk at room temperature. The results obtained suggest that milk glycosidases might partially degrade valuable glycans, which would result in lower recovery of glycans and thus represent a loss for the dairy ingredients industry if these activities are pronounced.

Celiac disease (CeD) is an autoimmune enteropathy triggered by immunogenic gluten peptides released during the gastrointestinal digestion of wheat. Our aim was to identify T cell epitope-containing peptides after ex vivo digestion of ancestral (einkorn, spelt and emmer) and common (hexaploid) wheat (Fram, Bastian, Børsum and Mirakel) using human gastrointestinal juices. Wheat porridge was digested using a static ex vivo model. Peptides released after 240 min of digestion were analyzed by liquid chromatography coupled to high-resolution mass spectrometry (HPLC-ESI MS/MS). Ex vivo digestion released fewer T cell epitope-containing peptides from the ancestral wheat varieties (einkorn (n = 38), spelt (n = 45) and emmer (n = 68)) compared to the common wheat varieties (Fram (n = 72), Børsum (n = 99), Bastian (n = 155) and Mirakel (n = 144)). Neither the immunodominant 33mer and 25mer α-gliadin peptides, nor the 26mer γ-gliadin peptide, were found in any of the digested wheat types. In conclusion, human digestive juice was able to digest the 33mer and 25mer α-gliadin, and the 26mer γ-gliadin derived peptides, while their fragments still contained naive T cell reactive epitopes. Although ancestral wheat released fewer immunogenic peptides after human digestion ex vivo, they are still highly toxic to celiac patients. More general use of these ancient wheat variants may, nevertheless, reduce CeD incidence.

Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.

The aim of the present study was to utilize the protein from pigeon pea milling waste by-product for the preparation of protein isolate (PPI). The PPI was exposed to pepsin hydrolysis to reveal functional aspects like increased antioxidant capacity along with gelling ability. Complexation study demonstrated the use of PPI as an encapsulating material for water insoluble bioactive compounds (curcumin). Fluorescence spectroscopy revealed the formation of a complex through hydrophobic interaction. Ex vivo digestibility of the major proteins of PPI resulted in many peptides after digestion with human gastrointestinal enzymes. The peptide sequences identified may have potential biological activities, as revealed by bioinformatic data analysis. The presence of bioactive peptides and essential amino acids in the ex vivo digest suggests the role of PPI as a nutraceutical protein. The study provides a new value addition to pigeon pea milling waste by-product, a novel nutraceutical protein that can be used in functional food formulations.

This work aimed to study the opioid peptide β-casomorphin-7 (BCM7) degradation or stability during digestion using human gastrointestinal (GI) juices and porcine jejunal brush border membrane (BBM) peptidases. Synthetic BCM7 was subjected to in vitro digestion by GI fluids obtained from human volunteers for 180 min, and to downstream degradation with porcine BBM vesicles. The BCM7 was sampled at 4 time points over 24 h after BBM addition. The digests were profiled by HPLC-electrospray ionization mass spectrometry (ESI/MS) to monitor BCM7 during GI digestion, and intact BCM7 through BBM digestion was quantified by reverse-phase (RP)-HPLC. We found that BCM7 was partly digested with human GI enzymes, as 3 proteolytic fragments in addition to f(60-66) YPFPGPI were detected: f(62-66) FPGPI, f(60-65) YPFPGP and f(61-66) PFPGPI. The RP-HPLC analysis revealed that 42% of the initial peptide was degraded after only 2 h of BBM digestion, and as much as 79% was degraded after 4-h digestion with supplementation of BBM. In conclusion, this study showed that most of BCM7 was degraded during GI and BBM digestion, although a small amount (5%) was still detected after 24-h digestion. It remains to be studied whether the small amount of intact BCM7 detected after in vitro digestion is transported via active transceptors in the human intestinal epithelial cells and enters blood circulation.

The formation of malondialdehyde (MDA), 4-hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE), and 4-oxo-2-nonenal (ONE) in cod liver-,anchovy-, krill-, and algae oil during in vitro digestion with human gastrointestinal fluids was investigated. Adding rabbit gastric lipase, lipase inhibitor (orlistat) and tocopherols to cod liver oil, lipolysis and oxidation was also studied. Among the marine oils, the highest aldehyde levels (18 mu M MDA, 3 mu M HHE and 0.2 mu M HNE) were detected after digestion of cod liver oil, while the lowest levels were detected in krill and algae oils. Addition of rabbit gastric lipase significantly increased the release of HNE during the digestion. Orlistat significantly reduced lipolysis and MDA formation. Formation of MDA and HHE was delayed by tocopherols, the tocopherol mix Covi-ox (R) T 70 EU being more effective than pure alpha-tocopherol.

This study explored the influence of different emulsification layers as mono- and bilayers on lipid digestion by using in vitro and in vivo digestion methods. The monolayer emulsion of rapeseed oil contained whey proteins and the bilayer emulsion, whey proteins and carboxymethyl cellulose. The in vitro digestion using human gastrointestinal enzymes showed that the lipid digestion as free fatty acids was slowed down in the bilayer emulsion compared with the monolayer. Droplet size was still low in the gastric phase and pseudoplasticity was well preserved (even though viscosity decreased) during in vitro gastrointestinal digestion. The in vivo studies confirmed a lower fat bioavailability from bilayer emulsions by a reduction in the triglyceride level in the blood of rats, fed by the bilayer emulsion. The results clearly showed that lipid digestion was slower in the bilayer emulsion than in the monolayer. These results provide bio-relevant information about the behavior of emulsions upon digestion. PRACTICAL APPLICATION: The layer-by-layer production approach that was presented here allows the preparation of emulsions with slower fat bioavailability. Such behavior of the bilayer emulsion made it interesting for the formulation of food products with low fat bioavailability.