Whole-Genome Sequence of Lactococcus lactis Subsp. lactis LL16 Confirms Safety, Probiotic Potential, and Reveals Functional Traits.

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.

Effect of sprouting on the proteome of chickpea flour and on its digestibility by ex vivo gastro-duodenal digestion complemented with jejunal brush border membrane enzymes.

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.

Naturally Occurring Glycosidases in Milk from Native Cattle Breeds: Activity and Consequences on Free and Protein Bound-Glycans.

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 ß-galactosidase, N-acetyl-ß-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.

Ancestral Wheat Types Release Fewer Celiac Disease Related T Cell Epitopes than Common Wheat upon Ex Vivo Human Gastrointestinal Digestion.

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.

Ex vivo Digestion of Milk from Red Chittagong Cattle Focusing Proteolysis and Lipolysis.

Ex vivo digestion of proteins and fat in Red Chittagong Cattle milk from Bangladesh was carried out using human gastrointestinal enzymes. This was done to investigate the protein digestion in this bovine breed's milk with an especial focus on the degradation of the allergenic milk proteins; αs1-casein and ß-lactoglobulin and also to record the generation of peptides. Lipolysis of the milk fat and release of fatty acids were also under consideration. After 40 min of gastric digestion, all the αs-caseins were digested completely while ß-lactoglobulin remained intact. During 120 min of duodenal digestion ß-lactoglobulin was reduced, however, still some intact ß-lactoglobulin was observed. The highest number of peptides was identified from ß-casein and almost all the peptides from κ-casein and ß-lactoglobulin were identified from the gastric and duodenal samples, respectively. No lipolysis was observed in the gastric phase of digestion. After 120 min of duodenal digestion, milk fat showed 48% lipolysis. Medium (C10:0 to C16:0) and long (≥C17:0) chain fatty acids showed 6% to 19% less lipolysis than the short (C6:0 to C8:0) chain fatty acids. Among the unsaturated fatty acids C18:1∑others showed highest lipolysis (81%) which was more than three times of C18:2∑all and all other unsaturated fatty acids showed lipolysis ranging from 32% to 38%. The overall digestion of Bangladeshi Red Cattle milk was more or less similar to the digestion of Nordic bovine milk (Norwegian Red Cattle).

The impact of meal composition on the release of fatty acids from salmon during in vitro gastrointestinal digestion.

We hypothesize that the rate of release of lipids from salmon muscle during in vitro digestion is altered by additional meal components. In vitro digestion of salmon was performed using a mixture of porcine gastrointestinal enzymes and bile salts. Broccoli and barley were also added to the digestion simulating a meal. The extent of lipolysis was determined by measuring the release of fatty acids (FAs) during sampling at the simulated gastric phase endpoint (60 minutes) and 20, 40, 60, 80, 110 and 140 minutes simulated small intestinal phase, using solid phase extraction and GC-FID. Adding barley resulted in a lower overall release of FA from salmon, whereas broccoli caused an initial delay followed by increased release from 80-140 min when lipid digestion of salmon alone plateaued. The impact of broccoli and barley on the release of peptides and digesta viscosity were also measured. The effect of different components in the meal shown by this in vitro study suggests that it would be possible to make dietary changes affecting the lipolysis, further triggering specific responses in the gastrointestinal tract. However, these observations need to be validated in vivo, and the mechanisms need to be further examined.

Structural characteristic, pH and thermal stabilities of apo and holo forms of caprine and bovine lactoferrins.

Apo and holo forms of lactoferrin (LF) from caprine and bovine species have been characterized and compared with regard to the structural stability determined by thermal denaturation temperature values (T (m)), at pH 2.0-8.0. The bovine lactoferrin (bLF) showed highest thermal stability with a T (m) of 90 ± 1°C at pH 7.0 whereas caprine lactoferrin (cLF) showed a lower T (m) value 68 ± 1°C. The holo form was much more stable than the apo form for the bLF as compared to cLF. When pH was gradually reduced to 3.0, the T (m) values of both holo bLF and holo cLF were reduced showing T (m) values of 49 ± 1 and 40 ± 1°C, respectively. Both apo and holo forms of cLF and bLF were found to be most stable at pH 7.0. A significant loss in the iron content of both holo and apo forms of the cLF and bLF was observed when pH was decreased from 7.0 to 2.0. At the same time a gradual unfolding of the apo and holo forms of both cLF and bLF was shown by maximum exposure of hydrophobic regions at pH 3.0. This was supported with a loss in α-helix structure together with an increase in the content of unordered (aperiodic) structure, while ß structure seemed unchanged at all pH values. Since LF is used today as fortifier in many products, like infant formulas and exerts many biological functions in human, the structural changes, iron binding and release affected by pH and thermal denaturation temperature are important factors to be clarified for more than the bovine species.