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Summary Background FGF-21 is described as a powerful metabolic regulator with beneficial effects including glucose-lowering and improvement of insulin sensitivity without hypoglycaemia. On the other hand, FGF-21 is activated when muscle and other tissues are stressed by external effects or internal cellular pathogens that lead to shortcomings in metabolic balance. Previous results suggested that FGF-21 could be a promising target to develop future metabolic therapeutics. Purpose The present study was performed to gain deeper insight into the regulation of FGF-21 by protein metabolism in obese human subjects. Methods FGF-21 serum concentrations were measured in a cohort of n = 246 obese humans ± type 2 diabetes mellitus (T2DM) (median age 53.0 [46.0; 60.0] years and BMI 40.43 [35.11; 47.24] kg/m2) and related to the nutritional protein intake. In addition, the effect of a novel oligopeptide purified from a β-casein hydrolysate on FGF-21 was examined in vitro in liver cells and in vivo in a human intervention study with the main focus on metabolic inflammation including 40 mainly obese subjects (mean age 41.08 ± 9.76 years, mean BMI 38.29 ± 9.4 kg/m2) in a randomized 20 weeks double-blind cross-over design. Main findings In the cohort analysis, FGF-21 serum concentrations were significant lower with higher protein intake in obese subjects without T2DM but not in obese subjects with T2DM. Furthermore, relative methionine intake was inversely related to FGF-21. While global protein intake in obesity was inversely associated with FGF-21, incubation of HepG2 cells with a β-casein oligopeptide increased FGF-21 expression in vitro. This stimulatory effect was also present in vivo, since in the clinical intervention study treatment of obese subjects with the β-casein oligopeptide for 8 weeks significantly increased FGF-21 serum levels from W0 = 23.86 pg/mL to W8 = 30.54 pg/mL (p Conclusion While the total nutritional protein intake is inversely associated with FGF-21 serum levels, a purified and well characterised oligopeptide is able to induce FGF-21 serum levels in humans. These findings suggest a differential role of various components of protein metabolism on FGF-21, rather than this factor being solely a sensor of total nutritional protein intake.

Whey protein-enriched cheese can be produced by means of a high-temperature treatment of a part of the cheese milk. In this way, the nutritional quality of the resulting cheeses can be increased while resources are conserved. High-performance thin-layer chromatography-immunostaining (HPTLC-IS) using specific β-lactoglobulin (β-LG) antibodies was applied to study the implementation and stability of β-LG in two different sample sets of whey protein-enriched Edam model cheeses, including industrial-scale ones. Two methods were compared for the extraction of the proteins/peptides from the cheese samples. By applying tryptic hydrolysis directly from a suspended cheese sample instead of a supernatant of a centrifuged suspension, a better yield was obtained for the extraction of β-LG. When applying this method, it was found that selected epitopes in the tryptic β-LG peptides remain stable over the ripening period of the cheese. For four of the tryptic β-LG peptides detected by immunostaining, the amino acid sequence was identified using MALDI-TOF-MS/MS. One of the peptides identified was the semi-tryptic peptide VYVEELKPTP. A linear relationship was found between the content of this peptide in cheese and the proportion of high-heated milk in the cheese milk. β-LG enrichment factors of 1.72 (

Bioactive milk oligosaccharide concentrates are not yet available as functional food ingredients. The aim of the present study was to develop an optimized nanofiltration process for the enrichment of milk oligosaccharides by achieving a better permeation of milk salts and residual sugars during nanofiltration in an acidic (pH 5) or neutral (pH 7) environment. A bovine retentate produced by nanofiltration with a 100-fold increase in the milk oligosaccharide content in relation to total sugar content was applied. In addition, the process should be suitable for the enrichment of milk oligosaccharides in caprine milk. Milk oligosaccharides were identified by high performance anion exchange chromatography (HPAEC). Generally, a greater enrichment of milk oligosaccharides in the final nanofiltration retentate (14 % milk oligosaccharides by dry mass, 4-fold higher than in the first nanofiltration retentate, 140-fold higher than in the raw material) and a better separation of salts and residual sugars were achieved in bovine milk by nanofiltration at pH 5. The high milk oligosaccharide content in relation to the total sugar content in the final nanofiltration retentate (92 %, 10-fold higher than in the first nanofiltration retentate, 900-fold higher than in the starting sample) indicated a nearly complete permeation of monosaccharides and disaccharides. Nanofiltration of caprine milk resulted in a 31-fold higher milk oligosaccharide content in relation to total sugar content than in the starting sample. In the present work, the influence of the pH on the degree of enrichment of milk oligosaccharides by nanofiltration was evaluated for the first time.

Oligosaccharides in milk (MOS) have been reported in the literature to exert various bioactivities and to modulate the immune system. However, processes to obtain milk oligosaccharides on industrial scale as food ingredients are currently not available. Therefore, the aim of the study on hand was the evaluation of different nanofiltration (NF) membranes for the enrichment of MOS from bovine milk. Moreover, a transfer of the NF process from laboratory to pilot plant and industrial scale was performed. The immunostimulatory effect of the MOS concentrates was studied by the activity of NFκB in human embryotic kidney cells (HEKnfκbRE-cells). NF was carried out with lactose hydrolyzed skimmed and ultrafiltered milk permeate by application of different membranes. The quantification of MOS was determined by high-pH anion-exchange chromatography with pulsed amperometric detection and parallel online electrospray ion trap mass spectrometry. The enrichment of MOS (3-sialyl-lactose, 6-sialyl-lactose, N-acetylgalactosaminyl-lactose) on laboratory, pilot plant and industrial scale was achieved by the retention of these oligosaccharides of at least 50 % in NF retentate. The content of MOS in relation to total sugar content in the retentate from NF on industrial scale was 100-fold higher than in the initial sample. The MOS retentates and the standards (3-sialyl-lactose, 6-sialyl-lactose) exhibited increased NFκB activity in HEKnfκbRE-cells. Although there exist a few studies about the enrichment of MOS by NF, this is the first report about a screening of the efficiency of different NF membranes for the enrichment of MOS on different technological scales of production.

BACKGROUND The aim of the present study was to generate and identify potential anti-inflammatory peptides from bovine β-casein with enzyme preparations from cod and hog. Furthermore, the potential of cod trypsin, derived from fishery by-products, to produce these bioactive peptides for replacement of non-food-grade tosyl phenylalanyl chloromethyl ketone (TPCK)-treated porcine trypsin enzyme preparation was evaluated. RESULTS Potential anti-inflammatory peptides were obtained by hydrolysis of β-casein with the tryptic enzyme preparations cod trypsin, porcine trypsin (TPCK-treated) and a porcine trypsin and chymotrypsin preparation (PTN 6.0 S). Proteolysates generated with enzyme preparations containing mainly chymotryptic activity (Cryotin, Cryotin F) did not exhibit any effect. CONCLUSION The more chymotryptic enzyme activity is present, the lower is the potential anti-inflammatory activity of the hydrolysates in HEKnfκb-RE cells. Comparable peptides were produced by application of porcine trypsin (TPCK) and cod trypsin. Therefore, the enzyme preparation cod trypsin can replace the non-food-grade porcine enzyme preparation trypsin (TPCK) for the generation of potential anti-inflammatory peptides from β-casein. © 2015 Society of Chemical Industry

Galacto-oligosaccharides (GOS) were synthesised by enzymatic transgalactosylation in UF-skimmed milk permeate fortified with lactose (40% w/w) using β-galactosidases (β-GAL) derived from Aspergillus aculeatus, Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. GOS yields were assessed arithmetically after photometric determination of the sugar profile. GOS composition was characterised by HPAEC-PAD/MS analysis. The GOS yields, expressed as a percentage of the initial lactose content, were 41%, 21%, 13% and 11% with β-GALs from B. circulans, A. oryzae, A. aculeatus and K. lactis, respectively, under optimal conditions. Thus, the B. circulans enzyme revealed the highest affinity to transgalactosylation relative to hydrolysis. HPAEC-PAD/MS results showed similarities in GOS composition, especially with β-GAL from A. oryzae, A. aculeatus and K. lactis with a preference towards β-galactosyl residues β(1-6). GOS generated are mainly di-, tri- and a few tetra-saccharides. Only the B. circulans β-GAL formed above all penta-saccharides, mainly via β1-4 glycosidic linkages.

Several bioactive peptides are encrypted within the sequence of major milk proteins, requiring enzymatic proteolysis for release and activation. The present study aimed at the identification of potential anti-inflammatory activities in tryptic hydrolysates of bovine β-casein. Inflammatory processes involve in most cases an activation of Nuclear factor Kappa-light-chain enhancer of activated B cells (NFκB), which is a pro-inflammatory transcription factor of several genes. Hence, a NFκB reporter cell line was established, and TNF-α mediated activation of NFκB was used as a measurement. Bovine β-casein (β-CN) was hydrolysed by trypsin and fractionated by ultrafiltration. Total proteolysate as well as the fraction containing peptides between 1 and 5 kDa showed an inhibitory effect in the cell-based assay, while the fraction containing molecules smaller than 1 kDa did not. This anti-inflammatory effect was ascribed to a group of large, hydrophobic peptides, which were identified using LC–MS. The main peptide was synthesised and showed a significant anti-inflammatory effect in HEK nfkb-RE -cells. Thus, for the first time, a casein-derived peptide having an anti-inflammatory effect in vitro has been identified.

Summary The potential of β-galactosidase (β-GAL) and glucose isomerase (GLI) to enhance the sweetening power of lactose and to generate galactooligosaccharides (GOS) and lactulose was studied. UF-permeates of skim milk, sweet whey, acid whey and lactose solutions were incubated with β-GAL and GLI. Lactose hydrolysis (LH) was 96–99%, glucose isomerisation (GI) about 53%. On a scale from 0 to 5, the intensity of sweetness increased from 1 to 3. The bi-enzymatic process was also applied to yoghurt manufacture. Because of the inhibiting effect of calcium on GLI, the incubation was performed in UF-permeates of skim milk. LH in yoghurt was 94%, whereas GI was 36% on average. Whey syneresis in yoghurt decreased from 12% to 5%. The sweetness increased from 1.4 to 2.9 on average. Applying the bi-enzymatic system to 40% lactose solutions led to synthesis of about 20% prebiotic GOS. Products, such as 6-galactobiose, allolactose and 6-galactosyllactose were identified. About 1% lactulose was generated. Lactulose-derived oligosaccharides were not found.

Thirty milk samples [high-temperature short-time (HTST) milk, extended shelf life (ESL) milk (directly heated, indirectly heated, microfiltered), ultra-high temperature milk] from 17 German dairies were analysed. Total viable counts of directly or indirectly heated ESL milk were significantly lower than those in microfiltered ESL and HTST milk. Evaluation of indigenous enzyme activity revealed sufficient heat treatments in all milk samples. The manufacturing processes were differentiated by estimating furosine and acid soluble whey proteins. Sensory examinations revealed a preference for HTST heated and microfiltered ESL milk. However, a significant discrimination of drinking milk types was not possible. Vitamin losses were not detected, and concentrations of vitamins in different types of milk were comparable.

This study compared the effects of isochrone heating with different temperature–time combinations on the residual activities of alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and lactoperoxidase (LPO) in bovine (Holstein Friesian Cow), ovine (East Friesian Dairy Sheep) and caprine (German Improved Fawn) milk. Averages of 774, 1413 and 67 U/l ALP were determined in raw milk from cows, sheep and goats, respectively. The GGT values averaged 4143, 1871 and 603 U/l and the LPO activities averaged 2015, 2796 and 5190 U/l. After a holder pasteurisation (62 °C for 30 min to 65 °C for 32 min), the ALP activity in milk from the three mammals was

Extended shelf life (ESL) milk was processed with integrated microfiltration (pore size 1.4 µm). The germ-enriched retentate was not used for the final whole milk. Microfiltration led only to a negligible change in the content of the main components of the ESL product compared with the source milk. The total protein was only slightly decreased (0.02–0.03%) and the ratio of the protein fractions was unchanged within the measurement accuracy. The furosine content of the isolated fat globuline membrane fraction could be used as a diagnostic to prove cream had been subjected to high-temperature treatment. The shelf life of the ESL milk was distinctly prolonged compared to HTST-pasteurized milk.

The aim of the present study was to generate and identify potential anti-inflammatory peptides from bovine β-casein with enzyme preparations from cod and hog. Furthermore, the potential of cod trypsin, derived from fishery by-products, to produce these bioactive peptides for replacement of non-food-grade tosyl phenylalanyl chloromethyl ketone (TPCK)-treated porcine trypsin enzyme preparation was evaluated.Potential anti-inflammatory peptides were obtained by hydrolysis of β-casein with the tryptic enzyme preparations cod trypsin, porcine trypsin (TPCK-treated) and a porcine trypsin and chymotrypsin preparation (PTN 6.0 S). Proteolysates generated with enzyme preparations containing mainly chymotryptic activity (Cryotin, Cryotin F) did not exhibit any effect.The more chymotryptic enzyme activity is present, the lower is the potential anti-inflammatory activity of the hydrolysates in HEK(nfκb-RE) cells. Comparable peptides were produced by application of porcine trypsin (TPCK) and cod trypsin. Therefore, the enzyme preparation cod trypsin can replace the non-food-grade porcine enzyme preparation trypsin (TPCK) for the generation of potential anti-inflammatory peptides from β-casein.

The paper describes studies on the influence of heat impact in reconstituted skim milk on chemical and functional properties of yoghurt products. Reconstituted skim milk was heated for 20 min at 85 degrees C, 90 degrees C, or 95 degrees C. Ropy (producing exopolysaccharides, EPS) or non-ropy strains of S. thermophilus and L. delbrueckii subsp. bulgaricus were used as starter culture for yoghurt manufacture. The studies have shown that the fermentation times decreased with increasing heat impact when the ropy starter culture was used, while they remained to a far extent unchanged if the non-ropy starter culture was applied. The lactic acid contents of the yoghurt products were in the same range when the milk was heated at 85 degrees C or 90 degrees C, while they were different when milk was heated at 95 degrees C. There was a tendency visible that an increase in preheating leads to increased L(+)- and decreased D(-)-lactic acid contents if the non-ropy culture was applied. Using the ropy culture, it was vice versa. A slightly decrease in proteolysis with increasing heat impact was to be noted with both starter cultures. Concerning the relation of proteolysis to acidification, the fermentation process could be subdivided into three sections with different slopes if the non-ropy starter culture was used, while a linear relation was found if the ropy starter culture was applied. Regarding final product characteristics it was found that the functional properties of yoghurt decreased with increasing heat impact when the ropy starter culture was applied, while they remained to a far extent unchanged when the non-ropy starter culture was used. It can be concluded from these studies that a preheating of milk at a temperature of 85 degrees C (20 min) is optimal in regard to final yoghurt product characteristics.

Till now, industrial fractionation and isolation processes for whey proteins have mainly been based on chemical process steps combined with membrane techniques. Actually, the reaction kinetics of thermal denaturation of α-lactalbumin and β-lactoglobulin allow to compute the remain- ing portions of native whey proteins in dependence of the selected temperature-time conditions. In the temperature range of 85-95 °C and corresponding holding times, the differences in reaction rates for both whey protein fractions are highest i.e. only moderate denaturation for α-lactalbumin but extensive denaturation for β-lactoglobulin. After such thermal treatment the ratio of native α-lactal- bumin to native β-lactoglobulin may increase from 0.3 (raw milk) to approximately 4. An effective separation of aggregated (denatured) whey proteins from soluble whey proteins is achieved by means of microfiltration (ceramic membranes / 0.1 μm).

During the heat treatment of protein-containing foods, the amino acid lysine is most prone to undergo chemical reactions in the course of amino acid cross-linking or Maillard reactions. Among the reaction products formed, lysinoalanine (LAL), N(epsilon)-fructoselysine (FL) and N(epsilon)-carboxymethyllysine (CML) are those which serve as sensitive markers for the heat treatment applied. From a nutritional perspective, these compounds are ingested with the diet in considerable amounts but information about their metabolic transit and putative in vivo effects is scarce. In the present study, casein-linked LAL, FL and CML were administered to rats in two different doses for 10 days. Quantitation of LAL, FL and CML in plasma, tissue and faeces samples revealed that the kidneys are the predominant sites of accumulation and excretion. The maximum percent of dietary LAL, FL and CML excreted in the urine was 5.6, 5.2 and 29%, whereas the respective recoveries in the kidneys were 0.02, 26 and 1.4%. The plasma and tissue analyses revealed that the endogenous load of either compound is increased by its dietary intake. But the dose-dependent utilisation of dietary protein-linked LAL, FL and CML in rats has been demonstrated for the first time to vary substantially from each other.