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In a screening for proteolytically active lactic acid bacteria, three strains, Lactobacillus delbrueckii ssp. lactis 92202, Lactobacillus helveticus 92201, and Lactobacillus delbrueckii ssp. bulgaricus 92059, showed the highest activities following growth in milk. All three strains degraded α- and β-casein, but did not hydrolyse κ-casein. HPLC analysis of skim milk fermentation revealed increasing amounts of peptides after 5 and 10 h with Lb. d. ssp. bulgaricus 92059. Hydrolysates obtained with Lb. d. ssp. lactis 92202 and Lb. d. ssp. bulgaricus 92059 revealed the highest angiotensin-converting enzyme-inhibitory effect. The effect was dose dependent. Almost no effect (<10%) was seen for Lb. helveticus 92201. For Lb. d. ssp. bulgaricus 92059, maximal inhibition of approx. 65% was reached after 25 h of fermentation. In an in vitro assay measuring potential immunomodulation, hydrolysates of the three strains yielded anti-inflammatory activities in the presence of TNF-α. However, the effects were more pronounced at lower hydrolysate concentrations. In the absence of TNF-α, slight pro-inflammatory effects were observed. The hydrolysate of Lb. d. ssp. bulgaricus 92059, when purified by means of solid-phase extraction, exhibited pro-inflammatory activity. Sour whey containing Lb. d. ssp. bulgaricus 92059 cells showed pro-inflammatory activity while cell-free sour whey was clearly anti-inflammatory. In the purified hydrolysate, 20 different α- and β-casein (CN)-derived peptides could be identified by LC-MS. Most peptides originated from the central and C-terminal regions of β-casein. Peptide length was between 9 (β-CN(f 59–67)) and 22 amino acids (β-CN(f 117–138)).

The proteinase-encoding prtB gene of Lactobacillus ( Lb .) delbrueckii ( d. ) subsp. bulgaricus 92059 was cloned and sequenced. Two soluble, secreted, C-terminally His-tagged derivatives were constructed and expressed in Lactococcus lactis by means of the NICE® Expression System. In both obtained derivatives PrtBb and PrtB2, the C-terminal, cell wall-binding domain was deleted. In addition, in derivative PrtB2, the C-terminal part of the B domain was deleted and the signal sequence was replaced by a lactococcal export signal. The affinity-purified derivatives were both proteolytically active. Peptide hydrolysates produced from casein with each of the derivatives showed identical peptide composition, as determined by liquid chromatography–mass spectrometry. Comparison of the peptides generated to those generated with living Lb . d . subsp. bulgaricus 92059 cells (Kliche et al. Appl Microbiol Biotechnol 101:7621–7633, 2017 ) showed that β-casein was the casein fraction most susceptible to hydrolysis and that some significant differences were observed between the products obtained by either the derivatives or living Lb. d . subsp. bulgaricus 92059 cells. When tested for biological activity, the hydrolysate obtained with PrtBb showed 50% inhibition of angiotensin-converting enzyme at a concentration of 0.5 mg/ml and immunomodulation/anti-inflammation in an in vitro assay of TNF-α induced NFκB activation at concentrations of 5 and 2.5 mg/ml, respectively. The enzymatically obtained hydrolysate did not show any pro-inflammatory or cytotoxic activity.

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.

Several technologies are available for incorporating whey proteins into a cheese matrix. However, there is no valid analytical method available to determine the whey protein content in matured cheese, to date. Consequently, the aim of the present study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of individual whey proteins based on specific marker peptides (‘bottom-up’ proteomic approach). Therefore, the whey protein-enriched model of the Edam-type cheese was produced in a pilot plant and on an industrial scale. Tryptic hydrolysis experiments were performed to evaluate the suitability of identified potential marker peptides (PMPs) for α-lactalbumin (α-LA) and β-lactoglobulin (β-LG). Based on the findings, α-LA and β-LG appeared to be resistant to proteolytic degradation during six weeks of ripening and no influence on the PMP was observed. Good levels of linearity (R2 > 0.9714), repeatability (CVs < 5%), and recovery rate (80% to 120%) were determined for most PMPs. However, absolute quantification with external peptide and protein standards revealed differences in model cheese depending on the PMP, e.g., 0.50% ± 0.02% to 5.31% ± 0.25% for β-LG. As protein spiking prior to hydrolysis revealed differing digestion behavior of whey proteins, further studies are required to enable valid quantification in various cheese types.

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.

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 (n = 3, sample set I) and 1.33 ± 0.19 (n = 1, sample set II) were determined for the cheese samples containing 30% high-heated milk compared to the non-enriched samples. The relative β-LG contents in the cheese samples with 30% high-heated milk were calculated to be 4.35% ± 0.39% (sample set I) and 9.11% ± 0.29% (sample set II) using a one-point calibration. It can be concluded that the HPTLC-IS method used is a suitable tool for the analysis of whey protein accumulation in cheese, being therefore potentially directly applicable on an industrial scale. For more accurate quantification of the whey protein content in cheese, an enhanced calibration curve needs to be applied.