Studies On The Preparation Of Low-phenylalanine Whey Hydrolysate And Its Regulation On Bone Formation

Phenylketonuria(PKU)is an amino acid metabolism disorder which due to a lack of enzyme required for the metabolism of phenylalanine(Phe).Amino acids-based lowPhe diets decrease the blood Phe of PKU patients,while it has limited effect on attenuating the imbalanced bone metabolism and bone loss,which are frequently occurred in PKU.Whey protein is a high-quality milk protein,and it has been widely used in infant formulas.The objectives of this study were to prepare a low-Phe whey hydrolysate(LPH)with osteoblastogenesis,and to identify osteogenic peptides from LPH.The main findings are as follows:(1)The preparation of LPH using enzymatic hydrolysis and resin adsorption.Thermolysin(T)combined with Protease A2SD(A2)released 94.13% Phe from whey protein concentrate(WPC);the D101 macroporous resin absorption further removed98.38% Phe from the TA2 hydrolysate(TA2H).The Phe content of LPH was 1.38 ±0.11 mg/g protein equivalent.(2)LPH is a peptide-based substance with abundant small peptides and mild flavor.The protein content of LPH is 60.41 g/100 g;the contents of branch chain amino acids and essential amino acids of LPH are 18.49 and 29.81 g/100 g,respectively.The percentage of peptides with molecular weight between 180?1500 Da in LPH was96.52%,while the increased short peptides also increased the intensity of bitterness and astringency.However,aroma-active compounds in TA2 H with lipid and rancid flavor were decreased after adsorption,contributing to a mild flavor of LPH.(3)TA2H and LPH promoted the osteoblastogenesis of MC3T3-E1 cells in a dosedependent manner.100 to 1000 ?g/m L TA2 H and LPH promoted cell proliferation,differentiation(enhanced activity of alkaline phosphatase(ALP)and expression of type-I collagen and osteocalcin),and mineralization(increased calcium deposits).The expression of a key transcription factor RUNX2 was also enhanced by LPH and TA2 H.Meanwhile,an increased expression ratio of osteoprotegerin(OPG)/receptor activator of nuclear factor kappa-B ligand(RANKL)suggested the potential of whey hydrolysates in inhibiting bone resorption.(4)p38/MAPK-Runx2 signaling pathway was responsible for LPH stimulated osteoblast differentiation.LPH activated the phosphorylation of p44/42 and Akt within1 h,while its activation on p38 was lasted for 24 h.Furthermore,co-treatment with a selective p38 inhibitor SB203580 decreased LPH stimulated RUNX2 phosphorylation as well as the increased activity of ALP.(4)Oral administration of LPH attenuated bone loss and imbalanced bone metabolism in ovariectomized(OVX)mice.OVX mice feed with different diets containing WPC,TA2 H,and LPH for 12 weeks did not affect body weights and uterus weights significantly.Whey samples improved bone mineral density and microarchitecture of distal femur,improved femoral ash content,improved serum calcium and phosphorus,improved bone morphological structure,increased femoral maximal load.LPH regulated bone metabolism by inhibiting the activation of ALP,bone resorption markers(TRACP5b,ICTP,and RANKL),and inflammatory markers(TNF-? and IL-6).(5)Peptide sequences of VIESPPEINT and SKVLPVPQK in LPH had strong osteogenic differentiation ability in MC3T3-E1 osteoblast cells.LPH was fractionated by HPLC and the resultant 16 fractions were examined for ALP activity in MC3T3-E1.The most active fraction LPH-F9 was further sequenced using LC-MS.A total of 15 peptides of Bos taurus were identified in F9.According to the peak area,7 peptides were selected and further chemically synthesized.Two LPH peptides showed the strongest ability in promoting osteogenic differentiation in MC3T3-E1 osteoblast cells.In summary,LPH with small peptides and mild flavor was successfully prepared using enzymatic hydrolysis and resin adsorption,indicating its use as a suitable protein substitute to make special diets for PKU patients.LPH,along with WPC and TA2 H,all showed osteoanabolic activities in MC3T3-E1 cells and ovariectomized mice.We also identified the peptides in LPH with capacity on promoting osteoblast differentiation.