The Effects of a Novel Series of KTTKS Analogues on Cytotoxicity and Proteolytic Activity

KTTKS is a matrikine that originates from the proteolytic hydrolysis of collagen. This peptide stimulates ECM production and types I and III collagen expression in vitro. A more stable form of KTTKS is pal-KTTKS, known as Matrixyl® or palmitoyl pentapeptide-3. A series of novel pentapeptides, analogues of KTTKS with the general formula X-KTTKS-OH(NH2), where X = acetyl, lipoyl, palmitoyl residues, was designed and synthesized. Their effect on amidolytic activity of urokinase, thrombin, trypsin, plasmin, t-PA, and kallikrein were tested. Cytotoxic tests on fibroblasts, as well as collagen and DNA biosynthesis tests for selected peptides, were also carried out. The test results showed that the most active plasmin inhibitors were palmitoyl peptides, whether in acid or amide form. No biological effects of lysine modification to arginine in the synthesized peptides were found. None of the synthesized peptides was not cytotoxic on fibroblasts, and three of them showed cell growth. These three compounds showed no concentration-activity relationship in the collagen and DNA biosynthesis assays.

Optimization for continuous overflow proteolytic hydrolysis of spent brewer’s yeast by using proteases

A large amount of spent yeast as by-product is annually generated from brewing industry and it contains about 50-55% protein with good balance of amino acids. The hydrolysate produced from spent brewer’s yeast may be used in food application. The yield of proteolylic hydrolysis for spent brewer’s yeast and amino acid contents of hydrolysates depend on factors such as temperature, pH value, type of used enzyme and ratio enzyme/substrate, time. Besides, applied hydrolysing methods (batch-, or continuous method) has effected on degree of hydrolysis. With the purpose of how proteolytic hydrolysis having effects on the spent brewer’s yeast for food application in industrial scale, continuous overflow method was used in this study. Bitterness of hydrolysate and the yield of continuous overflow proteolytic hydrolysis process are the two interested factors for protein hydrolysis. In this report, it is dealt with determination for optimal conditions to obtain the highest yield of hydrolysis process and the lowest bitterness of hydrolysate. Response surface methodology (RSM) was used to determine optimal condition for continuous overflow proteolytic hydrolysis of spent brewer’s yeast. The optimal conditions for obtaining high degree of hydrolysis and low bitterness are determined as followings: ratio of enzyme mixture (alcalase 7.5 U/g and flavourzyme 10 U/g), pH at 7.5, hydrolysis temperature at 51oC and hydrolysis time of 9 hours. Under the optimal conditions, the yield of hydrolysis was 59.62% ± 0.027 and the bitterness equivalently with concentration of quinine was 7.86 ± 0.033 μmol /ml.

OPTIMIZATION FOR BATCH PROTEOLYTIC HYDROLYSIS OF SPENT BREWER’S YEAST BY USING PROTEASES

The yield of proteolylic hydrolysis for spent brewer’s yeast by protease and aminoacid contents of hydrolysates (the main factors influencing in taste of hydrolysed product) depends on factors influencing in catalytic activities of enzymes as temperature, pH value, type of used enzymes and ratio enzyme/substrate. With the purpose to hydrolyse the spent brewer’s yeast for food application, bitterness of hydrolysate takes the firth consideration, and than the yield of hydrolysing process plays economic role. In this paper, it is dealt with determination of optimal conditions to obtain the highest yield of hydrolysis process and the lowest bitterness of hydrolysate (the bitterness is determined by sensory evaluation, expressed equivalently with concentration of quinine). Response surface methodology (RSM) was used to determine optimum condition for batch proteolytic hydrolysis of spent brewer’s yeast. The influencing factors were investigated as temperature (X1): 40 oC–60 oC; pH (X2): 6.0–9.0, ratio E (flavourzyme)/S (X3): 5–10 U/g and hydrolysis time (X4): 6–9 hours. The experimental responses including degree of hydrolysis (Y1) (%) and bitterness of hydrolysate (Y2) (μmol quinine/ml) are performed in second-degree model. The optimal conditions for obtaining high degree of hydrolysis and low bitterness are determined: Ratio of enzyme mixture (alcalase 7.5 U/g and flavourzyme 8.5 U/g), pH 7.5, hydrolysis temperature at 52oC and hydrolysis time 9 hours. Under the optimal conditions, the actual values obtained for the yield of hydrolysis was 40.81 ± 0.044 % and the bitterness equivalently with concentration of quinine was 16.37 ± 0.03 μmol quinine/ml.

Optimisation of indirect competitive ELISAs of a-, b-, and k-caseins for the recognition of thermal and proteolytic treatment of milk and milk products

Polyclonal antibodies were raised against six immunogens (three native and three thermally treated casein fractions: a+b-casein, k-casein and whole casein). Using these antibodies the procedures of an indirect competitive enzyme immunoassay (ELISA) were constructed, optimised and characterised for determination of each immunogen. It was found that ELISA of caseins is very specific without any interferences of whey proteins and with proportionally low cross-interactions between caseins. Detection limits for a-, b-, and k-caseins and whole casein were 110, 49, 58 and 505 ng/ml, respectively. The coefficient of variation was lower than 12% in intra-assay and lower than 9% in inter-assay. The developed ELISA format was used to study changes in casein immunoreactivity during heat-treatment and proteolytic hydrolysis. Heating below 100°C did not change the milk immunoreactivity but heating above 100°C caused its significant changes. Depending on type of proteolytic treatment (with enzyme preparation Pancreatin or with microbial cultures of Lactobacillus helveticus and Lactococcus) a decrease and an increase in casein immunoreactivity were observed. While Pancreatin reduced the casein immunoreactivity substantially (5–1000 times in dependence on the casein type), the more gentle proteolysis by bacteria caused not only its reduction (even 100 times at k-casein) but also its increase (1.5 times at a-casein).