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The purpose of this research was to find the best experimental conditions for glycosylation of lysozyme and casein with dextran, and to investigate the effect of glycosylation on the functional properties of these proteins. Glycosylation was performed by allowing proteins to react with dextran under Maillard reaction conditions. The extent of glycosylation was determined by sugar analysis, SDS-PAGE, gel filtration and cation-exchange chromatography. Glycosylation of lysozyme with a 1:5 weight ratio of protein to dextran, held at 60 • C for one week under a relative humidity of 79% resulted in coupling of 3.0 mole dextran to one mole lysozyme. In the case of casein, at 60 • C, 48 h and 79% relative humidity, 0.1 and 0.13 mole dextran was attached to one mole of casein when a weight ratio of protein to dextran of 1:5 and 1:7.5 was used, respectively. A decrease in the degree of glycosylation occurred when samples were incubated for 72 h. Enzymatic activity of glycosylated lysozyme was reduced by 20% compared with unmodified lysozyme (P < 0.05). Both proteins exhibited improved solubility at different pH values (3, 7 and 9), different temperatures (25, 40 and 60 • C) and increased heat stability, with a better emulsion activity and emulsion stability than with un-modified proteins (P < 0.05). These changes might increase the applicability of lysozyme as a natural antimicrobial and casein as a protein ingredient in different food systems.

Kluyveromyces marxianus protein hydrolysates were prepared by two different sonicated-enzymatic (trypsin and chymotrypsin) hydrolysis treatments to obtain antioxidant and ACE-inhibitory peptides. Trypsin and chymotrypsin hydrolysates obtained by 5 h, exhibited the highest antioxidant and ACE-inhibitory activities. After fractionation using ultrafiltration and reverse phase high performance liquid chromatography (RP-HPLC) techniques, two new peptides were identified. One fragment (LL-9, MW = 1180 Da) with the amino acid sequence of Leu-Pro-Glu-Ser-Val-His-Leu-Asp-Lys showed significant ACE inhibitory activity (IC = 22.88 μM) while another peptide fragment (VL-9, MW = 1118 Da) with the amino acid sequence of Val-Leu-Ser-Thr-Ser-Phe-Pro-Pro-Lys showed the highest antioxidant and ACE inhibitory properties (IC = 15.20 μM, 5568 μM TE/mg protein). The molecular docking studies revealed that the ACE inhibitory activities of VL-9 is due to interaction with the S2 (His513, His353, Glu281) and S'1 (Glu162) pockets of ACE and LL-9 can fit perfectly into the S1 (Thr345) and S2 (Tyr520, Lys511, Gln281) pockets of ACE.

Available online A B S T R A C TYeast protein hydrolysate may be considered as a good source of bioactive peptides. Yeast hydrolysate was prepared by two different physical-enzymatic and autolysis treatments to identify the most active angiotensin I-converting enzyme (ACE) inhibitory and antioxidant peptides. The most active hydrolysate was obtained after sonication-trypsin hydrolysis. The hydrolysate was subjected to fractionation by ultrafiltration. Fraction with molecular weight of <3 kDa exhibited the highest activity. Reverse phase high performance liquid chromatography (RP-HPLC) resolved this fraction into five fractions, one of which (fraction F3) with amino acid sequence of Tyr-Gly-Lys-Pro-Val-Ala-Val-Pro-Ala-Arg (MW:1057.45 Da) exhibited ACE inhibitory (IC50 = 0.42 ± 0.02 mg/ml) and antioxidant activities (26.25 ± 0.13 µM TE/µg protein). Taken together, the results of this study show that S. cerevisiae proteins contain specific peptides in their sequences which can be released by enzymatic hydrolysis. These peptides have excellent bioactive properties that can potentially replace the antioxidant and antihypertensive agents with chemical origin.

Enzymatic hydrolysis has been widely applied for production of protein hydrolysate from shrimp waste and for purification of chitin. In the present study, shrimp (P. semisulcatus) head waste was hydrolysed, using a commercial proteolytic enzyme, Alcalase. In order to improve protein extraction efficiency, certain chemicals such as sodium sulphite and Triton x-100 were used along with the enzyme. When Alcalase (12 AU/kg) used alone, the yield of protein extraction was 45.1% and by using Triton x-100 (0.01 g/kg) together with Alcalase, the yield was decreased to 39%, whereas the presence of sodium sulphite (200 mmol/L) with the enzyme or with the enzyme and Triton x-100 increased the level of protein extraction to 62% and 65.1%, respectively. The resulting protein powder contained sufficient amounts of essential amino acids to be used in feed formulations. By precipitating proteins from the resulting protein extract at pH 3.1, the residual sulphite in protein powder was decreased by 97% and thus the powder can be considered suitable for animal and/or aquaculture feed formulations.

In recent years much attention and interest have been directed toward application of natural antimicrobial agents in foods. Some naturally occurring proteins such as lactoperoxidase, lactoferrin, and lysozyme have received considerable attention and are being considered as potential antimicrobial agents in foods. Lysozyme kills bacteria by hydrolyzing the peptidoglycan layer of the cell wall of certain bacterial species, hence its application as a natural antimicrobial agent has been suggested. However, limitations in the action of lysozyme against only Gram-positive bacteria have prompted scientists to extend the antimicrobial effects of lysozyme by several types of chemical modifications. During the last 2 decades extensive research has been directed toward modification of lysozyme in order to improve its antimicrobial properties. This review will report on the latest information available on lysozyme modifications and examine the applicability of the modified lysozymes in controlling growth of Gram-positive and Gram-negative bacteria in foods. The results of modifications of lysozyme using its conjugation with different small molecule, polysaccharides, as well as modifications using proteolytic enzymes will be reviewed. These types of modifications have not only increased the functional properties of lysozyme (such as solubility and heat stability) but also extended the antimicrobial activity of lysozyme. Many examples will be given to show that modification can decrease the count of Gram-negative bacteria in bacterial culture and in foods by as much as 5 log CFU/mL and in some cases essentially eliminated Escherichia coli. In conclusion this review demonstrates that modified lysozymes are excellent natural food preservatives, which can be used in food industry.

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The purposes of this research were to glycosylate lysozyme with dextran under Maillard reaction conditions and assess the antimicrobial characteristics of the lysozyme-dextran conjugate in a culture medium and cheese curd. Solutions containing lysozyme and dextran were incubated at 60 degrees C and at 79% relative humidity. Gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to follow the glycosylation process. Under optimum conditions 3.7 mol of dextran were coupled to 1 mol of lysozyme. Lytic activity of the conjugate against the cell wall of Micrococcus luteus was about 62% of that of native lysozyme. Evaluation of the lysozyme-dextran conjugate against test microorganisms (Staphylococcus aureus and Escherichia coli) in culture media indicated a progressive increase in antimicrobial activity, with an increase in enzyme-conjugate concentration. The lysozyme-dextran conjugate was also effective against E. coli in a natural food system, as it reduced the bacterial count by 3 log in cheese curd after 40 days of storage. Unlike E. coli, the antimicrobial action of lysozyme against S. aureus was not improved by conjugation with dextran in both in vitro and in vivo tests. Antimicrobial activity of the lysozyme-dextran conjugate against gram-negative bacteria is probably related to the remaining lytic activity as well as the excellent surfactant properties of the lysozyme-dextran conjugate. These results might increase the applicability of lysozyme as a natural antimicrobial ingredient in different food products.