INFLUENCE OF NONENZYMATIC GLYCOSYLATION (GLYCATION) OF PEA PROTEINS (PISUM SATIVUM) ON THEIR SUSCEPTIBILITY TO ENZYMATIC HYDROLYSIS

The Maillard reaction, also referred to as nonenzymatically glycosylation (glycation), causes changes to protein structure and occurs in food. The aim of this study was to investigate changes in susceptibility of glycated pea proteins to enzymatic hydrolysis. A pea proteins extract was glycated using different sugars (glucose, fructose, lactose and glucosamine). Then, the physicochemical changes of proteins after glycation in ultraviolet spectra as well as the content of sugars and free amino groups were determined and also separated electrophoretically. Native and glycated pea proteins were hydrolyzed for 120 min with pepsin (one-step hydrolysis) or with pepsin and trypsin (in two-step process). Pea proteins glycated by glucose and lactose were less susceptible to pepsin hydrolysis than the pea proteins glycated by fructose and glucosamine in comparison with native pea proteins. Glycated pea proteins were more susceptible to hydrolysis by pepsin and trypsin in the two-step hydrolysis than the native ones.

PRACTICAL APPLICATION

Nonenzymatic glycosylation (glycation) of food proteins is used for the improvement of their functional properties. The formed glycoproteins can be characterized by changing biochemical properties, i.e., the susceptibility to the enzymatic hydrolysis or the influence on the growth and survival of the gut microflora. For this reason, investigations on the effect of the glycation of food proteins on their biological properties were necessary from the nutritional and health-promoting point of view.     

Antioxidant activities of enzymatic‐hydrolysed proteins of dromedary (Camelus dromedarius) colostrum

This work investigated the antioxidant activities of dromedary colostrum proteins before and after hydrolysis by pepsin, trypsin, α‐chymotrypsin, pancreatin and papain. The enzymatic hydrolysis affected the degrees of hydrolysis, electrophoretic profiles, molecular weight distribution and hydrophobic/hydrophilic properties of the generated peptides. The antioxidant activities were evaluated using four antioxidant assays, including 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) radical‐scavenging activities, ferric reducing power and ferrous ion chelating activity. Interestingly, the antioxidant activities of dromedary colostrum proteins were enhanced after enzymatic hydrolysis. The highest antioxidant potential was obtained by pancreatic hydrolysates (P ≤ 0.05). These results suggest that dromedary colostrum protein hydrolysates are an important source of natural antioxidant peptides.     

Sea buckthorn (Hippophae rhamnoides) proanthocyanidins inhibit in vitro enzymatic hydrolysis of protein

Abstract: Interactions of phenolics with other food constituents and digestive enzymes are likely to have interference with the digestion and bioavailability of food and phenolics. In this study the effect of sea buckthorn proanthocyanidins on in vitro digestion of protein was evaluated. Optimization of the extraction conditions showed that maximum recovery of sea buckthorn proanthocyanidins was obtained with acidified acetone; water mixture (60% to 70%, v/v). Crude proanthocyanidin extracts thus prepared were purified using sephadex gel column chromatography and their average degree of polymerization and the effects on enzymatic hydrolysis of bovine serum albumin as influenced by their protein precipitation capacities were studied. Average degree of polymerization of proanthocyanidins in berry pulp, kernel, seed coat, and leaves was 7.4, 5.6, 8.2, and 10.6, respectively. The EC50 values for the protein precipitation by the PA of berry pulp, kernel seed coat, and leaves were 44.2, 44.1, 65.8, and 39.8 μg, respectively. Relative enzymatic hydrolysis of the protein‐proanthocyanidin complexes was 44.1% to 60.3% for pepsin and 57.5% to 67.7% for trypsin. Interactions of sea buckthorn proanthocyanidins with food proteins and digestive enzymes might alter the protein digestibility and phenolic bioavailabilty.     

Antioxidant and nitric oxide inhibitory activities of tilapia (Oreochromis niloticus) protein hydrolysate: effect of ultrasonic pretreatment and ultrasonic‐assisted enzymatic hydrolysis

Ultrasound was incorporated to processing of fish protein hydrolysate to facilitate homogenate pretreatment and enzymatic hydrolysis of tilapia (Oreochromis niloticus) muscle protein. Their effects on Flavourzyme hydrolysis and biological activities of the tilapia hydrolysate were examined. The ultrasound‐assisted hydrolysis caused reduction in degree of hydrolysis ranging from 23% to 35% relative to that of the conventional process. The 70 W ultrasound‐assisted hydrolysis process increased DPPH radical‐scavenging activity and reducing power of tilapia hydrolysate prepared from the non‐pretreatment homogenate by 33% and 45%, respectively. All hydrolysates have no cytotoxicity on RAW264.7 cell lines at the maximum concentration of 20 mg protein mL^?1. The 70 W ultrasound pretreatment at 30 and 45 min combined with conventional hydrolysis is the suitable condition for producing tilapia hydrolysate with nitric oxide inhibitory and antioxidative activities on RAW264.7 cell lines, respectively. As a result, ultrasound could be applied to enzymatic protein hydrolysis either as pretreatment or during the hydrolysis.     

Effect of tannic acid on in vitro enzymatic hydrolysis of some protein sources

The pH‐stat system has been used to assess the effect of tannic acid (TA) on solubility and in vitro enzyme hydrolysis of different proteins. Added TA (from 10 to 50 g kg^?1) decreased the extent of hydrolysis of bovine serum albumin. Enzymic hydrolysis of casein, pea meal, soybean meal, and haemoglobin (HB) was increased, as measured by total amino acids released and by the degree of hydrolysis. SDS‐PAGE confirmed the results of the in vitro enzymatic hydrolysis. These findings suggest that, under in vitro conditions, when simulating the gastrointestinal environment of domestic mammals, the negative effects of TA described from in vivo experiments are not necessarily due to reduced hydrolysis of proteins. Copyright © 2003 Society of Chemical Industry     

Functional properties of protein hydrolysates from pea (Pisum sativum,L) seeds

The aim of this study was to investigate the effects of partial enzymatic hydrolysis on functional properties of two different pea protein isolates obtained from two pea genotypes, Maja and L1. Papain and commercial protease (Streptomyces griseus protease) were used for protein modification. Solubility, emulsifying and foaming properties were estimated at four different pH values (3.0, 5.0, 7.0 and 8.0). Papain increased solubility of L1 pea protein isolate at pH 3.0, 5.0 and 8.0, emulsifying properties and foaming capacity at all pH values. Otherwise, papain increased solubility of Maja pea protein isolate only at pH 8.0. This pea protein isolate modified with both enzymes formed emulsions with improved stability at lower pH (3.0, 5.0). The commercial protease‐prepared pea protein isolates showed generally low solubility and different emulsifying and foaming properties. Proper selection of enzyme, conditions of hydrolysis and genotypes could result in production of pea protein isolates with desirable functional properties.     

酶水解法提高大豆蛋白水解度的研究

用蛋白酶水解方法提高大豆分离蛋白水解度.通过对多种蛋白酶的对比分析可知,风味蛋白酶的水解效果好于其它蛋白酶,但正交试验结果表明,即使在优化条件下水解,单一的风味蛋白酶水解所得大豆分离蛋白水解度最高只达到43.12%.若先用胃蛋白酶水解再用风味蛋白酶水解则水解度最高可达68.81%,而风味蛋白酶与其它酶的联合应用效果略差;若先使用风味蛋白酶后使用胃酶则水解度只有47.89%.表明不同酶对大豆蛋白分子具有不同的水解特点.     

Hemoglobin hydrolysates from porcine blood obtained through enzymatic hydrolysis assisted by high hydrostatic pressure processing

The purpose of this work was to obtain discoloured hemoglobin (Hb) hydrolysates from porcine red blood cells fraction by combining enzymatic hydrolysis and high hydrostatic pressure (HHP) treatments. Samples of Hb were submitted to treatments of enzymatic hydrolysis by trypsin (EC 3.4.21.4) or pepsin (EC 3.4.23.1), under controlled conditions of temperature and pH, for 2, 6 and 24 h, and pressurization at 400 MPa, at 20 °C for 15 min, after the addition of the proteases. To evaluate the effects of the proteolysis reactions, the effectiveness of discolouration, the extent of hydrolysis as the percentage of non-protein nitrogen, and the SDS-PAGE electrophoretic profiles of the hydrolysates were determined. The results showed that HHP had a significant influence on enzymatic hydrolysis of Hb by trypsin, whereas did not improve the extent of proteolysis in the case of pepsin. The main conclusion derived from this study is that pressurization of Hb samples after the addition of trypsin enhanced enzymatic hydrolysis of the Hb, hence permitting a decrease in the incubation time.

Industrial relevance

The present study is focussed on assessing the effects of enzymatic hydrolysis with trypsin or pepsin assisted by HHP treatment on the discolouration of Hb from porcine blood. HHP treatment was able to increase the activity of trypsin and to enhance discolouration effectiveness of Hb with pepsin. The results from this work corroborate the potential of the application of HHP processing combined with enzymatic treatments on the development of food ingredients from porcine blood.     

STABILITY OF PEPSIN (EC 3.4.23.1) DURING IN VITRO PROTEIN DIGESTIBILITY ASSAY1,2

To maximize the efficiency of utilization of pepsin and estimate the contamination of pepsin for in vitro protein digestibility assays, the specific activity decay and peptide bond hydrolysis of pepsin incubated at different pH and concentration were studied with the bovine hemoglobin method and the o‐phthaldialdehyde method, respectively. It was found that increase of pH and concentration of pepsin increased pepsin's half‐life for both specific activity decay and peptide bond hydrolysis. The half‐life for specific activity decay was not extended by the presence of a substrate protein. The results indicated the time needed to maximize pepsin utilization depended on pH and the concentration of pepsin. At the time when all specific activity of pepsin was lost, the average size of pepsin autolysates was between 6.9 and 12.1 amino acid residues, suggesting most peptic protein would be fractionated as digestible protein.     

Impact of glycated pea proteins on the activity of free‐swimming and immobilised bacteria

BACKGROUND: Glycation (non‐enzymatic glycosylation), a spontaneously occurring process, is responsible for alteration of the structures and biological activities of proteins, making them highly active. Regrettably, information regarding the impact of glycated food proteins on intestinal bacteria still remains sparse. Pea seeds are considered to be a biological material of a high nutritional value, low content of anti‐nutritional substances and proven health‐promoting action and therefore they were used in this study. Since glycated pea proteins are proven to display a lowered susceptibility to the enzymatic digestion, their impact on the activity of both free‐swimming and immobilised bacteria was studied. RESULTS: In vitro model systems were used to prove the stimulatory impact of glycated pea proteins on the proliferation rate and survival, as well as on the metabolic activity of free‐swimming and immobilised bacteria. CONCLUSIONS: This phenomenon is of great importance because glycated food proteins are not only a source of nutrients and energy but also display new properties and increased biological activities. Additionally, they are able to modify the bacterial intestinal ecosystem, thus affecting the general health status of a consumer. Copyright © 2010 Society of Chemical Industry     

Effect of non-enzymatic glycosylation of pea albumins on their immunoreactive properties

The aim of this study was to determine the effect of non-enzymatic glycosylation of pea proteins on their immunoreactive properties. Extracted total pea albumins were glycated. No changes were found in molecular weight distribution of total pea albumins before and after glycation using size exclusion chromatography and SDS–PAGE methods. SDS–PAGE GLYCO test stained the glycated proteins and OPA method showed 15% progress in glycation. Glycated and unglycated pea albumins were orally and intraperitoneally administered to Balb/C mice. Serum specific IgG and IgA and sIgA were determined. No difference in serum specific IgG level was found after oral mice immunization with TA and GTA. In the presence of antigen SPL lymphocytes culture showed higher proliferation activity as compared to the culture without the antigen addition. The glycation does not change the immunoreactivity of proteins significantly. During the presented route of immunization with TA and GTA specific tolerance mechanism could be induced.     

Influence of enzymatic hydrolysis,pH and storage temperature on the emulsifying properties of canola protein isolate and hydrolysates

The aim of this work was to enhance emulsification properties of canola proteins through enzymatic proteolysis and pH variaton. Canola protein isolate (CPI) and hydrolysates (CPHs) were used to form emulsions at pH 4.0, 7.0 and 9.0 followed by storage at 4 or 25 °C for 7 days. Controlled enzymatic hydrolysis led to increased peptide bond cleavage with time (0.23 g/100 g in CPI to 7.18 g/100 g after 24‐h Alcalase hydrolysis). Generally, oil droplet sizes were smaller for emulsions made at pH 9.0, which suggest better quality than those made at pH 4.0 and 7.0. Trypsin hydrolysate emulsions were the most physically stable at pH 7.0 and 9.0; in contrast, the pepsin hydrolysate emulsions were unstable at all conditions. The results suggest that selective enzymatic hydrolysis could play an important role in enhancing successful incorporation of canola proteins and peptides into food systems as protein emulsifiers.     

乌鸡血酶解工艺条件的研究

以新鲜乌鸡血为主要原料,研究了乌鸡血的酶解工艺条件,结果表明对乌鸡血进行间歇式超声破碎处理,其效果比机械破碎效果更好;通过比较AS1.398中性蛋白酶、胃蛋白酶、木瓜蛋白酶、碱性蛋白酶对乌鸡血酶解作用能力大小,选取AS1.398中性蛋白酶作为酶解用酶;同时在单因素和正交实验基础上,确定了AS1.398中性蛋白酶的酶解最佳工艺条件为:酶解液初始pH7.5,酶加量为8000U/g,酶解温度为45℃,底物浓度为8%,酶解时间6h。此条件下的水解度达到22.22%,血红素含量为0.62mg/mL。      

Relationship between proportion and composition of albumins,and in vitro protein digestibility of raw and cooked pea seeds (Pisum sativum L.)

BACKGROUND: Peas provide an excellent plant protein resource for human diets, but their proteins are less readily digestible than animal proteins. To identify the relationship between composition and in vitro digestibility of pea protein, eight pea varieties with a wide range of protein content (157.3–272.7 g kg^?1) were determined for the proportion of albumins and globulins, their compositions using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and in vitro protein digestibility (IVPD) before and after heat treatment using a multi‐enzyme (trypsin, chymotrypsin and peptidase) method. RESULTS: The proportion of albumins based on total seed protein content decreased from 229 to 147 g kg^?1 as seed protein content increased from 157.3 to 272.7 g kg^?1, while the proportion of globulins increased from 483 to 590 g kg^?1. The IVPDs of eight raw pea seeds were 79.9–83.5%, with significant varietal variations, and those were improved to 85.9–86.8% by cooking. Albumins, including (pea albumins 2) PA2, trypsin inhibitor, lectin and lipoxygenase, were identified as proteolytic resistant proteins. Globulins were mostly digested by protease treatment after heating. CONCLUSION: The quantitative ratio of albumins and globulins, and the quantitative variations of albumin protein components, including lipoxygenase, PA2, lectins and trypsin inhibitors, appear to influence the protein digestibility of both raw and cooked pea seeds. Copyright © 2010 Society of Chemical Industry     

Biological activity of camel milk casein following enzymatic digestion

The aim of this study was to investigate the effects of enzymatic hydrolysis with digestive enzymes of camel whole casein and beta-casein (β-CN) on their antioxidant and Angiotensin Converting Enzyme (ACE)-inhibitory properties. Peptides in each hydrolysate were fractionated with ultra-filtration membranes. The antioxidant activity was determined using a Trolox equivalent antioxidant capacity (TEAC) scale. After enzymatic hydrolysis, both antioxidant and ACE-inhibitory activities of camel whole casein and camel β-CN were enhanced. Camel whole casein and β-CN showed significant ACE-inhibitory activities after hydrolysis with pepsin alone and after pepsinolysis followed by trypsinolysis and chymotrypsinolysis. Camel β-CN showed high antioxidant activity after hydrolysis with chymotrypsin. The results of this study suggest that when camel milk is consumed and digested, the produced peptides start to act as natural antioxidants and ACE-inhibitors.     

Influence of enzymatic treatment on the nutritional and functional properties of pea flour

The effect of enzymatic treatment on the nutritional value and functional properties of pea flour was investigated. Pea flour was hydrolyzed with acid protease from Aspergillus saitoi, to give two different hydrolyzed pea flours. This enzymatic treatment led to a significant (p < 0.05) decrease in crude and true protein and to an increase of free amino acids and non-protein nitrogen. The nutritional value decreased, but an increase in the avilability of protein was expected as result of lower trypsin inhibitor activity and phytic acid content in hydrolyzed pea flours. The amino acid profile of unhydrolyzed pea flour was slightly modified after enzymatic hydrolysis, increasing (significantly) the isoleucine, leucine, lysine, cystine, phenylalanine, threonine, alanine, arginine and aspartic acid contents as a result of the added enzyme. In addition, enzymatic treatment released hydrophobic amino acids, which significantly improved the protein solubility at acid pH, the oil absorption capacity and the emulsification capacity of pea flours. Protein solubility, foaming capacity, foam stability, water absorption capacity, gelation capacity and green colour decreased. It was thus confirmed that treatment with acid protease improves some functional properties of pea flour, but the effect on nutritional properties was unclear.     

Optimisation of enzymatic hydrolysis for concentration of squalene in palm fatty acid distillate

BACKGROUND: Squalene was concentrated from palm fatty acid distillate (PFAD) in this study using commercial immobilised Candida antarctica lipase (Novozyme 435^®). The PFAD was neutralised (NPFAD) using an alkali to liberate the free fatty acids and then hydrolysed at 65 ± 1 °C. The enzymatic hydrolysis on NPFAD was optimised using response surface methodology (RSM) before being neutralised again to obtain a concentrated squalene fraction. RESULTS: A five‐level, three‐factor central composite rotatable design was adopted to evaluate the effects of the enzymatic hydrolysis parameters reaction time (4–12 h), water content (50–70% w/w) and enzyme concentration (1.5–3.5% w/w) on the percentage yield of squalene concentration. The optimal reaction parameters for maximum yield of squalene concentration were identified from the respective contour plots. The optimal enzymatic hydrolysis conditions were a reaction time of 7.05 h, a water content of 61.40% w/w and an enzyme concentration of 2.23% w/w. CONCLUSION: RSM was used to determine the optimal conditions for enzymatic hydrolysis of NPFAD with C. antarctica lipase for maximum recovery of squalene which could be implemented on an industrial scale. Copyright © 2008 Society of Chemical Industry     

Acetylcholinesterase‐inhibitor hydrolysates obtained from ‘in vitro’ enzymatic hydrolysis of mannoproteins extracted from different strains of yeasts

In vitro inhibitory activity against acetylcholinesterase (AChE) of peptides obtained by enzymatic hydrolysis of mannoproteins extracted from strains of yeasts was investigated. Yeast mannoproteins were extracted from strains belonging to the genera Brettanomyces, Candida, Pichia and Saccharomyces isolated from dairy products. They were obtained by heat treatment in citrate buffer and purified by affinity chromatography with concanavalin A. Each purified extract was subsequently hydrolysed with proteolytic enzymes (trypsin, pepsin, chymotrypsin and proteinase K) applied individually or in combination, thus generating smaller peptides. Inhibitory activity of the latter against AChE was determined. The molecular weight of mannoproteins, determined by SDS‐PAGE, was between 6.5 and 30 kDa. As regards AChE inhibition, a preliminary screening of all hydrolysed extracts was performed, yielding variable results with 59% maximum inhibition. Subsequently, when inhibitory concentration 50 (IC50) was determined, the extracts showed higher inhibitory activity (between 6.75 and 12.3 mg mL^?1). Results showed that the mannoproteins separated from yeast strains of food origin generated bioactive peptides by enzymatic hydrolysis, which can be of interest to the manufacturing of food with potential functional properties.     

Changes in the carotenoid content of apricot (Prunus armeniaca,var Bergeron) during enzymatic browning: β‐carotene inhibition of chlorogenic acid degradation

Considering the numerous beneficial effects in human health ascribed to carotenoids, studies were performed to investigate the modification of carotenoid amount and composition during apricot enzymatic browning. First works on bruised apricot purees have shown a trans‐β‐carotene isomerisation (20%) induced by enzymatic browning. To clarify this isomerisation, oxidation of chlorogenic acid in presence of trans‐β‐carotene, catalysed by purified apricot polyphenoloxidase (PPO), was followed by HPLC and polarography. Isomerisation rate of trans‐β‐carotene in its cis isomer was found to increase with chlorogenic acid concentration. Moreover, trans‐β‐carotene was shown to be a potent inhibitor of phenol degradation. This inhibition was partially ascribed to PPO inhibition (non‐competitive inhibitor towards phenol with an apparent Ki close to 0.5 mM , a mixed type inhibitor towards oxygen with an apparent Ki close to 0.15 mM ). The additional inhibition was explained by non‐enzymatic reactions involving trans‐β‐carotene and chlorogenic acid o‐quinones and leading to phenol regeneration and carotene isomerisation. © 2000 Society of Chemical Industry     

Study on the self‐assembly property of type I collagen prepared from tilapia (Oreochromis niloticus) skin by different extraction methods

Type I collagen was prepared from tilapia (Oreochromis niloticus) skin by acetic acid and pepsin process at 4 °C, respectively (ASC and PSC), and hot‐water method separately at 25, 35 and 45 °C (C‐25, C‐35 and C‐45). Their structure and self‐assembly property were discussed. SDS‐PAGE patterns suggested that pepsin hydrolysis and the 35 and 45 °C extraction produced collagen with much reduced proportions of α‐ and β‐chains. Fourier transform infrared spectroscopy spectra revealed that pepsin hydrolysis did not change the conformation of collagen, but higher extraction temperature did. Self‐assembly curves and atomic force microscopy (AFM) observations showed that only ASC, PSC and C‐25 could self‐assemble into fibrils with D‐periodicity, but the reconstruction rate of C‐25 was lower. Besides, PSC had relatively higher resolution ratio compared with others. Overall, pepsin‐extracted collagen displayed higher solubility and better fibril‐forming capacity, having the potential of applying in biomaterials and food‐packaging materials.