Purification and identification of antioxidant peptides from walnut (Juglans regia L.) protein hydrolysates

Walnut proteins were hydrolyzed separately using three different proteases to obtain antioxidant peptides. The antioxidant activities of the hydrolysates were measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay. Among hydrolysates, pepsin hydrolysate obtained by 3 h exhibited the highest antioxidant activities, which could also quench the hydroxyl radical, chelate ferrous ion, exhibit reducing power and inhibit the lipid peroxidation. Then, 3-h pepsin hydrolysates were purified sequentially by ultrafiltration, gel filtration and RP-HPLC. The sequence of the peptide with the highest antioxidative activity was identified to be Ala-Asp-Ala-Phe (423.23 Da) using RP-HPLC-ESI-MS, which was identified for the first time from walnut protein hydrolysates. Last, the inhibition of the peptide on lipid peroxidation was similar with that of reduced glutathione (GSH). These results indicate that the protein hydrolysates and/or its isolated peptides may be effectively used as food additives.     

Investigation of Botanical Origin,Phenolic Compounds,Carotenoids, and Antioxidant Properties of Monofloral and Multifloral Bee Bread

Bee bread is a unique natural product made by bees and good for human health. It has many bioactive molecules that can treat or prevent diseases. In this study, melissopalynological methods were used to examine five bee bread samples. Major plant sources found in bee bread were Lotus spp., Trifolium spp., and Xeranthemum spp., which are from the Fabaceae and Asteraceae families. Then, the amount of phenolic compounds and major carotenoids in bee bread (BB) samples were quantified. Gallic acid, caffeic acid, quercetin, and kaempferol were found in all BB samples, with β-carotene being the most abundant carotenoid in all but BB1. In addition, the total phenolic/flavonoid content and antioxidant activities of all BB samples were determined. Total flavonoid, total phenolic, DPPH⋅, and ABTS⋅⁺ values were varied between 5.6–10.00 mg GAE/g DW, 1.2–4.3 mg QE/g DW, 1.2–5.5 mg TEAC/g DW, and 2.6–15.4 mg TEAC/g DW, respectively.     

Total monoamine oxidase (MAO) inhibition by chestnut honey,pollen and propolis

Abstract

Monoamine oxidase (MAO) inhibitors are generally used in the treatment of depressive disorders and some neurodegenerative illnesses, such as Parkinson’s disease and Alzheimer’s disease. The aim of this preliminary study was to investigate the MAO [MAO (E.C.1.4.3.4)] inhibiting effect of various apitherapeutic products, such as chestnut honey, pollen and propolis. Extracts’ MAO inhibition was measured using peroxidase-linked spectrophotometric assay in enzyme isolated from rat liver microsomes, and the values are expressed as the inhibition concentration (IC₅₀) causing 50% inhibition of MAO. The antioxidant activity of the bee products was also determined in terms of total phenolic content (TPC) and ferric reducing/antioxidant power in aquatic extracts. All samples exhibited substantial inhibition of MAO, propolis having the highest. Inhibition was related to samples’ TPCs and antioxidant capacities. These results show that bee products possess a sedative effect and may be effective in protecting humans against depression and similar diseases.     

Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis

To utilize fish processing waste, tuna backbone protein was hydrolyzed using different proteases (alcalase, α-chymotrypsin, neutrase, papain, pepsin and trypsin) for production of antioxidant peptide. Antioxidant activities of hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR) spectrometer. Among hydrolysates, peptic hydrolysate exhibited the highest antioxidant activity compared to other hydrolysates. To identify antioxidant peptide, peptic hydrolysate was purified using consecutive chromatographic methods, and the antioxidant peptide was identified to be VKAGFAWTANQQLS (1519 Da) by Q-TOF ESI mass spectroscopy. The antioxidant activities of antioxidant peptide from tuna backbone protein (APTBP) was evaluated, and the results show that APTBP significantly inhibited lipid peroxidation in linoleic acid emulsion system and also quenched free radicals (DPPH, hydroxyl and superoxide) in a dose-dependent manner. Moreover, APTBP did not show any cytotoxic effect against MRC-5 and ECV304 cell lines.     

乳清分离蛋白酶解物的抗氧化活性研究

研究了乳清分离蛋白(WPI)酶解物对DPPH.、超氧阴离子自由基和羟基自由基的清除效果,同时用还原法研究了其抗氧化活性。结果表明,WPI酶解物在体外具有较强的抗氧化能力。木瓜蛋白酶酶解物和胰蛋白酶酶解物对DPPH.、超氧阴离子自由基、羟基自由基的清除能力和还原能力强于胰凝乳蛋白酶酶解物和胃蛋白酶酶解物。     

Purification of a novel peptide derived from Mytilus coruscus and in vitro/in vivo evaluation of its bioactive properties

Excess oxidant can promote inflammatory responses. Moreover, chronic inflammation accompanied by oxidative stress is connected various steps involved in many diseases. From the aspect, we investigated an antioxidant peptide to prevent inflammatory response against oxidant overexpression. To prepare the peptide, eight proteases were employed for enzymatic hydrolysis, and the antioxidant properties of the hydrolysates were investigated using free radical scavenging activity by electron spin resonance (ESR) spectrometry. Papain hydrolysates, which showed clearly superior free radical scavenging activity, were further purified using consecutive chromatographic methods. Finally, a novel antioxidant peptide was obtained, and the sequence was identified as Ser-Leu-Pro-Ile-Gly-Leu-Met-Ile-Ala-Met at N-terminal. Oral administration of the peptide to mice effectively inhibited malondialdehyde (MDA) levels in a thiobarbituric acid reactive substances (TBARS) assay, and we also confirmed the antioxidative enzyme activities in superoxide dismutase (SOD) and glutathione-s-transferase (GST) assays. This is the first report of an antioxidant peptide derived from the hydrolysate of Mytilus coruscus, and also these results suggest that the peptide possesses potent antioxidant activity, and potential to enhance anti-inflammatory response.     

Temporal changes of nutrient composition from pollen patty to bee bread with special emphasis on amino and fatty acids composition

Bee bread is prepared from pollen sources and salivary secretions by honey bee workers to serves the nutritional purpose of colony members. However, changes in nutrient composition occurring in bee bread from the pollen sources including pollen patty are poorly understood. We, therefore, examined the nutrient contents of pollen patty, used as source pollen feed of honey bee colony, and bee bread prepared by honey bee from this source at different time interval. Results revealed that amino acid content was increased from pollen patty to bee bread but fatty acid content was decreased. Essential amino acids like valine, lysine, and nonessential amino acids such as proline and alanine increased significantly over time. Among fatty acids, oleic acid, linoleic acid and linolenic acid decreased significantly from pollen patty to bee bread. Minerals contents in bee bread did not show any significant change during the study period. These changes could be presumably justified due to addition of nectar, honey, gland secretions and also microbial symbionts which remains as a task of further study.     

Antioxidant Peptides from the Protein Hydrolysates of <Emphasis Type="Italic">Conus betulinus</Emphasis>

The current study aims at the isolation and characterization of the peptides, believed to have antioxidant activity, from Conus betulinus by using different types of enzymes. The body and viscera of C. betulinus were treated with three enzymes viz. trypsin, pepsin and papain to obtain peptide hydrolysates. The activities of the hydrolysates were analyzed by DPPH and hydroxyl radical assay by using electron spin resonance (ESR) device. Active hydrolysates were purified using ion exchange chromatography followed by gel filtration chromatography. The activity of the separated fractions was analyzed by ESR; in which the result showed that trypsin hydrolysate of body (28.48 and 76.00%) and viscera (38.45 and 83.00%) respectively have high activity than the other hydrolysates. The HPLC result of purified fraction showed, presence of active amino acids viz., metheonine, cystine, histidine etc. This purified peptide has more antioxidant activity that could reduce the excess free radicals in body in order to prevent free radical induced diseases.     

Flowers morphology and nectar concentration determine the preferred food source of stingless bee,Heterotrigona itama

Heterotrigona itama is a stingless bee species from Meliponini tribe. The bee collects nectar, pollen and resin to produce honey, bee bread, and propolis. The bee is also known to visit and collect nectar from various types of flowers but there are limited studies on why this species of bee prefers to visit certain types of flowers. This study was conducted to identify the nectar concentration in selected flowers favoured by H. itama and the relationship between the bee and the morphology of the flowers. Nectar was obtained from different species of flowers and the concentrations were measured using a digital refractometer. The tube length of each flower species and the tongue length of the bees were also measured. The results revealed that flowers preferred by H. itama have high nectar concentrations. The tube lengths of the preferred flowers were between 2.0 and 4.0 mm, which is compatible with the tongue length of the bee. This study revealed that both nectar concentration and flower morphology are important factors for the bees in choosing their food sources. The results from this study will benefit the beekeepers in the identification of flowers that should be planted in their farms to improve stingless bee beekeeping activities. Understanding the relationship between the bees and their flower preferences could also help us to understand the importance of conserving both the bee colonies and the various species of flowering plants to ensure the sustainability of flora and fauna in the ecosystem.     

Functional,antioxidant and antibacterial properties of protein hydrolysates prepared from fish meat fermented by Bacillus subtilis A26

Composition, functional properties and in vitro antioxidant and antibacterial activities of protein hydrolysates prepared with a proteolytic bacterium, Bacillus subtilis A26, through fermentation of fish proteins were investigated. Fermented fish meat protein hydrolysates (FPHs) were prepared from sardinelle (SPH), zebra blenny (ZPH) goby (GPH) and ray (RPH). The protein content of freeze-dried FPHs ranged from 74.3% to 81%. All fermented hydrolysates had an excellent solubility and possessed interfacial properties. The antioxidant activities of FPHs were evaluated by different methods, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power assay, β-carotene bleaching and DNA nicking assay. All hydrolysates showed dose-dependent antioxidant activities. Further, FPHs exhibited antibacterial activity and SPH was the most effective, particularly against Gram positive bacteria.     

长木蜂的筑巢和采粉贮粮行为

【目的】通过研究长木蜂Xylocopa tranquebarorum筑巢和贮粮行为, 为进一步查明独栖性蜂类行为特点、开发新的蜂类资源提供依据。【方法】采用目测和拍照等方法对长木蜂的整个筑巢过程进行了连续观察, 用游标卡尺对巢口大小进行测量, 采用室内解剖巢室对长木蜂贮蜂粮的大小和数量进行观测。【结果】长木蜂主要在竹子上筑巢, 偶尔也发现在芦苇上筑巢。最喜欢选择竹节直径1.2～2.5 cm的孝顺竹Bambusa multiplex和刚竹属 Phyllostachys的竹种上筑巢。其筑巢过程为：雌蜂寻找合适的筑巢地点, 咬巢口, 清理巢室, 采集花粉蜜制作蜂粮, 在蜂粮上产卵, 制作巢室隔板。筑巢地点主要位于离旧巢1 m以内的位置。雌蜂啃咬巢口平均用时（292±29）min, 制作一块蜂粮需采集粉蜜22～40次, 采集粉蜜平均用时（17.31±0.52）min/次, 携粉蜜回巢滞留时间平均为（16.45±1.08）min/次;巢中卸落粉蜜平均用时为（15.29±1.03）min/次, 一生贮蜂粮平均6块左右;蜂粮近长方形, 长12～18 mm, 宽6～10 mm, 平均重量（0.7140±0.0269）g。【结论】长木蜂雌蜂不同个体之间筑巢行为相似, 而采集粉蜜的次数和贮蜂粮所用时间均有显著差异。     

Bacterial communities associated with honeybee food stores are correlated with land use

Microbial communities, associated with almost all metazoans, can be inherited from the environment. Although the honeybee (Apis mellifera L.) gut microbiome is well documented, studies of the gut focus on just a small component of the bee microbiome. Other key areas such as the comb, propolis, honey, and stored pollen (bee bread) are poorly understood. Furthermore, little is known about the relationship between the pollinator microbiome and its environment. Here we present a study of the bee bread microbiome and its relationship with land use. We estimated bacterial community composition using both Illumina MiSeq DNA sequencing and denaturing gradient gel electrophoresis (DGGE). Illumina was used to gain a deeper understanding of precise species diversity across samples. DGGE was used on a larger number of samples where the costs of MiSeq had become prohibitive and therefore allowed us to study a greater number of bee breads across broader geographical axes. The former demonstrates bee bread comprises, on average, 13 distinct bacterial phyla; Bacteroidetes, Firmicutes, Alpha‐proteobacteria, Beta‐proteobacteria, and Gamma‐proteobacteria were the five most abundant. The most common genera were Pseudomonas, Arsenophonus, Lactobacillus, Erwinia, and Acinetobacter. DGGE data show bacterial community composition and diversity varied spatially and temporally both within and between hives. Land use data were obtained from the 2007 Countryside Survey. Certain habitats, such as improved grasslands, are associated with low diversity bee breads, meaning that these environments may be poor sources of bee‐associated bacteria. Decreased bee bread bacterial diversity may result in reduced function within hives. Although the dispersal of microbes is ubiquitous, this study has demonstrated landscape‐level effects on microbial community composition.     

Phenolics from Chilean Bee Bread Exhibit Antioxidant and Antibacterial Properties: The First Prospective Study

Bee bread (BB) is a beehive product generated upon fermentation of pollen combined with flower nectar and glandular secretions. The potential application of BB is related to its nutritional and functional components, including phenolic compounds. This is the first prospective study on palynological parameters, phenolics, antioxidant, and antibacterial activity of Chilean bee bread in vitro. The tested material exhibited high levels of phenolics (1340±186 mg GAE/100 g BB) and showed antioxidant capacity as determined by the FRAP (51±2 μmol Trolox equivalent/g BB) and ORAC-FL (643±64 μmol Trolox equivalent/g BB) and antibacterial activity against Streptococcus pyogenes. Furthermore, the phenolic acids and flavonoids was determined using liquid chromatography-mass spectrometry, and the concentration was determined using liquid chromatography with diode array detection. Kaempferol, quercetin, ferulic acid, and rutin were the main phenolics found. This study demonstrates the bioactive potential of Chilean BB and supports the evidence that this bee product is a promising source of antioxidants and antimicrobial compounds.     

Multiobjective optimization of the antioxidant activities of horse mackerel hydrolysates produced with protease mixtures

Fish protein hydrolysates (FPH) from horse mackerel were produced by employing an enzyme mixture of subtilisin and trypsin. The antioxidant activity of fish hydrolysates (DPPH scavenging activity, Fe²⁺ chelating activity and Fe³⁺ reducing power) was modelled as a function of the operating conditions for the hydrolysis (i.e. protein concentration, temperature and composition of the enzyme mixture). The antioxidant activities showed different behavior depending on whether their controlling pathway was the transference of electrons/protons (i.e. DPPH scavenging activity and Fe³⁺ reducing power) or metal chelation. In the first case, the antioxidant activities increased with the decrease of substrate concentration and temperature when pure trypsin (DPPH scavenging activity) or a mixture of enzymes (Fe³⁺ reducing capacity) was employed. Contrarily, hydrolysates showed higher Fe²⁺ chelating activities at moderate concentration and high temperature (i.e. 5 g/L and 55 °C) employing solely subtilisin. The conflictive behavior among the antioxidant properties suggested using a multiobjective optimization technique. The ε-constraint method was chosen for this purpose. This approach allows determining the most adequate operational conditions for producing hydrolysate with a specific antioxidant profile which is the first approximation to the production of taylor-made antioxidant hydrolysates.     

Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame protein by gastrointestinal digestion

To extract antioxidant peptide from hoki frame protein hydrolysate (APHPH), we employed six proteases (pepsin, trypsin, papain, alpha-chymotrypsin, Alcalase and Neutrase) for enzymatic hydrolysis, and the antioxidant activities of their hydrolysates were investigated using both lipid peroxidation inhibition assay and free radical scavenging assay by electron spin resonance spin-trapping technique. Among hydrolysates, peptic hydrolysate, having the highest antioxidant activity, further separated into four groups using ultrafiltration membranes and purified consecutive chromatographic methods. Finally, the purified peptide had a molecular mass of 1801 Da, and amino acid sequence was identified as Glu-Ser-Thr-Val-Pro-Glu-Arg-Thr-His-Pro-Ala-Cys-Pro-Asp-Phe-Asn. APHPH inhibited lipid peroxidation higher than that of alpha-tocopherol as positive control and efficiently quenched different sources of free radical: 1,1-diphenyl-2-pycryl-hydrazyl (IC(50)=41.37 microM), hydroxyl (IC(50)=17.77 microM), peroxyl (IC(50)=18.99 microM) and superoxide radicals (IC(50)=172.10 microM). Furthermore, APHPH decreased t-butylhydroperoxide-induced cytotoxicity on human embryonic lung fibroblasts and efficiently protected free-radical-induced DNA damage.     

Purification and identification of antioxidant peptides from egg white protein hydrolysate

Egg white proteins were hydrolysed separately using five different proteases to obtain antioxidant peptides. The antioxidant activity of egg white protein hydrolysates was influenced by the time of hydrolysis and the type of enzyme. Of the various hydrolysates produced, papain hydrolysate obtained by 3-h hydrolysis (PEWPH) displayed the highest DPPH radical scavenging activity. PEWPH could also quench the superoxide anion and hydroxyl radicals, effectively inhibit lipid peroxidation and exhibit reducing power. Then, PEWPH was purified sequentially by ultrafiltration, gel filtration, RP-HPLC and two fractions with relatively strong antioxidant activity were subsequently subjected to LC-MS/MS for peptide sequence identification. The sequences of the two antioxidant peptides were identified to be Tyr-Leu-Gly-Ala-Lys (551.54?Da) and Gly-Gly-Leu-Glu-Pro-Ile-Asn-Phe-Gln (974.55?Da), and they were identified for the first time from food-derived protein hydrolysates. Last, the two purified peptides were synthesized and they showed 7.48- and 6.02-fold higher DPPH radical scavenging activity compared with the crude PEWPH, respectively. These results indicate that PEWPH and/or its isolated peptides may be useful ingredients in food and nutraceutical applications.     

长木蜂蜂粮酿制过程中pH值和花粉活力的测定

测定手采新鲜的芍药花粉、芍药长木蜂Xylocopa tranquebarorum（Swederus）花粉和不同酿制时间蜂粮样品的平均pH值,结果分别是6．19、5．92和4．05（40日龄蜂粮）。随着酿制时间的延长,蜂粮的pH值下降,2日龄蜂粮到3日龄蜂粮的pH值从5．57降至4．82,下降速率明显。经测定,紫藤长木蜂花粉和不同日龄蜂粮样品中紫藤花粉的纯度均在98％以上;芍药长木蜂花粉和不同日龄蜂粮样品中芍药花粉的纯度均在93％以上;测定紫藤花粉的长木蜂蜂粮和芍药花粉的长木蜂蜂粮中的花粉活力。结果表明,在1日龄蜂粮中花粉活力基本丧失,3日龄后均失去萌发力,并且不同的花粉蜂粮其花粉活力表现一致。     

Quantitative analysis of the relationship between structure and antioxidant activity of tripeptides

Peptides from enzymatic hydrolysates of food proteins exhibit significant antioxidant activity. Several studies have attempted to determine the factors contributing to the antioxidant activity of peptides; however, the physicochemical properties and factors essential for the antioxidant activity of peptides are still unclear. In this study, in order to clarify the factors important for peptide antioxidant activity based on the properties of component amino acids, 55 tripeptides were synthesized from 20 natural amino acids and their antioxidant activity was measured using the Trolox equivalent antioxidant capacity (TEAC) assay system. The tripeptides were divided into two data sets: a training set comprising 50 compounds and a validated set comprising five compounds. The structure‐activity relationship of the training set was then analyzed using classical quantitative structure‐activity relationship (QSAR) analysis. The study findings demonstrate that the presence of a cysteine residue at any position, an aromatic amino acid at the C‐terminus, higher hydrophobicity of the N‐terminal residue, and smaller HOMO‐LUMO energy gap of the middle residue can significantly enhance the antioxidant activity. The activities of the five validated compounds were predicted using the constructed QSAR model, and a good correlation between measured and predicted activities was observed. The information obtained from the QSAR model could be useful for effective production of antioxidant peptides from food proteins such as egg white proteins.     

Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw

In the present study, a peptide having antioxidant properties was isolated from bullfrog skin protein, Rana catesbeiana Shaw. Bullfrog skin protein was hydrolyzed using alcalase, neutrase, pepsin, papain, alpha-chymotrypsin and trypsin. Antioxidant activities of respective hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR) spectrometer. Among hydrolysates, alcalase derived hydrolysate exhibited the highest antioxidant activities than those of other enzyme hydrolysates. In order to purity a peptide having potent antioxidant properties, alcalase hydrolysate was separated using consecutive chromatographic methods on a Hiprep 16/10 DEAE FF anion exchange column, Superdex Peptide 10/300 GL gel filtration column and highan octadecylsilane (ODS) C18 reversed phase column. Finally, a potent antioxidative peptide was isolated and its sequence was identified to be LEELEEELEGCE (1487 Da) by Q-TOF ESI mass spectroscopy. This antioxidant peptide from bullfrog skin protein (APBSP) inhibited lipid peroxidation higher than that of alpha-tocopherol as positive control and efficiently quenched different sources of free radicals: DPPH radical (IC(50)=16.1 microM), hydroxyl radical (IC(50)=12.8 microM), superoxide radical (IC(50)=34.0 microM) and peroxyl radical (IC(50)=32.6 microM). Moreover, MTT assay showed that this peptide does not exert any cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5).     

Antioxidant peptides isolated from the marine rotifer,Brachionus rotundiformis

Protein derived from the rotifer Brachionus rotundiformis was hydrolyzed using different proteases (Alcalase, α-chymotrypsin, Neutrase, papain, pepsin and trypsin) for production of antioxidant peptide. Antioxidant activities of hydrolysates were evaluated using DPPH radical scavenging activity. Peptic hydrolysate exhibited the highest antioxidative activity compared to other hydrolysates. To identify antioxidant peptides, peptic hydrolysate was purified using consecutive chromatographic methods, and antioxidant peptides were identified to be Leu-Leu-Gly-Pro-Gly-Leu-Thr-Asn-His-Ala (1076 Da), and Asp-Leu-Gly-Leu-Gly-Leu-Pro-Gly-Ala-His (1033 Da) by Q-TOF ESI mass spectroscopy. EC₅₀ values of purified peptides were 189.8 and 167.7 μM, respectively. Antioxidant activities of peptides purified from the rotifer protein hydrolysate were evaluated, with results showing that peptides significantly quenched free radicals.