Enhancement of antimicrobial activity of a lactoperoxidase system by carrot extract and β-carotene

Effect of carrot extract (CE) on the bactericidal activity of a bovine lactoperoxidase system (LPOS) was evaluated using Salmonella enteritidis (10⁶ cfu/ml). LPOS antimicrobial activity increased from 1.4 to 3.8 log units by addition of 20-fold diluted CE. β-Carotene, a major pigment of carrot, also induced the increase in the activity, indicating that β-carotene is one of the major enhancers of LPOS antimicrobial activity. Discolouration of β-carotene was observed by the co-localisation of LPOS with β-carotene. The discolouration was mainly caused by the oxidation of β-carotene through hypothiocyanate ion (OSCN^?), which is produced by LPOS. The storage test of LPOS in the presence of β-carotene indicated that oxidised β-carotene leads to the sustainable OSCN^? generation through an oxidised β-carotene/SCN^? redox cycling reaction. Concomitant use of LPOS and β-carotene or β-carotene-rich food components raises the possibility of development of an effective disinfectant.     

Effect of esterification with fatty acid of β-cryptoxanthin on its thermal stability and antioxidant activity by chemiluminescence method

β-Cryptoxanthin, a pro-vitamin A carotenoid of potential interest for health, was found in vegetables and fruits in both free and esterified forms. Stabilities of free β-cryptoxanthin (FCX) and its esters including β-cryptoxanthin laurate (CXL), β-cryptoxanthin myristate (CXM), and β-cryptoxanthin palmitate (CXP) against heat were studied by high performance liquid chromatography–mass spectrometry (HPLC–MS). All β-cryptoxanthin fatty acid esters were more stable against heat than FCX. The antioxidant activities of FCX and its esters on superoxide anion, hydroxide radical and singlet oxygen were investigated by the pyrogallol-luminol system, the CuSO₄–phenanthroline–Vc–H₂O₂ system, and OH–NaClO–H₂O₂ system, respectively, using chemiluminescence (CL) methods in vitro. The antioxidant activities of β-cryptoxanthin fatty acid esters were not significantly different in all methods, furthermore, no synergetic effects amongst them were obtained. These results suggest that esterification of OH groups with fatty acids might not affect antioxidant activity of β-cryptoxanthin but stabilize β-cryptoxanthin against heat degradation.     

Optimization of β-carotene production by a mutant of the lactose-positive yeast Rhodotorula acheniorum from whey ultrafiltrate

The present work investigated on carotenogenesis with high β-carotene content by a new isolated high-activity strain-producer Rhodotorula acheniorum mutant MRN in cheese whey ultrafiltrate. After a serial of UV, ethymethanesurfonate (EMS), and nitrosoguanidine (NTG) mutagenesis, a mutant named MRN of the red lactose-positive yeast strain R. acheniorum was obtained. Then, the effects of different growth medium factors on carotenoid production by this mutant at batch-scale level were identified and optimized by means of response surface methodology (RSM) in order to achieve high-level production of β-carotene. The optimum conditions required to achieve the highest level of β-carotene (262.12±1.01 mg/L) were determined as follows: whey ultrafiltrate (WU) lactose concentration 55 g/L, pH 5.85, ammonium sulfate concentration 3.5 g/L, temperature 23°C, and aeration rate 1.56 vvm. The medium optimization resulted in a 6.45-fold increase in volumetric production (262.12±1.01 mg/L) and a 4.62-fold increase in the cellular accumulation (10.69±0.19 mg/g) of β-carotene.     

Enhancement of the hydrolysis activity of β-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis

Thermostable β-galactosidase (BgaB) from Geobacillus stearothermophilus is characterized by its thermoactivity in the hydrolysis of lactose to produce lactose-free milk products. However, BgaB has limited activity toward lactose. We established a method for screening evolved mutants with high hydrolysis activity based on prediction of substrate binding sites. Seven amino acid residues were identified as candidates for substrate binding to galactose. To study the hydrolysis activity of these residues, we constructed mutants by site-saturation mutagenesis of these residue sites, and each variant was screened for its hydrolysis activity. The first round of mutagenesis showed that changes in amino acid residues of Arg109, Tyr272, and Glu351 resulted in altered hydrolysis activity, including greater activity toward ortho-nitrophenyl-β-d-galactopyranoside (oNPG). The mutants R109V and R109L displayed changes in the optimum pH from 7.0 to 6.5, and the mutant R109V/L displayed different substrate affinity and catalytic efficiency (k_cat/K_m). Mutant R109G showed complete loss of BgaB enzymatic activity, suggesting that Arg109 plays a significant role in maintaining hydrolysis activity. The optimum pH of mutant E351R increased from 7.0 to 7.5 and this mutant showed a prominent increase in catalytic efficiency with oNPG and lactose as substrates.     

Binding of curcumin to β-lactoglobulin and its effect on antioxidant characteristics of curcumin

The binding of curcumin (CCM) to bovine β-lactoglobulin (β-Lg) was investigated by Fourier transform infrared and fluorescence. The effect of binding on antioxidant activity of CCM was determined by using ABTS and hydroxyl radical scavenging capacity and total reducing ability. Our results showed that when CCM binds to β-Lg, it lead to a partial change in protein structure. In fact, CCM was bound respectively to two different sites of protein at pH 6.0 and 7.0 via hydrophobic interaction. CCM–β-Lg complex was formed by one molecule of protein combining with one molecule of CCM. Moreover, the average distance from one binding site to Trp residues in protein is similar with another. This result suggested that fluorescence resonance energy transfer cannot be used as unique method to study the characteristics of binding of ligands to proteins. The antioxidant activity of CCM might be improved by binding with β-Lg.     

Structure and antioxidant activity of β-lactoglobulin-glycoconjugates obtained by high-intensity-ultrasound-induced Maillard reaction in aqueous model systems under neutral conditions

Sonication is a new processing technology in the dairy industry. The aim of this study was to test glycation of β-lactoglobulin (BLG) in Maillard reaction (MR) induced by high-intensity ultrasound in aqueous solution under neutral conditions at 10–15 °C, which is not favourable for the MR. BLG was sonicated in the presence of glucose, galactose, lactose, fructose, ribose and arabinose. Formation of Maillard reaction products (MRPs) was monitored by mass spectrometry, spectrophotometry and fluorimetry. Ultrasound treatment resulted in formation of MRPs with all tested carbohydrates. Ribose induced the highest degree of modification resulting in 76% of BLG modified and an average of three anhydroribose units attached. Circular dichroism spectra analyses indicated only minor alterations in secondary and tertiary structures. MRP obtained by ultrasound exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity and possessed increased iron-chelating activity and reducing power. High-intensity ultrasound efficiently promotes BLG-glycoconjugates formation by MR in aqueous solutions under non-denaturing conditions.     

Enhancement of β-glucosidase activity on the cell-surface of sake yeast by disruption of SED1

We determined the genetic background that would result in a more optimal display of heterologously expressed β-glucosidase (BGL) on the cell surface of yeast Saccharomyces cerevisiae. Amongst a collection of 28 strains carrying deletions in genes for glycosylphosphatidyl inositol (GPI)-anchored proteins, the Δsed1 and Δtos6 strains had significantly higher BGL-activity whilst maintaining wild type growth. Absence of Sed1p, which might facilitate incorporation of anchored BGL on the cell-surface, could also influence the activity of BGL on the cell surface with the heterologous gene being placed under the control of the SED1 promoter. For the evaluation of its industrial applicability we tested this system in heterologous and homogenous SED1-disruptants of sake yeast, a diploid S. cerevisiae strain, in which either the SED1 ORF or the complete gene including the promoter was deleted by use of the high-efficiency loss of heterozygosity method. Evaluation of disruptants displaying BGL showed that deletion of the SED1 ORF enhanced BGL activity on the cell surface, while additional deletion of the SED1 promoter increased further BGL activity on the cell surface. Compared to heterozygous disruption, homozygous disruption resulted generally in a higher BGL activity. Thus, homozygous deletion of both SED1 gene and promoter resulted in the most efficient display of BGL reaching a 1.6-fold increase of BGL-activity compared to wild type.     

Determination of β-carotene content and vitamin A activity of vegetables by high-performance liquid chromatography and spectrophotometry

A fast and sensitive high-performance liquid chromatographic (HPLC) method for the determination of the provitamin A, β-carotene, in vegetables is described. After alkaline saponification and organic extraction β-carotene is separated from other carotenoids on a reversed-phase HPLC column and determined by measurement of its absorbance at 445 nm. The total amount of carotenoids is determined by measurement of the absorbance at 445 nm of the extract prior to HPLC.Results are presented of the analysis of β-carotene and total carotenoids in some vegetables bought at a local market in Dar es Salaam. The percentage β-carotene, of the total carotenoid content, showed a great variation. The vitamin A activity of the vegetables was calculated from the analytical results.     

LC-MS–MS characterisation of curry leaf flavonols and antioxidant activity

Curry leaf (Murraya koenegii) is a common flavouring agent in Indian foods. This study characterised the flavonol profile of curry leaf extracted with different solvents and the relative antioxidant capacity of these extracts by quantifying phenolic constituents. Flavonols were extracted using ethanol, methanol, or acetone prior to identification and quantification using liquid chromatography coupled to atmospheric pressure chemical ionisation (APCI) mass spectrometry in tandem mode (LC-MS–MS) with negative ion detection. Major curry leaf flavonols included myricetin-3-galactoside, quercetin-O-pentohexoside, quercetin-3-diglucoside, quercetin-3-O-rutinoside, quercetin-3-glucoside, quercetin-3-acetylhexoside, quercetin-O-xylo-pentoside, kaempferol-O-glucoside, and kaempferol-aglucoside. Lag-time and TBARS tests demonstrated that curry leaf phenolics prevent cupric-ion induced oxidation of LDL. The best extraction yield was obtained with 80% ethanol. Acetone extracts provided better antioxidant activity expressed as increased lag-time formation, than did ethanol or methanol extracts. Curry leaf is a rich source of flavonols that have biological activity in vitro and further studies are warranted in regards to the potential health benefits and identification of the novel flavonols whose identities remain unknown.     

Characteristics and antioxidant activity of ultrafiltrated Maillard reaction products from a casein–glucose model system

Maillard reaction products (MRPs) were prepared from casein–glucose by refluxing for 130 min at 102 °C and initial pH 12.0 without pH control to investigate the characteristics of casein–glucose Maillard reaction and the antioxidant activity difference among different fractions of MRPs. Browning and intermediate products increased, however, the pH of the system decreased with increase in the heating time. Free amino group content decreased 78% during first 10 min and did not change nearly thereafter. Amino acid analysis indicated that lysine and arginine decreased significantly, and casein was partially hydrolysed to peptides or free amino acid. High molecular weight compounds were dominant in the MRPs, determined by high performance gel-filtration chromatography. After ultrafiltration, antioxidant activity of each MRPs fraction was investigated by DPPH radical-scavenging activity, reducing power, Fe²⁺ chelating activity and lecithin oxidation assay. MRPs of different molecular weight exhibited distinctly different antioxidant activities.     

Pulsed high-pressure processing of barley-based non-dairy alternative milk: β-carotene retention,protein solubility and antioxidant activity

Emerging technologies have been investigated as smart process options for manufacturing plant-based non-dairy alternative milk. However, few studies have evaluated the effects of these non-conventional technologies on the preservation of bioactive and nutritional compounds. In this regard, this study aimed to examine the impact of pulsed high-pressure processing at non-thermal and thermal conditions on the quality parameters of barley-based non-dairy milk enriched with β-carotene. The effects of the pressure (100, 300, and 600 MPa), number of pulses (1, 2, and 3 at 100 and 300 MPa), and temperature (40 and 80 °C) on the soluble protein content, β-carotene retention, total phenolic content, and flavonoid content were assessed. Also, we examined the antioxidant capacity of the samples using in vitro assays. The samples were exposed to pressure for 2 min, but the total treatment time varied from 2.5 to 9 min, according to the pressure and pulse conditions. Higher pressure pulses at 100 MPa favored the homogenization of the system. The solubility of the barley proteins increased by increasing the number of pulses from 1 to 3 at 100 MPa. β-Carotene was thermally degraded up to 20% by the thermal treatments at 80 °C. Additionally, the treatments performed at 80 °C provided samples with lower phenolic and flavonoid contents. On the other hand, the rise in pressure up to 300 MPa promoted the disruption of the cell membranes of barley particles, favoring the extraction of bonded phenolic and flavonoid compounds. Regardless of the number of pulses, samples treated at 300 MPa and 40 °C presented the highest phenolic and flavonoid contents. Antioxidant capacity results presented similar behavior as observed for phenolic and flavonoid contents. Therefore, the different combinations of pressure and temperature promoted distinct effects on the protein solubility and bioactive compounds of the barley-based non-dairy milk.     

Statistical optimization,partial purification,and characterization of coffee pulp β-glucosidase and its application in ethanol production

Extracellular β-glucosidase was produced using coffee pulp as a sole carbon source by Penicillium verrucosum by solid state fermentation and 897.36±59 U/g enzyme activity was obtained. Increase in 2.21-fold of enzyme activity on optimizing the bioprocess parameters by response surface methodology based on central composite rotatable design is illustrated. Maximum production level of 1,991.17 U/g was obtained with optimum values of pH 4.2, moisture 66.8%, and fermentation duration of 56 h. The enzyme was partially purified and the enzyme activity was optimum at 50°C temperature and at pH 6. The metal ions such as Mg²⁺, Zn²⁺, Ca²⁺, K⁺, detergents, and chelator such as EDTA were effective and further increased the β-glucosidase activity. On application of β-glucosidase for simultaneous saccharifiation and fermentation, 3.3% ethanol was obtained. Thus, this study provides insight on exploitation of P. verrucosum for synthesis of of β-glucosidase using coffee pulp which is available abundantly in coffee processing industries.

     

Structure and antioxidant activity of Maillard reaction products from α-lactalbumin and β-lactoglobulin with ribose in an aqueous model system

Maillard reaction products (MRPs) were prepared from aqueous model mixtures containing 3% (w/w) ribose and 3% (w/w) of the dairy proteins α-lactalbumin (α-LA) or β-lactoglobulin (β-LG), heated at 95 °C, for up to 5 h. The pH of MRPs decreased significantly during heat treatment of α-LA-Ribose and β-LG-Ribose mixtures from 8.4 to 5.3. The amino group content in MRPs, derived from the α-LA-Ribose and β-LG-Ribose model system, was decreased noticeably during the first hour and did not change thereafter. The loss of free ribose in MRPs was higher for β-LG-Ribose than for α-LA-Ribose. During the Maillard reaction, the concentration of native and non-native α-LA, or β-LG, decreased and the formation of aggregates was observed. Fluorescence intensity of the β-LG-Ribose MRPs reached maximum within 1 h, compared to 2 h for α-LA-Ribose MRPs. Meanwhile, modification of the UV/vis absorption spectra for α-LA and β-LG was mainly due to a condensation reaction with ribose. Dynamic light scattering showed a significant increase in the particle size of the MRPs. Size exclusion chromatography of MRPs revealed the production of both high and low molecular weight material. Electrophoresis of MRPs indicated polymerization of α-LA and β-LG monomers via inter-molecular disulfide bridge, but also via other covelant bonds. MRPs from α-LA-Ribose and β-LG-Ribose exhibited increased antioxidant activities, therefore theses MRPs may be used as natural antioxidants in food products.     

Antioxidant activity of β-cyclodextrin inclusion complexes containing trans-cinnamaldehyde by DPPH,ABTS and FRAP

This study was carried out to observed β-cyclodextrin (β-CD) inclusion complexes containing trans-cinnamaldehyde (CIN) by using DPPH, ABTS and FRAP assay. Antioxidative ability was compared between pure CIN and β-CD-CIN inclusion complexes and particle size, encapsulation efficiency, and temperature-dependent release of inclusion complexes were investigated. High concentration of β-CD (1.8%) as well as guest oil 1:3 molar ratio (β-CD:CIN) influenced on particle size bigger during self-assembly process. And particle sizes were increased as storage period. In the antioxidant capacity results, pure β-CD (1.8%) was antioxidative without CIN especially at FRAP assay. Antioxidant activity dramatically increased after 1:1 molar ratio (1.8% β-CD:CIN), especially at DPPH assay and ABTS^•+ assay. In this study, β-CD complexation enhanced CIN solubility and affected increase the antioxidant activity of the CIN. Moreover, we need to consider that molar ratio of between β-CD concentration and CIN is effective manufacturing condition to improve antioxidant activity of β-CD-CIN inclusion complexes.     

Molecular structure of large-scale extracted β-glucan from barley and oat: Identification of a significantly changed block structure in a high β-glucan barley mutant

Health effects of β-glucan are typically related to dose, size and viscosity without taking the specific molecular structure into account. High β-glucan mutant barley, mother barley and oat β-glucans were large-scale extracted by comparable protocols using hot water, enzyme assisted hydrolysis and ethanol precipitation leading to similar molecular masses (200–300 kDa). Multivariate data analysis on all compositional, structural and functional features demonstrated that the main variance among the samples was primarily explained by block structural differences as determined by HPSEC–PAD. In particular the barley high β-glucan mutant proved to exhibit a unique block structure with DP3 and DP4 contributions of: 78.9% and 16.7% as compared to the barley mother (72.1% and 21.4%) and oat (66.1% and 29.1%). This unique block structure was further confirmed by the ¹H NMR determination of the β-1,4 to β-1,3 linkage ratio. Low solubility of the barley samples was potentially an effect of substructures consisting of longer repetitive cellotriosyl sequences. FT-Raman and NMR spectroscopy were useful in measuring sample impurities of α-glucans and prediction of β-linkage characteristics.     

Digestibility and immunoreactivity of soybean β-conglycinin and its deglycosylated form

IgE-mediated allergy to soybean may be connected to an incomplete protein digestion causing an inappropriate immune response in the gut. The aim of this study was to gain insight on the digestibility and immunoreactivity of β-conglycinin and its deglycosylated form. To achieve this goal both proteins were digested mimicking gastrointestinal physiological conditions. Digests were characterised by SDS–PAGE, MALDI-TOF-MS and immunological assays. Data on IgG binding western blot provided key information related to the composition of the digests indicating that whereas deglycosylated β-conglycinin was completely removed by gastrointestinal digestion, α and β subunits of β-conglycinin glycoprotein partially survive the process of digestion. Such differential digestive behaviour might be influenced by glycan moieties, at least to some extent, and should be an additional factor to be considered on its potential allergenicity. Results also support the existence of linear epitopes in the three subunits (α, α′, and β) of both protein forms.