Water Dilution Method Research Articles

Growth-Inhibitory Effect of Chicken Egg Yolk Polyclonal Antibodies (IgY) on Zoonotic Pathogens Campylobacter jejuni, Salmonella spp. and Escherichia coli, In Vitro

The overuse of antibiotics in animal husbandry has driven the search for alternative strategies to combat zoonotic pathogens. Foodborne zoonotic diseases caused by pathogenic bacteria pose a significant threat to human health, and therefore food safety should be a priority. This study investigates the in vitro inhibitory effects of chicken egg yolk immunoglobulin Y (IgY) on the growth and viability of three major foodborne pathogens: Campylobacter jejuni, Salmonella spp., and Escherichia coli. IgY was isolated from immunized hen egg yolks using a modified water dilution method, and its antigen-specificity confirmed through agglutination assays. Growth inhibition was evaluated across multiple doses and time points, revealing a dose-dependent bacteriostatic effect against all tested pathogens. A single dose of IgY (0.5 mg/mL) significantly reduced C. jejuni counts by up to 7 log, while repeated doses were required for Salmonella spp. and E. coli. These findings highlight egg yolk immunoglobulin’s potential as a source of sustainable, effective, ethical, readily available, and inexpensive antibiotic substitutes in livestock management. Future research will focus on validating these results in vivo and exploring large-scale production of IgY for practical application in animal healthcare.

Abstract P201: Adherence to Time-Restricted Eating is Related to Long-Term Weight Changes Among Women

Introduction: Chronic disruption of circadian rhythms is linked to weight gain and metabolic dysregulation. Time-restricted eating (TRE), a form of intermittent fasting, has shown effectiveness in improving short-term weight loss and energy homeostasis. However, associations between habitual adherence to TRE and long-term weight change remain understudied. Hypothesis: We tested whether adherence to TRE assessed by the gold-standard seven-day dietary records (7DDRs) was related to 5-year change in body weight among women. Methods: The present analysis included 650 women (mean [SD]: age 63 [9] y; BMI 26.4 [5.3] kg/m 2 ) without cardiovascular disease at baseline who completed 7DDRs in a sub-study of the Nurses’ Health Studies (NHS/NHSII), the Women’s Lifestyle Validation Study (WLVS) (2010-12). TRE was indicated by 4-12 hours of daily eating window (EW). The adherence to TRE was assessed by summing up the TRE days in a week. We also calculated 7-day averaged values of EW hours, last/first time of eating occasion (EO), and within-person variability of these variables. Weight change from baseline to a follow-up survey (NHS: 2016-18; NHSII: 2015-17) were analyzed. Total energy expenditure (TEE) and physical activity expenditure (PAEE) were measured at baseline using the doubly labeled water dilution method. Results: Longer averaged hours of EW were related to greater weight gain (β 0.7 [0.3] kg per 2 hours) after adjusting for covariates of demographic factors, total energy intake, physical activity, alcohol, within-person variation of EW, the averaged time of last EO, and the initial body weight ( p =0.02). Consistently, adherence to TRE was associated with less weight gain (β -0.25 [SE 0.1] kg per day increment; p =0.03) in a model adjusting for these covariates. We found that lower adherence to TRE and longer hours of EW were associated with weight gain ( p <0.05 for both) when the last eating time occurred at a later time in the day than at an earlier time. The baseline energy metabolism (TEE and PAEE) modified the associations between the hours of EW and weight changes ( P interaction-EW-TEE = 0.006; P interaction-EW-PAEE =0.01), showing significant relationships particularly among women with higher levels of adiposity and greater energy expenditure at baseline. Conclusions: Adherence to TRE and fewer habitual hours of eating were related to long-term weight changes among middle-aged and elderly women. The last eating time and energy homeostasis may partly modify the associations.

Production of anti-tetanus toxin IgY and study of its protective effects in a mouse model

ABSTRACT Tetanus is an acute and often fatal infectious disease caused by Clostridium tetani. Tetanus toxin (TT) is responsible for spastic paralysis observed in tetanus. Anti-tetanus antibodies obtained from horses and humans are the most antitoxins used for tetanus treatment, although some clinical side effects and disadvantages have been reported in their application. The aim of this study is the production of anti-TT IgY and evaluation of its protective effects in a mouse model. Anti-TT IgY was purified from the egg yolk using PEG6000 precipitation and water dilution methods, and its purity was verified by SDS-PAGE. Finally, the potency of purified anti-TT IgY in neutralizing the lethal effects of TT was studied in vivo using a mouse model. PEG6000 precipitation method had better results. Animal studies showed that the purified IgY neutralized the toxic effects of 100 MLD of TT and multiple intravenous-dose injections of anti-TT IgY also had a continuous effect of TT neutralization. The purified anti-TT IgY was effective in neutralizing the lethal activity of TT in a mouse model. Our results suggested that IgY could be an alternative therapeutic source for the management of tetanus in the future. Abbreviations Anti-TT, Anti-tetanus toxin; ELISA, Enzyme-linked immunosorbent assay; IgY, Immunoglobulin Y; MLD, Minimum lethal dose; PBS, Phosphate buffer solution; PEG, Polyethylene glycol; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; TIG, Tetanus immune globulin; TT, Tetanus toxin; WD, Water dilution; RT, Room temperature.

A new approach of immunotherapy against Crotalus snakes envenoming: ostrich (Struthio camelus) egg yolk antibodies (IgY-technology)

Crotalid envenomation is a neglected collective health problem involving many countries in America, which need secure and inexpensive snake anti-venom treatments. Here, high antibody titers (IgY) were raised in the Ostrich (Struthio camelus) egg yolk by immunizing with the venom of Venezuelan venomous Crotalus snakes. Ostriches were immunized with a pool of venoms from common rattlesnake (Crotalus durissus cumanensis), Uracoan rattlesnake (Crotalus vegrandis), Guayana rattlesnake (Crotalus durissus ruruima) and black rattlesnake (Crotalus pifanorum). The anti-snake venom antibodies were prepared from egg yolk by the water dilution method, enriched by the addition of caprylic acid (CA) and precipitation with ammonium sulfate at 30% (W/V). The purity and molecular mass of the final product was satisfactory, yielding a single ∼ 175 kDa band in SDS-PAGE gels ran under non-reducing conditions. In the immunoblot analysis, specific binding of the antivenom was observed with most venom proteins. The LD50 was 16.5 g/mouse (825 µg/kg body weight). High titers of IgY against Crot/pool venom were shown by ELISA. The median effective dose (ED50) was 19.66 mg/2LD50. IgY antibodies neutralized efficiently the Crot/pool venom lethality. As far as we know, this is the first anti-snake venom produced in ostriches, which could make this technology an affordable alternative for low-income countries, since it is likely to produce about 2-4 g of IgY per ostrich egg. Hence, almost 400 g of IgY can be purified from only one ostrich during a year. In addition, there are enormous differences in the cost of investment in the maintenance of horses, from the points of view of infrastructure, feeding and veterinary care, in which the cost can reach USD 100 per animal per day, compared to a maintenance cost of USD 146 per month per producing bird. These results are encouraging and could easily be extrapolated to the manufacturing of other antivenoms and antitoxins as well, as they could be applied to the manufacturing of potential diagnostic tools.

Comparative Study of the Efficacy of Different Artemisia Cina Extracts and their Nanoparticulated Forms against A549 Lung Cancer Cell Line

The present study aimed to investigate the possible antiproliferative effects of the plant extracts of Artemisia cina on lung cancer, and whether the nanoencapsulation of the plant extracts in the form of nanoemulsion would potentiate their therapeutic efficacy. Soxhlet extraction of the freshly collected shade-dried plant using n-hexane and methanol was performed, and the two extracts were loaded within nanoemulsion using the water dilution method. The nanoemulsion was characterized for its particle size, zeta potential, polydispersity, physicochemical stability under refrigeration conditions, and was morphologically visualized using transmission electron microscopy. The antiproliferative activity of the extracts and their nanoforms was tested in A549 lung cancer cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results revealed the successful preparation of the plant extract-loaded nanoemulsions with suitable particle size from 15-16 nm, a homogenous dispersion with polydispersity index (PDI) ranging from 0.23-0.31, and neutral surface charge. The nanoemulsions displayed insignificant changes in their physicochemical properties after storage for 3 months, and their spherical morphology was confirmed using transmission electron microscopy. Both n-hexane and methanol extracts di splayed antiproliferative activity against A549 cells, with IC50 values of 35.96 ± 1.7 and 41.6 ± 2.8 μg/ml, respectively. On the other hand, their respective nanoparticulated forms displayed superior antiproliferative activity, with IC50 values of 12.59 ± 0.7 and 5.6 ± 0.4 μg/ml, respectively. Therefore, it can be concluded that nanoencapsulation of plant extracts would significantly potentiate their antiproliferative activity, which paves the way towards a more effective anticancer therapy.

Preparation, Characterization and Cytotoxic Activity of New Oleuropein Microemulsion Against HCT-116 Colon Cancer Cells

The work was aimed to elucidate whether the nanoencapsulation of oleuropein would potentiate its anticancer activity against colon cancer or not. Oleuropein microemulsion was prepared using the water dilution method, in which oleic acid, tween 20, and ethanol were selected as the oily phase, surfactant, and cosurfactant, respectively. The microemulsion was characterized in terms of particle size, zeta potential, polydispersity index, morphology, in vitro drug release, stability, and anticancer activity on HCT-116 colon cancer cells. Results showed that oleuropein microemulsion was successfully prepared with a particle size of 10 nm, polydispersity index of 0.283, and a neutral charge. Moreover, it was able to sustain the release of drug for 24 h with a cumulative percentage release of 99%. Transmission electron microscopic examination of the microemulsion confirmed its small particle size and revealed spherical particle morphology. The as-prepared microemulsion was stable, with no significant changes in particle size, polydispersity or charge observed upon storage. Moreover, the oleuropein microemulsion was 106 times more cytotoxic than oleuropein alone on HCT-116 colon cancer cells. Therefore, it can be concluded that the proposed microemulsion is a promising delivery carrier for oleuropein, which is worthy of further experimentation on animals to examine the drug bioavailability.

Transdermal delivery enhancement of carvacrol from Origanum vulgare L. essential oil by microemulsion.

Carvacrol has been reported for analgesic and anti-inflammatory activity by cyclooxygenase inhibition but it could induce gastrointestinal toxicity because of its non-selective inhibition. Therefore, the present study aimed to develop transdermal microemulsion from Origanum vulgare essential oil to deliver carvacrol into and through the skin which would overwhelm the gastrointestinal problems. O. vulgare essential oil was extracted by hydrodistillation and its carvacrol content was determined using high performance liquid chromatography. Pseudoternary phase diagrams were constructed using water dilution method to investigate the suitable microemulsion components. Microemulsions were then characterized for external appearance, particle size, size distribution, zeta potential, electrical conductivity, refractive index, viscosity, transmittance, pH, and stability. Additionally, the irritation property of microemulsions were investigated by hen's egg on the chorioallantoic membrane assay. The release profile, percutaneous absorption, and skin retention were investigated using dialysis bag and Franz diffusion cell, respectively. The interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were investigated using the enzyme-linked immunosorbent assay. The results remarked that carvacrol was a major component of O. vulgare essential oil with high concentration of 83.7%. The most suitable microemulsion (ME 1), composing of 5% w/w O. vulgare essential oil, 25%w/w Tween 60, 25%w/w butylene glycol, and 45%w/w deionized water, had the smallest internal droplet size (179.5±27.9nm), the narrowest polydispersity index (0.30±0.07), the highest transmittance (93.13±0.04%), and Newtonian flow behavior with low viscosity of 0.30±0.07 Pas. ME 1 could reduce the irritation effect of O. vulgare essential oil since ME 1 (IS=3.1±0.10) exhibited significantly lower irritation effect than its blank formulation (IS=4.8±0.02) and O. vulgare oil solution (IS=5.0±0.01) (p<0.05). Furthermore, ME 1 sustain released carvacrol from the formulation, remarkedly deliver more carvacrol through the skin layer (2.6±2.2%) and significantly retained carvacrol in the skin layer (2.60±1.25%). Additionally, ME 1 significantly enhanced IL-6 inhibition of O. vulgaris oil and carvacrol (p<0.05). Therefore, O. vulgaris oil microemulsion was suggested to be used for the transdermal delivery and anti-inflammatory activities enhancement of carvacrol.

Fabrication of nano-structured lipid carrier for encapsulation of vitamin D3 for fortification of ‘Lassi’; A milk based beverage

A phase inversion based cold water dilution method was developed to encapsulate Vitamin D3 (Vit D) in nano-structured lipid carrier (NLC) by blending caprylic-/capric triglyceride, Leciva S70 and Kolliphor HS®15, Vit D and sodium chloride. To optimize the process; a total of forty one formulations prepared by varying in their composition were tested for presence of NLC. Out of forty one formulations, only thirteen formulations resulted in NLC formation which were further evaluated for their physico-chemical attributes (particle size, zeta potential, transmittance, encapsulation efficiency and Vit D release). During principal component analysis using XLstats it was found that NLC-19, fabricated with 20% (v/v) Kolliphor, 20% (v/v) CCTG and 60% (v/v) water, 2.5% (w/v) Leciva, 2% (w/v) Vit D and 5% (w/v) sodium chloride was the most suitable for purpose of encapsulating Vitamin D. Hence, NLC-19 formulation was further taken up for stability studies under the following environmental stress conditions: (a) Temperature and humidity: accelerated condition: 45 ± 2 °C and RH 75 ± 5%, ambient condition: 25 ± 3 °C and RH 65 ± 5% and refrigerated condition: 6 ± 2 °C and RH 55 ± 5%, (b) pH: 3, 4, 5, 6, and 7, and (c) Ionic strength (NaCl concentration): 0 mM, 250 mM, 500 mM and 750 mM. The sensory evaluation of ‘Lassi’ (fortified with NLC-19) and its acceptability further confirmed the suitability of NLC-19 for the purpose of fortification of Vitamin D3 in ‘Lassi’ (A milk based beverage).

Exploring the unique anticancer properties of curcumin nanoparticles

The aim of the current work was to elucidate whether the encapsulation of curcumin in nanoparticles (of microemulsion type) would allow its exertion of anticancer activity on several cancer cell lines or not. Curcumin microemulsion was prepared using the water dilution method, in which oleic acid constituted the oily phase, and tween 20, ethanol constituted the surfactant and cosurfactant respectively. The microemulsion was tested for its particle size, charge, morphology using transmission electron microscopy, in vitro curcumin release, stability and anticancer activity on HCT-116 colon cancer line, A549 lung cancer cell line, and PC3 prostate cancer cell line. The safety of the formulation was tested on fibroblasts cell line. Results revealed that curcumin microemulsion was successfully prepared with a particle size of 11.45±0.21 nm, and a negative charge of -9.07 mV, and it was able to sustain the release of drug for 24 hours with a cumulative percent released of 66.72%. Its spherical morphology was confirmed using transmission electron microscopy. No significant changes in particle size or charge were observed upon storage, suggesting the physical stability of the microemulsion. Curcumin microemulsion was proven safe on the normal fibroblasts cell line, and it preserved the anticancer activity of curcumin against A549, HCT116, and PC3 cell lines. Results of this study delineate that microemulsion is a suitable nanodelivery system for curcumin, which needs to be tested on animal tumor models for verification of the anticancer properties in vivo.

Synergistic potential of Juniperus communis and Helichrysum italicum essential oils against nontuberculous mycobacteria.

The present study evaluated the possible synergistic antimycobacterial interactions of Juniperus communis and Helichrysum italicum essential oils (EO). Antimycobacterial potential was tested against Mycobacterium avium and Mycobacterium intracellulare using broth and water dilution method and checkerboard synergy method. Antiadhesion and antibiofilm effect of EOs was evaluated on biotic (HeLa cells) and abiotic surface (polystyrene). To evaluate the possible mechanisms of action, cellular leakage of proteins and DNA was tested and structural changes were visualized with a transmission electron microscope. MIC, minimum bactericidal concentration (MBC) and minimal effective concentration (MEC) were 1.6 mg ml-1 for J. communis EO and 3.2 mg ml-1 for H. italicum EO against both mycobacteria. All combinations of EOs in checkerboard synergy method produced fractional inhibitory concentration index values ranging from 0.501 to 1.5, corresponding to synergistic, additive or indifferent effects. Mycobacterium avium showed a greater tendency to create biofilm but these EOs at subinhibitory concentrations (sMIC) effectively blocked the adhesion and the establishment of biofilm. The exposure of both mycobacteria to MICs and sMICs lead to significant morphological changes: acquired a swollen form, ghost-like cell, disorganized cytoplasm detached from the cell wall. OD value of supernatant for both mycobacteria exposed to EOs have confirmed that there is a leakage of cellular material. The leakage of the cellular material is noticeably higher in sMIC, which is probably due to cell wall damage. sMIC of both EOs have an additive or synergistic effect, reducing MICs, limiting adhesion and preventing the formation of biofilms.

Fat content of striped mice decreases during the breeding season but not during the food-restricted dry season.

Individuals that are capable of accumulating appropriate fat stores are assumed to have selective advantages when food becomes scarce. Similarly to species from temperate zones, some species inhabiting arid areas accumulate fat stores prior to periods of food limitation. Yet, we have little knowledge concerning seasonal variation in body composition and the relationship between fat store size and disappearance risk in species from arid habitats. Using the water dilution method, we examined the body composition of African striped mice (Rhabdomys pumilio) living in a seasonal habitat with a long food-restricted dry season. We tested for seasonal changes in body composition (N=159 measurements of 113 individuals) and whether dry season survival was related to fat mass (N=66 individuals). Fat stores were similar in size at the onset and the end of the dry season, but surprisingly smaller at the onset of the moist breeding season. Fat stores showed a negative relationship with food availability. Individual variation in fat stores was not associated with disappearance risk, but there was a positive association of disappearance risk with body mass. Increased disappearance risk of heavy individuals suggests elevated dispersal rates in competitive individuals. This study suggests that non-breeding philopatric striped mice do not accumulate large fat stores prior to the food-limited dry season but that they might mobilize fat stores at the onset of the breeding season to satisfy the energetic demands of reproduction and/or to decrease costs associated with larger fat stores, such as increased predation risk.

Development of a new approach of immunotherapy against scorpion envenoming: Avian IgYs an alternative to equine IgGs

Antivenom treatment has been largely used against scorpion stings. Despite their efficacy, the use of mammalian antivenoms may cause adverse effects due to the immune system activation. IgYs from hyperimmunized laying hens against venoms could be a promising alternative to equine IgGs due to the various benefits that these antibodies can provide. Here we report the preparation of specific IgYs by immunizing laying hens with Aah (Androctonus australis hector) scorpion venom.IgYs were isolated from egg yolks by water dilution and salt precipitation methods; they were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, western blot and ELISA. The efficiency of these immunoglobulins on the pathophysiological effects induced by Aah venom was assessed by histological and metabolical analysis of the aorta and the heart. The inflammatory response was assessed by evaluating the granulocyte tissue infiltration and oxidative/nitrosative status.Results revealed high IgYs titers against Aah venom by ELISA. Overall, these IgYs seem to protect efficiently mice against envenomation and neutralized the lethal effects of scorpion venom with a high efficacy; the median effective dose (ED50) was 221 μl/2 LD50; i.e. an amount of 79.23 mg of IgY scan neutralize 1 mg of Aah venom. IgY antibodies neutralize effectively the Aah venom lethality and could prevent severe pathological effects induced by scorpion venom and could be used as an effective alternative to equine IgGs against scorpion envenoming.

Longitudinal Study of Rhesus Monkeys Determines That Amylase and Lipase Levels Are Significant Risk Factors for Type 2 Diabetes Mellitus

Decreased levels of amylase have been reported in type 2 diabetes (T2DM), however, longitudinal within subject study ranging from healthy normal levels to preT2DM and overt DM has not previously been conducted in humans or in nonhuman primates. Rhesus monkeys provide the ideal subjects for this study as they are maintained under constant dietary and environmental conditions, and have, to date, been shown to have the same gastrointestinal/pancreas physiology and pathophysiology as humans. They frequently develop T2DM spontaneously and naturally, with no experimental intervention, and with life time rates of T2DM estimated to be 20 to 30 %. Adult rhesus monkeys were studied for a minimum of 3 years and a maximum of 20 years, with a minimum of 2 metabolic parameter measurements per year. These parameters included fasting plasma glucose (FPG), fasting plasma insulin (FPI), complete blood chemistry, and lipid profiles. Body fat was determined by the tritiated water dilution method or by the dual energy xray absorptiometry (DEXA). Insulin sensitivity was quantified by the euglycemic hyperinsulinemic clamp method. These parameters were used to identify the phases in the progression of monkeys from normal to overt diabetes as follows: normal = phases 1–3; metabolic syndrome = phases 4–6, impaired glucose tolerance= phase 7, and overt type 2 diabetes = phase 8. Diabetic monkeys requiring insulin treatment were excluded from this study. Human criteria were used to define MetSyn, IGT and T2DM. Normal healthy serum amylase levels were determined for the first time and shown to be 253 ±5.1 U/L for adults 7 to 11 yrs old (“human years” about 21 to 35 yrs), with normal lipase levels of 29.7± 1.6 U/L. Serum amylase levels declined with age by about 25%, with no change in serum lipase levels with age. In those that progressed through phases 4 to 8 reaching overt type 2 diabetes, serum amylase showed significantly greater declines from ages 13 to 20 yrs with progressive and parallel declines in metabolic parameters, including insulin sensitivity, and HDL cholesterol levels and increases in serum triglycerides and subsequently increases in fasting plasma glucose. The significant rise in serum triglycerides and the decline in amylase together serve as excellent early predictors of the progression to overt T2DM. These risk factors were highly predictive of both early and late onset diabetes. The lower amylase levels observed previously in people with diabetes were clearly not a consequence of diabetes, but were in fact markers of impending diabetes. Such biomarkers are likely to be useful in predicting the development of T2DM in patients years before the disease becomes overt.This study was carried out in accord with the FASEB principles for the use of animals in research.Support or Funding InformationThe work was supported by NIA N01AG31012 and NIA HHSN2532008002C to BCH, and the Department of Health Research, Government of India Human Resources Development Fellowship to UKC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Production of Chicken Egg Yolk Antibody (IgY) Against Recombinant Cholera Toxin B Subunit and Evaluation of Its Prophylaxis Potency in Mice.

Cholera toxin (CT), responsible for the harmful effects of cholera infection, is made up of one A subunit (enzymatic), and five B subunits (cell binding). The release of cholera toxin is the main reason for the debilitating loss of intestinal fluid. Inhibition of the B subunit (CTB) may block CT activity. To determine the effect of anti CTB-IgY against oral challenge with V. cholera in suckling infant mice. The binding domain of cholera toxin was amplified and ligated into pET28a vector. The pET28a (+)/ctb expression vector was confirmed by endonuclease digestion and sequence analysis. The expression of recombinant CTB in E. coli was performed by induction with IPTG. After immunizing the chickens with recombinant CTB, IgY was purified by water dilution method and NaCl precipitation and analyzed by SDS-PAGE. Moreover, the activity and specificity of the IgY antibody were assessed by ELISA. The SDS-PAGE and western blot techniques showed that CTB protein was successfully expressed and specifically recognized by polyclonal antibodies against the cholera toxin. The oral administration of anti- (V. cholera+CTB) in infant mice in challenge with active V. cholera bacterium demonstrated high rate of survival. The increase in the number of antibiotic resistant bacteria implies the necessity of finding novel antibiotics. Our results suggest the possibility of passive protection from purified IgY, hence implying that anti CTB-IgY may be useful in the treatment of cholera infections.

IgY extraction and purification from chicken egg yolk

The interest for immunoglobulin Y (IgY) isolation arises from the possible applications of these immunoglobulins in diagnostics and therapeutics. Powered whole eggs or yolks have been used in veterinary medicine as an inexpensive immunoglobulin Y source for the treatment of enteric diseases. Incorporating feed grade egg yolk antibodies into animal diets has been examined extensively in an attempt to limit pathogenic diarrhea causing by Escherichia coli in swine, and limit Salmonella establishment in calves and mice, as well as Campylobacter, Clostridium, and Salmonella in poultry. Thus, therapeutic administration of the IgY might reduce the clinical use of antibiotics, and minimize the risk of developing bacterial antibiotic resistance. Methods of immunoglobulins isolation and purification from hen yolk are reviewed. For a large-scale production, one of the problems is separating the water-soluble protein fraction from the lipids and other hydrophobic substances. Precipitation or aggregation of lipid occurs under various conditions: (1) by use of water dilution under acidic conditions; (2) by means of acids; (3) combination of solvents (chloroform, aceton) which selectively solubilize the lipids; (4) using of 3.5% polyethylene glycol and (5) by applying natural gums (polyanionic polysaccharides). Further purification of IgY after crude extraction can be achieved by selective precipitation. IgY precipitation can be carried out by salt precipitation using saturated solutions of sodium or ammonium sulphate, which dehydrate proteins. Furthermore, selective precipitation is achieved by using 8.8% sodium chloride or at 12% of polyethylene glycol. The methods comparison on the basis of IgY yield, showed that the mean IgY yield obtained by water dilution method or precipitation with salts, or by using polyethylene glycol was 5.6 mg/ml of egg yolk, 6.3 mg/ml of egg yolk and 8.7 mg/ml of egg yolk correspondingly and did not significantly differ between them. Water dilution method seemed to offer the best IgY recoveries. Moreover, this method in combination with chromatography and filtration can be applied easily in an industrial environment. Filtration technology offers the best opportunities for industrial applications while precipitation with polyethylene glycol or salts provides a cheap and easy methodology for laboratory use. In the production of safer foods, an important strategy is to exploit natural antimicrobial agents as alternatives to conventional synthetic chemical preservatives. In this regard, the IgY is of much interest for its potential application in fortified foods, such as administration for prevention of enteric diseases. The use of IgY is cost-effective.

A novel and efficient immunoglobulin Y extraction method using poloxamer-polyethylene glycol

ABSTRACTAlthough many IgY extraction methods (such as polyethylene glycol (PEG) precipitation method, octanoic acid method, water dilution method, etc.) have been established, there is still industrial drive and real need in developing scale-up IgY production methods. Some previous studies have reported that poloxamer degreasing method shows very good result in IgY extraction from egg yolk with high degreasing speed, harmlessness, simpleness in operation and minimal effect on antibody titer. In this study, we developed a new method, poloxamer-PEG method, to obtain functional IgY with high purity and yield. First, the delipidation solution was added into the diluted yolk samples, and then the filtrates were collected from the diluted yolk samples after 3 hr in room temperature. PEG-6000 was added into the collected filtrates and the mixture was centrifuged after shaking on the roller mixer for 45 min at room temperature. Last, the precipitates were resuspended in 1 mL phosphate buffered solution (PBS) buffer and dialyzed overnight. The results showed that the total protein concentrate of extractive could reach at 30 mg/mL and the purity of the IgY could reach at 92.71% with the novel method, which was superior to the PEG precipitation method and water dilution method.

Simplified Method for Groundwater Treatment Using Dilution and Ceramic Filter

Groundwater is one of the natural resources that is not susceptible to pollutants. However, increasing activities of municipal, industrial, agricultural or extreme land use activities have resulted in groundwater contamination as occured at the Research Centre for Soft Soil Malaysia (RECESS), Universiti Tun Hussein Onn Malaysia (UTHM). Thus, aims of this study is to treat groundwater by using rainwater and simple ceramic filter as a treatment agent. The treatment uses rain water dilution, ceramic filters and combined method of dilute and filtering as an alternate treatment which are simple and more practical compared to modern or chemical methods. The water went through dilution treatment processes able to get rid of 57% reduction compared to initial condition. Meanwhile, the water that passes through the filtering process successfully get rid of as much as 86% groundwater parameters where only chloride does not pass the standard. Favorable results for the combination methods of dilution and filtration methods that can succesfully eliminate 100% parameters that donot pass the standards of the Ministry of Health and the Interim National Drinking Water Quality Standard such as those found in groundwater in RECESS, UTHM especially sulfate and chloride. As a result, it allows the raw water that will use clean drinking water and safe. It also proves that the method used in this study is very effective in improving the quality of groundwater.

Trace-moisture generator designed for performance tests of trace-moisture analyzers

The National Metrology Institute of Japan (NMIJ) developed a trace-moisture generator, named the “simplified trace-moisture generator (STMG),” which was designed particularly for effectively testing the performance of trace-moisture analyzers. The STMG produces trace moisture in nitrogen gas from approximately 10nmol/mol (ppb) to 10μmol/mol (ppm) in amount fraction (mole fraction) of water, using diffusion-tube and two-stage dilution methods. In contrast to the magnetic suspension balance/diffusion-tube humidity generator (MSB/DTG) developed at NMIJ in 2007, the STMG does not require an MSB to measure the evaporation rate of water from the diffusion cell (hence, the name “simplified”). Instead, a moisture analyzer based on cavity ring-down spectroscopy (CRDS) calibrated in a manner traceable to the International System of Units (SI) measures the amount fraction of water in the generated gas. By introducing a sonic nozzle placed at the outlet of the generation chamber, the pressure stability in the chamber is maintained even after the rapid change in the flow rate of the diluent nitrogen gas. This enabled smooth moisture-concentration switching within 1min for 90 % response to the step change between 11nmol/mol and 8.8μmol/mol. The controllability and short-term stability of the moisture content in the generated gas were demonstrated successfully. The long-term stability of moisture generation was evaluated by estimating evaporation rate from the flow rate of nitrogen and the amount fraction of water measured by the CRDS trace-moisture analyzer. It was confirmed that the STMG can generate trace moisture in nitrogen with good stability and repeatability for five consecutive months.

Physicochemical properties of leftover egg yolk after livetins removal

Isolation of immunoglobulin Y (IgY) from egg yolk using water-dilution method generates large quantities of leftover pellet as the co-product. Although egg yolk is well known for its great emulsion property, there is a lack of understanding on how livetins removal would affect the emulsion and rheological properties of the pellet. Therefore, the objective of this study was to determine the effect of soluble protein removal on emulsifying and rheological characteristics of the leftover egg yolk. Egg yolk pellet exhibited distinct structural and physicochemical properties after soluble protein removal. Emulsions prepared from pellet were more vulnerable to coalescence instability than that of egg yolk, although both had a similar oil droplet size. Egg yolk displayed a Newtonian behaviour, compared to a shear-thinning behaviour of pellet. Pellet showed a higher apparent viscosity as well as higher viscoelastic moduli than those of egg yolk, probably due to increased hydrogen bond and hydrophobic interactions in the pellet. Therefore, the changes on the emulsion stability and the rheological properties of egg yolk after soluble proteins removal should be considered in food formulation and processing. Further study is needed to improve the emulsion property of pellet for uses in the food industry.

Preparation of antibodies type IgY against Salmonella typhi lipopolysaccharide in chicken eggs

ipopolysaccharide was extracted from local isolate Sallmonella typhi (previously isolated and characterized) by hot EDTA method, and the extract was partially purified by gel filtration chromatography on sepharose Cl-6B gel. The results showed that the percentage of the carbohydrates amount in the partially purified LPS extract was 43.7%, while the percentage of binding proteins in the same extract was 0.7% with no nucleic acids was found. The molecular weight for the LPS was measured by the gel filtration chromatography method using Sepharose Cl-6B gel and was equivalent to 263000 Dalton. The LD¬50 of LPS was determined by injection of chicken embryos type Ice Brown in the charioallantoic membrane, and was 14.66 µg/Kg. In order to obtain anti S. typhi IgY antibodies, chickens were immunized with the partially purified S. typhi subcutaneously. The IgY antibodies were extracted from eggs yolk by water dilution method and the extract was partially purified by ammonium sulphate precipitation at ratio 60% saturation, and gel filtration chromatography on sepharose Cl-6B gel. The results showed that the protein amount was equivalent to 23.5 mg/ml; specific activity was 0.268, and an overall yield of 70%. The molecular weight for the IgY antibodies was measured by the gel filtration chromatography method using Sepharose Cl-6B gel and was found to be 178000 dalton. The concentration of anti S.typhi LPS IgY antibodies in chicken eggs were investigated by ELISA and was found to be 6.3 mg/ml, and there is a significant differences (P&lt;0.01).