Color and structural modifications of alkaline extracted sunflower protein concentrates and isolates using L-cysteine and glutathione.

Alkaline sunflower protein extraction can be performed along with de-phenolization of sunflower seed proteins if greening is unwanted. This greening is promoted at alkaline pH when chlorogenic acid (CGA) oxidizes and reacts with amino acids such as lysine. Thiol-containing dough conditioners: L-cysteine hydrochloride and glutathione (GSH) were investigated as an alternative de-greening strategy to acidification and de-phenolization. Greening and browning inhibition of thiols (GSH and Cysteine) were modeled by a combination of additive and interaction effects of extraction pH (7.0 to 11.0) and thiol concentration (0.00 to 5.60 mM) randomly assigned by Response Surface Methodology (RSM). The powders with the highest greening were the controls (pH 8.9-9.3 and no added thiols) and powders at pH 10.41 with 0.82 mM thiols. From RSM, the maximum greening inhibition was achieved at pH 8.71 and 4.23 mM cysteine, and pH 8.51 and 3.78 mM GSH. However, cysteine caused more browning at alkaline pH than GSH. Furthermore, fluorescence spectroscopy showed that cysteine had a protective effect against alkaline unfolding, whereas GSH quenched fluorescence in a concentration-dependent manner. Overall, de-greening of alkaline extracted sunflower protein was achieved by adding cysteine or glutathione, but the thiols differed in their contribution to the browning and unfolding effect.

Blood Parasite Load as an Early Marker to Predict Treatment Response in Visceral Leishmaniasis in Eastern Africa.

BACKGROUND: To expedite the development of new oral treatment regimens for visceral leishmaniasis (VL), there is a need for early markers to evaluate treatment response and predict long-term outcomes. METHODS: Data from 3 clinical trials were combined in this study, in which Eastern African VL patients received various antileishmanial therapies. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative polymerase chain reaction (qPCR) before, during, and up to 6 months after treatment. The predictive performance of pharmacodynamic parameters for clinical relapse was evaluated using receiver-operating characteristic curves. Clinical trial simulations were performed to determine the power associated with the use of blood parasite load as a surrogate endpoint to predict clinical outcome at 6 months. RESULTS: The absolute parasite density on day 56 after start of treatment was found to be a highly sensitive predictor of relapse within 6 months of follow-up at a cutoff of 20 parasites/mL (area under the curve 0.92, specificity 0.91, sensitivity 0.89). Blood parasite loads correlated well with tissue parasite loads (ρ = 0.80) and with microscopy gradings of bone marrow and spleen aspirate smears. Clinical trial simulations indicated a > 80% power to detect a difference in cure rate between treatment regimens if this difference was high (> 50%) and when minimally 30 patients were included per regimen. CONCLUSIONS: Blood Leishmania parasite load determined by qPCR is a promising early biomarker to predict relapse in VL patients. Once optimized, it might be useful in dose finding studies of new chemical entities.

Cysteine's effects on chlorogenic acid quinone induced greening and browning: Mechanism and effect on antioxidant reducing capacity.

Formation of green trihydroxy benzacridine (TBA) derivatives when chlorogenic acid (CGA) quinones and amino acids react can be unappealing for some consumers. Cysteine was studied as an anti-greening strategy, given that cysteine-CGA conjugates are colorless. Buffered 2.55 mM CGA: 5.09 mM lysine: (0-5.09) mM cysteine solutions at pH 8 and 9 were prepared and incubated for a maximum of 48 h at 22 C. Color intensity and conjugate formation was monitored spectrophotometrically, and by HPLC and LC-MS respectively, while antioxidant capacity was measured by Folin-Ciolcateau and Trolox equivalent antioxidant capacity assays. Green TBA formation was promoted at higher pH and inhibited as cysteine concentration increased. Concentration-dependent cysteine inhibition of CGA-lysine greening was attributed to redox diphenol regeneration and formation of cysteinyl-CGA conjugates, which also contributed to antioxidant capacity. pH had a greater effect on antioxidant capacity than added cysteine. Results suggested a potential anti-greening approach for alkaline CGA quinone-amine greening.

Acidulant effect on greening, reducing capacity, and tryptophan fluorescence of sunflower butter cookie dough during refrigerated storage.

BACKGROUND: Sunflower seed derived butter can be a source of protein and phenolic antioxidants in refrigerated dough. Chlorogenic quinone-amino acid induced greening can however occur at alkaline pH, which could result in less bioavailable conjugated phenol-amino acids. Acidulants were tested as potential anti-greening ingredients in refrigerated chemically leavened cookie dough. Effect of refrigerated storage time, leavening agents and acidulants on tryptophan fluorescence (λex = 280 nm, λem = 300-500 nm), color (hunter L*, a*, b* color scale), reducing capacity [1,1'-diphenyl-2-picryl-hydrazyl (DPPH) and Folin-Ciocalteu reducing capacity (FCRC)], and hydroxycinnamic acids were measured. RESULTS: The pH range of acidified doughs was 4.83-6.98 compared to 7.65-9.18 in non-acidified leavened doughs after 24 days. Greening was higher in baking soda dough control (a* = -0.54) than baking powder dough control (a* = 2.98) after 24 days, attributed to higher pH (9.18) of the former compared to pH 7.14 in the later. Tryptophan fluorescence intensity in baking soda dough decreased in the order: control > glucono-delta lactone ≈ citric acid after 24 days. The DPPH and FCRC of acidified doughs were greater than corresponding control doughs. CONCLUSION: The use of acidulants would prevent greening in sunflower dough without lowering its phenolic concentration, making use of sunflower butter in refrigerated dough for baked goods feasible. © 2018 Society of Chemical Industry.

Pharmacokinetics, Safety, and Efficacy of an Allometric Miltefosine Regimen for the Treatment of Visceral Leishmaniasis in Eastern African Children: An Open-label, Phase II Clinical Trial.

BACKGROUND: Convenient, safe, and effective treatments for visceral leishmaniasis in Eastern African children are lacking. Miltefosine, the only oral treatment, failed to achieve adequate efficacy, particularly in children, in whom linear dosing (2.5 mg/kg/day for 28 days) resulted in a 59% cure rate, with lower systemic exposure than in adults. METHODS: We conducted a Phase II trial in 30 children with visceral leishmaniasis, aged 4-12 years, to test whether 28 days of allometric miltefosine dosing safely achieves a higher systemic exposure than linear dosing. RESULTS: Miltefosine accumulated during treatment. Median areas under the concentration time curve from days 0-210 and plasma maximum concentration values were slightly higher than those reported previously for children on linear dosing, but not dose-proportionally. Miltefosine exposure at the start of treatment was increased, with higher median plasma concentrations on day 7 (5.88 versus 2.67 µg/mL). Concentration-time curves were less variable, avoiding the low levels of exposure observed with linear dosing. The 210-day cure rate was 90% (95% confidence interval, 73-98%), similar to that previously described in adults. There were 19 treatment-related adverse events (AEs), but none caused treatment discontinuation. There were 2 serious AEs: both were unrelated to treatment and both patients were fully recovered. CONCLUSIONS: Allometric miltefosine dosing achieved increased and less-variable exposure than linear dosing, though not reaching the expected exposure levels. The new dosing regimen safely increased the efficacy of miltefosine for Eastern African children with visceral leishmaniasis. Further development of miltefosine should adopt allometric dosing in pediatric patients. CLINICAL TRIALS REGISTRATION: NCT02431143.

Greening in sunflower butter cookies as a function of egg replacers and baking temperature.

Chlorogenic acid (CGA) binding to proteins in alkaline conditions results in the production of green trihydroxy benzacradine (TBA) derivatives. The formation of TBA derivatives could decrease product quality due to the potential losses in soluble protein and antioxidants and the production of an undesirable green color. To determine how cookie formulation affected the formation of TBA derivatives in sunflower butter cookies, two egg replacers (chia and banana) and two baking temperatures (162.8 and 190.6 °C) were used. Moisture, greening intensity, CGA content and antioxidant capacity were measured. Cookies made with egg and baked at 162.8 °C had the highest moisture, internal greening intensity, and TBA derivative formation, in addition to lower CGA content and antioxidant capacity. Cookies made with banana baked at 190.6 °C produced the opposite outcome with 35, 4, and 23% less internal greening, moisture, and TBA derivatives, respectively, and 90 and 76% higher CGA and antioxidant capacity. Internal greening was positively correlated with moisture and adduct concentration, and negatively correlated with spread factor and CGA content. Moisture had a significant impact on greening, which indicates that baking temperature and cookie dough formulation can be modified to produce a less green cookie with more unreacted antioxidants and protein.

Lowering greening of cookies made from sunflower butter using acidic ingredients and effect on reducing capacity, tryptophan and protein oxidation.

Lowering of greening formed from oxidized chlorogenic acid (CGA) and amino groups, and favoured at alkaline pH, was investigated using acidic ingredients (sour cream, buttermilk, yoghurt, and honey) in sunflower butter cookies. Cookies with maple syrup added were used as a positive control. Changes in greening intensity, greening reactants (total phenols, CGA, protein), antioxidant capacity, tryptophan and Schiff base fluorescence were measured. Percentage greening, pH and aw of cookies followed the same order: maple syrup > sour cream ≥ buttermilk > yoghurt > honey. pH was positively correlated with greening intensity (r = 0.77) and negatively correlated with CGA (r = -0.96). Total phenolic content, antioxidant capacity, tryptophan and Schiff bases were similar among cookies. The results suggest it is possible to decrease greening by minimizing storage time and using acidic ingredients. Minimal greening with acidic ingredients can extend the application of sunflower butter as a baking ingredient without loss of free radical-scavenging capacity, or higher protein oxidation.

Chlorogenic acid oxidation-induced greening of sunflower butter cookies as a function of different sweeteners and storage conditions.

Sunflower butter use as an allergen-free alternative to tree and legume nut butter in baking is limited by chlorogenic acid induced greening that occurs at alkaline pH. Limited information is available on controlling this greening in a food matrix. This study examined how different liquid sweeteners and relative humidity influenced greening of sunflower butter cookies. Doughs had similar initial pH (7.52-7.66) which increased to 8.44-9.13 after baking as ranked: xylitol>maple syrup>corn syrup>honey>agave syrup. Cookies made with maple syrup had the highest moisture and greening corresponding with lowest free chlorogenic acid. The % greening followed the same trend as greening intensity, and was positively correlated (r=0.9101) with chlorogenic-lysine adduct content. Our findings provide an ingredient solution to controlling greening, as results demonstrate that greening can be promoted with high relative humidity storage, and use of high moisture and pH ingredients. Unwanted greening can be inhibited by simply changing the liquid sweetener.

Lipid and protein antioxidant capacity of dried Agaricus bisporus in salted cooked ground beef.

Dried Agaricus bisporus powder (DAB)'s antioxidant capacity was tested in refrigerated cooked ground beef (CGB) containing 0, 1 or 1.5% NaCl. Lipid and protein oxidation products were monitored over time and correlated with changes in phenolic content. On day 16, 88-94% lower malondialdehyde (MDA) was found in CGB with DAB compared to control (1.15mg MDA/kg samples). Volatile aldehydes were up to 99% lower on day 16 in CGB with DAB than controls. In unsalted CGB, thiols dropped by 82% in control compared to <60% in CGB with DAB. On day 16, tryptophan fluorescence decline in unsalted control was higher (28%) than that in CGB with rosemary or DAB (2.4-5.5%) while Schiff bases declined in control and CGB+1% DAB, but increased in CGB+2% and 4% DAB. DAB's extension of shelf life was concentration dependent. Phenolic compounds had moderate to strong negative correlations with MDA up to day 10 indicating a possible role of DAB phenolics in preventing malondialdehyde production.

Chlorogenic Acid Oxidation and Its Reaction with Sunflower Proteins to Form Green-Colored Complexes.

Sunflower seeds are used to produce oil for human consumption, but its protein meal by-product has long been used as animal feed. Formation of green-colored complexes through oxidized chlorogenic acid(CGA)-protein interactions is a primary reason why defatted sunflower protein has not been widely utilized by the food industry. Sunflower protein possesses many properties that make it an appealing alternative protein source from both a marketing and formulation perspective, including its low cost, absence of major allergens, low antitrypsin inhibitors, and its status as both vegan and "clean" label friendly. With the global demand for sunflower oil and novel protein sources expected to increase and waste recovery a concern for many, providing uses for the sunflower meal and its fiber and polyphenol components would provide added value to by-products from sunflower oil processing. This review addresses the unique green pigmentation associated with the interaction of sunflower protein and oxidized CGA by outlining the sunflower oil and protein meal market, CGA reactions contributing to greening, methods for CGA extraction, and the effect of processing on sunflower protein quality and the greening reaction. This review also addresses potential food applications of sunflower protein-based ingredients, such as addition of texturized protein to food products; a microencapsulation matrix for antioxidants; edible, flexible biodegradable films; and even use of sunflower butter as an alternative to peanut butter where the green color is not considered undesirable. Continued studies are needed to make sunflower-based products and CGA-extraction processes available across the global marketplace.

Breakdown of simple female genital fistula repair after 7 day versus 14 day postoperative bladder catheterisation: a randomised, controlled, open-label, non-inferiority trial.

BACKGROUND: Duration of bladder catheterisation after female genital fistula repair varies widely. We aimed to establish whether 7 day bladder catheterisation was non-inferior to 14 days in terms of incidence of fistula repair breakdown in women with simple fistula. METHODS: In this randomised, controlled, open-label, non-inferiority trial, we enrolled patients at eight hospitals in the Democratic Republic of the Congo, Ethiopia, Guinea, Kenya, Niger, Nigeria, Sierra Leone, and Uganda. Consenting patients were eligible if they had a simple fistula that was closed after surgery and remained closed 7 days after surgery, understood study procedures and requirements, and agreed to return for follow-up 3 months after surgery. We excluded women if their fistula was not simple or was radiation-induced, associated with cancer, or due to lymphogranuloma venereum; if they were pregnant; or if they had multiple fistula. A research assistant at each site randomly allocated participants 1:1 (randomly varying block sizes of 4-6; stratified by country) to 7 day or 14 day bladder catheterisation (via a random allocation sequence computer generated centrally by WHO). Outcome assessors were not masked to treatment assignment. The primary outcome was fistula repair breakdown, on the basis of dye test results, any time between 8 days after catheter removal and 3 months after surgery. The non-inferiority margin was 10%, assessed in the per-protocol population. This trial is registered with ClinicalTrials.gov, number NCT01428830. FINDINGS: We randomly allocated 524 participants between March 7, 2012, and May 6, 2013; 261 in the 7 day group and 263 in the 14 day group. In the per-protocol analysis, ten (4%) of 250 patients had repair breakdown in the 7 day group (95% CI 2-8) compared with eight (3%) of 251 (2-6) in the 14 day group (risk difference 0·8% [95% CI -2·8 to 4·5]), meeting the criteria for non-inferiority. INTERPRETATION: 7 day bladder catheterisation after repair of simple fistula is non-inferior to 14 day catheterisation and could be used for management of women after repair of simple fistula with no evidence of a significantly increased risk of repair breakdown, urinary retention, or residual incontinence up to 3 months after surgery. FUNDING: US Agency for International Development.

Effect of coffee filtrate, methylglyoxal, glyoxal, and caffeine on Salmonella typhimurium and S. enteritidis survival in ground chicken breasts.

The antimicrobial effect of roasted coffee filtrate (CF) and dicarbonyls on Salmonella Typhimurium and Salmonella Enteritidis in raw ground chicken breast meat (GCB) was investigated. Coffee was brewed and filtered before addition to GCB. Coffee filtrate with and without added caffeine, methylglyoxal, and/or glyoxal was added to GCB and then inoculated with Salmonella Typhimurium and Salmonella Enteritidis. Ground chicken samples were stomached with peptone water at days 1, 3, 5, and 7, plated on XLD agar with a TSA overlay, and Salmonella survivors were enumerated. CF alone gave less than a 1 Log reduction in all runs compared to control GCB with no treatment. Methylglyoxal (2.28 mg/g GCB) had the greatest antimicrobial effect against Salmonella Typhimurium and Salmonella Enteritidis in GCB with average Log reductions of 2.27 to 3.23, respectively, over the 7 d duration of the experiment compared to control GCB with no treatment. A 1 Log reduction was observed in GCB with CF, 0.93 mg glyoxal, and 1 mg caffeine/g chicken compared to the control and GCB with only CF. Heat-produced coffee compounds could potentially reduce Salmonella in retail ground chicken and chicken products.

Anti-Listeria monocytogenes activity of heat-treated lyophilized pomegranate juice in media and in ground top round beef.

Heat treatment can affect antimicrobial activity of plant by-products by altering phenolic content and composition and forming melanoidins. The antilisterial efficacy of heat-treated and unheated lyophilized pomegranate juice (LPJ) was determined. The LPJ was heated at 100 degrees C for 0, 30, 60, or 120 min and added at 2% (wt/wt) to ground top round beef, which was then cooked and inoculated with individual L. monocytogenes strains. Samples of meat stored at 5 degrees C were taken at days 1, 8, 14, and 21 and plated onto Oxford medium for enumeration of bacteria. The MIC of LPJ was determined, and agar well diffusion assays were conducted. Against five L. monocytogenes strains, LPJ had a MIC of 1.50 to 1.75% (wt/vol) and 16.8- to 20.0-mm zones of inhibition. In general, no significant differences in L. monocytogenes levels between the various treatments, including the commercial sodium lactate-sodium diacetate combination, were detected at days 1 and 8. The LPJ (0, 30, 60, and 120 min of heating) significantly inhibited growth of all five L. monocytogenes strains in refrigerated ground cooked beef by 1.80 to 4.61 log CFU/g at day 21. Heating did not negatively impact LPJ antilisterial activity. Addition of LPJ lowered pH values by 0.3 units. The L*, a*, and b* values of cooked ground beef with LPJ changed during the study by 3.4 to 4.43, 0.44 to 0.8, and 0.57 to 1.36 units, respectively, compared with the control. This is the first investigation to confirm pomegranate's antilisterial activity in vitro and in ground beef.

Capillary liquid chromatography-mass spectrometry for the rapid identification and quantification of almond flavonoids.

A rapid negative ion ESI high-performance capillary liquid chromatography-mass spectrometry method was developed to identify and quantify flavonoids (e.g., flavanols, flavonols, flavanones and glycosides). Fifteen standards and two varieties of almond skin extract powder (Carmel and Nonpareil) were used to demonstrate the chromatographic separation, reproducibility and accuracy of the method that employed a 150 mm x 0.3 mm ChromXP 3C18-EP-120 column. All standards eluted in less than 10 min, providing a 9-12x reduction in analysis time compared to existing methods (90-120 min). However, isomers (e.g., catechin/epicatechin and galactosides/glucosides) were not resolved and, therefore, identified and quantified collectively. RSDs for retention time and peak area reproducibility (mass spectrometry data) were <0.5% and <5.0%, respectively. Peak area reproducibility was greatly improved (from a RSD>10%) after the implementation of a low-flow metal needle in the ESI source. Quantitation by mass spectrometry also afforded a % error less than 5% for most compounds.

Inhibition of lipid oxidation in refrigerated and frozen salted raw minced chicken breasts with electron beam irradiated almond skin powder.

Antioxidant effects of electron beam irradiated almond skin powder (ASP) in raw minced chicken breasts (MCB) during refrigerated and frozen storage were studied. MCB samples were treated with BHT, non-irradiated ASP (0kGy), irradiated ASP (10kGy, 20kGy and 30kGy) and compared to MCB without antioxidants. Colour was determined on initial and final day of analysis while conjugated dienes (CD), peroxide values (POV), TBARS and hexanal content were evaluated periodically for 12 days of refrigerated storage and seven months of frozen storage. ASP addition lowered L* values compared to MCB without ASP or BHT. During refrigerated storage, MCB containing ASP had decreased formation of lipid oxidation products ranging from 0 to 66%, 7 to 24%, 0 to 37% and 4 to 71% reduction in POV, CD, TBARS and hexanal content, respectively, as compared to MCB without antioxidants over duration of study. A 15-65%, 3-25%, 14-50% and 28-82% reduction in POV, CD, TBARS and hexanal content, respectively, for frozen MCB was detected.

Encapsulation of nisin and lysozyme in liposomes enhances efficacy against Listeria monocytogenes.

The efficacy and stability against Listeria monocytogenes of nisin and lysozyme encapsulated in phospholipid liposomes was evaluated. Antimicrobial-containing liposomes were prepared by hydrating dried lipids with buffer containing nisin, nisin plus the fluorescence probe calcein, or calcein and lysozyme. Mixtures were then centrifuged and sonicated, and encapsulated liposomes were collected using size-exclusion chromatography. Antimicrobial concentration in liposomes was determined by bicinchoninic acid assay prior to determination of antimicrobial activity against strains of L. monocytogenes. When nisin was encapsulated in liposomes, protein concentrations of 0.39, 0.27, and 0.23 mg/ml for phosphatidylcholine (PC), PC-cholesterol (7:3), and PC-phosphatidylglycerol (PG)-cholesterol (5:2:3), respectively, were obtained. Encapsulation of nisin with calcein yielded protein concentrations of 0.35, 0.39, and 0.28 mg/ml for PC, PC-cholesterol, and PC-PG-cholesterol, respectively. Encapsulation of calcein with lysozyme resulted in protein concentrations of 0.43, 0.26, and 0.19 mg/ml for PC, PC-cholesterol, and PC-PG-cholesterol, respectively. Encapsulated nisin in 100% PC and PC-cholesterol liposomes inhibited bacterial growth by >2 log CFU/ml compared with free nisin. Growth inhibition with liposomal lysozyme was strain dependent, with greater inhibition observed for strains 310 and Scott A with PC-cholesterol and PC-PG-cholesterol liposomes. Inhibition of L. monocytogenes indicated the potential of liposomes to serve as delivery vehicles for antimicrobials in foods while improving stability of antimicrobials.

Size, stability, and entrapment efficiency of phospholipid nanocapsules containing polypeptide antimicrobials.

The effect of lipid composition [phosphatidylcholine (PC), phosphatidylglycerol (PG), and cholesterol] on size, stability, and entrapment efficiency of polypeptide antimicrobials in liposomal nanocapsules was investigated. PC, PC/cholesterol (70:30), and PC/PG/cholesterol (50:20:30) liposomes had entrapment efficiencies with calcein of 71, 57, and 54% with particle sizes of 85, 133, and 145 nm, respectively. Co-encapsulation of calcein and nisin resulted in entrapment efficiencies of 63, 54, and 59% with particle sizes of 144, 223, and 167 nm for PC, PC/cholesterol (70:30), and PC/PG/cholesterol (50:20:30), respectively. Co-encapsulation of calcein and lysozyme yielded entrapment efficiencies of 61, 60, and 61% with particle sizes of 161, 162, and 174 nm, respectively. The highest concentration of antimicrobials was encapsulated in 100% PC liposomes. Nisin induced more calcein release compared to lysozyme. Results demonstrate that production and optimization of stable nanoparticulate aqueous dispersions of polypeptide antimicrobials for microbiological stabilization of food products depend on selection of suitable lipid-antimicrobial combinations.