Learning disability varies by month of conception. The underlying mechanism is unknown but vitamin D, necessary for normal brain development, is commonly deficient over winter in high latitude countries due to insufficient ultraviolet radiation. We linked the 2007–2016 Scottish School Pupil Censuses to Scottish maternity records and to sunshine hours and antenatal ultraviolet A/B radiation exposure derived from weather stations and satellites respectively. Logistic regression analyses were used to explore the associations between solar radiation, then ultraviolet B, and learning disabilities, adjusting for the potential confounding effects of month of conception and sex. Of the 422,512 eligible, singleton schoolchildren born at term in Scotland, 79,616 (18.8%) had a learning disability. Total antenatal sunshine hours  and ultraviolet B exposure  were inversely associated with learning disabilities with evidence of a dose-relationship. The latter association was independent of ultraviolet A exposure. Significant associations were demonstrated for exposure in all three trimesters. Low maternal exposure to ultraviolet B radiation may play a role in the seasonal patterning of learning disabilities. Further studies are required to corroborate findings and determine the effectiveness of supplements. Identifying and addressing modifiable risk factors could reduce the burden on health and social care. We previously demonstrated seasonal patterning whereby the risk of learning disabilities varied by month of conception;2 with seasonality accounting for 11.4% of all cases of learning disabilities.Learning disabilities have profound, long-term effects on affected individuals, their families and society as a whole. The lifetime cost of affected children born over one year in the USA has been estimated at over $51 billionDuring the antenatal period, the fetus undergoes rapid development and growth making it susceptible to environmental exposures with the potential of long-term consequences.3. Exposure to UVB radiation promotes vitamin D production. As a result of low levels of UVB radiation, vitamin D deficiency is common over winter months in high latitude countries4. Therefore, seasonal variation in maternal vitamin D concentrations over the critical period of fetal development is a biologically plausible mechanism underlying the seasonal patterning of learning disability.Maternal serum concentrations of vitamin D are important for normal brain developmentIn order to test this hypothesis, we linked Scotland-wide education and maternity records with satellite data to explore the associations between total, and trimester-specific, sunshine hours and UVB exposure, and learning disability. Sunshine hours are a proxy of total UV radiation but the amount of radiation reaching Earth’s surface varies with solar activity and the solar cycle. Therefore, adjustment of the sunshine hours with sunspot data provides a better proxy of women’s total UV exposure. UV levels are strongly correlated with season; however, superimposed on this annual cycle, there is an 11-year solar cycle over which changes in the sun’s oscillatory magnetic field result in variations in the number of sunspots and, therefore, solar radiation. As a result, UV levels vary between years and their associations can be disentangled from other seasonally patterned phenomena such as temperature and lifestyle by adjusting for month of conception.5. The actual amount of UVB reaching the earth’s surface varies greatly with weather conditions, such as cloud cover6. Therefore, whilst levels of UVA and UVB are correlated, there is sufficient variation to determine if associations with solar radiation are driven by UVA or UVB.Furthermore, whilst UVA radiation covers longer wavelengths and is not absorbed by the ozone layer, UVB radiation covers shorter wavelengths and, therefore, is mostly absorbed before reaching the earth’s surfaceWe explored whether overall UV radiation (sunshine hours adjusted for sunspots) was associated with learning disabilities before examining UVB specifically.Overall, 1,023,592 singleton children who attended Scottish schools at some point between 2007 and 2016 were born in Scotland. Of these, 601,066 were ineligible for inclusion: 57,687 were born <37 weeks gestation; 38,010 were born >41 weeks gestation, 1,058 had missing data on gestation; 72 had a birthweight <400 g; 279 had a birthweight >6,500 g; one was <4 years old; four were >19 years old; 486,403 were conceived prior to 2000 or born after 2011; and 17,552 could not be linked to sunshine hours data. The remaining 422,526 were eligible for inclusion. A further 14 had to be excluded because the child’s sex was missing. Therefore, the final study population comprised 422,512 schoolchildren. Of these 79,616 (18.8%) had a learning disability: 49,770 (23.1%) boys and 29,846 (14.4%) girls.2 (range 0.40 kJ/m2 to 0.62 kJ/m2) in December to 19.84 kJ/m2 (range 17.93 kJ/m2 to 20.01 kJ/m2) in June.The crude prevalence of children with learning disabilities varied by month of conception; ranging from 16.5% among children conceived in July to 21.0% among those conceived in February, March and April  from north to south; this equates to a relatively small range of latitudes (from 58.4° North to 58.6° North). Our calculation of UV exposure assumed that women resident in Scotland remained in Scotland during their pregnancy. No data were available on changes in UV exposure due to holidays and trips aboard. Similarly, we had no data on individual differences in exposure to UV due to the proportion of time spent outdoors, choice of clothing, or use of sunblock. Finally, whilst our findings are consistent with a possible role of vitamin D, other photo-active compounds may also be relevant.In conclusion, low UVB exposure in winter months may be contributing to the patterning of learning disability by month of conception. Further investigation is required to corroborate this hypothesis including measurement of serum vitamin D levels in pregnancy cohorts and intervention studies of vitamin D supplements.The School Pupil Census is conducted annually by all local authority run primary, secondary and special schools in Scotland and the data are collated by the Scottish Exchange of Educational Data (ScotXed). The census records whether the child has a special educational need; defined as being unable to benefit fully from school education without help beyond that normally given to schoolchildren of the same age. We defined learning disability as a record of special educational need attributed to: intellectual disabilities, language or speech disorder including dyslexia, physical or motor impairment, autistic spectrum disorder or social, emotional or behavioural difficulties.24. The SMR02 records the child’s sex and date of birth, gestation at delivery, and whether it was a multiple delivery. Since the early 1990s, gestational age has been confirmed by ultrasound conducted in the first half of pregnancy in more than 95% of pregnant women in the United Kingdom. If there is more than seven days difference between gestational age calculated from ultrasound and from last menstrual period, the former is used28. Date of conception was derived from date of delivery minus gestational age at delivery plus two weeks. Trimester 1 comprised the first three months of pregnancy from month of conception; trimester 2 comprised months four, five, and six; and trimester 3 comprised all subsequent months until delivery.We used probabilistic matching to link individual children included in any School Pupil Census conducted between 2007 and 2016 inclusive, to their maternity record (Scottish Morbidity Record 02 (SMR02)). The linkage methodology has been described in detail previously and demonstrated to be 99% accurate for singleton children29. https://www.metoffice.gov.uk/climate/uk/data/ukcp09/faq#faq10. These data are modelled estimates based on measured sunshine values from the network of static sensors (Kipp & Zonen and Campbell-Stokes sensors) across Scotland. Monthly mean sunspot numbers for the northern hemisphere are produced by the Royal Observatory of Belgium in Brussels and were obtained from the Sunspot Index and Long-term Solar Observations (SILSO) website30. Global 5 km UV radiation irradiance data were produced by the Japan Aerospace Exploration Agency (JAXA) using measurements from the moderate resolution imaging spectroradiometer (MODIS) instrument onboard NASA’s aqua and terra satellites. Downward irradiance values  for UVA (315nm-400nm) and UVB (280–315 nm) are available at a daily resolution from 2000 to 2010 inclusive. We downloaded every binary file available from 1 January 2000 to 31 December 2010 from the publicly accessible JAXA FTP site . We extracted the contents of each binary file, which gave us a UVA and UVB value for each latitude and longitude on earth. We then deleted any points that were not contained within a Great Britain bounding box . The Great Britain points were then interpolated, via inverse weighted distance, to raster images. The rasters were projected to Ordnance Survey Great Britain (OSGB) 1936/British National Grid. Finally, we took the Northing and Easting centroids of each postcode unit in Scotland (Ordnance Survey codepoint ver 2017.4.0) and used these to extract the UVA and UVB values from only those grid squares covering residential areas in Scotland. Scotland-wide monthly means were then calculated resulting in a monthly time-series of UVA and UVB irradiance values measured in W/m2. These were converted to kJ/m2 by multiplying by 86,400 (seconds in the day) and dividing by 1,000 to be consistent with other epidemiological studies. For the four environmental exposures  we calculated exposure over each trimester of pregnancy and total exposure over the whole of pregnancy. Each was then categorised into overall and trimester-specific quintiles.Monthly mean, 5 km × 5 km gridded, sunshine hours were downloaded from the Meteorological OfficeInclusion in the study was restricted to singleton children born in Scotland at term; defined as 37–41 weeks gestation inclusive. We excluded children whose age was recorded as <4 years or >19 years in the School Pupil Census. Since UV data were only available for 2000–2010 inclusive, children conceived prior to 2000 or born after 2011 were excluded as they did not have UV exposure data for the whole of their intra-uterine period.Firstly, we tested whether there was evidence of any association between overall solar radiation and learning disabilities using sunshine hours as a proxy measure of the former. A series of univariate logistic regression analyses were performed of the associations between trimester specific and overall sunshine hours and learning disability. The models were then adjusted sequentially for month of conception then child’s sex then number of sunspots. The trimester specific models were then adjusted for the number of sunshine hours in the other two trimesters. Secondly, we explored whether there were associations specific to UVB exposure.The same models were run using UVB as the exposure and adjusting for month of conception, then child’s sex, then UVA exposure and, finally, the trimester specific models were adjusted for UVB exposure in the other two trimesters. All analyses were undertaken using Stata v14.


Phlebotomus neglectus males. The virus was provisionally named the Drin virus after a river near the locality of Kukës, where the infected sand flies were trapped. Genetic and phylogenetic analysis revealed that the Drin virus is closely related to the Corfou (CFUV) virus, isolated in the 1980s from Phlebotomus major sand flies on the eponymous island of Greece, and may also be involved in human infections because of its similarity to the sand fly fever Sicilian virus. The latter justifies further studies to specifically address this concern. Together with recent findings, this study confirms that Albania and the Balkan peninsula are hot spots for phleboviruses.Phlebotomine sand flies are generalist vectors with significant implications for public health. They are able to transmit phleboviruses that cause sand fly fever, headaches, or meningitis in humans. Albania is a country in Southeast Europe with a typical Mediterranean climate which provides convenient conditions for the presence of sand flies. Hence, the circulation of phleboviruses, such as the Toscana and Balkan viruses, has been recently described in the country. We followed a virus discovery approach on sand fly samples collected in 2015 and 2016 in seven regions of Albania, with the aim to investigate and characterize potentially circulating phleboviruses in phlebotomine sand flies. A presumed novel phlebovirus was detected in a pool consisting of 24  Phlebovirus, family Phenuiviridae, order Bunyavirales) can be classified into three serological complexes, which are also regarded as taxonomic species or tentative species—the Sandfly fever Naples phlebovirus species (SFNV), the Salehabad phlebovirus species, and the Sandfly fever Sicilian phlebovirus tentative species (SFSV). The SFNV includes the following viruses: Arrábida (ARRV), Balkan (BALKV), Fermo (FERV), Gordil (GORV), Granada (GRAV), Massilia (MASV), Punique (PUNV), Saddaguia (SADV), Saint-Floris (SAFV), sand fly fever Naples (SFNV), Tehran (THEV), Toscana (TOSV), and Zerdali (ZERV) . The Salehabad phlebovirus species includes Adana (ADAV), Adria (ADRV), Alcube (ALCV), Arbia (ARBV), Arumowot (AMTV), Medjerda Valley (MVV), Odrenisrou (ODRV), Olbia (OLBV), Salehabad (SALV), Bregalaka (BREV), and Zaba (ZABAV) . The SFSV tentative phlebovirus species is a serocomplex which includes several phlebovirus species that are yet to be recognized by the International Committee on Taxonomy of Viruses (ICTV). The SFSV and Corfou (CFUV) phlebovirus tentative species are antigenically and genetically close to each other and may be grouped together as a unique species [According to their antigenic relationships, old world sand fly-borne phleboviruses , followed by P. perfiliewi (14.4%), P. papatasi (4.6%), P. tobbi (3.6%), P. similis (1.8%), P. simici (<1%), Sergentomyia dentata (<1%), and Sergentomyia minuta (<1%) [Albania has a typical Mediterranean climate that provides convenient environmental conditions for the active circulation of sand flies between May and November. To date, a total of eight sand fly species have been described in Albania, ta (<1%) ,12.Salehabad phlebovirus species [Recently, several phleboviruses were identified in specific areas of Albania—the BALKV virus, belonging to the SFNV species and the ADRV virus, belonging to the  species ,13.Our aim was to investigate and characterize the phleboviruses in phlebotomine sand flies collected in more than one region in Albania from June to September of 2015 and 2016.2 baited traps  were used to collect sand flies in seven regions of Albania from June to September of 2015 and 2016 ) with 3 mm tungsten beads added into tubes. Sand fly tissues were homogenized by a Mixer Mill MM300 , and the mixture was centrifuged at 5800× g for 10 min.A volume of 600 μL of Eagle’s minimal essential medium (EMEM) , 1% penicillin–streptomycin, 1% (200 mM) 2 atmosphere. The nucleons were examined daily for the presence of a cytopathic effect (CPE) and passaged 8 times. In each passage, 200 μL of supernatant medium was collected from the nucleons and tested by reverse transcription PCR (RT-PCR) with stratifin (SFN), Nphlebo, and Lambert primers [A volume of 50 μL homogenate supernatant was inoculated into 450 μL Vero cells in enriched EMEM without FBS. The cells were then incubated at room temperature for 1 h. After 1 h, 2.5 mL of fresh EMEM with 5% FBS was added and the nucleons incubated at 37 °C in a 5% CO primers ,15,16,17A volume of 2 μL of the original homogenate supernatant was used in an attempt to infect 2 to 3 day-old Oncins France 1 (OF1) mice intracerebrally. The mice were observed for signs of disease and sacrificed on days 6 to 14 of the experiment. These animal experiments were carried out according to the animal experiment regulations of Aix-Marseille Université under the license A1301309. The RNA was extracted from the brain tissues of the mice by using a QIAcube HT system with a QIAamp 96 Virus QIAcube HT Kit  according to the manufacturer’s instructions and studied for the presence of the Drin virus.A volume of 200 μL homogenate supernatant was used for nucleic acid extraction. For the 2015 sand fly samples, the extraction was performed by a BioRobot EZ1-XL Advanced  with a Virus Extraction Mini Kit (Qiagen). The samples from 2016 were extracted using QIAcube HT system with a QIAamp 96 Virus QIAcube HT Kit .RT-PCR was performed with the Super Script III (Thermo Fisher Scientific) kit using three different systems: (1) the Nphlebo1S/1R system targeting the polymerase gene in the L RNA segment, (2) the SFNV1S/1R system targeting the nucleoprotein gene in the S RNA segment, and (3) the Lambert Phlebovirus system, also targeting the S RNA segment ,15,16,17Nested PCRs were performed with a DreamTaq kit  with Nphlebo2 and SFNV2 primers. The following cycle was used for the Nested-PCRs: 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, the annealing temperature for 45 s, and 72 °C for 30 s, with a final elongation step at 72 °C for 7 min.Real-time quantitative RT-PCR (RT–qPCR) was performed with the EXPRESS qPCR SuperMix Universal  kit and the Quant Studio 12K Flex System. The cycling program of the RT-qPCR reaction consisted of 50 °C for 5 min and 94 °C for 20 min, followed by 40 cycles at 95 °C for 3 s, then 60 °C for 30 s. Specific primers were used to screen for the SFSV group viruses and TOSV ,18.The Drin virus was identified as closely related to the Corfou virus based on the partial sequence obtained from the Nphlebo primers in the polymerase (L RNA).Because of the closeness between the polymerase partial sequences of the Drin and CFUV viruses, we designed new primers using the CFUV virus sequences as a reference. We assumed that these primers would also amplify the Drin virus segments .Additionally, we designed real-time RT-qPCR assays specifically targeting the Drin virus L-, M- and S-segments to test all the pools to check whether the Drin virus sequences were present in any of the other analyzed pools . The samThe positive PCR products were purified using a QIAquick PCR purification kit  and sequenced with next-generation sequencing (NGS) using an AU4c Ion-Torrent PGM platform as described .The sequence data was analyzed through CLC Genomics Workbench version 11 software . Homologous sequences from phleboviruses were aligned with homologous sequences of interest using Clustal W in Molecular Evolutionary Genetics Analysis 6 (MEGA6) software . The evoc oxidase I (COI) and cytochrome b (Cytb) genes, respectively, as previously described [To identify the sand fly species constitutive of the Drin virus positive pool, barcoding PCR was performed with 3 μL of nucleic acid to amplify partial regions of the cytochrome escribed ,22. The COI gene and 397 reads for the Cytb gene. All 7307 reads 100% matched the P. neglectus sequences, and thus demonstrated that the 24 sand flies contained in Pool #17 belonged to the P. neglectus species.A total of 2502 sand flies were tested for phlebovirus presence, 533 collected in 2015 , and 1969 in 2016  in seven different regions of Albania . They weVero cells inoculated with supernatant corresponding to Pool #17 were examined daily for CPE. Eight blind passages were done, but no CPE was observed. In addition, each passage was tested for the presence of Drin virus RNA using the SFN, Nphlebo, and Lambert sets of primers; the results were consistently negative, indicating that the Drin virus was not isolated in cell culture. For this reason, we decided to try virus isolation by using intracerebral (IC) inoculation into suckling mice. A total of 25 baby mice were injected with sand fly homogenate supernatant with the attempt to isolate the Drin virus. None of the mice developed clinical signs, which suggested that the Drin virus was not replicating in their brain tissues. Baby mice were euthanized at 7 days post-IC injection, the brain was then collected postmortem, with nucleic acid extraction performed as described above and tested with different PCR assays. All results were negative, showing that the Drin virus had not been isolated.Partial sequences of the three RNA segments  were amplified and sequenced. In all cases, the Drin virus sequences were most closely related to, but clearly distinct from, those of the CFUV virus .Regardless of the gene used for analysis, the Drin virus is grouped with the CFUV and Toros viruses (with 99–100% bootstrap support), the three viruses forming a sister cluster to the group including the Dashli virus and strains of SFSV .All these viruses form a solid monophyletic cluster which is consistent with the proposal that they should all be placed within a single species, one that is yet to be recognized by the ICTV as a SFSV species.Phlebovirus. Recently, the isolation of the Dashli virus in Iran has been an opportunity to propose to group SFSV, CFUV, and Dashli viruses within a single species putatively named Sandfly fever Sicilian phlebovirus species [The viruses belonging to the SFSV and CFUV tentative phlebovirus species have previously been described in Italy, Turkey, Cyprus, Greece, Iran, and Ethiopia, but they have never been detected in Albania or the other countries of the Western region of the Balkans ,24,25,26 species .P. neglectus species which is the predominant species in that area. Other studies have demonstrated that phleboviruses were potentially transmitted by several distinct species of sand flies [COI and Cytb genes of sand flies is an efficient approach for species identification within a pool of insects. Here, all sand flies included in Pool #17 were P. neglectus, suggesting that this species is capable of transmitting the Drin virus between sand flies and possible vertebrate hosts. As P. neglectus is widespread in the Balkan peninsula, in Eastern Europe, and in Turkey [In this study we identified a novel phlebovirus, tentatively named the Drin virus after the eponymous river in the Kukës region of Albania. The virus was identified in a pool containing only male sand flies belonging to the nd flies . The secnd flies ,28,29,30nd flies . Assuminnd flies . There ind flies . Sand flnd flies , NGS anan Turkey , and becn Turkey  and Toscn Turkey , competeGene by gene comparative distance analysis showed that pairwise distances of the Drin virus versus CFUV and Toros viruses were consistently lower than the lowest distances observed between Drin virus and SFSV–Dashli virus. Detailed genetic distance analyses have been done for the SFSV and CFUV phlebovirus tentative species by Alkan et al. .Larroussius). Evidence pointing to the Drin and CFUV viruses as two distinct viruses—they have been discovered in different species of sand flies, they were discovered in different countries, and the Drin virus was found in sand flies trapped in the northern part of Albania which has no contact with Corfou island that faces the Ionian coasts of Albania. It is not known whether cross-neutralization exists for the Drin and CFUV viruses, and the isolation of the Drin virus is required to conduct the proper experiments to address this question.The limited genetic divergence between partial sequences of the Drin and CFUV viruses questions whether they are two distinct viruses or two strains of the same virus. Since taxonomy does not regulate delineation below the species level, there may be contradictory elements. In favor of considering that the Drin and CFUV viruses are two strains of the same virus—they are genetically very close, and they have been detected in two species of sand flies belonging to the same subgenus  showing that SFSV was circulating in Portugal, but there is not any seroprevalence survey to document this in Albania .Sandfly fever Naples phlebovirus species, Adria, Zaba and Bregalaka viruses for the Salehabad phlebovirus species, CFUV and Drin viruses for the tentative SFSV phlebovirus species.As a consequence, there are representatives of the three main species of sand fly-borne phleboviruses in Albania  and in the Balkan as well: Toscana and Balkan viruses for the SFSV has been described in an extended geographic area from southwestern Europe to the Middle East including northern Africa. SFSV epidemics were recorded in Italy, Cyprus, Turkey, and Ethiopia ,27. Furt


Additionally, the structure–activity relationship of isolated HMW melanoidin fractions were analyzed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The results show that roasting at a temperature of 150 °C and a relative air humidity of 0.3% effectively enhances the total phenolics content and the antioxidant potential of almost all HMW cocoa melanoidin fractions. The ATR-FTIR analysis revealed that the various mechanisms of action of HMW melanoidins isolates of different types of cocoa beans related to their structural diversity. Consequently, the results clearly demonstrated that HMW cocoa fractions isolated from cocoa beans  roasted at higher temperatures with the lower relative humidity of air possess high antioxidant properties in vitro.Melanoidins from real foods and model systems have received considerable interest due to potential health benefits. However, due to the complexity of these compounds, to date, the exact structure of melanoidins and mechanism involved in their biological activity has not been fully elucidated. Thus, the aim of this study was to investigate the total phenolic content, antioxidant properties, and structural characteristics of high-molecular weight (HMW) melanoidin fractions isolated by dialysis (>12.4 kDa) from raw and roasted cocoa beans of Criollo, Forastero, and Trinitario beans cultivated in various area. In vitro antioxidant properties of all studied HMW cocoa fractions were evaluated by four different assays, namely free radical scavenging activity against DPPH Bifidobacteria in the gut [Enterobacter and Escherichia. More recent studies have reported that LMW (<10 kDa), intermediate molecular weight (10–30 kDa), and HMW (>30 kDa) fractions isolated from cocoa powder by ultrafiltration have a dose-dependent inhibitory activity against α-glucosidase [In recent years, a number of biological properties and potential health benefits of consuming cocoa-derived products have been investigated, e.g., antioxidant, anti-inflammatory, anti-carcinogenic, and antifungal properties. Much research has focused on the cocoa phenolic compounds, such as flavonoids , as a potential health-promoting compounds due to its antioxidant capacity and abundance in the cocoa beans. However, the bioactive compounds composition of cocoa beans is influenced by many factors including variety, climatic and agronomic conditions, post-harvest practices, and storage conditions ,2,3. Mor the gut ,11. Curr the gut . Phenoli the gut ,14. Howe the gut ,11,15, o the gut ,17 invescosidase . This accosidase . In our cosidase ,20. In ocosidase . In thatTheobroma cacao L. types. To the best of our knowledge, this is the first report concerning the determination of in vitro antioxidant activity and ATR-FTIR structural characterization of isolated HMW melanoidin fractions of Criollo, Forastero, and Trinitario beans from different regions of Africa.In continuation with our previous study, this work was designed to carry out a comparative investigation of total phenolic content, antioxidant activity, and structural characteristic of HMW melanoidin fractions isolated from cocoa beans, both raw and roasted at different temperatures  and relative air humidity levels (RH 0.3% and 5.0%) of three Gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1- hydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), sodium acetate, ferric chloride hexahydrate, ferrozine, and ammonium acetate were purchased from Sigma-Aldrich . All other reagents were of analytical grade and were purchased from Chempur . Ultrapure water (resistivity 18.2 MΩ cm), obtained from a Milli-Q purification system , was used for all analyses.T. cacao groups: Criollo and Trinitario originating from Madagascar, and Forastero from Ghana. All cocoa beans were harvested at their technological maturity in 2013 and purchased from commercial sources. Cocoa fruit reaches physiological maturity around 5 months after flowering and is harvested at 5–6 months. Cocoa pods harvested at 5–6 months are considered to be technologically matured as the beans have developed optimum quality [Analyses and experiments were conducted on samples of fermented and dried cocoa beans of the three main  quality . The desRaw cocoa beans of each group after removal of impurities and broken or chipped beans were convectively roasted in batches of 200 g in a tunnel with the forced air flow without circulation (adapted for either dry or humid air). Process air humidity was gradually increased using 4.0 MPa saturated steam from a generator. Roasting was performed at four temperatures  and two relative air humidity levels (0.3% or 5.0%). The heat treatment parameters were chosen to obtain a range of roasted beans with acceptable physico-chemical and sensory properties. Usually, during roasting, the raw cocoa beans are exposed to temperatures that range from 135 to 150 °C, whereas the “fine or flavor” varieties require lower temperatures than the “bulk” ones . It was g). Subsequently, pooled extracts were cooled to room temperature and filtered through Whatman no. 4 filter paper to remove insolubles. An aliquot of the filtrate was dialyzed using a dialysis tubes  for 1 day under running tap water and for 2 days against 2000 mL of water at 4 °C with constant stirring. The water was changed 4–5 times until no further color was visible in the dialysate. The dialysate containing LMW compounds was removed. After dialysis, the retentate containing the HMW fraction was frozen at −20 °C and lyophilized  using a DELTA 1-24LSC Christ freeze drier . All lyophilized HMW materials were then stored in plastic bags at −20 °C to prevent hydration until used.The HMW melanoidin fraction was obtained from raw and roasted cocoa beans by dialysis according to our previously research . Briefly2CO3 solution was added to the mixture. The final volume was adjusted to 10 mL with high-purity deionized water. The solution was then mixed vigorously and allowed to stand at room temperature in the dark place for 60 min. The absorbance of reaction mixture was measured at 765 nm using a UV-1800 spectrophotometer . For each sample, experiments were conducted in triplicate. The results were expressed as mg gallic acid equivalents (GAE) per gram of lyophilized HMW cocoa fraction dry weight (mg GAE/g dw).Total phenolic contents (TPC) of HMW isolates from raw and roasted cocoa beans were determined using the Folin–Ciocalteu method, as described by Belšcak et al. . BrieflyThe free radical-scavenging activity of the HMW isolates from raw and roasted cocoa beans was determined by the DPPH and ABTS assays as previously described by Oracz and Nebesny . All ana3·6H2O and then warmed at 37 °C for 30 min before using. Aliquots (0.1 mL) of the suitably diluted lyophilized HMW cocoa samples with high-purity deionized water (1.5 mg/mL) were mixed with 4 mL of fresh FRAP solution. The solution was then mixed vigorously and allowed to stand for 30 min at 37 °C. The absorbance of reaction mixture was measured at 593 nm using a UV-1800 spectrophotometer . For each sample, experiments were conducted in triplicate. The results were expressed as µmol Trolox equivalents per gram of lyophilized HMW cocoa fraction dry weight (μmol TE/g dw).The FRAP assay was performed according to the protocol described by Pastoriza and Rufián-Henares  with sli2. After mixing, the solution was allowed to stand at room temperature for 30 s, followed by the addition of 100 μL 5 mM ferrozine. The reaction mixture was then vortexed and left to stand at room temperature for 15 min. The absorbance of the solution was measured spectrophotometrically at 562 nm with UV-1800 UV-VIS Spectrophotometer . Worth noting, that low absorbance of the resulting solution indicated a strong ferrous ion chelating ability. A reaction mixture containing 1 mL of high-purity deionized water instead of sample solution served as the blank. The results are expressed as the percentage chelating activity (%).Chelating activity of the HMW isolates from raw and roasted cocoa beans was evaluated using the method of Gu et al. . Briefly−1 with 40 scans and a resolution of 4 cm−1. Around 5 mg of lyophilized HMW cocoa fractions was deposited on the diamond platform prior to measurement. Background and sample spectra were acquired at 4 cm−1 resolution with 40 scans from 400 to 4000 cm−1The infrared spectroscopy is based on the absorption of radiation due to vibrations bonds of molecules ,26. The p ≥ 0.05, the data were subjected to the analysis of variance (ANOVA). The effects of variety, roasting temperature, roasting air humidity, and their interaction on total phenolic content and antioxidant activity of HMW melanoidin fractions were tested by means of two-way ANOVA. The significant differences among the means were estimated through Tukey’s HSD test. For all statistical analysis, p < 0.05 was considered as statistical significance. The error bars in all figures correspond to the standard deviations. The correlation coefficients between investigated parameters were assessed by means of the Pearson correlation test using Microsoft Office Excel 2016 .The results are presented as mean ± standard deviations of three replicates. Statistical tests were evaluated by using the Statistica 13.0 software . The obtained data were tested for normal distribution (Shapiro-Wilk test) and equal variances (Levene’s test). As all values showed normal distribution and homogeneity of variance, considering T. cacao groups and origins. In this work, the total phenolic content and the antioxidant activity of the HMW melanoidin fractions (>12 kDa) from raw and roasted, at different temperatures and relative air humidities, cocoa beans of different groups were evaluated by using different in vitro spectrophotometric assays.The present study is a continuation of our previous work ,20 explop < 0.05) in TPC of the HMW fractions were found between the three cocoa types. The HMW fractions from both raw and roasted Criollo beans were characterized by the highest TPC, ranging from 121.99 to 149.90 mg GAE/g DW. Less phenolics were observed in HMW isolates from Forastero beans (117.46–139.12 mg GAE/g DW), while HMW materials from Trinitario beans contained the lowest amounts of these compounds (93.02–129.00 mg GAE/g DW). In order to assess the influence of cocoa variety, roasting temperature, roasting air humidity, and their interactions on TPC content of HMW melanoidin fractions, a two-way ANOVA was carried out. The results showed that the cocoa variety and roasting process parameters significantly affected the TPC of HMW melanoidins isolated from the studied cocoa beans (p < 0.01). Generally, more phenolics were contained in the almost all of HMW fractions isolated from roasted beans than from the raw ones. The greatest increase in TPC  of the HMW fractions was observed when cocoa beans of the Trinitario type were roasted at 110 °C. Moreover, it was also noticed that thermal processing of Criollo and Forastero beans at 150 °C with RH of 0.3% led to the highest increase in the TPC . As can be seen from the results, the changes in the TPC of the HMW fractions also depended on the RH and in almost all samples were slightly less advanced when the air humidity was increased from 0.3% to 5.0%. Thus, it was confirmed that influence of the roasting parameters on TPC of the HMW fractions is complex, and huge differences are observed depending on applied cocoa bean variety and process conditions, like temperature and relative air humidity level. According to Pérez-Martínez et al. [The total phenolics content (TPC) of HMW melanoidin fractions from the three cocoa types is shown in z et al. , the Folz et al. ,28.Summa et al.  reportedT. cacao groups and origins were assessed against DPPH and ABTS radicals , roasting temperature (p < 0.001), roasting air humidity (p < 0.01), and interaction of variety and roasting conditions (p < 0.001) for the DPPH radical-scavenging activity (p < 0.05) activity against DPPH (689.16 μmol TE/g dw) but the similar against ABTS radical cations (806.22 μmol TE/g dw). The lowest radical scavenging abilities against the ABTS and DPPH assays was exhibited by HMW isolates from unroasted Trinitario beans, which is consistent with the results of TPCs discussed above. These results showed that, depending on the cocoa type and roasting conditions, an increase or decrease in the antioxidant capacity of HMW melanoidin fractions was observed. Roasting of all types of cocoa beans at temperatures ranging from 110 to 150 °C caused a significant increase (p < 0.05) in the DPPH radical-scavenging activity of HMW fractions. However, the changes in the free radical-scavenging activity of HMW fractions were considerably smaller at the higher RH. Interestingly, we observed that HMW fractions from Criollo beans roasted at 150 °C with lower RH exhibited a higher antioxidant activity by DPPH scavenging assay (1475.61 μmol TE/g dw) compared to all other melanoidin samples. Roasting of Forastero and Trinitario beans at temperatures ranging from 110 to 150 °C caused a significant increase (p < 0.05) in the ABTS radical-scavenging activity of their HMW melanoidin fractions. It was also observed that the HMW fractions from Criollo beans, roasted at temperatures of 110, 120, and 150 °C, displayed the significantly increased ABTS scavenging capacity, while after roasting at 135 °C, exhibited a slightly lower antioxidant activity by this method. Nevertheless, among HMW melanoidin fractions isolated from roasted beans of the three cocoa varieties, the highest ABTS radical-scavenging capacity (1172.37 μmol TE/g dw) was exhibited by the fraction from Criollo beans roasted at 120 °C and RH of 0.3%. Based on the results presented in • than ABTS•+ radicals in the case of chocolate melanoidins. Our results corroborated also with those reported by Summa et al. [• radical involve transfer of a hydrogen atom, while the reactions with ABTS•+ radicals involve electron transfer process [2), such as phenolic compounds, quinones, and low molecular weight MRPs, in the HMW materials. These compounds might be attached to the structure of melanoidins via non-covalent bonds and influence their biological properties [2). This was a clear indication that the synergistic effect between different bioactive compounds present in the structure of melanoidins could determine their biological properties. Since, the HMW cocoa melanoidins have the ability to scavenge free radicals, thereby preventing lipid peroxidation chain reactions that cause damage a wide range of molecules found in living cells, they could serve as potential nutraceuticals and functional ingredients.However, there were significant differences between HMW melanoidin fractions from both the raw and roasted cocoa beans regarding their antioxidant abilities. Two-way ANOVA revealed significant effect of cocoa variety  complex to TPTZ–Fe (II). In order to identify the influence of cocoa variety, roasting temperature, roasting air humidity, and their interactions on ferric reducing antioxidant power (FRAP), a two-way ANOVA was performed. As demonstrated in p < 0.05) in the reducing properties of HMW fractions were found between the three cocoa types. The HMW fractions from Criollo beans exhibited the highest FRAP values, ranging from 622.42 to 1077.18 μmol TE/g dw, followed by isolates of Forastero beans (533.90–947.46 μmol TE/g dw), while HMW materials of Trinitario beans had the lowest reducing power (440.49–779.49 μmol TE/g dw). The results also showed that the roasting process parameters significantly (p < 0.05) affected the reducing properties of HMW materials isolated from the studied cocoa beans. Generally, all of HMW fractions isolated from unroasted beans showed a lesser reducing power compared to the isolates from the roasted samples. The greatest increase in the ferric reducing ability  of HMW fractions was observed when cocoa beans of all studied cocoa groups were roasted at 135 °C with RH of 0.3%. As can be seen from the results, the changes in the reducing power of HMW fractions depended on the RH and in almost all samples was slightly more pronounced when the air humidity was decreased from 5.0% to 0.3%. We can conclude that all studied HMW cocoa melanoidin fractions behaved as reductants and inactivators of oxidants due to the presence of electron-donors in their structure. The observed increase in the reducing capacity of HMW cocoa fractions could be linked to the formation of new substances able to donate electrons or to terminate radical chain reactions, during roasting of cocoa beans. As described above, the HMW cocoa melanoidin fractions contained different amounts of free and bound phenolic compounds and the presence of these polyphenols along with compounds formed in the advanced and final stages of Maillard reaction could contribute to the antioxidant properties observed by FRAP method. These residues due to the presence of hydroxyl and pyrrole groups can act as reducing agents via their redox potential of transferring electrons [The reducing capacity may serve as a significant indicator of potential antioxidant activity. Thus, the HMW melanoidin fractions of unroasted and roasted cocoa beans of tree different lectrons ,33. In alectrons , it was p < 0.001), roasting temperature (p < 0.001), roasting air humidity (p < 0.01), and their interactions (p < 0.001).Our results suggest that the antioxidant capacity of HMW fractions from raw and roasted beans depend on the cocoa type and roasting conditions, wherein no significant correlation was observed between DPPH, ABTS, and FRAP values. This may be explained by the fact that different constituents in the HMW fractions would have different mechanisms of action regarding their antioxidant activities. According Tabart et al. , the wei2+), being the most powerful pro-oxidants among various species of metal ions. The ferrous ion in the Fenton reaction can catalyze the generation of potentially toxic reactive oxygen species (ROS), such as hydroxyl radicals (•OH) that initiate lipid peroxidation [p < 0.001). The results of this study demonstrate that the chelating activity of HMW fractions isolated from beans of the three cocoa types was increased by roasting at temperatures in the range of 110 to 150 °C, which may be ascribed to structural changes in melanoidins and phenolic compounds. Moreover, the changes in the iron binding ability greatly depended on the roasting air humidity (0.3–5.0%) and were considerably smaller when the RH was elevated. The chelating activity of the HMW isolates obtained from roasted samples varied from 45.21% to 92.07%. The increase in the metal-chelating activity was more pronounced in HMW isolates from Forastero and Trinitario beans, while the lowest changes were observed in the Criollo samples. Irrespective of the cocoa type, the greatest increase in the iron chelating ability occurred when cocoa beans were roasted at 150 °C and the lower relative humidity of air (RH of 0.3%) whereas the lowest rise was caused by roasting at 110 °C with humid air (RH of 5.0%). These results are consistent with the described above differences in the free radical scavenging activities and the reducing properties of HMW cocoa. However, unlike to the WAAC, the melanoidin fraction of Forastero beans roasted at higher temperatures exhibited the highest ferrous ion chelating potential, compared to other cocoa groups (even in the relation to Criollo samples).The changes in ferrous ion chelating activity of HMW fractions, caused by roasting of beans of all tested cocoa groups, are presented in xidation . It was Such differences could be explained by variations in the composition of studied HMW melanoidin fractions that are strongly determined by cocoa variety and roasting conditions ,20. In a−1 to 4000 cm−1 wavenumber region) of the cocoa melanoidins are given in To provide a comprehensive explanation about the antioxidant properties of HMW cocoa fractions, it was necessary to precisely characterize the chemical structure of these materials. We report here for the first time the ATR-FTIR spectroscopic characterization of HMW cocoa melanoidins. The FTIR spectra of HMW isolates obtained from raw and roasted cocoa beans were collected with FTIR spectrometer equipped with an ATR sample accessory. The ATR-FTIR spectra , C=C, or C=N [−1 due to the stretching vibrations of the entire anhydroglucose ring. All mentioned bands confirms the presence of a phenol group sensitive to hydrogen bonding [−1 in the FTIR spectra were attributed to the stretch of the C–H of aromatic ring and would be due to stretching vibrations of CH2 and CH3 groups that can originate from fatty acids present in the cocoa melanoidin fractions [−1 clearly reflects adsorption of aliphatic compounds in the melanoidins structure. Some reports revelated that interactions between reactive carbonyl compounds arising from lipid oxidation reactions and amino acids or proteins might play an important role in the formation of brown HMW macromolecular compounds upon high-temperature processing of cocoa beans [−1 were ascribed to N–H bending vibrations from amine or amide groups, and C=O stretching vibrations from flavonoids, phenolic acids and its derivatives, quinones, and lipids [−1 mainly corresponds to the C=O stretching vibrational mode of the different structures of the protein backbone. The band at 1515 cm−1 was attributed to C=C stretching vibrations from aromatic rings of phenolic compounds [−1 is closely correlated with the antioxidant activity of plant extracts. In this study, in the FTIR spectrum of HMW fractions of Trinitario beans, the intensity of the 1630 cm−1 peak is significantly lower compared to the Criollo and Forastero melanoidins. This behavior corroborates the results obtained in DPPH˙ and ABTS scavenging assays and could be a consequence of the higher number of phenolic groups in the HMW melanoidin fractions of Criollo and Forastero groups compared to those of Trinitario type. Moreover, the relative intensity of this peak increases during roasting of almost all cocoa beans, which may be the result of adsorption of phenolic compounds and/or its derivatives in the melanoidin structures. This was a clear evidence that the studied HMW melanoidin fractions possessed residues (OH or NH2 groups) that could act simultaneously as hydrogen and electron donors. Our previous study on UHPLC-DAD-ESI-HR-MSn analysis of HMW melanoidin fractions derived from two different types of cocoa beans revealed that both free and bound phenolic compounds, including three flavan-3-ols, seven phenolic acids, one phenolic aldehyde, and four n-phenylpropenoyl-L-amino acids (NPAs), are present in these fraction [−1 is called the fingerprint region. This region of the infrared spectrum contains vibrations that are specific for the large number of infrared bands, including C–O, C–C, and C–N single bond stretches, C–H bending vibrations, and some bands due to benzene rings [−1 corresponded to C–O–C and C–O stretching vibrations of the glycoside linkage and C–O bond stretching vibration in glycerol. According to the literature, the occurrence of this band is an evidence of polysaccharide and lipids in samples [The heterogenic nature of the cocoa melanoidins has been confirmed by Fourier transform infrared spectra of HMW fractions isolated from different types of , or C=N  and char bonding . Moving  bonding . Therefpractions . A igheroa beans ,45. Bandd lipids . The banompounds ,26,45. Mompounds  noticed fraction . Most ofne rings ,26. The  samples ,26.Our results demonstrate that ATR-FTIR spectroscopy may be used as a direct and nondestructive method for the rapid investigation of the structural characteristics and functional properties of the HMW melanoidin fractions. According to our findings, the presence of different types of compounds in HMW melanoidin fractions is responsible for their bioactive properties  and its different mechanisms of action.T. cacao groups. The results showed that the cocoa type and roasting conditions affect the total phenolic content and antioxidant properties of HMW melanoidin fraction isolated from the studied beans. The ATR-FTIR analysis revealed the presence of different bioactive compounds with various mechanism of action in HMW cocoa melanoidin fractions. We found that, both for TPC and in vitro antioxidant activity, HMW cocoa melanoidin fractions of Criollo beans showed significantly higher values than those of Fosrastero and Trinitario samples. Moreover, we observed that that roasting at higher temperatures with the lower relative humidity of air effectively enhances in vitro antioxidant potential of almost all HMW fractions isolated from cocoa beans. Consequently, irrespective of the cocoa type, the thermal processing at 150 °C and RH of 0.3% can be recommended to obtain HMW materials with the highest total phenolic content and strong in vitro antioxidant potential. This fact could indicate that the thermal processing of cocoa beans enhances the concentration of bioactive molecules in HMW fractions that contribute to the antioxidant response observed in in vitro tests. Our findings suggest, in general, that optimization of the roasting conditions and choosing an appropriate cocoa variety may provide functional advantages by enhancing in vitro antioxidant properties of HMW fractions of cocoa beans.In conclusion, the present study, to the best of our knowledge, was the first time a comprehensive study was carried out on the total phenolic content and antioxidant activities, as well as structure–activity relationships of HMW cocoa melanoidin fractions isolated from raw and roasted, under different temperature and relative air humidity conditions, cocoa beans of different


These and other related markers  have been linked with cognitive health. This study explored the efficacy of WPI for improving cognitive function via delivery of vitamin B12. Moderately vitamin B12-deficient participants aged between 45 and 75 years (n = 56) were recruited into this randomised controlled crossover trial. Participants  consumed 50 g whey  or soy protein isolate  for eight weeks. Following a 16-week washout phase, they consumed the alternative supplement. Consumption of WPI significantly improved active B12 and folate status but did not result in direct improvements in cognitive function. However, there was evidence of improvement in reaction time (p = 0.02) and reasoning speed (p = 0.04) in the SPI condition for females. Additional analyses showed that changes in active B12, HcY and folate measures during WPI treatment correlated with improvements in cognitive function . Results indicate that WPI itself did not result in improved cognitive function but some evidence of benefit of SPI for females was found. However, consistent with previous research, we present further evidence of a role for active B12, HcY and folate in supporting cognitive improvement in adults with low B vitamin status.Whey protein isolate (WPI) is high in vitamin B The impact of cognitive decline in a dramatically ageing population is one of the biggest challenges of the future, given its impact on both the individual and society . Not onl12, low levels (together with low folate levels) are a common cause of elevated total homocysteine (HcY), which has been shown to be an independent risk factor for cognitive decline and Alzheimer’s disease [12 deficiency is common in older adults, with prevalence increasing substantially with age [12 [12 levels, with prevalence increasing to ~30% for men aged ≥70 years and women ≥80 years [Epidemiological and observational studies suggest a relationship exists between nutrient deficiencies and cognitive decline in ageing adults ,4,5. In  disease ,10,11,12with age ,16,17,1880 years . 12 and its association with cognitive impairment in older adults [12 concentrations and cognitive decline. However, it was noted that the four studies that used alternate biomarkers of vitamin B12, that is, methylmalonic acid  and holotranscobalamin  did find evidence of an association with cognitive status [12 deficiency appear to display a stronger relationship with cognition than serum B12 [Over the past few decades, studies have produced inconsistent findings regarding vitamin Br adults  found noe status ,21,22,23erum B12 ,24,25,2612 associated with a 30% slower rate of decline in individuals aged over 65 years. Furthermore, a 5-year prospective study of 107 community-dwelling participants aged 61–87 years without cognitive impairment at enrolment showed that the decrease in brain volume was greatest among those with lower serum vitamin B12 and active B12 levels and higher plasma total homocysteine (HcY) and MMA levels at baseline [A 10-year longitudinal cohort study of 1648 individuals  found thbaseline . These rbaseline , in whic12, which may support brain health in diverse ways [12, equivalent to 100% of the Recommended Dietary Allowance (RDA). Acid whey powder contains high amounts of lactose (60–70 g per 100 g), which is problematic given the increased risk of lactose malabsorption in older adults [12 (6 µg/100 g). Additionally, WPI has an 80% lower level of sodium and 14-fold higher level of natural folate relative to acid whey powder. While WPI has already been shown to be beneficial for improving cognitive performance in the context of memory tasks in stress-vulnerable subjects [12 from WPI remains relatively unexplored, particularly in relation to age-related cognitive decline. A recent comprehensive review of the literature on the association of dairy foods and cognitive function in older adults concluded that “low-fat dairy products, when consumed regularly as part of a balanced diet, may have a number of beneficial outcomes for neuro-cognitive health during ageing” and identified the whey fraction as being particularly rich in a range of bioactives, including proline-rich polypeptides, α-lactalbumin and Brse ways . Whey isr adults . Whey prsubjects , the bio12 status and related factors in individuals with subclinical B12 levels at baseline have already been reported [12 status  and changes in cognitive function during the course of the active intervention.The purpose of the present study was to consider the role that dairy-based products such as WPI may have on nutritional markers associated with age-related cognitive decline. Primary outcomes including the impact of WPI supplementation on vitamin Breported  but are The study was advertised in local newspapers in the Adelaide metropolitan area and on the CSIRO clinic website. Interested participants contacted the clinic to arrange an appointment for screening. Fifty-six eligible participants attended an information session and read the study information sheet before providing written informed consent to participate in the study. 12/choline/antioxidant vitamins at doses that exceed 25% of the Recommended Dietary Allowance, subclinical vitamin B12 deficiency as defined previously [12 in the range of 100–350 pmol/L, plasma MMA >0.20 µmol/L and serum creatinine concentration of 120 µmol/L or less) and willing to consume the quantities of WPI or SPI specified for the trial.Inclusion criteria were: healthy subjects aged between 45 and 75 years, not taking vitamin Beviously   randomised controlled crossover intervention, which was approved by the CSIRO Human Research Ethics Committee. Participants were randomised to daily intake of 50 g WPI or 50 g SPI (control) for eight weeks. Following an intervening 16-week washout phase, participants crossed over to the alternative supplement for the final eight weeks. The randomisation scheme was generated by the clinical trials manager using the online resource website: 12  and also refrain from taking supplements containing vitamin B12/choline/antioxidants). Dietary restrictions included limiting consumption of vegetables or pulses (2 servings/day), fruits or juices (3 servings/day), black tea or coffee (2 cups/day), chocolate (50 g/day), wine (200 mL/day) and/or beer (375 mL/day) in order to avoid an excessive intake of antioxidants to minimise variation between subjects and groups. Participants completed 3-day food and beverage intake records at baseline and endpoint to monitor their adherence to these restrictions and were supported throughout the study by a dietitian. Participants were asked to maintain their habitual diet during the intervention trial but refrain from eating foods high in vitamin BThe WPI or SPI was consumed daily as 25 g blended in 200 mL fruit juice or water twice a day so that the total daily intake was 50 g WPI or 50 g SPI each day. The WPI and SPI were provided in 25 g sachets (two per participant for each day of the intervention phases). A compliance checklist for use of the WPI and SPI sachets was completed by the participants. 12 or foods fortified with vitamin B12. Serum B12 was checked mid-way during the washout period. Those exceeding 350 pmol/L serum B12 concentration were required to restrict intake of foods rich in B12 until they achieved a < 350 pmol/L serum concentration before starting the second treatment phase. During the washout period, participants were allowed to return to their habitual dietary habits and were required to avoid consumption of supplements containing vitamin B12, respectively. Independent analysis by PathWest Laboratory Medicine WA  showed that WPI contained 5.64 µg of vitamin B12 per 100 g, whereas the amount of vitamin B12 in SPI was less than 0.34 µg/100 g. The nutritional composition of WPI and SPI is shown in 12, active B12 , methylmalonic acid (MMA), plasma total homocysteine levels (HcY) and serum folate. Complete descriptions of these measures and associated laboratory processing and results have been described elsewhere [Blood samples were collected at the beginning and end of each of the intervention phases and half-way through the washout phase, that is, at 0, 8, 16, 24 and 32 weeks, within the clinical research unit. Blood samples were used fresh or biobanked frozen at −80 °C depending on the requirements of each assay. The biochemical outcome measures included in the present analyses were: serum Blsewhere .The CSIRO Cognitive Assessment Battery (C-CAB) comprised of 10 individual cognitive tasks that measure cognitive domains including processing speed, reaction time/attention, reasoning speed, verbal and numeric working memory, immediate and delayed word memory. The tasks utilised obtain latency/response time and accuracy data with performance outcomes for each measure reflecting both of these components. The test battery was programmed to run in the Inquisit version 4 environment  and the Participants were presented with a list of 15 words obtained from the MRC Psycholinguistic Database , which iThe first task assessing processing speed was adapted from previous research . A code The second task was a computerised string search task adapted from the ETS Kit of Factor Referenced Tests . In thisTwo-choice and four-choice reaction time tasks were used to measure this domain and were adapted from our previous studies . To begiThis was measured using a letter memory task adapted from previous studies . In thisThis was measured using a number memory task adapted from previous studies  and was Approximately 20 minutes into the cognitive battery, participants were tested again on the 15 words presented at the start of the session. The test consisted of 30 trials involving the original 15 words, plus 15 new distracters not used in the immediate recognition test. All other presentation parameters and scoring parameters were as per the immediate recognition test described above. Two tasks adapted from the odd-man-out reaction time paradigm were used to assess this domain . The tesThe second task is procedurally equivalent to the first task. However, in this version, trials consisted of eight mixed shapes  arranged horizontally. The target stimuli were triangles as opposed to illuminated circles and participants indicated to which side—left or right—of the two closer triangles the odd-man-out  is located. Outcomes measures were as per above.n = 74 participants were required for a repeated-measures model assuming a small within-between interaction effect (η2 = 0.02) with 90% power. For crossover designs, as used herein, this figure is halved, requiring a minimum of n = 37 participants. The number of recruited participants who completed this study was n = 44, thus satisfying statistical power requirements.The required sample size for this study was estimated using GPower. Results showed a minimum of × % correct). Finally, performance was averaged across tasks measuring the same domain  to arrive at a composite measure.Transformations were undertaken to normalise distributions of cognitive tasks for analysis purposes. In line with previous studies , work raCognitive data were analysed at the Intention-to-Treat level and therefore included all participants who commenced the trial (whether or not they withdrew). Change in cognitive function was assessed using linear mixed effects models—capable of handling missing data—and the repeated nature of the measurements were accounted for by permitting random slopes and intercepts for each participant for each treatment. Fixed effects included time, treatment and time by treatment interactions. An unstructured covariance matrix was specified for random effects and all models controlled for participant’s sex, age and the order of assigned treatments. The assumption of normally distributed residuals was assessed and was satisfied for all models.12 and related parameters occurring during the WPI intervention period only and corresponding changes in cognitive function using first-order correlations adjusted for sex and age covariates. The calculation of change scores was thus based only on those who attended final follow-up (n = 44) and who therefore had complete data.The final analyses assessed the relationship between changes in vitamin Bp ≥ 0.42). Descriptive statistics regarding changes in cognition show that it was generally neutral or positive.Participants in this sample had a mean age of 60 years (SD = 8.5 years) and were reasonably well balanced in regard to sex . p ≤ 0.001). In addition, there were significant time by treatment interaction effects observed for reasoning speed (p = 0.04) and reaction time (p = 0.02). In both cases, performance increased over the duration of the intervention with a significantly greater increase observed during the SPI compared to WPI treatment. Additional three way interactions (time by treatment by sex) showed that these effects were only evident in females during SPI treatment (both p < 0.05). As shown in Analysis of data using mixed effects models showed significant overall time effects for reasoning speed, verbal working memory and numeric working memory  in homocysteine levels were related to improvements in processing speed, numeric working memory and verbal working memory. In the SPI condition, homocysteine changes were related to reasoning speed with higher levels equating to reduced performance. We conducted the same analyses using combined B12 (cB12), a combined indicator of corrected B12 status [12.The results of this intervention on various biochemical scores have been reported in full previously . However2 status  and the 12 status in low B12 individuals. Cognitive function was measured for a variety of important domains using a robust cognitive assessment battery. Overall, there were no effects of WPI on cognitive function, which generally remained stable across visits during this treatment. On the contrary, SPI appeared to have benefits for the cognitive domains of reasoning speed and reaction time in females only. Given the age of participants in this sample (45–75 years) and the presence of this effect true for females only, this finding possibly reflects the impact of soy isoflavones on cognitive function in post-menopausal women [This study sought to examine the effects of WPI and SPI on cognitive function and vitamin Bal women . A recenal women . 12 status during WPI treatment and changes in cognitive function. Specifically, increases in active B12 and serum folate were associated with corresponding improvements in some cognitive domains. Reductions in homocysteine levels were also consistently related to improvements in cognitive performance, including working memory. Whilst there were no clear treatment effects linking WPI specifically to improved cognitive function, the changes observed in these key biochemical markers and the relation of these to improved cognition were consistent with the broader research literature regarding the role of B12, folate and homocysteine in helping to maintain good cognitive function [Despite the absence of a main treatment effect for WPI in terms of its ability to improve cognitive performance, positive findings were evident regarding changes in biometric measures of vitamin BThe finding of direct associations between changes in biochemical and cognitive measures despite the absence of any effect directly attributable to WPI likely reflects the choice of the control treatment. More specifically, although SPI is a useful comparison given its different nutritional profile to WPI in terms of levels of B-vitamins and folate, the isoflavones present can have an impact on cognitive function, most notably in females . Therefo12 related markers and improved cognitive performance, future studies should consider the potential for dairy-based supplements to slow the rate of decline in individuals with quantifiable Age-Associated Memory Impairment (AAMI). These individuals are particularly interesting given the absence of underlying pathology  explaining their decline and the fact that they do not yet meet criteria for MCI [In addition to using a relatively active control treatment, this study employed a relatively short intervention duration (eight weeks). Prior research has proposed that interventions involving otherwise healthy participants, as used herein, require durations as long as 2-to-5 years in order to demonstrate an impact on cognitive function . Thus, i for MCI .The potential utility of using WPI or SPI for extended periods of time as a means of supplementing diets must be balanced against any potential negative impacts of these. For example, dairy (WPI) has been linked with self-reported gut discomfort and subsequent avoidance of such products . Questio12 and folate status having a role in supporting cognitive health. Specifically, important vitamin B12 and folate related markers were improved as a result of WPI supplementation and these changes were correlated with improvements in cognitive performance. However, the findings from this study are limited to similarly aged adults with subclinical vitamin B12 deficiency and further research is needed to explore how these findings may relate to other groups and populations. In conclusion, although this intervention did not demonstrate a direct effect of WPI supplementation on cognitive function, the results provide some support to the notion of vitamin B


Studies were undertaken to investigate the effects of ultraviolet (UV) irradiation on the gel strength, color, thermal properties, protein molecular masses, and functional groups of commercially available fish gelatin samples. Commercially available tilapia skin gelatin powder was used as the raw material to investigate the functional properties of fish skin gelatin powder treated with UV irradiation for different durations (0–6 h). The functional properties of fish gelatin and the optimum irradiation treatment conditions were determined through gel strength testing, color characterization, differential scanning calorimetry, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy. UV irradiation treatment increased gel strength and thermal stability, and significantly degraded the macromolecules. FTIR and Raman spectroscopy data indicated that UV irradiation treatment did not significantly change the molecular structure of fish gelatin powder, but these methods could discriminate the molecular structure of gelatin from various sources. Irradiation for 2 h yielded the highest gel strength and melting peak temperature, and the lowest chromatic aberration. Gelatin, a partially hydrolyzed protein product derived from animal collagen, is soluble in hot water and forms a reversible gel widely used in the food, medicine, beauty, and photography industries ,2,3.Commercial gelatin is a raw material sourced from collagen and bone (or related byproducts) of pigs, cattle, and other animals. However, in recent years, animal diseases among cattle and pigs, such as mad cow disease  and foot and mouth disease (FMD), have brought into question the safety of the source materials used for gelatin ,5. DurinAccording to the 2015 data of the Fisheries Agency of the Executive Yuan Agricultural Committee , the tilTo improve the functional properties of fish gelatin, studies have examined whether adding various agents, such as sugars, salts, and glycerol, can modify the functional properties of fish gelatin ,15. HoweThis study used commercially available tilapia fish skin gelatin powder as a raw material to investigate the effects of UV irradiation treatment on fish skin gelatin powder (UVFGP) for durations of 0 to 6 h and to determine the optimum processing conditions, with the objective of using UVFGP to replace mammalian gelatin. The functional properties of gelatin and the optimum irradiation treatment conditions were analyzed through gel strength analysis, colorimetry, differential scanning calorimetry (DSC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy.2, the gel strength of the control and irradiated samples (218.9 ± 1.4 Bloom exposed for 1 h and 235.9 ± 1.7 Bloom exposed for 2 h) were significantly higher than that of the control sample (208.8 ± 1.5 Bloom). These results are consistent with those of Bhat and Karim [The study examined the effect of various doses of UV irradiation on the gel strength of gelatin solution. Fish gelatin powder was treated for 0, 1, 2, 3, 4, 5, and 6 h, and the results are presented in Color is an essential indicator of raw food materials, and can be used to deduce the basic physical properties of irradiated gelatin powder. The changes in the color of gelatin powder after irradiation at different doses are presented in With increase in the dose of UV radiation, the color of the powder progressively changed from bright white to pinkish yellow. Chromatic aberration degree (ΔE) is a quantification of perceived difference in color and is calculated using L*, a*, and b* values; this parameter can be used to compare color differences between samples. When ΔE is 1.5–3.0, chromatic aberration is small yet noticeable, and when ΔE is 3.0–6.0, the aberration is significant . On irraGelatin powder treated with different doses of UV irradiation was transformed into gels . The colThe effect of UV irradiation dose  on fish gelatin powder was examined through DSC; the results are presented in The results indicate that the UV-treated fish gelatin powder underwent radiation-induced chemical changes, and that the melting peak temperature of the powder can be increased by exposure for 2 h. The gel strength of the modified fish gelatin did not increase significantly when the irradiation duration exceeded 3 h.Similarly, the pattern of the irradiated sample containing 15% acrylamide exhibited a blurred and widened band and low color brightness relative to the non-irradiated fish gelatin powder at the relative molecular weight of 28 kDa B. These The effect of UV irradiation dose on the gelatin functional groups were examined through the FTIR spectrometry of fish gelatin powders exposed to 0, 1, 2, 3, 4, 5, and 6 h of irradiation . The spe2 undergoes a functional change in its molecular structure, namely a slight difference in the amide B group. The molecular structure of fish gelatin powder did not change significantly when the energy of the UV radiation energy was low.This result is similar to that of Bhat and Karim , but is Per the FTIR spectra in −1. The δ (CH2) deformations band is at 1480–1430 cm−1, the amide III band is at 1280–1100 cm−1, and the aromatic ring breathing band is at 1070–950 cm−1. In this study, fish gelatin powder was characterized through Raman spectra  related to CH2 deformations indicate that the data obtained for the PGP and FGP groups differed significantly, whereas that for the UVFGP group did not. Second, per Sarbon et al. [−1/1246 cm−1). Finally, the results indicate similarity with the aforementioned data by selecting the tyrosine-related intensity ratio (855 cm−1/830 cm−1).n et al. , the dat2 deformations and tyrosine in the PGP and FGP groups can be used to distinguish the groups, whereas the data obtained using proline cannot. However, the data of UVFGP obtained through this method did differ significantly. The Raman experiments confirmed that treating fish gelatin powder with low-energy UV irradiation did not significantly affect the molecular structure of the powder. Nevertheless, this method can be used to differentiate between the molecular structures of gelatin powder from different sources.The final results indicate that the data analysis of CHYang and Wang  noted thTilapia skin gelatin and porcine skin gelatin were purchased from the Jellice Pioneer Provate Limited Taiwan Branch . Each 100 g gelatin sample was sealed in a 1 kg polyethylene (PE) bag. All chemicals, namely acrylamide/bis-acryamide 30% solution, ammonia persulfate (APS), 0.5 M Tris-buffer (pH 6.8), 1.5 M tris-buffer (pH 8.8), sodium dodecyl sulfate (SDS), and N, N, N0, N0-tetramethylethylenediamine (TEMED), for the electrophoresis were purchased from Bio Basic Inc. .This study adopted the method used by Sung and Chen , with fe2) measurements were performed using UV light at nine points across the stainless sheet plate and averaged. UV radiation dose measurements were performed per manufacturer instructions. Using a UV light meter  with a spectral range of 220–275 nm, illuminance (mw/cmw/v) colloidal solution; it was then poured into a test bottle (3.8 cm diameter × 2.7 cm high). The prepared solution was placed in a 10 °C refrigerator for cooling and kept for 16–18 h. After completing the sample preparation, it was immediately removed for testing of its gel strength using a texture analyzer  and a P/0.5 cylindrical probe of diameter 1.27 cm. The test speed was 0.5 mm/s and the penetration distance was 4 mm. These gel strength measurements were performed by analyzing the force–time curve of the gelatin solution in triplicate.This study followed the method developed by the British Standard Institute in 1975 . The gelThe CIELAB color  and total color difference (ΔE) of tilapia skin gelatin powder was measured using a colorimeter . After using a whiteboard for calibration, the fish gelatin powder was placed in a scanning spectrophotometer sample tray, followed by L*, a*, and b* determination. L* values range from 0 to 100 and represent the lightness of the color; L* = 0 represents black and L* = 100 represents white. Values for a* and b* range from −60 to 60, with negative and positive a* signifying green and red, respectively, and negative and positive b* signifying blue and yellow, respectively . All tes2, which was released at 50 mL/min. The thermal stability analysis of the gelatin powder was performed in triplicate to record the change of heat enthalpy (ΔH) during the test; the onset temperature (To), peak temperature (Tp), and enthalpy data values were also recorded.The DSC  analysis of gelatin powder was conducted using a method adopted from Rahman et al. , but witv/v)) and stacking gel (4% (v/v) of acrylamide). After the gel solidified, 20 μg of protein was pipetted into each well, and a voltage of 60 volts was applied. Subsequently, the gel was stained using Coomassie brilliant blue R250, and the molecular weight was determined using a molecular-weight size marker. The molecular size was relatively small because it was a fast protein.This study adopted a method reported by Lin et al. . The gel−1; scanning was performed 32 times. The spectral wave number was in the IR range of 650–4000 cm−1.This study adopted the method reported by Benjakul et al. . Fish ge−1, and a 785 nm laser was used as the excitation source. The samples were analyzed at a laser power of 100 mW, and all assays were performed in triplicate.This study adopted a version of the method reported by Sarbon et al. . This anp < 0.05.All experiments were repeated at least thrice. The results were analyzed using one-way analysis of variance (ANOVA), and Duncan’s new multirange tests by using SPSS  with the significance level set at According to the gel strength and color analysis, the color of the fish gelatin powder gradually changed from bright white to slightly yellowish after irradiation with UV light. The gel strength and the melting peak temperature of gelatin increased significantly and was the highest when irradiated for 2 h; the color underwent noticeable chromatic aberration. When the samples were irradiated for more than 2 h, the increase in gel strength and melting peak temperature was insignificant and was accompanied by a substantial yellowing phenomenon. Thus, irradiation for 2 h is optimal.The peak temperature of melting and cracking, as determined through DSC analysis, indicated that fish skin gelatin has poor thermal stability compared with commercially available porcine gelatin powder. Nevertheless, fish gelatin powder modified through optimal UV irradiation exhibited improved melting peak temperature and thermal stability similar to that of porcine gelatin powder, but the cracking peak temperature did not change significantly.Gel electrophoresis and optical spectral analyses revealed that with increased dose of UV radiation, the macromolecules of the gelatin powder degraded and accumulated at the bottom. The FTIR and Raman spectra also confirmed this finding. Thus, FTIR and Raman analyses can be used to differentiate between gelatin powders from different sources.


Magicicada spp.) are well-known for their long life cycles of 13 and 17 years and their mass synchronized emergences. Although periodical cicada life cycles are relatively strict, the biogeographic patterns of periodical cicada broods, or year-classes, indicate that they must undergo some degree of life cycle switching. We present a new map of periodical cicada Brood V, which emerged in 2016, and demonstrate that it consists of at least four distinct parts that span an area in the United States stretching from Ohio to Long Island. We discuss mtDNA haplotype variation in this brood in relation to other periodical cicada broods, noting that different parts of this brood appear to have different origins. We use this information to refine a hypothesis for the formation of periodical cicada broods by 1- and 4-year life cycle jumps.The periodical cicadas of North America ( Magicicada) adults emerge synchronously, predictably, and in overwhelming numbers; their black bodies, orange wing veins, striking red eyes, and loud acoustical choruses are unmistakable. Although early European settlers in North America mistook these insects for migratory locusts  and has a reported range whose various parts are in close proximity to or in contact with Broods I, VIII, IX, X, and XIV  periodical cicada sightings from the general public in May 2016. We used these records to inform our decisions about areas to map in detail. Individual crowdsourced records are not necessarily reliable; thus, we weighted crowdsourced records by assigning higher confidence to records that were clustered or that were in close proximity to verified records as described in http://www.cicadamania.com/ which we then investigated.The website 1 V2 V3 I4 V5 I6 V7 I8 I9 I10 I11), then we constructed a single linear feature connecting (I4 V5 I6 V7). We extracted the lengths of all linear features as conservative estimates of overlap. We did not use records of single individuals as endpoints of these features.Because roads are sparse in the parts of Virginia where Brood V contacts Brood I, direct measurements of overlaps are impractical. Instead, we made conservative estimates of overlaps by constructing linear features in Arc GIS 10.5 that linked verified presence observations of Brood I with verified presence records of Brood V. To do so we looked for instances in which series of verified records of one brood are clearly within territory occupied by the other brood; for example, if we collected 11 records along a route occupied by Broods I and V in the order  and from unpublished field notes and records in the http://www.magicicada.org database. To verify the historical presence of the newly discovered disjunct population in Carbon County, Pennsylvania, we gathered data from archived copies of newspapers in the Carbon County area for the Brood V emergence years 1883, 1897, 1931, 1948 and 1965 and 1999. We searched for the terms “cicada” or “locust”  for 64  I and V  to deter I and V . DetailsM. septendecim in Carbon County, Pennsylvania, associated with the Lehigh River in and around the town of Jim Thorpe; and (4) a previously known disjunct population of M. septendecim on eastern Long Island containing only M. septendecim (Brood V consists of four distinct parts: (1) a large body of the brood extending from the Ohio Valley to Lake Erie; (2) a nearby but separate, southeastern portion associated with the Shenandoah Valley and in close contact with Brood I; (3) a small disjunct population of tendecim –5. We fotendecim .Magicicada). Crowdsourced records suggest that we did not miss any significant populations . The UMMZ collection also contains four M. septendecim collected 5∕13∕1982 in Lehigh Gorge. Jim Thorpe is the gateway to the Lehigh Gorge.Even though the Carbon County Pennsylvania disjunct of Brood V  has not Carbon Advocate, of Leighton, predicted that periodical cicadas would appear that summer “in accordance with long established customs;” the same paper later reported on May 29 that a substantial emergence had occurred. In June 1897, the Allentown Morning Call noted the emergence of periodical cicadas in Schuylkill County, which is immediately to the southwest of Carbon County. While we did not find any periodical cicadas in Schuylkill County in 2016, we did not search the county exhaustively, and we did find periodical cicadas in Carbon County within 7 km of the Carbon/Schuylkill County line. In June 1931, the same newspaper reported periodical cicadas in Leighton and Mauch Chunk. The Allentown Morning Call also reported periodical cicadas in June 1948 and June 1965, with specific mentions of Mauch Chunk and Carbon County. In sum, these newspaper reports seem largely credible, since they describe cicadas in approximately the correct locations in Brood V emergence years; thus, these records support the existence of this disjunct at least as far back as 1880. Complete details of Pennsylvania Brood V historical records are included in the  and our survey in 2016, and anecdotal reports from residents of Calverton suggest a steep decline in Magicicada density both in protected and developed areas of north eastern Long Island. No records of Brood XI on Long Island exist beyond those recorded by W.T. Davis in the early 1900’s and reported in Brood XIV is the main brood on Long Island and only small pockets of Broods I, V, and IX exist in a restricted area of northeastern Long Island . The LonCrowdsourcing has been a key component of periodical cicada mapping ever since C. L. Marlatt constructed maps by enhancing older datasets with reports from state entomologists, agricultural experiment station bulletins, weather service observers, post offices, and entomological enthusiasts . Our croAlthough some morphometric studies suggested that broods are single-origin monophyletic assemblages , MarlattM. septendecim from Midwestern localities belong to a central US mtDNA clade (Am) shared with all other Midwestern M. septendecim brood populations, while Brood V M. septendecim from eastern portions of the range belong to an eastern mtDNA clade (Ae), shared with all other eastern M. septendecim brood populations  to join the Brood I schedule, followed by another 4-year deceleration to adopt their current Brood V schedule [XIV]+4 → [I]+4 →[V]. In the second scenario, the ancestors of this Brood V population were Brood II cicadas that underwent a 1-year acceleration to join the Brood I schedule followed by a 4-year deceleration to become Brood V [II]-1 →[I]+4 → [V]. Alternatively, the ancestors could have been Brood II cicadas that underwent a 4-year deceleration (joining the Brood VI schedule) followed by a 1-year acceleration [II]+4 → [VI]-1 → [V]. These scenarios, involving either a Brood XIV or a Brood II ancestor, each require at least one 4-year deceleration. Of these scenarios, a Brood II ancestor seems more plausible because a) no populations of Brood XIV are currently found nearby , and b) In one sense, the brood concept is a bookkeeping tool; periodical cicada broods are numbered sequentially with an arbitrary start date of 1893 . In anot10.7717/peerj.5282/supp-1Supplemental Information 1Click here for additional data file.