Propionibacterium freudenreichii is a commercially relevant bacterium with probiotic potential. This bacterium can exert protective effects particularly against colorectal cancer (CRC), via the production of short chain fatty acids (SCFA), namely acetate and propionate. In this work, we aimed to evaluate the performance and adaptation capacity of P. freudenreichii to a simulated digestive stress using different culture media, namely YEL, Basal medium, Mimicking the Content of the Human Colon medium (MCHC) and DMEM. The effect of the fermented culture broth on CRC cells survival and of CRC cells conditioned media on the bacteria performance was also evaluated. Basal medium was found to be the best for P. freudenreichii to produce SCFA. MCHC medium, despite being the medium in which lower amounts of acetate and propionate were produced, showed higher acetate and propionate yields as compared to other media. We also observed that the presence of lactate in CRC cells conditioned growth medium resulting from cell metabolism, leads to an increased production of SCFA by the bacteria. The bacterial fermented broth successfully inhibited CRC cells proliferation and increased cell death. Our results showed for the first time that P. freudenreichii performance might be stimulated by extracellular lactate produced by CRC metabolic switch also known as “Warburg effect,” where cancer cells “ferment” glucose into lactate. Additionally, our results suggest that P. freudenreichii could be potentially used as a probiotic in CRC prevention at early stages of the carcinogenesis process and might help in CRC therapeutic approaches. P. freudenreichii resists the harsh physical and chemical stresses exhibiting a high resistance to the digestive stresses which is essential for its survival in the gut and also for exerting its beneficial effects  constitutes a major concern in developed countries being associated to lifestyle which is emerging as a critical element for its prevention . Evidenc effects , 12. P. opionate , 13–15. opionate . Severalopionate –23. SCFAopionate –26. In topionate . Moreoveh groups , 27, 28.h groups  demonstrPropionibacterium freudenreichii subsp. freudenreichii DSM 20271 was purchased from DSMZ (German collection of microorganisms and cell cultures). Freeze dried bacteria were reactivated and routinely cultivated on yeast extract-lactate (YEL) medium at 37°C without agitation. A P. freudenreichii tolerant to a simulated digestive stress (adapted bacteria) was also used. P. freudenreichii adaptation was performed as described by Lan et al. , the adapted bacterial culture was transferred to fresh liquid MCHC media to reactivate its metabolism and was characterized regarding growth and SCFAs production in different media.n et al.  with somP. freudenreichii were grown under anaerobic conditions at 37°C without agitation and monitored at defined time points  by measuring the absorbance of cell suspensions at 650 nm in a spectrophotometer . The culture media used to grow the bacteria were: YEL medium . Trace elements H+ solution contains HCl (1.8 g L−1), H3BO3 (61.8 mg L−1), MnCl2 (61.3 mg L−1), FeCl2 (1.0 g L−1), CoCl2 (64.5 mg L−1), NiCl2 (12.9 mg L−1), and ZnCl2 (67.7 mg L−1). Trace elements OH− solution contains NaOH (0.4 g L−1), Na2SeO3 (17.3 mg L−1), Na2WO4 (29.4 mg L−1), and Na2MoO4 (20.5 mg L−1). The salts and vitamins solution contains NH4Cl (24 g L−1), NaCl (24 g L−1), MgCl2.6H2O (8 g L−1), CaCl2.2H2O (8.8 g L−1), biotin (20 mg L−1), nicotamid (0.2 g L−1), p-aminobenzoic acid (0.1 g L−1), thiamin (0.2 g L−1), panthotenic acid (0.1 g L−1), pyridoxamine (0.5 g L−1), cyanocobalamine (0.1 g L−1), riboflavin (0.1 g L−1), folate (50 mg L−1), and lipoate (50 mg L−1). The bicarbonate solution contains NaHCO3 (80 g L−1), and Na2S.9H2O (5 g L−1). Basal medium (BM) is similar to YEL medium but without tryptone and containing 1 g L−1 of yeast extract. The medium “mimicking the content of the human colon” (MCHC) (−1), xylan (0.5 g L−1), mucin (0.5 g L−1), starch (0.5 g L−1), peptone (0.5 g L−1), tryptone (2.5 g L−1), yeast extract (0.5 g L−1), bile salts (0.05 g L−1), K2HPO4 (2.0 g L−1), NaHCO3 (0.2 g L−1), NaCl (4.5 g L−1), MgSO4.7H2O (0.5 g L−1), CaCl2.2H2O (0.45 g L−1), MnCl2 (0.2 g L−1), haemin (50 mg L−1), FeSO4.7H2O (5.0 mg L−1), CoCl2.6H2O (50 mg L−1), tween 80 (2.0 mL L−1), sodium lactate (2.3 g L−1), thiamine HCl (4.0 μg L−1), calcium pantothenate (10 μg L−1), nicorinic acid (5.0 μg L−1), p-aminobenzoic acid (5.0 μg L−1), biotin (2.0 μg L−1), vitamin B12 (0.5 μg L−1), and cysteine (0.8 g L−1). Dulbecco‘s Modified Eagle‘s Medium  supplemented with 10% (v/v) heat inactivated fetal bovine serum  was also used to grow bacteria cultures. Bacteria were also cultured in CRC cells conditioned medium which consists in the supernatants collected after CRC cells growth. CRC cells were cultivated in 75 cm2 tissue culture flasks  at a density of 20 × 104 cell mL−1, using DMEM medium without antibiotic. The CRC conditioned medium was collected after 72 h of incubation and was then diluted (1:1) in fresh DMEM medium.Cultures of normal and adapted , Na2WO4 .4 mg L−12SO4 as the mobile phase at an 0.6 mL min−1 flow rate. Glucose concentration was measured using the same HPLC system but equipped with a Metacarb 87H column  and a RI detector  operated using the same conditions of temperature and mobile phase and a flow rate of 0.7 mL min−1. Samples for further analysis were centrifuged at 10 000 g for 5 min , filtered with 0.2 μm filters . Standard solutions of acetate, propionate and glucose were used in a wide range of concentrations (0–10 g L−1) was used to elaborate the calibration curves. An area of “x” represents ,  and  g L−1 (for x > 0) of acetate, propionate and glucose, respectively.Lactate, acetate and propionate were analyzed by high-performance liquid chromatography (HPLC) equipped with an UV detector  using a Metacarb 67H column  operated at 60°C with 0.01 N HBiomass was determined from a standard curve of optical density vs. dry weight. Dry weights for the standard curve were obtained by filtering the culture through 0.2 μm filters. After filtration, the filters were dried and weighed. The standard curve was plotted from the mean values of two determinations, and the biomass was obtained from the least-squares regression line. All fermentations were performed in triplicated, except the DMEM fermentation which was performed in duplicated.2, in DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin . RKO cells were kept in exponential growth phase and subcultured once or twice a week. For the assays, subconfluent cells, in exponential growth phase, were detached with trypsin/EDTA  and ressuspended in fresh medium at the appropriate density.The human CRC derived cell line RKO (CRL-2577) was kindly provided by IPATIMUP. Cells were grown as monolayers at 37°C in a humidified incubator with 5% CO5 cell mL−1 in a final volume of 0.5 ml per well, excluding the cell cycle analysis in which cells were cultured in 6-well plates at a cellular density of 1.5 × 105 cell mL−1 in a final volume of 1.5 mL per well. Cells were incubated at 37°C in a 5% CO2 atmosphere during 24 h, to allow cell adhesion. After adhesion, the culture medium was removed and replaced by different treatment conditions.In all treatments performed in this work, cells in exponential growth phase were cultured in 24-well plates at a cellular density of 1 × 1050)  and 30% maximal inhibitory concentration (IC30)  of acetate and propionate  at 37°C for 15 min. After incubation, 50 μL propidium iodide (PI) staining solution [0.5 mg mL−1 in sodium citrate 1% (w/v)] was added and the cells were mixed in a vortex and were incubated at room temperature for 30 min in the dark. Cells with red fluorescence [FL-3 channel (488/620 nm)] were analyzed in Epics XL flow cytometer  with an average of 20,000 counts per sample. Data were analyzed with the Flowing software program  to generate DNA content frequency histograms and quantify the amount of cells in the individual cell-cycle, including sub-G1 population assumed as death cells.After 48 h of treatment, cells were harvested by scraping and the medium was collected and centrifuged at 500 g for 3 min. The pellet was resuspended in 500 μL PBS 1x and incubated on ice for 15 min. After this period, 1.5 ml of 96% (v/v) cold ethanol (stored at −20°C) was added to the pellet and was incubated on ice during 15 min, to allow cell fixation. Cells were then washed with 4 ml PBS 1x, centrifuged at 500 g for 3 min at 4°C and the pellet again washed with PBS 1x. The final pellet was resuspended in 500 μL PBS 1x and was incubated with 50 μL of RNase A solution [200 μg mLp ≤ 0.05 were regarded as statistically significant. All statistical tests were performed using the software Graphpad Prism 5.Results correspond to the mean ± standard error of the mean (SEM) of at least three independent experiments. The data were analyzed using an analysis of variance (one-way ANOVA) followed by Tukey's Test to compare all the experiments or Dunnett‘s test to assess the differences between control and different conditions tested. The confidence interval used was 95% and Propionibacterium freudenreichii was grown and characterized regarding biomass and SCFA production in different culture media. The results obtained are illustrated in Figure −1 in YEL, BM, MCHC, and DMEM media, respectively. The acetate production was found to be around 1.65 ± 0.01; 1.81 ± 0.01; 0.58 ± 0.02, and 0.79 ± 0.02 g L−1 in YEL, BM, MCHC, and DMEM media, respectively; while the propionate production was around 4.69 ± 0.01; 4.81 ± 0.02; 1.37 ± 0.03, and 2.60 ± 0.05 g L−1 in YEL, BM, MCHC, and DMEM media, respectively. These results showed that higher amounts of biomass production were not correlated with higher amounts of SCFA production. For instance, in DMEM, the bacteria exhibited higher amounts of biomass production; however, lower amounts of SCFA were produced. In MCHC, the bacteria displayed lower amounts of both biomass and SCFA production. The higher production of SCFA was obtained using BM, followed by the YEL medium.Biomass production reached ~0.86 ± 0.02; 0.53 ± 0.01; 0.25 ± 0.01, and 1.55 ± 0.01 g LP. freudenreichii were obtained in MCHC medium, the acetate and propionate yields are high as compared to other media.Growth rates, yields and productivities were determined for all culture media tested and are summarized in Table P. freudenreichii depends on its capacity to survive the digestive tract stresses and to remain metabolically active in the gut. In order to obtain bacteria tolerant to this type of stress, a digestive stress challenge was performed. P. freudenreichii was exposed to acidic pH for 1.5 h and bile salts concentrations for 2 h, mimicking the digestion process. The performance of adapted P. freudenreichii was evaluated and the results obtained are presented in Table The probiotic efficiency of P. freudenreichii displayed an increase in the biomass concentration and growth rate, in DMEM and MCHC media, when compared to the performance of the non-adapted P. freudenreichii. However, the production of SCFA, particularly propionate production, were negatively affected by the digestive stress challenge. Significant statistical differences could be observed between YEL and BM medium (p < 0.001) when comparing the results obtained for the normal and adapted bacteria . This effect was also observed for acetate production. Interestingly, the adapted P. freudenreichii acetate yields in MCHC media were positively affected by the digestive stress challenge showing a slight increase (Table Adapted  p < 0.00 when comP. freudenreichii was studied through simulation using medium where CRC cells were grown (conditioned medium). The diluted and non-diluted conditioned medium containing 2.7 and 1 g L−1 of glucose, respectively, were used to simulate the effect of CRC cells in the growth of normal (non-adapted) P. freudenreichii and on SCFAs production. Diluted and non-diluted conditioned medium containing 3.2 and 1.8 g L−1 of glucose, respectively, were used with the same purpose for the adapted bacteria. As controls, fresh DMEM medium with the same concentrations of glucose present in the conditioned medium were used. The results presented in Figure P. freudenreichii growth in conditioned medium exhibited similar behaviors. Growth and SCFA production parameters are summarized in Table The effect of co-culture of CRC cells with probiotic bacteria in the production of SCFA in normal and adapted The increased SCFA production in conditioned medium could be due to the presence of some metabolites produced by CRC cells that are positively assimilated by bacteria. In order to identify such compounds, present in conditioned medium which could explain the improved SCFA production, CRC cells conditioned medium diluted and non-diluted with fresh DMEM medium were analyzed by HPLC.P. freudenreichii was composed of 2.4 and 0.9 g L−1 of lactate, respectively, while 1.6 and 0.8 g L−1 of lactate was quantified in non-diluted and diluted conditioned medium used to culture adapted P. freudenreichii. The presence of lactate in CRC cells conditioned medium diluted and non-diluted with fresh DMEM can explain the improvement of the biotransformation performance by probiotic bacteria given that lactate is the favorite carbon source for these bacteria.The HPLC analyzed showed that lactate was detected in the conditioned medium but not in the controls, what is in accordance to the production of lactate by CRC cells metabolism switch, also known as “warbourg effect” where cancer cells “ferment” glucose into lactate. Non-diluted and diluted conditioned medium used to culture normal 50 and IC30 values of acetate  and propionate  alone were previously determined in our laboratory  led to a decrease of 12% in cell proliferation when compared with the negative control. The combination of acetate and propionate exhibited a higher effect in cell proliferation. The IC50 of acetate and propionate together inhibited cell proliferation almost completely. The IC30 of acetate and propionate together reduced cell proliferation by ~80%, being higher than the effect of the IC30 of the acids alone. Proliferation of CRC cells incubated with 0.79 g L−1 of pure acetate and 2.6 g L−1 of pure propionate (i.e. the SCFA concentrations present in DMEM fermented broth by P. freudenreichii) was reduced by ~21% being significantly different from the negative control.The ICP. freudenreichii on the CRC cells, the influence of the fermented broth was also analyzed. Among the four fermented broths used, BM and DMEM were selected since they showed no toxic effect to the CRC cells (data not shown). BM diluted in fresh DMEM medium and CRC cells conditioned medium diluted in fresh DMEM medium were used as a negative control of BM and DMEM fermented broths, respectively. Fermented broths were deproteinized  to eliminate any possible cytotoxic effect from contaminating proteins. Diluted fermented broths contained 0.9 g L−1 of acetate and 2.4 g L−1 of propionate for P. freudenreichii and fermented broth of adapted P. freudenreichii, containing 0.6 g L−1 of acetate and 1.5 g L−1 of propionate were used to treat colorectal cancer cells. Cells were also treated with pure SCFA at the concentrations previous described (SCFA control). Results of BM and DMEM fermented broth by adapted and normal bacteria and the respective controls are represented in Figure In order to evaluate the effect of the SCFA produced by adapted and normal p < 0.01) in BM fermented broth from normal P. freudenreichii. All conditions of DMEM fermented broth  was observed when cells were treated with BM fermented broth than when cells were treated with DMEM fermented broth (20–35%). Cells treated with DMEM fermented broth also seem to be arrested in the G2/M phase of the cell-cycle (10–30%).Cell cycle distribution of CRC treated with fermented broths (BM and DMEM) and the respective controls was also studied by flow cytometry measurement of the DNA content of cells stained with PI Figure . SCFA prP. freudenreichii, which are present in several dairy products frequently consumed  and therefore indicate that the digestive stress affects the probiotic capacity of propionibacteria, although the production yield showed for acetate is high in comparison to other media. Thus, further developments and optimizations of P. freudenreichii for the envisaged use must be explored.−1, while other strains produced lower amounts, thus suggesting that the propionate production rate is strain-dependent. P. freudenreichii subsp. freudenreichii, named TL3, produced 1.32 g L−1 of propionate, which is similar to the propionate concentration obtained in this study (1.4 g L−1). Jan et al. (P. freudenreichii subsp. freudenreichii), that propionibacteria produced 2.7 g L−1 of propionate and 0.75 g L−1 of acetate. Therefore, the results presented are in accordance with the literature.Lan et al.  culturedn et al.  using thP. freudenreichii subsp. freudenreichii DSM 20271 to the digestive stress by culturing cells in a simulated digestive stress media. Several authors have performed digestive stress challenges to test the adaptation and tolerance of propionibacteria  similarly to what was reported in our own, as well as in others studies (To study the potential antineoplastic effect of propionibacteria fermented culture media on CRC cells, cells were treated with DMEM and BM fermented broth by adapted and normal  studies , 27. HowP. freudenreichii may be able to grow in co-culture with CRC cell lines since a slower growth with an increased SCFA production was observed. Moreover, our results demonstrated that this biotransformation performance induced cell death of CRC.Cancer cells are known for its biological capabilities acquired during the multistep development of human tumors that makes them act differently from normal cells . One of P. freudenrechii fermentation, leading to high propionate and acetate yields (P. freudenrechii has a complex metabolism that involves several metabolic pathways. Lactate consumption yields pyruvate which can be reduced to produce propionate via the transcarboxylase cycles, or oxidized to yield acetate and CO2 (The improvement of SCFAs production in conditioned medium may be due to the metabolites excreted by RKO cell lines, namely lactate. Lactate is an ideal carbon source for e yields . P. freu and CO2 .P. freudenreichii promotes a cytotoxic effect on CRC cells, via their metabolites, and that the CRC cells increases the acetate and propionate production by P. freudenreichii, which in turn will lead to an increased cytotoxic effect on the CRC cells.CRC is known to rely on glycolysis for energy production and this activity leads to the production of important amounts of lactate, which are exported into the extracellular milieu, thus contributing to the acidic microenvironment , 42. ThiPropionibacterium freudenreichii was able to produce high amounts of acetate and propionate in BM and YEL media. Adaptation of P. freudenreichii to digestive stress was successfully obtained and its behavior was similar to the normal bacteria. Interestingly, we observed a great improvement in the bacteria performance using the MCHC and DMEM media which are similar to the real scenario of the GIT. The effect of CRC cells conditioned medium rich in lactate on the bacterial performance showed that bacteria were able to grow remaining metabolically active and produced more SCFA in the presence of lactate. Furthermore, the exposure of CRC cells to SCFA produced by Propionibacteria inhibited their proliferation and induced cell cycle arrest. Our results suggest the use of P. freudenreichii as a probiotic in the prevention and/or as therapeutic adjuvant in CRC.The experimental work was developed by MC. All authors contributed to the planning, discussion, and writing of the manuscript and have given approval to its final version. Both senior authors designed the study, planed the experimental work and contributed equally to this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Parthenium hysterophorus weed biomass was evaluated as a substrate for cellulase production. The cellulose in the biomass was used as the main source of carbon. Solid-state fermentation was carried out using Trichoderma reesei, and optimization of cultural conditions was done for maximization of cellulase production. The results revealed that highest cellulase production was achieved on the 8th day of incubation, at 30 °C, keeping solid-to-liquid ratio 1:2 when two discs of inoculum were used per gram of the substrate. The optimized inoculum age was 96 h for CMCase and 120 h for FPase. On studying the enhancing effect of different carbon and nitrogen sources, lactose and ammonium molybdate were found suitable, respectively. The optimized concentration of lactose for the highest CMCase and FPase activities was 1.5 and 1%, respectively. Ammonium molybdate was best at 1% concentration for both CMCase and FPase. Maximum CMCase and FPase activities obtained were 20.49 and 2.42 U/gds, respectively.The potential of untreated  The biomass has been used in the raw form, without any chemical pretreatment. The pretreatment of lignocellulosics is an energy intensive and costly affair. Therefore, Parthenium lignocellulose was used in the untreated form for the production of cellulases cost effectively.In the present study, Parthenium hysterophorus biomass was used as a substrate for producing cellulases. It was procured from local areas, including road sides and the abandoned land. The biomass was washed thoroughly with tap water followed with its chopping and then oven drying at 50–60 °C for around 24 h (until complete drying). It was finally ground to a particle size of 0.5 mm.3 for 20 min  were calculated from the difference in weight before and after acid hydrolysis.For cellulose content determination, 1 gm of dried biomass was fluxed with 10 ml of 80% acetic acid and 1.5 ml of HNOTrichoderma reesei (NCIM 992), which was obtained from NCIM, Pune, India. It was maintained on potato dextrose agar (PDA) medium and preserved at 4 °C. The purity of the culture was tested by observing the growth of the culture on agar medium as individual colony followed with its microscopic examination.The microbial culture selected for the production of cellulases was 7 spores/ml) was inoculated per gram of cellulosic biomass.For inoculum preparation, the culture was grown for 5 days in potato dextrose agar plates, at 30 °C. For enzyme production, 10 mm disc of fungal mycelium (≈2 × 10−1) urea, 0.3; peptone, 0.1; (NH4)2SO4, 1.4; KH2PO4, 2.0; CaCl2·2H2O, 0.3; MgSO4·7H2O, 0.3 and trace elements (mgL−1): FeSO4·7H2O, 5; MnSO4·H2O, 1.6; ZnCl2·7H2O, 1.4, and CoCl2·6H2O, 2.0. The contents of the flasks were sterilized in an autoclave at 121 °C at 15 psi for 30 min. The flasks were inoculated with fresh mycelium followed with their incubation at 30 °C. All the experiments were performed in triplicates, and the data were expressed as mean ± SD of three replicate samples. In addition, the analysis of variance was performed statistically using spss 16 according to Tukey’s test at 5% probability (p ≤ 0.05).For solid-state fermentation, the dried and ground biomass was taken in the Erlenmeyer flasks and solid-to-liquid ratio was maintained 1:2.5 using Mandel’s medium (pH 6.0) as a moistening agent. The Mandel’s medium was prepared with the following composition (gLThe enzyme was extracted using tenfolds (v/w) of sterilized distilled water. The contents were mixed thoroughly followed with the separation of liquid from the solid portion using a muslin cloth and then subjected to the centrifugation at 4 °C at 10,000 rpm for 20 min. The crude enzyme was finally obtained by filtering the supernatant through Whatman filter paper no. 1.Endoglucanase activity  and FPase activity  were assayed according to the methods by Ghose . The reaT. reesei using Parthenium hysterophorus biomass.Optimization of cultural conditions Table  was doneT. reesei was cultivated under solid-state conditions in different flasks and one flask at a time was taken out after incubation for 4, 6, 8, 10, 12, and 14 days. The crude enzyme was extracted and used for enzyme assays.To study the effect of the incubation period, During the solid-state fermentation conditions, the initial moisture level in different flasks was varied by keeping solid-to-liquid ratios 1:1.25, 1:1.5, 1:1.75, 1:2, 1:2.25, and 1:2.5. The incubation was done for time period optimized in above experiment.T. reesei at different temperature conditions, i.e., 20, 25, 30, 35, and 40 °C.The effect of temperature was studied by producing cellulase from T. reesei grown on potato dextrose agar medium.The amount of the inoculum was optimized by inoculating the fermentation flasks with the different number of discs, i.e., 1–5, of The effect of inoculum age, i.e., 48, 72, 96, 120, 144, 168, and 192 h old culture, was studied.To study the effect of carbon sources, Mandel’s medium was supplemented with 1% of different carbon sources, i.e., glucose, fructose, galactose, lactose, sucrose, cellobiose, starch, CMC, mannitol, and sorbitol. The effect of concentration of optimal carbon source was determined by varying its concentration from 0.25 to 2.5% at an interval of 0.25%.3, KNO3, casein, peptone, beef extract, yeast extract, malt extract, and meat extract were incorporated in Mandel’s medium, replacing other nitrogen sources. The effect of concentration of optimal nitrogen source was determined by varying its concentration from 0.25 to 2.0% at an interval of 0.25%.1% of different nitrogen sources (inorganic and organic), i.e., urea, ammonium chloride, ammonium nitrate, ammonium sulphate, ammonium molybdate, NaNOParthenium biomass was found to have 36.11% cellulose content, 22.8% lignin, and 26% hemicellulose. Other studies have also revealed the presence of 28%  showed maximum production upon incubation for 10 days when water hyacinth was used as substrate for SSF  to 14 days at intervals of 2 days Table , inhibitT. reesei showed maximum activities for both CMCase (11.65 U/gds) as well as FPase (1.29 U/gds) when solid-to-liquid ratio was 1:2  inoculum was used in solid-state conditions using coir waste as a substrate  and FPase (1.65 U/gds) activities were highest when two discs were used for inoculating per gram of the substrate  and 120 h (1.79 U/gds FPase) old fungal culture was used as inoculum, respectively (Table Aspergillus sp. SU14-M15 under SSF conditions using wheat bran. Aspergillus hortai produced maximum levels of cellulase when 144 h old inoculum was used for liquid state fermentation (El-Hadi et al. Age of the inoculum, a factor representing the physiological state of the fungus, affects biomass production (Bhargav et al. ly Table  have fouParthenium biomass supports the growth of T. reesei as well as the cellulase production. However, the rate of the cellulases synthesis is generally slow when insoluble sugars in the lignocellulosic biomass are used as a substrate (Chen and Wayman T. reesei (Dashtban et al. Acremonium cellulolyticus. In a study by Sun et al. (Trihoderma species cultivated on apple pomace under SSF conditions produced the highest levels of cellulase when supplemented with lactose. In Acrophialophora nainiana also, the highest CMCase production was induced in the presence of lactose (Barros et al. Apsegillus hortai, enhanced CMCase production was observed in the presence of lactose (El-Hadi et al. Cellulose in the n et al. , TrihodeIn studying the effect of carbon source concentration Fig. , 1.5% laT. reesei, grown on Parthenium biomass, has been shown in Fig. Trichoderma (Mukhopadhyaya and Nandi Apsergillus fumigatus (Stewart and Parry Aspergillus terreus (Vyas et al. Bacillus pumilus EWBCM1. Inductive effect of ammonium salts can be attributed to the direct entry of ammonium in protein synthesis (Vyas et al. Choice of an appropriate nitrogen source for the cultivation of cellulolytic microbes is an important factor determining the yield of the process. Like carbon sources, the nitrogen sources have an inducible effect on the cellulases. The effect of different nitrogen sources on cellulase production by Parthenium hysterophorus weed biomass can successfully be utilized for cellulase production. Optimization studies have demonstrated that significant enhancement in enzyme production is possible using suitable cultural conditions. Many of the discussed parameters showed marked effect on improvement in cellulase synthesis by T. reesei grown on the Parthenium biomass. Optimum conditions recorded were 30 °C incubation temperature, an incubation time of 8 days. and 1:2 initial solid-to-liquid ratio. Supplementation with 1.5% lactose and 1% ammonium molybdate further increased both CMCase and FPase activities. Appropriate inoculum age and inoculum level, i.e., 96–120 h and 2 discs per gram of biomass, respectively, were also found important factors affecting cellulase production.The present study indicates that Parthenium biomass was not subjected to any physicochemical pretreatment. This was intended to minimize the cost of cellulase production from Parthenium biomass using it in untreated form, because pretreatment is known to increase the production cost many folds. Moreover, the current work included the preliminary study to first test the potency of weed biomass for cellulase production. However, this work can subsequently be extended to bioethanol production. In addition, management of Parthenium hysterophorus has been a concern for many years. A lot of research inputs and financial investments in this regard have been unsuccessful in controlling this weed in an eco-friendly manner. The utilization of Parthenium hysterophorus biomass for cellulase production does not only suggest the potential of this weed for various lignocellulose-based applications, but also the solution for controlling this noxious weed to manageable levels.The


MPMO was synthesized from MC that reacted with meta-chloroperbenzoic acid and, after separation and purification, was identified by 1H and 13C NMR and GC-MS. The antioxidant activity was investigated by DPPH, cooxidation β-carotene/linoleic acid, and thiobarbituric acid assays. With the use of colorimetric determination, the antilipase effect on the pancreatic lipase was tested, while the molecular interaction profiles were evaluated by docking molecular study. MC (IC50 = 312.50 ± 2.28 μg/mL) and MPMO (IC50 = 8.29 ± 0.80 μg/mL) inhibited the DPPH free radical. The inhibition of lipid peroxidation (%) was 73.08 ± 4.79 and 36.16 ± 4.11 to MC and MPMO, respectively. The malonaldehyde content was significantly reduced in the presence of MC and MPMO. MC and MPMO inhibited the pancreatic lipase in 58.12 and 26.93%, respectively. MC and MPMO  produced a binding affinity value lower than did diundecylphosphatidylcholine . These findings show that MC and MPMO present antioxidant and antilipase activities, which may be promising molecular targets for the treatment of diseases associated with oxidative damage and lipid metabolism.This study investigated the  The imbalance between “prooxidant” and “antioxidant” chemical species produces oxidative stress, which causes lipidic peroxidation, aggression to proteins, and damage to DNA and RNA and triggers mechanisms associated with inflammatory, cardiovascular, and neuro-degenerative diseases; allergies; accelerated aging; hemorrhage; cataracts; immunological dysfunctions; and cancer , 2. Amonp-allylanisole, is a special metabolite belonging to the class of phenylpropanoids found in essential oils of medicinal and food plants oxirane (MPMO) was synthesized from methyl chavicol, which showed the physical appearance of a brown oily liquid of molecular formula Cphy (GC) .1H NMR, 500 Hz, (CDCl3): δ (ppm): 6.85 ; 6.74 ; 3.89 ; 2.70 ; 2.56 ; and 2.11  : δ (ppm): 171.395; 145.782; 145.608; 130.553; 120.612; 115.412; 110.935; 60.610; 56.193; and 52.774 (m/z = 164 (M+); 121; 108; 91; 77; and 65  . 13C NMRd 52.774 . MS: m/z; and 65 .The kinetic profile showed that MC has a slow antioxidant capacity with reaction time greater than one hour. The percentage of unreacted DPPH radicals with MC in relation to the time is shown in 50 values of the samples ranged from 0.01 ± 0.01 to 312.50 ± 2.28 mg/mL and were significantly different from each other (P < 0.001). Considering the concentration of 50 mg/mL, MPMO was more effective than MC in inhibiting DPPH, since it produced an activity percentage (%) of approximately 80% of inhibition.The antioxidant potential of MC, MPMO, and BHT against DPPH is presented in β-carotene/linoleic acid method. MC was more effective in inhibiting lipid peroxidation, since this compound presented a lower decay when compared to MPMO. After 15 minutes, the absorbances of the compounds are different from those of the negative control (P < 0.001).With the data in P < 0.001).The concentration of malonaldehyde (MDA) decreased in the homogenate treated with BHT, MC, and MPMO when compared to that of the negative control . On the The inhibitory activity of MC and MPMO on the pancreatic lipase was 58.12 and 26.93%, respectively. Orlistat, the positive control, was effective by 76.80% of inhibition .In this investigation, the parameters were validated using the redocking method to reproduce a protein-ligand crystallographic complex with a root-mean-square deviation (RMSD) of less than 2 Å. The redocking of the crystallographic ligand, diundecylphosphatidylcholine (PLC) (1.3232 Å) and orlistat (1.84 Å) , at the −1, which was greater than that of orlistat  and lower when compared to that of PLC  (The molecular interactions between MC or MPMO and pancreatic lipase are of hydrogen bonding type that exhibited Ser153 and His264 residues as target amino acids . The moll·mol−1) .meta-chloroperbenzoic acid as an epoxidizing reagent. In addition, the structure of MC allows the functionalization of the olefin with peracids and occurs by a possible electrophilic biomolecular mechanism, where the peracid would be in a cyclic structure, stabilized by an intramolecular hydrogen bond to form a chelate  was about 38 times lower than that of MPMO (8.29 ± 0.80 mg/mL). The presence of the epoxide in MPMO can justify the higher antioxidant action when compared to MC, since this group increases its polarity and allows the electron donation to the DPPH radical. In addition, components of essential oils containing hydroxyl groups attached to the aromatic ring, unsaturations, and availability of electrons are associated with the antioxidant activity [Tagetes lucida, which contains 95.7% of MC, produced IC50 of 37.9 μg/mL [50 of 1.092 ± 0.066 mg/mL [The results of the antioxidant activity showed that MC and MPMO presented an antioxidant potential in trials that differ in relation to the evaluated mechanism. In the DPPH free radical sequestration assay, the ICactivity . However.9 μg/mL , while s66 mg/mL . Probabl66 mg/mL , 42.β-carotene/linoleic acid system and thiobarbituric acid assays are in vitro tests that reproduce physiological situations of oxidative stress, which can lead to cell death in extreme cases [β-carotene. Thus, these findings show that MC is a molecule with antioxidant potential against lipid peroxidation, since it inhibited 73.08% of the oxidative process, while BHT (positive control) presented 59.66%, and MPMO was able to inhibit 36.16%. In addition, the results of the thiobarbituric acid assay corroborate the data on cooxidation of β-carotene/linoleic acid system, since MC was more active in inhibiting the formation of malonaldehyde in the homogenate. Lipoperoxidation is a process that involves the initiation, propagation, and termination steps, and antioxidants can block the first step (initiation) by neutralizing reactive oxygen species and/or inhibiting the propagation by suppressing the peroxyl radicals [Lipid peroxidation by the cooxidation of me cases , 44. As radicals . Probablp-nitrophenol palmitate in palmitic acid and p-nitrophenol. The release of these substances results in a yellow-colored chromogen that is intensified at high pH  [2 decrease the solvation layer of the enzyme, which implies a better dissolution, increases the ionic strength of the medium, and enables the formation of the ligand-protein complex [in vitro pancreatic lipase by 58.12%  . A surfa complex . Our resy 58.12% , and they 58.12% . Althougy 58.12% . FurtherNatural compounds, such as alkaloids, carotenoids, glycosides, polyphenols, polysaccharides, saponins, and terpenoids, are described as pancreatic lipase inhibitors . In partS. typhimurium. Both estragole and its 1′-hydroxy metabolite produced unscheduled DNA synthesis in rat hepatocytes in vitro and estragole also in vivo. The formation of hepatic DNA adducts has also been demonstrated in mice. In this sense, the toxicity of methyl chavicol and its analogue with generation of toxic metabolites [Although methyl chavicol is found in essential oils of medicinal plants widely used by the population , the carabolites –15 may pin vitro and in silico assays. The results suggest that these compounds may be promising for the development of new therapeutic options for the treatment of diseases associated with oxidative processes and metabolic alterations.MC and MPMO have an antioxidant activity and are capable of inhibiting the pancreatic lipase enzyme using the


Glyphodes pyloalis Walker was studied under laboratory conditions at constant temperatures of 12, 16, 20, 24, 28, 30, 32, and 36 °C. No development occurred at 36 °C. Although eggs hatched at 12 ºC, no larvae were capable of developing to adult stage. At 16 ºC, survival rate was low (4%) and prepupal stage lasted 101.68 ± 11.03 d. Larvae completed development through six stadia at 16, 30, and 32 °C. Developmental time of overall immature stages varied from 46.62 d at 20 °C to 22.04 d at 30 °C and increased at 32 °C. The lower temperature thresholds of 10.30 and 11.22 °C, and thermal constants of 429.18 and 401.88 DD were estimated by traditional and Ikemoto–Takai linear models, respectively. The Tmin values estimated by Analytis, Briere-2, Lactin-2, and Sharpe–Schoolfield–Ikemoto (SSI) for overall immature stages were 12.40, 12.92, 9.00, and 13.04 °C, respectively. The fastest development temperatures (Tfast) of 31.1, 31.1, 30.8, and 30.7 °C were estimated for overall immature stages based on Analytis, Briere-2, Lactin-2, and SSI, respectively. The intrinsic optimum temperature (Topt) estimated from the thermodynamic SSI model for total developmental time was 24.63 °C, in which the maximal active state enzymes involved in developmental process. The nonlinear models of Analytis, Lactin-2, Briere-2, and SSI estimated the upper temperature thresholds (Tmax) at 36.66, 35.97, 38.88, and 34.05 °C, respectively. These ﬁndings could be used to predict the population dynamics of G. pyloalis for an effective management.Development of  Glyphodes pyloalis Walker , is a specialist pest on mulberry (Morus spp.) and is widely distributed throughout Asia, where the species causes serious damage to sericulture not only by its larval grazing on leaves but also by transmission of some viral diseases infectious to the silkworm  were kept inside the oviposition containers (50 × 50 × 50 cm) with a 10% honey solution on cotton wool for feeding and mulberry leaves for oviposition.The larvae of After mating and oviposition, one hundred to 300  freshly laid eggs (< 24 h old) were transferred to plastic boxes (18 × 15 × 7 cm) with wet cotton wool in growth chambers at eight constant temperatures of 12, 16, 20, 24, 28, 30, 32, and 36 ± 1 °C at 70 ± 10% R.H. and photoperiod of 16:8 L:D h. Changing in shape and color of eggs was monitored daily under the stereomicroscope. Incubation period and hatching rate were recorded.The newly hatched larvae were placed individually in plastic containers (7 × 8 × 3 cm) with a hole in their lids covered by a ﬁne mesh to provide ventilation. The petioles of mulberry leaves were kept in tubes containing water to keep the cutting leaves as fresh as possible. The leaves were replaced every other day for larvae at 16–24 °C and daily for those larvae at 28–32 °C because plant desiccation occurred faster than lower temperatures. The larvae were checked and the instars were regularly recorded using the exuviae of larval head capsules. The matured larvae changed color from green to purple and began making fine cocoons considering prepupal stage. After pupation, they were slipcovered and sexes were determined based on morphological characters of pupal last abdominal segment. After that they were replaced in their fine cocoons. The cocooned pupae were checked and the emerged adults recorded daily. Developmental time of different and overall immature stages was recorded based on regular observations with 24 h intervals.G. pyloalis was calculated to obtain the developmental rate. Two linear, traditional and Ikemoto–Takai models were applied to estimate the temperature-dependent development of egg, larval, prepupal, pupal, and total immature stages of G. pyloalis on mulberry leaves. The traditional and Ikemoto–Takai models are as follows, respectively:D is the duration of development (days), T is the ambient temperature, Tmin is the lower temperature threshold, and K is the thermal constant .The reciprocal of developmental time for different stages of Tmin and larger K, hence, The latter function is a new linearized formula was proposed by Tmin is the lower temperature threshold, Tmax is the upper temperature threshold, a, d, n, m, p, λ, and ΔT are ﬁtted coefﬁcients (Topt) in which the population size is maximal with a low mortality (Topt) should represent a temperature at which the mortality of insects is very low, and that the net reproductive rate is generally highest. In fact, Topt is different from Tfast that make insects develop fastest within shortest duration:Φρ is the mean developmental rate at Topt (1/d), Topt is the intrinsic optimum temperature at which the probability of an enzyme being in the active state is maximal. ΔAH, ΔLH, and ΔHH are the enthalpy of activation of the reaction that is catalyzed by the enzyme , the change in enthalpy associated with low temperature inactivation of the enzyme , and the change in enthalpy associated with high temperature inactivation of the enzyme , respectively, R is the gas constant , LT is the temperature at which the enzyme is 1/2 active and 1/2 low temperature inactive, and HT is the temperature at which the enzyme is 1/2 active and 1/2 high temperature inactive (Both in Kelvin degrees).It should be considered that the relationship between temperatures and developmental rate is curvilinear near lower and upper temperature thresholds. To describe the developmental rate over a wider temperature range, four nonlinear models including Analytis, Briere-2, Lactin-2, and Sharpe–Schoolfield–Ikemoto (SSI) were chosen . These ffﬁcients . In addiortality . Ikemotoortality  demonstrSince running the SSI model through G. pyloalis were estimated by above-mentioned models.Critical temperatures and thermal requirement of Tmin), the temperature below which different stages did not develop. The standard error (SE) of Tmin calculated from the linear models isThe lower temperature threshold , the temperature above which the life cannot be maintained for any significant period (The upper temperature threshold (t period . This vaTfast), defined as the temperature at which the highest developmental rate was recorded. However, the ﬁtness of population is usually not maximal because of the higher mortality at Tfast.The fastest development temperature (K), the amount of thermal energy (DD) needed to complete development of different stages. The thermal constant can be estimated only by the linear equation. The SE of K was estimated by using the following equation . The linear models were analyzed using statistical software MINITAB 16.0 and nonlinear models analyzed using linear and nonlinear platforms of JMP, v 7.0 . As far as prepupal stage is concerned, at 16 ºC G. pyloalis required 101.68 ± 11.03 d to develop to pupal stage maybe due to stop developing (or diapause occurrence) in prepupal stage. For these two reasons, too long duration and low survivorship of prepupal stage both occurred at 16 °C, developmental times of prepupal, pupal, and total immature stages were ignored and comparing the mean duration of above-mentioned stages  was done without considering of the temperature at 16 °C , larval , prepupal , pupal , and overall immature stages   . The larG. pyloalis at six constant temperatures revealed that the survival was highest (65%) at 28 °C, followed by 24 °C . Although egg hatching occurred at 12 °C, all neonate larvae died due to the exposure to low temperature. Furthermore, survival of total larval stage of by 24 °C .G. pyloalis increased linearly within the examined temperature range (20–30 °C). Developmental time at >30 °C (32 °C) was outside the linear segment of the growth curve and therefore excluded from the linear regression. Results of parameter estimation of linear models , coefficients of determination (R2 and R2adj), lower temperature thresholds and thermal constants are presented in The developmental rate of G. pyloalis at the temperature range from 20 to 32 °C (R2adj were used to determine the goodness of fit the models. The adjusted coefficients of determination (R2adj) in all tested models for overall immature stages were higher than 0.95. The curves of the relationship between temperature and developmental rate of total immature stages fitted by mentioned models are depicted in Four nonlinear models  were fitted to the data on developmental rate of egg, larval, prepupal, pupal, and overall immature stages of to 32 °C . The valTmin) for overall immature stages estimated by the Analytis, Briere-2, and Lactin-2 were 12.4, 12.92, and 9 °C, respectively. Moreover, the SSI model estimated LT for overall immatures stage at 13.04 °C, in which the hypothetical enzyme was half active and half inactive. Based on current study, the Analytis, Briere-2, Lactin-2, and SSI models estimated Tfast for overall immature stages at 31.1, 31.1, 30.8, and 30.7 °C, respectively. In estimated Tfast developmental time is shortest but the ﬁtness of population is usually not maximal because of the higher mortality. Whereas the estimates of Topt using SSI model for different stages varied from 22.66 to 28.21 °C which represented the optimal temperature for G. pyloalis population to develop with a low mortality.The results of present study showed that the lower developmental thresholds  and thermal requirements of the lesser mulberry pyralid. The biology of the pest was examined by G. pyloalis was 4.06 d at 24 ± 1 °C. Similarly working with G. pyloalis on four mulberry varieties, G. pyloalis at same temperature agreed with those reported by G. pyloalis required five stadia, however, an extra (sixth) stadium was observed at 16, 30, and 32 °C at present study. These differences may be due to larval body size depending on very high- or low-developmental rate at extreme temperatures. G. pyloalis at extreme temperatures.The estimated temperature thresholds and thermal constants are potential indicators for developing a phenology model of G. pyloalis under field conditions. G. pyloalis during 1933–1934 and stated that the matured larvae of last generation made the cocoons and hibernated inside the leaves on the ground from mid-autumn. Based on G. pyloalis as a distinct stage, this assumption seems to be presumable. No more information is currently available on overwintering of G. pyloalis, hence probability of hibernation or diapause occurrence in prepupal stage could be proposed cautiously.According to the present study, it should be considered that prepupal developmental times were 4.64, 2.28, 1.94, 2.11, and 2.25 d at 20, 24, 28, 30, and 32 °C, respectively, whereas at 16 °C prepupal developmental time prolonged 101.68 d. It seems that this longest prepupal developmental time observed at 16 °C may be related to less tolerance to low temperatures which could stop development and prolong this stage. Few researchers have focused on overwintering or diapause of G. pyloalis were 28.77 and 29.21 d for female and male, respectively. It seems that our findings for total developmental time were consistent with those of G. pyloalis completed the development from 35.04 to 37.64 d on different mulberry varieties.The results of the current study indicated that the egg-to-adult developmental time was completed in 29.34 d at 24 °C. Based on G. pyloalis decreased with increasing temperature from 16 to 30 °C, and came out from linear mode at 32 °C. Assuming that developmental rate of all immature stages is a linear function of temperature within the 20–30 °C range, whereas a nonlinear response occurred at extreme temperature 32 °C. Therefore, the data deviated from linearity at 32 °C was excluded from linear regressions. Moreover, developmental rate of G. pyloalis at 16 °C was omitted because very low survivorship (4%) was observed at this temperature.As temperature exerts noticeable inﬂuence among the climate factors, by directly affecting insect phenology and distribution, most of the models that describe insect development are temperature driven . SeveralG. pyloalis was estimated at 10.30 and 11.22 °C based on traditional and Ikemoto–Takai linear models, respectively. Both linear models had high values of R2 and R2adj, indicating a high degree of conﬁdence. It should be noted that the higher Tmin values were estimated by traditional (13.58 °C), Ikemoto–Takai (14.93 °C), Analytis (15.98 °C), Briere-2 (16.04 °C), Lactin-2 (14.8 °C), and SSI (16.96 °C) models for prepupal stage compared with other stages, suggesting that the prepupal stage showed sensitivity to lower temperatures. With regard to higher Tmin values for prepupal stage of G. pyloalis compared with other  stages, the prepupal stage might be assigned as critical stage for diapause or hibernation. Generally, low temperature might enable to stop development and induce hibernation at prepupal stage of G. pyloalis as G. pyloalis, as well as physiological experiments for understanding the hormonal mechanisms responsible for.According to the results of the present study, the lower developmental threshold for overall immature stages of G. pyloalis required high thermal constant for completion of entire immature stages, the late incidence of G. pyloalis during the post commercial season of mulberry could be justifiable. G. pyloalis were distinct during the late season and the pest damage was limited in spring crop of silkworm. In fact, those results could support our findings on high thermal constant of G. pyloalis, explaining some reasons for the pest prolonger in the late spring and summer. So far no information exists on temperature-dependent development of G. pyloalis and in the current study its critical temperatures and thermal requirements were estimated for the first time. Hence, further physiological and ecological studies would warrant to quantify the phenology of G. pyloalis based on thermal requirements.The obtained results of the present study revealed that the thermal constants for overall immature stages were 429.18 and 401.88 DD estimated by traditional and Ikemoto–Takai linear models. Considering that R2adj) in all mentioned nonlinear models fitting to overall developmental rate were higher than 0.95, suggesting the high degree of confidence in estimated parameters. Nonetheless, to select the models which provide satisfactory fit to observed data, the R2adj is not sufficient. It should be noticed that although the Lactin-2 gave a good fit to the observed data for total developmental times as indicated by the high values R2adj, the model underestimated the Tmin values. Comparing the Tmin estimated by Lactin-2 using observed total developmental rate under laboratory conditions indicated that the Lactin-2 did not provide a realistic estimate of this critical temperature. In fact, the Lactin-2 underestimated Tmin at 9 °C, whereas failure of G. pyloalis development was observed at 12 °C. Furthermore, Tmin of 12.09, 12.85, and 11.13 °C for egg stage estimated by the Analytis, Briere-2, and SSI models were strongly provided by experimental observations, in which, eggs of G. pyloalis could hatch at 12 °C but no neonate larvae survived and developed to next stages. The survivorship of larvae at higher temperatures compared with egg stage resulted in estimating the higher Tmin values for larval stage.Since the linear models is unsecure in predicting development in extreme temperatures, several nonlinear models provide critical temperatures such as lower and upper temperature thresholds, fastest development temperature and intrinsic optimum temperature . To descTmax  for total immature stages which are consistent with those of experimental observations. Whereas the Lactin-2 overestimated Tmax values for overall immature stages of G. pyloalis at 38.88 °C.Our findings revealed that the Analytis, Briere-2, and SSI models approximately provided satisfactory estimates of Tfast for overall immature stages at which the highest developmental rates were estimated, ranged 30.7–31.1 °C using the Analytis, Briere-2, Lactin-2, and SSI models. These four models seem to provide realistic values of Tfast because the shortest developmental time of G. pyloalis was recorded at 30 °C under laboratory conditions. Many earlier researchers documented that the temperature, at which the developmental time is shortest, should be considered as the optimal temperature (Topt) has different concepts from Tfast. In fact, the temperature at which the population size reaches its maximum is not the temperature (Tfast) that can make insects develop fastest with low survival and net reproductive rate. The current study showed that the values of Topt estimated by SSI model for overall immature stages of G. pyloalis was at 24.63 °C, although the highest developmental rate was estimated at 30.7 °C. Based on thermodynamic concepts of SSI model, at Topt of 24.63 °C determined for overall immature stages, the maximal active state enzymes involved in the developmental process (G. pyloalis at different temperatures because of lack of such information. In that case, the seasonal prediction of the occurrence as well as severity of the pest would be accurately clarified.Based on current study, perature , ignorin process . The intG. pyloalis in relation to temperature. The results of the present study could provide essential information on temperature-dependent development of G. pyloalis and its critical temperatures. Using those valuable information with other ecological data such as intrinsic rate of increase, survival rate and climate factors would enable researchers to predict the population dynamics of G. pyloalis for applied IPM implementation.Accordingly, the importance of the seasonal occurrence prediction of the pest for developing management strategies has led to different linear and nonlinear models that describe the developmental rate of


Apisin, a protein that is unique to royal jelly (RJ), is known to compose the greater part of the RJ proteins and to exist as a heterooligomer containing major royal jelly protein 1 and apisimin. However, few reports on the methods for quantifying apisin have been published. Thus, we attempted to quantify apisin using HPLC, a widely used analytical technique, as described below. Isoelectric precipitation and size-exclusion chromatography were used to obtain the purified protein, which was identified as apisin by SDS-PAGE and LC-MS analyses. The purified apisin was lyophilized and then used to generate a calibration curve to quantify apisin in RJ. The apisin content was fairly constant  in natural RJ. This study is the first to describe a simple, standardized method for quantifying apisin using HPLC and suggests that apisin can be used as a benchmark for future evaluations of RJ quality. Queen honeybees have an approximately 2-fold larger body size and 10-fold longer lifespan than worker bees. Royal jelly (RJ) is a special food for queen bees, and royalactin, an RJ protein, has been reported to play an important role in the differentiation of queen bees –7, among Apis mellifera L. [A 350 kDa glycoprotein from RJ exhibiting high-mannose-type N-glycosylation was first reported in 1995 . This 35ifera L. , 12. Subifera L. . In factifera L. , coincidifera L. . Therefoifera L. –16. Howeifera L.  or ultraifera L.  which isIn the present study, we attempted to establish a simple method for quantifying apisin by HPLC based on a standard curve. Then, we quantified the apisin content in various RJ products using the developed HPLC method. Apis mellifera L. in China, were obtained from API Co., Ltd. , and stored at −20°C and 4°C, respectively, until further use. Fresh RJ was also collected from the Kawashima Apiary of API Co., Ltd. Briefly, female honeybee larvae (approximately 1 mm in length) were gently placed into individual artificial queen cells (diameter 10 mm × height 12 mm). After 72 h, the larvae were removed from the cells, and then fresh RJ was collected with a soft spoon. The fresh RJ was aliquoted into tubes containing 150 mg each and used immediately or stored at −20°C, 4°C, 25°C, and 40°C.Natural and lyophilized RJ, which originated fromμL, a flow rate of 0.3 mL/min, a column temperature of 30°C, and a wavelength of 280 nm. The column was calibrated using Gel Filtration Calibration Kit High Molecular Weight Proteins . The degree (%) of purity of each sample was assessed by the ratio of the apisin peak area to the total peak area in the chromatograms.Natural RJ samples (3 g) were suspended in 100 mL of ultrapure water, and then the pH of the RJ suspension was increased from 4.0 to 6.0 by 0.2 by adding 10% (w/v) sodium hydroxide solution. Each pH-controlled suspension was centrifuged at 1,880 ×g for 20 min at 4°C to separate the supernatant and pellet. The supernatants were diluted to 100 mL with 0.1 M phosphate buffer containing 0.3 M NaCl (pH 7.2) by reference to a previous study , and theThe apisin-rich fraction was prepared by suspending the pellet in the buffer solution and was purified by size-exclusion column chromatography using Sephacryl S-300HR  with a mobile phase of 0.1 M phosphate buffer containing 0.3 M NaCl (pH 7.2). The purity of the apisin fraction was further analyzed on an anion-exchange chromatography using a TSK gel DEAE-5PW column . The apisin fraction was collected, desalted by ultrafiltration with an Amicon Ultra-15 unit , added to ampoules with a 9 : 1 ratio of trehalose (D-(+)-trehalose anhydrous Tokyo Chemical Ind., Co., Ltd., Tokyo, Japan) to apisin, and then lyophilized. The net amount of apisin was calculated by subtracting the trehalose weight from the total weight, including the weight of the container.In the stability test, the lyophilized apisin was preserved at −20°C or 4°C for 1, 3, 6, 9, and 12 months and was quantified using an internal standardization method at each point. The purified apisin and ovalbumin , which was used as an internal standard, were dissolved in the mobile phase solution consisting of 0.1 M phosphate buffer and 0.3 M NaCl (pH 7.2) and analyzed by size-exclusion HPLC. The residual amounts of apisin are expressed as a percent of the initial (day 0) value.μg of the protein was loaded onto 10–20% acrylamide SDS-PAGE gels  and electrophoresed with a constant current of 25 mA/gel for 70 min. A buffer consisting of 0.025 M Tris, 0.192 M glycine, and 0.2% (w/v) SDS was used as the running buffer. The proteins were visualized with Oriole Fluorescent Gel Stain . The gel was calibrated by measuring the migration distances for standard proteins of known molecular masses .The fractionated, purified protein was identified as apisin using SDS-PAGE, followed by LC-MS analysis. The test samples were dissolved in SDS sample buffer  containing 10% (v/v) glycerol, 2% (w/v) SDS, and 50 mM dithiothreitol), vortexed, and then boiled for 3 min at 95°C. After measuring the protein concentrations using a bicinchoninic acid (BCA) protein assay kit  and adding the tracking dye bromophenol blue, 5 4HCO3) in 50% acetonitrile (ACN), dehydrated with 100% ACN, reduced with 50 μL of 10 mM dithiothreitol/100 mM NH4HCO3 at 56°C for 30 min, and then alkylated with 50 μL of 55 mM iodoacetamide/100 mM NH4HCO3 for 45 min at room temperature in the dark. The gel slices were then washed with 100 mM NH4HCO3 and dehydrated with 100% ACN, and the proteins were digested with 50 μL of 0.01 g/L lysyl endopeptidase/100 mM NH4HCO3  or with 50 μL of 0.005 g/L aspartic protease/100 mM NH4HCO3  at 37°C for 16 h (overnight). The peptides were extracted by sonication for 3 min at ambient temperature, followed by the addition of the extraction buffer (50% ACN containing 0.5% formic acid), and vortexed twice for 10 min each. The extracted peptides were dried in a speed vacuum, dissolved in 20 μL of 0.1% formic acid by sonication, and then subjected to LC-MS analysis.Each protein band on the gel was excised, washed with 100 mM ammonium bicarbonate  by binary gradient elution (from 0.1% formic acid/water to 0.1% formic acid/ACN in 80 min) at a flow rate of 0.2 mL/min via an ultraperformance liquid chromatograph (UPLC) connected to a liquid chromatograph-electrospray ionization quadrupole/time-of-flight mass spectrometer (LC-ESI-QTof MS) . The LC-MS was operated in MSE (positive/sensitivity) mode. The MSE spectra were searched against the NCBI Taxonomy database, in which 64,296 proteins were registered with “Apis” (ID: 7459), using the ProteinLynx Global SERVER software . The parameters used in this analysis were as follows: (1) Lys-C or Asp-N as the protease, (2) one missed cleavage site, (3) carbamidomethylation of cysteine as a fixed modification, and (4) oxidation of methionine and tryptophan and phosphorylation of serine, threonine, and tyrosine as variable modifications. To identify the protein, at least 3 different theoretical y or b ions were assigned to one unique peptide, and a total of 7 different unambiguous y or b ions were necessary for the successful manual verification of the protein. The MS and MSE ion intensity thresholds were 150 counts and 50 counts, respectively.The peptides were separated on a Protein Separation Technology BEH300 C18 column  using a gradient elution with a water/ACN mobile phase (80 : 20 to 80 : 20 in 10 min) containing 0.1% formic acid at a flow rate of 0.2 mL/min. The MS results were analyzed using MAXENT I software (Waters).The protein bands on the SDS-PAGE gel were extracted using the alkaline extraction method . Brieflyμm), and apisin was quantified by size-exclusion HPLC analysis according to the methods described in Natural RJ (150 mg) or lyophilized RJ (50 mg) was dissolved in 50 mL of 0.1 M phosphate buffer containing 0.3 M NaCl at pH 7.2 and centrifuged at 12,500 ×g for 5 min at 4°C. The supernatant was filtered (0.2 Apis mellifera L.) keeping farm. The fresh RJ was immediately used to test the stability of apisin at −20°C, 4°C, 25°C, and 40°C. The residual quantity of apisin at each temperature was measured by the standard curve method using size-exclusion HPLC after 1, 4, 7, 14, and 32 days. The experiments were performed in triplicate.As described above, fresh RJ was prepared from our bee  precipitated proteins, and the purified proteins are shown in We demonstrated that apisin could be accurately quantified with a standard curve using HPLC. The apisin content in natural and lyophilized RJ was 3.93 to 4.67% (w/w) and 10.93 to 11.86% (w/w), respectively , indicatWe assessed the residual quantity of apisin in fresh RJ after preservation at various temperatures. The quantity of apisin was unchanged at −20°C and 4°C for at least 32 days but decreased by approximately 10% and 80% at 25°C and 40°C, respectively .Here, we successfully demonstrated the first HPLC-based method for the accurate quantitation of the RJ protein apisin. As shown in http://web.expasy.org/compute_pi/) based on their amino acid sequences. Based on these isoelectric points, the RJ proteins were precipitated at pH values ranging from 4.0 to 6.0, whereas the apisin-rich protein fractions were obtained at pH values from 4.4 to 5.2; thus, as expected, apisin has an isoelectric point of approximately 4.6. The apisin-rich fraction was also purified by size-exclusion gel chromatography, which is frequently used to separate various biomaterials, such as proteins and polysaccharides, according to their molecular sizes. Ultimately, we were able to derive apisin, which was detected as a single peak in the HPLC chromatogram, from the RJ proteins. To the best of our knowledge, this is the first report of the isolation of apisin using isoelectric precipitation followed by size-exclusion gel chromatography. Isoelectric precipitation is a commonly used, rapid, and simple method for fractionating individual proteins. In addition, this relatively easy method provides a large amount of the apisin-enriched fraction, which could be used for additional functional, physicochemical, and analytical studies of RJ.Initially, we attempted to fractionate crude apisin by isoelectric precipitation at the optimum pH values. Apisin is a heterooligomer containing MRJP1 and apisimin, and their theoretical isoelectric points were computed to be 5.03 and 4.55, respectively, using the ExPASy Compute pI/Mw tool  the sequences of the mature protein and the protein in the database, which includes a signal peptide sequence, differ. (2) Only one or two detectable peptides can be generated by the LC-MS analysis because two cleavage sites—Lys-C and Asp-N—exist in the apisimin sequence. (3) The detectable peptides include Ile, which can be distinguished from Leu by LC-MS analysis . Thus, wThird, we tried to measure the apisin content in various RJ products. The apisin fraction that was purified by gel chromatography was desalted and lyophilized with trehalose to obtain a dried apisin powder, which was used as a standard reference material. Trehalose is typically used to prevent the lyophilization-induced denaturation of proteins . Indeed,Currently, the content of 10-hydroxy-2-decenoic acid (10-HDA), which is a chemically stable fatty acid unique to RJ, is required for the specification of RJ products. However, a potential disadvantage of using 10-HDA is that the amount of 10-HDA in RJ decreases during the production process and can be manipulated by adding an extraneous 10-HDA-rich RJ fraction. In contrast, the content of apisin was fairly constant among RJ products, for example, the fresh RJ from our bee farm, the imported RJ from China, and the lyophilized RJ. Thus, there is a possibility that the MRJP1 oligomer apisin can be used as a benchmark for future evaluations of RJ quality in addition to 10-HDA, although there are a few critical issues that must be overcome relating to achieving a secure supply of the apisin reference standard for quantitative analysis.In conclusion, we developed the first method for the rapid isolation and purification of the RJ protein apisin and then quantified this protein by HPLC using a standard curve. This analysis revealed that the apisin content is fairly constant among natural RJ samples.


Long-term fertilisation has a large influence on soil physical and chemical properties in agro-ecosystems. The effects on the distribution of aggregates, however, are not fully understood. We determined the dynamic change of the distribution of aggregates and soil organic carbon (SOC) content over time in a long-term field experiment established in 1998 on the Loess Plateau of China and illustrated the relationship between them.Dv).We determined SOC content and the distribution of aggregates in nine fertiliser treatments: manure (M); nitrogen (N); phosphorus (P); M and N; M, N, and P; M and P; N and P; bare land; and an unfertilised control. These parameters were then used for a path analysis and to analyse the fractal dimension (Dv), however, did not differ among the treatments.The organic fertiliser increased SOC content. The proportions of 0.1–0.25 mm microaggregates and 0.25–0.5 mm macroaggregates were higher and the proportion of the 0.01–0.05 mm size class of the silt + clay fraction was lower in the treatments receiving organic fertiliser  than that in the control, indicating that the addition of organic fertiliser promoted aggregation. The distribution of aggregates characterised by their fractal dimension (Dv was strongly correlated with the proportion of the <0.002 mm size class of the silt + clay fraction that did not differ significantly among the treatments. The change in the distribution of aggregates was strongly correlated with SOC content, which could produce organic polymer binding agents to increase the proportion of larger particles. Long-term application of organic fertiliser is thus necessary for the improvement and maintenance of soil quality in semi-arid agricultural land when residues are removed. Restorase gases . SOC conThe distribution and stability of soil aggregates are important indicators of soil physical quality, highlighting the importance of soil management on particle aggregation and disaggregation . AggregaThe long-term application of organic fertiliser often increases SOC content  and the Dm) for studying the fractal characteristics of soil structures. The calculation of Dm, however, assumes that particles of different sizes have the same density, but this assumption has been challenged by Dv) based on laser diffraction to characterise soil-particle and aggregate distribution  to describe the dynamic changes in SOC content and Dv over 15 years.Long-term experiments can provide more realistic scenarios for observing changes in soil properties and processes  and are −1, total nitrogen (N) content of 0.57 g kg−1, total phosphorus (P) content of 0.63 g kg−1, available N content of 28.99 mg kg−1, available P content of 2.49 mg kg−1, available potassium (K) content of 84.86 mg kg−1, pH 8.6, and bulk density of 1.5 g cm−3.This study was part of an on-going long-term field fertilisation experiment established in 1998 at the Ansai National Field Scientific Observation and Research Station for Farmland Ecosystems, Shaanxi province, China . The station is at an altitude of 1,068–1,309 m a.s.l. and has a temperate semi-arid climate with a mean annual temperature of 8.8 °C and a mean annual rainfall of 500 mm. The soil is a Huangmian soil, which is classified as a Calcic Cambisol , origina2 for each plot. Each block contained nine treatments: manure (M), N, P, M and N (MN), M, N, and P (NMP), M and P (MP), N and P (NP), unfertilised bare land (BL), and an unfertilised control (CK)  were seeded on 29 April 2012 at a rate of 52.5 kg ha−1, and the plant density was about 51,000 plants ha−1. The crops were manually harvested, and the aboveground residues were removed in October.The long-term experiment had a triplicate randomised complete block design with an area of 14 mrol (CK) . BL had Dv, and SOC content. Visible plant residues were removed, and the samples were manually broken into fragments <10 mm and air-dried at room temperature. Each sample was passed through a 1-mm sieve for determining the distribution of the aggregates, and a subsample was then ground to pass through a 0.25-mm sieve for the determination of total SOC content.The soil was sampled annually in October from 1998 to 2012 to a depth of 20 cm. Three replicate soil samples were randomly excavated in each plot using a soil drill  and then mixed to produce a composite sample. The samples for 1998–2012 were used for the analysis of changes over time. The samples for 1998–2011 (126 samples) had been stored in the station’s soil library and were collected from one of the three replicate treatment plots. The samples for 2012 (27 samples) were used to examine the effects of fertilisation on aggregate distribution, The soil samples were soaked in distilled water for 24 h and mechanically dispersed by ultrasonication for 5 min . The samSOC content was determined by Walkley and Black dichromate oxidation .Dv was calculated as:r is the particle diameter, Ri is the diameter of size class i, V (r<Ri) is the total volume of particles with diameters <Ri, VT is the total volume of particles, Rmax is the maximal particle diameter, and Dv is the volume fractal dimension. Logarithms were derived for both sides of the equation, and Dv was obtained from the slopes of the double-logarithmic curves that fit the data.Path analysis is a supplement and extension of regression analysis that partitions simple correlation coefficients into direct and indirect effects through definite path coefficients among the variables and distinguishes between correlation and causation .Dv, with criteria for the stepwise regression of probability-of-F-to-enter ≤0.05 and probability-of-F-to remove ≥0.10. The size classes that did not significantly contribute to Dv at P = 0.10 were therefore not included in the regression model.The aggregates were categorised into nine size classes: <0.002, 0.002–0.005, 0.005–0.01, 0.01–0.05, 0.05–0.1, 0.1–0.2, 0.2–0.25, 0.25–0.5, and 0.5–1 mm. These nine size classes were categorised into three fractions: macroaggregates (0.25–1 mm), microaggregates (0.05–0.25 mm), and a silt + clay fraction (<0.05 mm). Backward-stepwise regression was used to identify the size class that explained most of the variation in Dv , and rijPij is the indirect effect of aggregate size class on Dv. Subscript designations 1–9 represent the <0.002, 0.002–0.005, 0.005–0.01, 0.05–0.1, 0.1–0.2, 0.2–0.25, 0.25–0.5, 0.5–1 mm aggregates and Dv, respectively.U, is an unmeasured variable in the path model that represents the unexplained part of an observed variable and is calculated as   .Dv did not differ significantly among the nine treatments.SOC content was significantly higher in the treatments receiving organic fertiliser  than in the treatments receiving mineral fertiliser  or in BL or CK. SOC content did not differ significantly among BL, CK, N, NP, and P. The 0.01–0.05 size class of the silt + clay fraction and 0.05–0.1 mm microaggregates represented >50% of the total soil aggregates . The proDv was correlated positively with the proportion of the <0.01 mm size class of the silt + clay fraction and negatively with the proportions of the 0.01–0.05 mm size class of the silt + clay fraction and 0.2–0.25 mm microaggregates. SOC content was correlated negatively with the proportion of the <0.05 mm size class of the silt + clay fraction and positively with the proportions of micro- and macroaggregates (0.05–1 mm).The proportions of all size classes of the silt + clay fraction were positively correlated with each other, except between the 0.01–0.05 mm and <0.002 mm and 0.005–0.01 mm size classes, and were negatively correlated with the proportions of micro- and macroaggregates (0.05–1 mm), except between the 0.2–0.25 and 0.002–0.005 and 0.01–0.05 mm size classes . The proU (uncorrected residue) of 0.054 and an R2 of 0.997 indicated a small unexplained part of the observed variable in the path model, and the path analysis explained 99.7% of the variability associated with Dv (Dv (P < 0.01). The magnitude of the path coefficients indicated that silt + clay fraction (<0.002 mm) (P19 = 0.887) was the most important causal factor in predicting Dv, followed by the 0.002–0.005 mm size class of the silt + clay fraction (P29 = 0.368). The direct effect of the <0.002 mm size class of the silt + clay fraction (0.724) on Dv was larger than its total indirect effects (−0.132), and the direct and total indirect effects of the 0.002–0.005 mm size class of the silt + clay fraction on Dv were comparable . The total indirect effects of the 0.005–0.01 mm size class of the silt + clay fraction (0.586) on Dv, however, were larger than the direct effects (0.055). The indirect effects of the 0.002–0.01 mm size classes of the silt + clay fraction on Dv due to the <0.002 mm size class were large (r12P19 = 0.749 and r13P19 = 0.732).A  with Dv . The pat with Dv  indicateDyx1x1 = 0.787), followed by correlations between the <0.002 mm size class of the silt + clay fraction and 0.25–0.5 mm macroaggregates (Dyx1x7 = 0.674), the <0.002 and 0.002–0.005 mm size classes of the silt + clay fraction (Dyx1x2 = 0.653), and the <0.002 and 0.01–0.05 mm size classes of the silt + clay fraction (Dyx1x8 = 0.486). The other coefficients of determination were low  and the total indirect effect of the 0.002–0.01 mm size classes on Dv due to the <0.002 mm size class matched the highest positive correlation coefficient between the <0.002 mm size class and Dv, corresponding to the highest coefficient of determination of the <0.002 mm size class (Dyx1x1 = 0.787), the large coefficients of determination of the correlations between the <0.002 mm size class of the silt + clay fraction and 0.25–0.5 mm macroaggregates (Dyx1x7 = 0.674), between the <0.002 and 0.002–0.005 mm size classes of the silt + clay fraction (Dyx1x2 = 0.653), and between the <0.002 and 0.01–0.05 mm size classes of the silt + clay fraction (Dyx1x8 = 0.486). Our result was in agreement with previous studies by Dv was positively correlated with the proportion of the <0.002 mm size class of the silt + clay fraction.ccession , becausefraction  that didfraction . The proThe proportion of the silt + clay fraction (<0.05 mm) was generally negatively correlated with the proportions of micro- and macroaggregates (0.05–1 mm), but the proportions of some size classes in the 0.05–1 mm fractions (micro- and macroaggregates) were positively correlated, indicating that larger aggregates formed at the expense of the silt and clay fraction and vice versa and that aggregates >0.05 mm may stimulate each other to reaggregate. This phenomenon was demonstrated by SOC content in our study was correlated negatively with the proportion of the silt + clay fraction (<0.05 mm) and positively with the proportions of micro- and macroaggregates (0.05–1 mm), indicating that the increase in SOC content facilitated aggregation and that micro- and macroaggregates played an important role in stabilising the SOC content . MucilagThe long-term application of organic fertiliser increased SOC content from 1998 to 2012, in agreement with the findings by Dv could not distinguish among the various treatments, because Dv was mainly determined by the proportion of the <0.002 mm size class of the silt + clay fraction that did not differ significantly among the treatments. SOC content was significantly higher in the treatments receiving organic fertiliser  compared to those receiving mineral fertiliser, BL, and CK and tended to increase over time, which promoted the formation of larger aggregates and the sequestration of SOC. The application of organic fertiliser can thus contribute greatly to the improvement and sustainability of soil quality in semi-arid agricultural land when residues are removed.Treatments receiving organic fertiliser increased the proportions of 0.1–0.25 mm microaggregates and 0.25–0.5 mm macroaggregates and correspondingly decreased the proportion of the 0.01–0.05 mm size class of the silt + clay fraction relative to CK, indicating that the application of organic fertiliser was favourable to the formation of larger aggregates. 10.7717/peerj.4758/supp-1Data S1Click here for additional data file.


To determine longitudinally the relationship between serum 25-hydroxyvitamin D (vitamin D) and vitamin D-binding protein (DBP) levels in mother-neonate pairs and evaluate the efficiency of prophylactic vitamin D on lactation days 45–60.Mother-neonate pairs whose serum calcium (Ca), phosphorus (P), magnesium (Mg), alkaline phosphatase (ALP), and parathyroid hormone (PTH) levels were in normal ranges on postpartum/postnatal days 5–10 were classified into two groups by their serum vitamin D concentrations (Group A: < 10 ng/ml and Group B: > 20 ng/ml). Both maternal and neonatal Ca, P, Mg, ALP, and PTH concentrations in group A and B were not different. Maternal and neonatal serum DBP levels were measured in two groups. The mother-neonate pairs in both groups were given 400 IU/d vitamin D orally. The same biochemical markers in group A were remeasured on days 45–60 of the lactation period.P = 0.000; P = 0.000 and P = 0.04; P = 0.004, respectively). On lactation days 45–60, the maternal and neonatal DBP concentrations were not different from those on postpartum/postnatal days 5–10. However, the maternal and neonatal vitamin D levels were significantly increased , while the neonatal PTH concentrations were significantly decreased (P = 0.000). The maternal and neonatal vitamin D concentrations were negatively correlated with their DBP concentrations .In group A, the mean maternal and neonatal vitamin D levels on postpartum/postnatal days 5–10 were significantly lower and the DBP levels were significantly higher than those in group B  is indicated for mothers and their infants. There are a growing number of reports on the high prevalence of vitamin D deficiency in pregnant women and their neonates in many developed and developing countries from areas of different sun exposure . In thosVitamin D-binding protein (DBP) is a multifunctional and highly expressed plasma protein. One of the major functions of DBP is to bind and transport vitamin D, the major circulating metabolite, and 1,25-dihydroxyvitamin D (active vitamin D) . PregnanWe hypothesized that high maternal and neonatal DBP levels may lead to an incorrect low estimate of the true Vitamin D level and that a prophylactic vitamin D dose (400 IU/d) would be sufficient for these mothers and their infants during the early lactation period. Therefore, the goal of the present study was to assess the relationship between serum vitamin D and DBP levels in both mothers and their neonates and evaluate the effectiveness of prophylactic vitamin D at lactation days 45–60.The University of Ataturk, Faculty of Medicine Ethics Committee approved the study. Written informed consent was obtained from all participants. The study was longitudinal and was conducted in winter and spring months between 2013 and 2016. Serum vitamin D and DBP concentrations of mothers and their neonates were investigated during the first 60 days of the lactation period. First, the mothers were selected from Department of Obstetrics and Gynaecology, Faculty of Medicine, Ataturk University, Erzurum, Turkey, which offers tertiary healthcare for a population of more than 4.000.000 individuals. Exclusion criteria for the mothers were multiple gestation, different ethnicity, delivery within the previous 2 years, pre-existing maternal disease, pre-eclampsia, BMI > 35.0 at enrolment, hypertension, smoking, hormonal or drug therapy, and lactation failure. Second, the neonates of the mothers that met the study criteria were evaluated. Inclusion criteria for neonates were term deliveries with appropriate for gestational age (AGA) after uncomplicated pregnancies. Neonates classified as large for gestational age (LGA) or small for gestational age (SGA) and having any fetal malformation, supplementation with formula, and failure for visit 2 were excluded from the study together with their mothers. Then, serum Ca, P, Mg, ALP, PTH and vitamin D concentrations in the mothers selected for the study were measured on postpartum days 5–10. The mothers having normal serum Ca (8.8–10.8 mg/dl), P (2.7–4.2 mg/dl), Mg (1.2–1.6 mg/dl), ALP (130–560 IU/l), and PTH (1–55 pg/ml) were classified into two groups by their serum vitamin D concentration (Group A: < 10 ng/ml and Group B: > 20 ng/ml), whereas those whose serum vitamin D concentration was 10–20 ng/ml were excluded from the study. Finally, the same biochemical markers in the neonates of the mothers included in the study were measured on postnatal days 5–10. Among the neonates having normal serum Ca (7.6–11.2 mg/dl), P (4.8–8.2 mg/dl), Mg (1.2–1.6 mg/dl), ALP (140–450 IU/l), and PTH (1–43 pg/ml), those having similar vitamin D concentrations as their mothers were included the study, whereas those whose serum vitamin D concentration was 10–20 ng/ml were excluded from the study together with their mothers. The mean maternal and neonatal serum Ca, P, Mg, ALP, and PTH concentrations in group A and B were not significantly different . Length (±0.1 cm) was measured using a body-length measurer by a pediatrician. Head circumference was measured by a tape measure.In addition to maternal age and clothing style (covered: Muslim style clothing and uncovered), the duration and the dose of the vitamin D supplementation and the duration of exposure to sunlight during the gestational period were recorded based on self-assessment of the mothers.The serum samples were maintained in a refrigerator overnight with the purpose of thawing before analysis. Serum Ca, P, Mg, and ALP levels were determined by spectrophotometric methods, using the Beckman Coulter- AU5800 chemistry analyzer. Serum PTH and vitamin D levels were measured by the immunoassay method, using the Beckman Coulter-DXI800 analyzer. We used a human vitamin D binding protein kit . The limit of sensitivity for the kit was 0.01 μg/ml. The intraassay and interassay coefficients of variation were < 10% and < 12%, respectively.The total sample size for the study was calculated by a formula below.r = 1. Zα/2 and Z1-β are 1.96 for 5% level of significance and 1.28 for 90% of statistical power, respectively. δ and d represent the pooled standard deviation and difference of the mean for DBP, respectively. According to that formula, the total sample size for each group with 90% of statistical power and 5% level of significance was calculated as 29.r is a ratio (n1/n2) of sample sizes in Group A (n1) and B (n2). We planned that group A and B had equal sample size. Thus, P < 0.05.All the calculations were made using SPSS (version 15.0 for Windows). The Kolmogorov-Smirnov test was used for normality. The differences between group A and group B were examined with Student’s t test and Chi square test. Longitudinal changes in the serum parameters in group A were analyzed with the paired t test. Correlations between two variables were tested by Pearson’s correlation co-efficient. The results were expressed as the means ± SD, and statistical significance was set at P = 0.001 and P = 0.02, respectively)    Table . In grouP = 0.000 and P = 0.004, respectively)   Table . In grouy) Table .Table 4Nr = − 0.239, P = 0.048 and r = − 0.401, P = 0.002)  and of 20–50 ng/ml 50–125 nmol/l) are considered to represent vitamin D deficiency and sufficiency, respectively –7. Accor5 nmol/l It has been shown that ethnicity and DBP genotype, age and gender, pregnancy and estrogen status, obesity, liver and renal diseases, diabetes, primary hyperparathyroidism, cancers, inflammation, and some therapeutic procedures such as plasma exchange and peritoneal dialysis may affect serum DBP and vitamin D concentrations . The DBPIn the follow-up of group A, both maternal and neonatal serum DBP concentrations were not different from those of lactation days 5–10, while serum vitamin D concentrations increased significantly. Vitamin D supplementation (400 IU/d) might have contributed to these higher concentrations. In addition, because vitamin D supplementation may just maintain the serum vitamin D concentration at a similar level, we can also speculate that the increase in serum vitamin D levels may be related to the release of vitamin D from DBP overtime.A high prevalence of subclinical vitamin D deficiency in pregnant women and neonates has been reported in many countries. In a review, 18% of pregnant women in the United Kingdom, 25% in the United Arab Emirates, 80% in Iran, 42% in northern India, 61% in New Zealand and 60–84% of pregnant non-Western women in the Netherlands have been shown to have serum vitamin D concentrations < 10 ng/ml [Our study has several limitations: 1) The number of subjects was relatively small and therefore, the power of the study is relatively limited; 2) All biochemical parameters in group B were in normal range. Therefore, they were measured only once for ethical reasons. Thus, the change in the biochemical parameters with time could not evaluated for group B; 3). We used an automated immunoassay method to measure serum vitamin D levels since only mothers and their infants whose serum Ca, P, Mg, ALP and PTH levels were normal were included in the study. However, it should be emphasized that, due to the analytical problem in available vitamin D assay, the serum vitamin D levels could also be measured by LC-MS/MS method.This is the first study that investigated the relationship between maternal and neonatal serum vitamin D and DBP during lactation. The findings of our study suggest that the high maternal and the neonatal serum DBP levels may be associated with falsely low vitamin D concentrations based on the normal serum Ca, P, Mg, ALP and PTH levels. Even if maternal and neonatal serum vitamin D concentrations are consistent with each other in terms of low serum vitamin D levels (< 10 ng/ml), this finding alone is not sufficient to diagnose vitamin D deficiency, without taking into consideration the serum Ca, P, Mg, ALP, and PTH levels. The lack of expected changes in serum Ca, P, ALP, and PTH levels together with low concentrations of vitamin D deficiency and a normal serum Mg may result from the measurement method and/or from high DBP levels, and may not reflect a true vitamin D deficiency. In this case, only prophylactic vitamin D (400 IU/d) is indicated for mothers and their infants.