Pinctada margaritifera, produces valuable pearls as a result of the biomineralisation process of a mantle graft from a donor inserted together with a nucleus into the gonad of a recipient oyster. To explore the respective roles of donor and recipient in pearl formation, a uniform experimental graft was designed using donor and recipient oysters monitored for their growth traits. At the same time, phenotypic parameters corresponding to pearl size and quality traits were recorded. Phenotypic interaction analysis demonstrated: 1) a positive correlation between recipient shell biometric parameters and pearl size, 2) an individual donor effect on cultured pearl quality traits. Furthermore, the expressions of biomineralisation biomarkers encoding proteins in the aragonite or prismatic layer showed: 1) higher gene expression levels of aragonite-related genes in the large donor phenotype in the graft tissue, and 2) correlation of gene expression in the pearl sac tissue with pearl quality traits and recipient biometric parameters. These results emphasize that pearl size is mainly driven by the recipient and that pearl quality traits are mainly driven by the donor.Grafting associates two distinct genotypes, each of which maintains its own genetic identity throughout the life of the grafted organism. Grafting technology is well documented in the plant kingdom, but much less so in animals. The pearl oyster,  The partners in a graft are known as the scion and root in plants and the donor and recipient in pearl oysters. The use of grafting technology in both plant cultivation and pearl production provides a means of improving quality in these industries. Grafting is a well-established practice that has many horticultural and biological uses. The numerous applications of plant grafting include vegetative propagation, avoidance of juvenility, cultivar change, creation of unusual growth forms, repair, size control, biotic and abiotic stress resistance and physiological improvement1. Some studies have examined the contribution of the scion; for example, a scion of a red-flowering rose grafted on a white rose stock will continue to produce red roses1. Grafting is commonly employed in agronomy to indirectly manipulate scion phenotype2. As for the host organism, rootstocks are commonly used to propagate selected scions to improve fruit tree tolerance to environmental stress, and to control tree size4. Roots anchor plants in the ground to provide water and nutrients from the soil and can serve as storage organs. The root system is a crucial component in coordinating response to a range of abiotic and biotic stressors6. The influence of rootstocks on yield productivity is being increasingly recognized to be as important as grafted scions. In the plant system, the genetic characteristics of the root appear to drive vegetative growth and the overall growth of the grafted system depending on environmental conditions7.From a genetic point of view, grafting creates a chimera of a single individual made up of genetic material issued from two (or more) distinct genomes, with each genome maintaining its own distinct genetic identity throughout the life of the grafted organism. Typically, grafting associates two tissues from different individuals, the recipient and graft, which form vascular connections and survive as a genetic chimera due to a unique symbiotic relationshipPinctada sp.), similarities to the scion–root system were suggested by studies on the contributions of the recipient and donor to the expression of the overall phenotype . In Pinctada maxima, results indicated low donor-related heritability of pearl size8, and xenografts between P. maxima and P. margaritifera demonstrated that individual donors significantly influence the color and the surface complexion of the pearl9. In P. margaritifera, one study demonstrated the differential influence of individual donors with black or red outer shell phenotypes, combined with green or yellow inner shell phenotypes, on pearl darkness level, color categories and luster10. Another study examined the phenotypic correlation between recipient shell weight and size of pearls in P. fucata, revealing a limited positive relationship whereby recipient oysters with heavier shells produced bigger pearls11. These results indicate potential underlying genetic correlations between the recipient or donor growth and pearl size, whereby selection for faster growing oysters might further improve pearl size.In the pearl oyster grafting process , we followed the kinetics of the recipient–donor influence on pearl formation by monitoring pearl size and quality parameters over 12 months. This information provides basic knowledge to help us to understand the donor-recipient correlations and their relative contributions in pearl oyster grafts.In the present study, the goal was to improve our understanding of the mechanisms driving the phenotypic variability arising from the recipient and the donor. The working hypothesis was that the recipient oyster confers quantitative traits to the pearl while the individual donor oyster drives qualitative traits of the pearl. In other words, everything related to growth and transfer of energy would be driven by the recipient oyster, and everything related to the quality of the nacrein deposit would be driven by the donor of the graft. Within this framework we defined four specific experiments to determine respective donor and recipient effects. (1) We sought to discern whether the donor or recipient was the main driver of pearl growth rate by comparing different recipient-donor phenotype combinations. (2) We evaluated drivers of the pearl phenotype by comparing effects of different recipient–donor phenotype combinations on nacre deposit quality. (3) We measured biomineralisation potential through a comparison of quantitative gene expression in the mantle graft (donor only effect) and the pearl sac (donor + recipient effect). For this approach, a representative panel of eight genes encoding proteins involved in aragonite biomineralisation , calcite biomineralisation  or proteins implicated in both layers (Nacrein) was screenedOverall, a total of 289 cultured pearls were harvested and analyzed. Donor pearl oysters were divided into two distinct groups according to their shell size : small donor oysters, denoted “QL”, and large ones, denoted “TL”. The distribution of QL and TL oysters are shown in Table p < 0.0001) with 79.8 ± 6.3 mm for shell height, 79.9 ± 6.9 mm for shell width and 21.0 ± 2 mm for shell thickness.At grafting time, the donor oyster width, height and thickness were recorded, showing average measurements of 48.6 ± 5.2 mm, 48.6 ± 5.2 mm and 12.7 ± 1.4 mm, respectively, for the QL donor group. The TL donor group was significantly larger for each parameter  . In the TL donor group, the average nacre weight and thickness were 0.34 ± 0.20 g and 0.70 ± 0.04 mm (N = 147). No significant effect of donor oyster shell size phenotype was detected for either nacre weight or thickness  except for color categories Fig. , where tp = 0.045), but no significant individual donor effect was shown for nacre weight (p = 0.059). Pearls from donor “Q21” (N = 7 pearls) showed the greatest average nacre thickness (1.16 ± 0.50 mm) and nacre weight (0.566 ± 0.298 g) compared with donor “Q17”, which represented the minimum (N = 7 pearls) . The influence of donor size and the evolution of this relationship through time on qualitative pearl traits at harvest are illustrated in Figure A significant individual donor effect was detected for nacre thickness (p = 0.002), color category (p < 0.0001), cultured pearl luster (matte or shiny/glossy/highly glossy) (p = 0.0002) and the surface defect classes (p < 0.0001). However, no significant difference was recorded between the individual donors for the absence/presence of circles (p = 0.218) or for the three shape categories (p = 0.156).A significant individual donor effect was detected for darkness level (p < 0.0001). In addition, there were significant positive correlations of nacre thickness and weight with shell width  , Spearman rho coefficients ranged from 0.51 to 0.65 and 0.49 to 0.62, respectively. The highest significant positive correlations were found for nacre thickness and pearl weight with recipient pearl oyster weight  Fig. .Figure 3Pif-177, MSI60 and Perl1, showed significant higher gene expression levels in the TL group (p < 0.0001) in the graft tissue compared with the QL group. Shem5, Shem 9 and Calc1 also showed significant higher gene expression levels in the TL group (p < 0.0001) compared with the QL group.The relative expression levels of the eight biomineralisation genes analyzed in the donor oyster mantle graft tissue and pearl sacs from the QL and TL groups are shown in Fig. p < 0.0001); only Prism was not correlated. Nacre thickness and pearl weight were not correlated with the relative expression level in the graft of eight biomineralisation genes  . Prism, Asp, Shem5 and Shem9 showed significantly greater expression in “QL” phenotypes compared with “TL” ones .Among the eight candidate genes studied in the pearl sac at the three harvest times, the expressions of six were significantly affected by donor phenotype Fig. . MSI60 aNo significant differences in relative expression of the panel of genes in the graft were found for pearl quality traits. In the pearl sac, however, significant effects were observed for the majority of the panel of genes, depending on grade, level of surface defects, luster and color (Supplementary Table p < 0.0001). Mean nacre thicknesses at harvest times T1, T2, T3 and T4 were 0.02 ± 0.01, 0.08 ± 0.03, 0.09 ± 0.03 and 0.11 ± 0.03 cm, respectively. Nacre was significantly thinner at T1 than at T2, T3 and T4. T3 and T4 were not significantly different from one another. Pearl nacre weight was also highly significantly different between harvest times (p < 0.0001). Mean pearl weights at harvest times T1, T2, T3 and T4 were 0.12 ± 0.07, 0.35 ± 0.14, 0.44 ± 0.17 and 0.54 ± 0.20 g, respectively. T1 pearls were significantly lighter in weight than T2, T3 and T4 ones. As with nacre thickness, T3 and T4 were not significantly different from one another , as shown in Fig. Analyzing kinetics data over the 12 months of the study, we observed, as expected, a very highly significant difference in nacre thickness between harvest times (p = 0.215). After 3 months of culture, the pearl shapes were distributed as follows: Baroque 29.6%, N = 21; Round 52%, N = 37; Oval 18%, N = 13. After 12 months of culture, the distribution had remained similar: Baroque 38%, N = 26; Round 31%, N = 21; Oval 31%, N = 21.Comparison between harvest times showed no significant difference between the proportions of the three shape categories (p = 0.005) between each of the harvest times. The T3 and T4 harvest times yielded about three times more circled pearls than T1.Figure p < 0.0001). T1 pearls were significantly more “matte” and less “glossy” than T2, T3 and T4 pearls. T1 had significantly less grade B pearls (12.7%) and more Reject pearls (43.7%) than the other harvest times T2 , T3 (19.7% and 11.3%) and T4 (38.2% and 2.9%).Data analysis showed a highly significant difference in pearl luster and grade between harvest times (p = 0.0004). T1 harvest time had significantly more lighter-toned pearls (32.4%) than the later harvest times T2 (7.6%), T3 (18.3%) and T4 (12.7%). T3 and T4 had significantly more dark pearls  than T1 (7%) and T2 (7.6%)  .P. fucata11 and in P. maxima15. Furthermore we observe a significant positive correlation between the expression of the aragonite gene in the pearl sac (Perl1) and one recipient oyster biometric parameter (shell height). This result suggests that the recipient oyster affects the activities of the biomineralising tissues, which are mainly dependent on calcium metabolism in the epithelial tissue of the pearl sac21. The recipient oyster regulates the metabolism of the pearl sac by supplying nutrients during the period of culture and thus regulates the expression of the biomineralisation genes, especially those implicated in the aragonite layers22. High biomineralisation capabilities may have contributed to a greater nacre deposition, as already observed in P. maxima25 and P. fucata26. The recipient oyster can be compared to the rootstock of a grafted plant, with recipient oyster and rootstock each corresponding to a grafted organism. In the plant system, rootstocks are the driving component of production systems and studies have reported that they can influence tree growth, yield and fruit size, weight, and rind thickness33. However, it is not fully understood how the rootstock controls scion growth. The grafted organism seems to have a key role in growth owing to its nutritive function. Interestingly, the same principle applies in the animal system we studied. The positive relationship between pearl size (nacre weight and thickness) and weight of the recipient is valuable information because it can serve as a basis for selective breeding strategy for fast growing recipient pearl oysters. For mollusk species, between 10% and 20% improvement in selected growth traits can be obtained per generation35.In the present study, we showed that pearl size phenotype (nacre weight and thickness) was highly correlated with the biometric parameters of the recipient oyster, while those of the donor oyster (QL/TL groupings) had no impact. This positive correlation confirms results observed in 36. A recent study in P. maxima found significant individual donor effects for color, nacre deposition, luster, shape and defects; however, each of these traits was grouped into broad categories in this previous study, making specific comparisons between the two studies difficult15. In plant models, research on rootstock–scion interaction demonstrated that yield and quality are conferred by the scion cultivar. Scion quality clearly affects final yield and fruit quality in grafted plants, but rootstock effects can also impact these characteristics37.A strong individual donor effect was observed on pearl quality traits, such as color  and quality parameters . Pearl quality traits are influenced by a number of highly variable factors, including genetics, the environment and their interaction, as well as surgical technique and graft hygienePif-177, MSI60, and Perl1). The protein genes Pif-177 and MSI60 regulate growth, nucleation and the organization of the aragonite crystal38. Perline is the protein equivalent of the N14 protein identified in P. maxima39 and seems to be specifically involved in the formation of the nacreous layer and promotion of aragonite crystal nucleation23. Aragonite-related gene expression levels were significantly higher in the graft tissue from the TL group than from the QL group. Some other genes (Shematrin 5 and 9 and Nacrein) showed significantly higher expression levels in the graft originating from the TL group. Shematrin 5 and 9 are implicated in the calcite mineralisation process, in which they facilitate the formation of calcite prisms40. These results seem to show the opposite trend to the pearl phenotype observations, since the TL and QL groups did not differ in nacre weight and thickness. The significantly higher expression levels of some genes in the TL group correspond to the biometric parameters of the donor oyster but not to the quantitative traits of the pearl. A study in P. fucata examined the differences of gene expression in large and small oysters. RNA-seq analysis revealed that the number of genes up-regulated in small oysters was greater compared to large oysters and could be explained by catch-up growth41. In the pearl sac analyses, we observed a relationship between relative gene expression in the pearl sac and grade, surface defects, luster and pearl color. The role of the pearl sac in nacreous layer biomineralisation is thought to mirror that of the oyster mantle. The gene-expression patterns of shell matrix proteins in pearl sacs were measured relative to the biomineralisation process of the nacreous layer42. The genome of the individual donor oyster and its influence on the pearl biomineralisation process remain active in the pearl sac tissue during the entire pearl culture period right up until harvest time45, confirming the influence of individual donors on quality pearl traits.Expression analysis in the pearl oyster graft, based on a panel of genes encoding proteins implicated in the shell biomineralisation process, indicated significant differences of gene expression level between the QL and TL donor groups for the three genes of the panel implicated in aragonite formation . Shell growth is influenced by two major factors: microalgal concentration and temperature, both of which regulate the expression of most of the shell matrix protein genes implicated in the biomineralisation process22. However, recipient growth had a linear pattern over the 12 months of culture. Thus, the growth of the recipient seems to be an important driving factor of pearl growth but not the only influence implicated. The high influence of the recipient and the weak influence of the donor on pearl size at harvest suggest that their respective roles are not totally revealed here.Our examination of pearl formation over time (12 months of rearing) clearly showed that nacreous deposition is not linear. By the half-way point of the experiment (6 months), 69.7% of the final nacre thickness and 65% of the nacre weight had already been reached. At this point, nacre deposition slowed down. No previous study has examined the deposition of the nacreous layer on the pearl over culture time. However, some studies did examine the beginning stage (35 days) of nacreous layer deposition during pearl formation in 48, who focused on surface color and pigmentation, the quality of nacreous pearls is determined by the ratio of the thickness of the lower prismatic layer to that of the upper nacreous layer. It was therefore logical that we found an improvement of quality grade over time, because nacre deposition increased with time of culture49. Individual donor oysters influence quality traits such as color, luster, amount of surface defects and grade but do not affect shape or the presence of circles. The differences observed between quality traits underline the complexity of their genetic basis and that of the donor/recipient interaction.In our experiment, we also recorded that luster, grade and darkness of pearls improved with duration of culture, but we also noted an increase in the proportion of circled pearls. According to WadaP. margaritifera. It was demonstrated that 1) pearl size characters (nacre weight and thickness) are influenced by the recipient oyster phenotype and 2) the major qualitative traits  are influenced by individual donor oysters. The recipient oyster shell parameters are correlated with pearl size, with the largest recipient giving the largest pearls. Similarly to other examples in the living world, the recipient organism seems to have a more important role than expected. Donor/recipient combinations should therefore be selected carefully. For the pearl industry, the contribution of the recipient found here suggests it would be beneficial to develop breeding programs on growth of recipient oysters in order to obtain larger and thus more valuable pearls. In parallel, a breeding program could be developed for quality and color of donor oysters in order to obtain high quality pearls in desired colors. Furthermore breeding program could be based on pedigree. In fact, genealogy information is essential for genetic traceability. The importance of maintaining pedigree records could aim to increase effective population sizes and minimise inbreeding to ensure long-term genetic gain, viability of aquaculture breeding programs and productivity by the development for example of a set of multiplex PCRs as in C. gigas oyster51 or in the abalone Haliotis midae52. The present work could serve as a basis for future studies exploring the donor/recipient relationship in greater depth.The present study is the first to identify the relative impact of donor/recipient oyster size on pearl size and quality traits during culture time in the marine bivalve Pinctada margaritifera were collected as spat using commercial collectors in the lagoon of Takaroa atoll  in March 2013. Nine months later (December 2013), 20 of these collectors were transferred to Ifremer facilities in Vairao on Tahiti Island . Pearl oysters (N = 1535) were then removed from the collectors and divided into two distinct groups according to their shell size : small “QL”,  and large “TL” . The distribution and phenotypes of QL and TL oysters are shown in Table 53, where they were left to grow, protected using plastic mesh to prevent predation, until they reached a sufficient size to start the grafting experiment. These oysters were then transferred to Regahiga Pearl Farm  to be used as donors. Prior to nucleus implantation and graft surgery, oysters from the QL and TL groups were collected from the rearing station, detached, washed with a high pressure spray (to remove epibiota) and stored until the grafting operation.Wild All grafts were performed under standard production conditions by a single expert at the Regahiga Pearl Farm so as to minimize the grafter effect on pearl quality traits described in ref. 17.Prior to the grafting operation, shell height, width and thickness of the 40 donor oysters were measured using Vernier calipers. All recipient oysters were washed with a high pressure spray to remove epibionts and the following biometric measurements were taken: shell height, width, thickness, and total weight (soft tissue parts + shells) (to 0.01 g) Fig. 17.keshi  were also harvested but these were not graded in the present study. The size of the cultured pearls was assessed by measuring nacre thickness and weight49.Once harvested, cultured pearls were cleaned by ultrasonication in soapy water with a LEO 801 laboratory cleaner ; they were then rinsed in distilled water. Some rebuts). Briefly, the five grades are mostly based on surface purity and luster, from A  to D (cultured pearls showing many highly visible defects over more than two thirds of their surface and having poor luster) and Rejects (cultured pearls that have too many defects to be graded). The latter are consequently discarded and ultimately destroyed. Finally, surface defects and luster (components of cultured pearl grade) were determined separately so that they could be studied independently. Quality traits were evaluated as described in ref. Surface defects, luster and grade category of the cultured pearls were evaluated. Cultured pearl shape was characterized in two ways: the presence/absence of circle/s and the shape category . Two kinds of color evaluation were made on the cultured pearls: the darkness of color and the visually-perceived color category . Cultured pearl grade was determined for each sample according to the official Tahitian classification  from the most to least valuable quality: A, B, C, D and Rejects  and tissues of the pearl sac during harvest  to compare relative gene expression through screening aragonite-related genes , calcite-related genes  and one gene implicated in both layers (Nacrein) (Table P. margaritifera tissue: SAGE (SAGES: AGCCTAGTGTGGGGGTTGG/SAGER: ACAGCGATGTACCCATTTCC)  or pearl sac samples (n = 240), using TRIzol® reagent (Life Technologies) according to the manufacturer’s recommendations. RNA was quantified using a NanoDrop ND-1000 spectrophotometer . Total RNA of each individual was then treated with DNAse I using a DNA-free Kit (Ambion). First, strand cDNA was synthesized from 500 ng total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and a mix of poly (dT) and random hexamer primers. Real-Time PCR amplifications were carried out on a Roche Light Cycler® 480. The amplification reaction contained 5 μL LC 480 SYBR Green I Mast (Roche), 4 μL cDNA template, and 1 μL of primer (1 µM), in a final volume of 10 μL. Each run included a positive cDNA and a blank control for each primer pair. The run protocol was as follows: initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 60 s. Lastly, the amplicon melting temperature curve was analyzed using a melting curve program: 45–95 °C with a heating rate of 0.1 °C s71 as follows:Relative gene expression levels were calculated using the delta–delta method, normalized with two reference genes, to compare the relative expression results72. PCR efficiency (E) was estimated for each primer pair by determining the slopes of standard curves obtained from serial dilution analysis of a cDNA to ensure that E ranged from 90 to 110%. The primers used for amplification are listed in Table Here, the ΔCt calibrator represents the mean of the ΔCt values obtained for the tested gene. The delta threshold cycle (ΔCt) is calculated by the difference in Ct for the target and reference genes. The relative stability of the GAPDH and SAGE combination was confirmed using NormFinder44 and placed the pearl in a numbered box. While our technique does not completely remove the potential for gonad tissue contamination within pearl sac samples, it does significantly reduce the possibility for gonad contaminations. In addition, the potential for contamination is further reduced because the gonad itself cannot mineralise and thus poses very little risk, as confirmed by Wang et al.73 who found MSI60 was not expressed within the gonads of P. fucata. A total of 80 pearl sacs were sampled every 3 months, giving a total of 240 pearl sac tissue samples over 9 months . Biomineralisation potential for 40 grafts and 217 pearl sacs that contained pearls was screened via gene expression as described in section ‘Biomineralisation gene expression analysis’  layer of tissue surrounding the pearls remained. At this point, only the pearl sac and the pearl remain. Next, we made an incision in the pearl sac, removed the pearl, and transferred the pearl sac into a 2.0 ml tube with RNAlater® until RNA extraction74 with the R© software. Qualitative classes based on cultured pearl surface defects, grade and darkness level were re-encoded into ordered categorical response variable. Scores from 0 to 4 were attributed to the different classes from the least to the most valuable. Analyses of ordered categorical response variables  were analyzed using cumulative link mixed models implemented in the ordinal package75 with the R© software. The significance of fixed variables was tested by comparing the fit of an all-inclusive model with that of a reduced model for each variable using a fisher test or chi-squared test. For the cultured pearl “color categories” and shape categories, donor and time of harvest effect were compared using χ2 tests.The normality of the data distribution and homogeneity of variance were tested for pearl size, donor, recipient oyster biometric parameters and relative gene expression data using the Shapiro‒Wilk test and Bartlett’s test. When necessary, transformations were used to adjust data to this distribution (logarithm or square roots). Group donor size (QL vs. TL) and time harvest were treated as fixed variables for statistical analysis, with donor treated as a random variable unless otherwise stated. A linear mixed effects model was used to assess the effect of the fixed and random variables on nacre weight, thickness and relative gene expression, and a generalized linear mixed effects model  was used for the presence or absence of circles and luster, both using packages lme4 and nlme77.Spearman’s correlation coefficient was used to evaluate the correlations between nacre weight and thickness, and the four biometric measures of the recipient and donor oysters, and gene expression. We have adjusted the p-value with Bonferroni correction  to compare pairwise correlation in multiple comparison. The gene expression values of each graft donor and pearl sac were associated with the corresponding nacre weight and thickness of each pearl. The statistics and the visualization for the correlation matrix Fig.  were donp values were lower than 0.05. Statistical analyses were performed using XLSTAT (version 2009.4.02) and R© software (version 3.2.1).In all cases, the differences were considered statistically significant when Supplementary Table S1


Results obtained showed that most of the parameters were majorly influenced by steeping and drying duration. Steeping duration significantly (p < .05) increased the moisture, protein, and ash content of the sorghum with a corresponding decrease in pH values. The obtained experimental and predicted values of the investigated parameters were similar, with statistical indices indicating the relative validity of the generated models . The varying values of the parameters obtained indicates the potential use of the sorghum starches as thickeners, starch substitutes, and for other desired roles in food processing.The quest for high‐quality starch that would meet the needs of manufacturers is ever increasing. This study investigated the effect of steeping duration, drying temperature, and duration on the chemical properties of sorghum starch, to possibly alter the characteristics of sorghum starch for food applications. Steeping duration, drying temperature, and drying time of starch isolation were optimized using a central composite design and nine parameters including  It is the 5th most important cereal crop in the world after rice, wheat, maize, barley  grains used for the study were purchased from Lafenwa market in Abeokuta , Ogun State, Nigeria. The grains were subsequently sorted and cleaned. Damaged grains, stones, and other extraneous materials removed and discarded.Sorghum (2.2X1), drying temperature (X2), and drying duration (X3). The three‐factor design gave a total of 15 experiments (Table Y1), amylose content (Y2), moisture content (Y3), protein content (Y4), ash content (Y5), crude fiber (Y6), fat content (Y7), carbohydrate content (Y8), and total energy (Y9) were evaluated. The mathematical model describing the relationship between the independent variables in terms of their linear, quadratic and interaction effects is described by a second‐order polynomial equation, presented in Equation o, α1–α3, α11–α33, and α12–α13 are the equation regression coefficients for intercept, linear, quadratic, and interaction coefficient, respectively, x1–x3 are coded independent variables.Using a central composite design (CCD) on MATLAB statistical software , experimental sets were obtained to investigate the influence of three independent variables, steeping duration X, drying 2.3The cleaned sorghum grains were steeped for different times Table  using th2.4The percentage yield of the starch was determined according to the method described by Akanbi, Nazamid, and Adebowale . Starch 2.52 g of the sample was dispersed in 20 ml of distilled water. The pH was, thereafter, measured using a pH meter (WPH CD70).2.6Amylose content was determined using the methods of Williams, Kuzina, and Hlynka  and Udac2.7Moisture content of the samples was determined according to the method described by AOAC . The sam2.8F tests at (p < .05) levels of probability are reported. Statistical models were generated using Minitab 16 statistical software  and were also used to execute ANOVA on the models at 5% confidence level. To validate the model equations obtained, the average absolute deviation (AAD), bias factor (Bf), and accuracy factor (Af) were calculated using Equations R2), was also obtained to compare the experimental and calculated values given by the models.All analyses were done in triplicate and results presented represent the average of triplicate determinations, expressed as mean and standard deviation. The data obtained were analyzed by analysis of variance (ANOVA) using SPSS Statistics 22 software . Significant 33.1p < .05) increase with steeping duration . This could mean that as steeping duration increased, there was more degradation of large molecular structures , drying temperature (X2), and drying time (X3)] on the production of starch from sorghum. Parameters determined were pH (Y1), amylose content (Y2), moisture content (Y3), protein content (Y4), ash content (Y5), crude fiber (Y6), fat content (Y7), carbohydrate content (Y8), and total energy (Y9) and the different models representing each provided in Equations This study investigated the effects of independent process variables [steeping duration , fat content (Y7), and total energy (Y9)  and accuracy factor (Af) to unity (1) and that of average absolute deviation (AAD) to zero indicates reasonable agreements between the predicted and observed parameters  (Table X1) and quadratic effect (X12) of steeping duration had respective negative and positive significant effect (p < .05) on the pH of sorghum starch. The surface plots on Figure p < .05) effect on the pH because all sample are still in the same medium.It was observed that as the steeping duration increased, the pH value of the sorghum starch samples decreased Table . This su1) Table , only thp < .05) influence the amylose content. This is also in agreement with the studies of Claver et al. (X2) and the quadratic interaction effects of steeping and drying duration (X1X3) and drying temperature and duration (X2X3) significantly (p < .05) influenced the amylose content. This is also reflected in the surface plot presented in Figure Amylose is an important parameter and component of starches. They play major and significant role in pasting, gelatinization, swelling, gel firmness and viscosity, contributing to the strength, and behavior of the sorghum starch. The amylose content of the sorghum starches ranged from 20 to approximately 30% Table . The higr et al.  that sugr et al. . Heat mir et al. , 2010 anr et al.  might har et al.  and can 3.4X3) was significant (p < .05) on the moisture content of the sorghum starch, suggesting that an increase in drying duration yield a decrease in moisture content and vice versa  had significant (p < .05) effect on protein content (Table The proximate composition of the sorghum starch samples is presented in Table nt Table .X1 and X3) had significant (p < .05) effect on the ash content, steeping duration, and drying temperature (X1 and X2) on carbohydrate and only that of drying duration (X3) on the energy values (Table It was further observed that the linear effects of steeping and drying duration (es Table , higher es Table .4Sorghum is an inexpensive, readily available source of food and an alternative starch source. Results obtained in this study suggest the susceptibility of the investigated sorghum starch parameters to steeping duration, drying temperature, and duration. Changes in the various parameters determined may be attributed to structural changes, molecular disruption and disintegration, probable modifications in particle sizes, breakage of bonds, and intermolecular interactions. The results showed that sorghum starch exhibited interesting properties, essential for food formulations, potential functional ingredient, and for possible use in other industrial applications where certain characteristics are desired. The optimal processing conditions for the processing of sorghum starch in this study was steeping time of 48 hr, drying temperature of 70°C and a drying duration of 180 min. At these conditions, desirable values for all the investigated parameters were obtained. Nonetheless, since the applications of starch are majorly guided by their physicochemical properties, subsequent use and purpose would influence the choice of sorghum starch obtained in this study. Further characterization of these starches is still needed to further understand their similarities and differences, for potential use in the food industry.None declared.


Lassa virus (LASV) is an arena virus causing hemorrhagic fever and it is endemic in several regions of West Africa. The disease-causingvirus records high mortality rate in endemic regions due to lack of appropriate treatment and prevention strategies. Therefore, it is ofinterest to design and develop viable vaccine components against the virus. We used the Lassa virus envelope glyco-proteins as avaccine target to identify linear peptides as potential epitopes with immunogenic properties by computer aided epitope predictiontools. We report a T-cell epitope 'LLGTFTWTL' and a B-cell epitope 'AELKCFGNTAVAKCNE' with predicted potentialimmunogenicity for further in vivo and in vitro consideration. Lassa virus is a single-stranded, enveloped, bi-segmented,ambisense RNA virus belonging to Arenaviridae virus family .It causThe incubation period of Lassa fever ranges from 6-21 days .About 8LASV genome contains two RNA segments coding for twoproteins each. The larger segment is approximately 7.2kb andencodes a small zinc-binding protein regulating transcription,replication and RNA polymerase . The smaInnovative stride towards the development of LASV vaccine wasmade earlier in this century and it was reported that trials onprimates were successful with the vaccine, but there were noreports on the human trials or further advancement of thatapproach . CurrentAn outline of the methodology used in this study shown inThe 12 sequences for glyco-proteins of different isolates of LASVwere retrieved from NCBI  database. These sequences were obtained fromdifferent strains of LASV at different time frame  in different endemic regions ofAfrica.Retrieved sequences were subjected to multiple sequencealignment using MEGA 7.0.26 software package(http://www.megasoftware.net). The CLUSTALW algorithmalong with 1000 bootstrap value and other default parameterswere adopted to fabricate the alignment. The sequences wereanalyzed to recognize the immunologically relevant regions thatwere achieved by predicting epitopic peptides. An amino acid stretch must be of a minimum length for being considered as anepitope that we are aiming to design. Due to representativelength of peptide that binds to HLA molecules, nonamers wereselected as the minimum length of the conserved sequences forthe prediction of epitope-based peptide in the current study.The conserved regions from the glyco-proteins were analyzed todistinguish their soluble and membrane regions. Each selectedconserved sequence was subjected to transmembrane topologyprophecy using TMHMM v0.2 server to identify the inner, outerand transmembrane portion with high degree of accuracy . ItshouThe cytotoxic T lymphocyte (CTL) epitopes from the conservedpeptides were predicted using the NetCTL 1.2 server, which isbased on neural network architecture and predicts candidateepitopes based on the processing of the peptides in vivo .During Linear B-cell epitope prediction was done with ABCpred v2.0server, which uses artificial neural network . This weNetNGlyc 1.0 Server and NetOGlyc 4.0 Server were used to checkthe glycoprotein sequences respectively. The epitopes, which hadno glycosylation site, were considered for further analysis.The immune epitope database and analysis resource (IEDB)MHC class I binding prediction tool was used to calculate IC50 values for peptidesbinding to specific MHC I molecules. This tool is based onstabilized matrix method-based prediction (SMM) . EpitopePrior to predicting epitopes interacting with MHC class II, theywere first checked for their antigenic properties like antigenicityand hydrophilicity by Kolaskar-Tangaonkar antigenicityprediction tool and Parker hydrophilicity prediction toolrespectively , 24. TheSelected T-cell and B-cell epitopes were used for populationcoverage analysis using population coverage tool from the IEDBanalysis resource . The allThe 3D structure of HLA-A*02:01 (PDB ID: 3UTQ) and HLADRB1*01:01 (PDB ID: 1AQD) were downloaded from the ProteinData Bank (PDB) database. Prior to docking, all the watermolecules were removed from the 3D structure of epitope freeHLA-A*02:01 and HLA-DRB1*01:01.The 3D structures of epitopes were designed using the PEPFOLDpeptide structure prediction server at the RessourceParisienne en Bioinformatique Structurale Mobyle Portal .The AutoDock tool from the Molecular Graphics Laboratorysoftware package (version 1.5.6) was employed for dockingpurpose. Both the proteins and ligands (epitopes) files wereconverted to PDBQT format for using in this docking study. Thegrid/ space box center for B-cell epitope and HLA-DRB1*01:01was set at 32.4206, -0.6947 and 6.2001 Å in the x, y and z axesrespectively, so that the epitope could bind at the binding grooveof HLA-DRB1*01:01. The grid/ space box center for T-cell epitopeand HLA-A*02:01 was set at 11.9650, 0.3823 and 16.3061 Å in thex, y and z axes respectively, so that the epitope could bind at thebinding groove of HLA-A*02:01. The size was set at 104.8110,83.3764 and 133.0247 Å in the x-, y- and z-dimensions,respectively.All the analysis was done at 1.00 Å spacing. The number ofoutputs was set at 10, while the exhaustiveness was kept at thedefault 8.00. AutoDock Vina program was used to perform theactual docking based on these parameters .Outputs were again visualized in the PYMOL molecular graphicssystem and the best output was selected based on higher bindingenergy. The binding affinity or the docking results were collectedin .csv format which opens in Microsoft excel. The non-bondinteraction between proteins and the ligands were visualizedusing Accelrys Discovery Studio Visualizer (version 2017 R2).The types of bonding, their distance and the categories ofbonding were also visualized. The hydrophobic and ionizablereceptor surfaces were determined using this software.Absorption, metabolism and toxicity profiles of the peptideepitopes were predicted using "admetSAR" prediction server(http://lmmd.ecust.edu.cn/admetsar1/).A total of 12 sequences of glycoproteins from different isolates ofLASV have been retrieved from GenBank at NCBI .The CLUTMHMM v2.0 prediction analysis revealed that 6 (out of 10)conserved regions of envelope glycoproteins fulfilled the criteriaof outer membrane characteristics .NetCTL tool predicted 66 possible interactions with MHC supertypes,which were, determined with a combinatorial score of TAPscore, MHC I binding score, proteasomal cleavage score andantigen processing score.ABCpred server was used to predict sixteen-mer epitopes fromthe selected conserved sequences. This tool found 29 epitopes.The Bepipred linear epitope prediction tool was used to analyzeconserved regions for epitope identification. This is followed byaccuracy crosscheck for epitope prediction using ABCpred.11 N-glycosylation sites were found but there were no sites for Oglycosylationin the target protein sequence . AmongtAll the 51 T-cell epitopes were then analyzed with anstabilization matrix method in IEDB MHC-I binding predictiontool having IC50 score of less than 250 nM (IC50< 250 nM)matching 81 possible MHC-I allele interactions with 17 differentT-cell epitopes . Top 6 eThe 22 B cell epitopes that were selected by ABCpred server anddid not contain any glycosylation site within them were checkedwith Kolaskar-Tongaonkar antigenicity prediction tool andParker hydrophilicity prediction tool for predicting theantigenicity and the hydrophilicity of the epitopes . 7epitoFor the epitopes of class I MHC molecules, Europe showed acoverage of 59%, North America showed almost 50%, WestAfrica showed 22.14% and the worldwide coverage was 50.04%. 'LLGTFTBinding models of the good epitopes  was completed usingglycoprotein (Accession Number NP_694870.1) withoutconsidering other isolates specific to LASV endemic regions indifferent time frame . The avaOur interest in designing B cell epitopes is because of its role toinduce the production of antibodies synthesized by B cells andmediates its effector functions . HoweverNetCTL tool was used to predict cytotoxic T cell epitopes as itintegrates prediction of peptide MHC class I binding,proteasomal C terminal cleavage, TAP transport efficiency. Thebest 66-epitope candidates were chosen based on a combinedscore. While projecting the B-cell epitopes, ABCpred server wasused and it was found that 29 epitopes cross the threshold levelof 0.51. Moreover, BepiPred was also used to predict B cellepitopes with other tools so that everybody can compare theresults showed by different prediction tools. This study usingdifferent tools will make the study more concrete and increasethe probability of epitope as a favorable candidate for furtherwet-lab verifications.Another important concern with glycoprotein is to elicitimmunity against protein antigen attached with sugar moiety.Protein glycosylation have crucial influence on uptake andproteolytic processing of protein antigens by sterically blockingthe action of proteases . This inInteraction analysis between different MHC supertypes/HLAalleles and epitopes in IEDB, SMM method was used amongothers (like NetMHCpan and CombLib) in this study. This isbecause both input and output are quantitative in these tools. Theoutput is easy to interpret and specific computational strategiesare built to handle experimental noise .Predicted T cell epitope ('LLGTFTWTL') showed adequate MHCI antigenicity score (0.41022). It is also found to interact withMHC-I alleles with IC50 score of less than 250 nM having highpopulation coverage of 38%. Moreover, this T cell epitope boundto the binding groove of HLA-A*02:01 with a binding energy of -10.5 kcal/mol similar to the control peptide's binding affinity .However, the B cell epitope (288-303), 'AELKCFGNTAVAKCNE'showed adequate score like ABCpred Score (0.95), antigenicityscore (1.04) and hydrophilicity score (2.431). It also showeddominant interactions with MHC II alleles with IC50 score of lessthan 250 nM having high population coverage of 18%. Thebinding energy for the epitope at the binding groove of HLADRB1*01:01, was estimated to be -14.1 kcal/mol. Meulen et al.(2004) reported a highly conserved region having an overlap oftwo CD4+T-cell epitopes defined by amino acids (289-301) and(282-294) with this epitope (288-303). The epitope betweenresidues 289-301 is 100% conserved in known arena virusesimplying its evolutionary and functional importance . Thus,tIdentification of epitopes in LASV is non-trivial towards thedevelopment of a peptide based vaccine formulation. We report aT-cell epitope 'LLGTFTWTL' and a B-cell epitope'AELKCFGNTAVAKCNE' with predicted immunogenicity forfurther in vivo and in vitro consideration.The authors declare no conflict of interests.


To compare the clinical outcomes after implantation of AcrySof Toric IOLs with different clear corneal incisions.Sixty cataract patients with regular corneal astigmatism who underwent phacoemulsification combined with implantation of an AcrySof Toric IOL were enrolled. They were divided into two groups according to the position of the clear corneal incision: steep-axis and non-steep-axis. Main outcome measurements included visual acuity, residual astigmatism and changes of corneal astigmatism 3 months postoperatively. Deviation of IOL axis according to the re-calculation using the actual surgically induced astigmatism (SIA) and visual function 3 months after surgery were also evaluated.P < 0.05). Besides, more patients in non-steep-axis group were with irregular corneal astigmatism after the surgery . The deviation of IOL axis according to the re-calculation using the actual SIA was significantly larger in non-steep-axis group than that of steep-axis group (P < 0.001). Moreover, the postoperative visual function was better in eyes of steep-axis group in various measurements, including point spread function, modulation transfer function and high-order aberrations.The corneal astigmatism decreased significantly in steep-axis group 3 months postoperatively (Steep-axis clear corneal incision could reduce the cylinder power of toric IOL and lower the chance of postoperative irregular astigmatism, which may consequently improve the postoperative visual quality. In virtue of improvements in surgical technology, the purpose of cataract surgery has advanced to the achievement of perfect visual quality postoperatively. However, 20%–30% of cataract patients, with corneal astigmatism of more than 1.25 diopters (D) before surgery [Toric IOLs were first made and put into clinical practice by Shimizu et al. in 1994 . The AcrTherefore, the purpose of this prospective study is to compare the clinical outcomes in terms of visual acuity, residual astigmatism, accuracy of preoperative calculation of toric IOL and visual quality in eyes undergoing cataract surgery with AcrySof Toric IOLs implantation using these two types of incisions. Our results should provide guidance of future cataract surgery with toric IOLs implantation.www.clinicaltrials.gov (accession number NCT02182921). All procedures adhered to the tenets of the Declaration of Helsinki. Each patient involved in this study has written an informed consent.This study was approved by the Institutional Review Board of the Eye and ENT Hospital of Fudan University, Shanghai, China, and was registered at Sixty patients who underwent uneventful phacoemulsification combined with AcrySof Toric IOL  implantation between September 2014 and April 2015 in Eye and ENT Hospital of Fudan University were enrolled. Inclusions were cataract with regular corneal astigmatism more than 1.0D. Exclusion criteria were ophthalmic pathology that might affect postoperative visual function, such as corneal diseases, glaucoma, ocular trauma et al. Patients with intraoperative or postoperative complications were also ruled out. The patients were divided into two groups (30 eyes of 30 patients in each group): steep-axis incision group, and non-steep-axis incision group.http://www.acrysoftoriccalculator.com) was utilized to calculate the IOL cylinder power, taking into account keratometry data , SIA and the position of the incision.Routine preoperative examinations were performed on every patient enrolled in this study, including uncorrected visual acuity (UCVA) and best corrected visual acuity (BCVA), slit-lamp examination, corneal topography , B-scan ultrasonography, IOL spherical power calculation . Online toric calculator software  under topical anesthesia in a standardized manner. For patients in steep-axis incision group, a clear corneal incision of 2.6 mm was made on the steep axis, while for patients in non-steep-axis incision group, an incision of the same size was made 15–75° deviating from steep axis. Subsequent procedures were all the same between the two groups as follows: injection of viscoelastics into anterior chamber, 5.5 mm continuous curvilinear capsulorhexis, hydro-dissection, phacoemusification, irrigation and aspiration of cortex, implantation of toric IOL, aspiration of viscoelastics, clockwise rotation of IOL towards planned axis, and finally incision hydration. No suture was used. The post-op therapies for all eyes were levofloxacin  and prednisolone acetate eye drops  four times a day for 2 weeks, and pranoprofen  four times a day for 1 month.http://sia-calculator.com) and toric IOL rotation. Toric IOL axis was re-calculated according to actual SIA and its deviation from implanted axis was assessed. Visual function including point spread function (PSF), modulation transfer function (MTF) and high-order aberrations (HOAs) were also investigated. Toric IOL rotation was assessed as the method we used before [Postoperative examinations were conducted 3 months after surgery to assess visual acuity, residual astigmatism, corneal astigmatism, SIA  Table P > 0.05, Chi-square test).Table P = 0.412, Student’s t test). No difference was either found in toric IOL rotation between the two groups .Residual astigmatism of steep-axis and non-steep-axis incision group were −0.61 ± 0.27D and −0.66 ± 0.37D, respectively 3 months after surgery, which was not statistically different . However, in non-steep-axis incision group, no significant change was found in corneal astigmatism. .In steep-axis incision group, the corneal astigmatism reduced significantly 3 month postoperatively compared with preoperative one .Besides, the anterior corneal surfaces of only 10.00% (3/30) of patients in steep-axis incision group were irregular while the proportion was as high as 43.33% (13/30) in non-steep-axis incision group after surgery .Angular variation of corneal steep meridian was significantly bigger after surgery in non-steep-axis incision group than in steep-axis incision group . However, when taking actual SIA and incision position into consideration to re-calculate the toric IOL axis, the gap between the original and the theoretical axis was significantly larger in non-steep-axis incision group .At 3 month after surgery, SIA of steep-axis and non-steep-axis incision group were 0.50 ± 0.21D and 0.54 ± 0.25D respectively with no statistical difference . No significant differences were found in visual acuity between the two groups after surgery .As shown in Table P = 0.006; Student’s t test). MTFs of patients in the two group were significantly different in all spatial frequency except 90 c/deg. .However, performances of visual function were better in steep-axis group as shown in Fig. P < 0.05, Student’s t test). For ocular HOAs, total HOAs, 3rd order HOAs, trefoil and tetrafoil aberrations were significantly different between the two groups at both 4 mm and 6 mm pupil size and the differences in 4th order HOAs were also significant at 6 mm pupil size .At 3 month after surgery, both corneal and ocular HOAs were greater in non-steep-axis incision group. For corneal HOAs, total HOAs and 3rd order HOAs were significantly different between the two groups at both 4 mm and 6 mm size and the differences in trefoil and tetrafoil aberrations were also significant at 6 mm size (Fig. Toric IOL has been proved to have perfect astigmatism correction effect. However, its efficacy is being affected by many factors, among which incision location and SIA are of great importance. Relatively, steep-axis corneal incision might be preferable due to its decrease of corneal astigmatism without changing the astigmatism axis. Nevertheless, owing to personal surgical practice, some surgeons tend to choose a habitual incision location. Studies comparing different incision locations’ influences on astigmatism correction after toric IOL implantation were also scant, let alone studies concentrating on postoperative visual quality. Therefore, we comprehensively assessed the clinical outcomes after implantation of AcrySof Toric IOLs with steep-axis incision and non-steep-axis incision. And we found that compared to patients with non-steep-axis incision, significantly smaller postoperative corneal astigmatism, lower postoperative irregular astigmatism, and better visual quality were found in patients with steep-axis incision.Incision’s influences on clinical outcomes of toric IOL implantation majorly depend on its influences on corneal astigmatism. Incisions of different sizes, directions, and types will lead to SIA of various vector magnitudes –16. AlthPrevious studies hypothesized that incision at the steep axis could flatten the steep meridian and steepen the flat meridian , and theNon-steep-axis incision was found to give rise to irregular astigmatism in our study, which corresponded with the results of visual quality. Since incisions nowadays are small or even micro in cataract surgery, the value of SIA is not large enough to affect visual acuity or refractory status obviously but may affect visual quality, and the increase of irregularity of corneal shape may also worsen the corneal aberration and consequently the overall visual quality. Previous studies showed that wavefront analysis might predict visual complaints. For example, glare was found to be associated with total HOAs and spherical aberration –23. In oIn conclusion, steep-axis incision could reduce the cylinder power of toric IOL and induce less irregular astigmatism, which brings about better postoperative visual quality. Therefore, steep-axis incision may be an ideal incision choice for toric IOL implantation.


Editor-in-chief of the Journal of Intensive Care. To date, this Journal, which was launched in October 2013, as the official journal of the Japanese Society of Intensive Care Medicine (JSICM), has been outstandingly led by Professor Satoshi Gando, the founding Editor-in-chief. Being an international journal, the members of Advisory and Editorial Board embrace many worldwide leaders in the intensive care field. Based on their continuous and persevering efforts, in addition to the enthusiastic contribution by all authors and/or researchers, we are proud of a number of remarkable and significant articles published in the first stage of this newborn journal. In the first 2 years after its launch, more than 55% of the submissions came from Japanese researchers and/or intensivists; now, almost 75% of the submissions come from researchers in other areas of the world, including North America and Europe, as well as Asia, Middle East, and Oceania , implying that the journal is close to obtaining Social Sciences Citations Index value .Owing to the countless journals that have been launched in the last decade, international journals must now compete with each other within the same field of medicine. By the end of 2017, there were 57 and 137 journals including the term, intensive care and critical care, respectively. To prevail and thereby step forward in these types of struggles between the journals, we must continue to work hard on improving both the academic and clinical quality of this journal by publishing high-quality articles and simultaneously providing more constructive peer-review processes. To date, all articles published in the Towards the Second Stage as an international journal, the Journal of Intensive Care shall work hard to contribute to the development of state-of-the-art intensive care and to thereby amend both the short- and long-term outcomes of critically ill patients. Our Editorial Board members earnestly look forward to a number of submissions from all over the world that will further improve the state of intensive care better than today.


Bacillus sp., depends on fermentation conditions such as pH, temperature, concentration of nutrients, carbon and nitrogen sources, among others. Bacillus megaterium CGTase produces those three types of CDs, however, β-CD should prevail. Although, waxy corn starch (CS) is used industrially to obtain CGTase and CDs because of its high amylopectin content, alternative sources such as amaranth starch (AS) could be used to accomplish those purposes. AS has high susceptibility to the amylolytic activity of CGTase because of its 80% amylopectin content. Therefore, the aim of this work was evaluate the AS as carbon source for CGTase production by B. megaterium in a submerged fermentation. Afterwards, the CGTase was purified partially and its activity to synthesize α-, β-, and γ-CDs was evaluated using 1% AS as substrate. B. megaterium produced a 66 kDa CGTase , from the early exponential growth phase which lasted 36 h. The maximum CGTase specific activity (106.62 ± 8.33 U/mg protein) was obtained after 36 h of culture. CGTase obtained with a Km = 0.152 mM and a Vmax = 13.4 μM/min yielded 40.47% total CDs using AS which was roughly twice as much as that of corn starch . High costs to produce CDs in the pharmaceutical and food industries might be reduced by using AS because of its higher α-, β- and γ-CDs production  in a shorter time than that needed for CS.Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins (CDs) from starch and related carbohydrates, producing a mixture of α-, β-, and γ-CDs in different amounts. CGTase production, mainly by  Cyclodextrins (CDs) are synthesized from starch and related carbohydrates such as amylose, amylopectin and maltooligosaccharide by cyclodextrin glycosyltransferase  which is a bacterial extracellular enzyme  as an alternative carbon source. Other strategy could be that used by Zhou et al. . Sivakumar and Shakilabanu  under aerobic conditions. However, the growth kinetic parameters of bacteria and enzyme activity at fermentation conditions were not evaluated. Hence, production of CGTase using AS as carbon source has not been assessed yet. Therefore, the aim of this work was, in the first part of the study, to characterize the CGTase production by B. megaterium in a SmF using starch of Amaranthus hypochondriacus L. as carbon source and CS was used as comparation. In the second part, CGTase obtained was used for study the production of CDs.Amaranth is a pseudo-cereal consumed mainly in Mexico and in Central and South America. Its starch content is around 58–66% and contain lysine at similar level to that of milk casein. AS is of a waxy or glutinous kind and consists of spherical, angular or poligonal granules with an exceptionally small size, ranging from 0.5 to 3 μm in diameter, which gives it high dispersibility. Amylose content in amaranth starch is exceptionally low, in the range 0–14%. Therefore, amaranth starch granules have high susceptibility to amylases because of their exceptionally high amylopectin content The yield and recovery of the starch obtained were estimated according to the following formulae:Amylose content was analyzed using an amylose/amylopectin Assay Kit  based on concanavalin A (Con A) method. Briefly, starch samples were completely dispersed by heating in dimethyl sulphoxide. Lipids were removed by precipitating the starch in ethanol, recovering the precipitated starch. After dissolution of the precipitated sample in an acetate/salt solution, amylopectin was specifically precipitated by adding Con A and then it was removed by centrifugation. The amylose was enzymatically hydrolyzed at D-glucose, which was analyzed using glucose oxidase/peroxidase (glucose oxidase plus peroxidase and 4-aminoantipyrine (GOPOD)) reagent. The total starch amount, in a separate aliquot of the acetate/salt solution, was also hydrolyzed at D-glucose and was measured colorimetrically by glucose oxidase/peroxidase. The concentration of amylose in the starch sample was estimated as the ratio of absorbance of GOPOD at 510 nm of the supernatant of the precipitated sample with Con A, regard to the total starch sample.L*a*b color parameters of AS were measured using a Hunter Lab Color Flez EZ  iluminante D65, 10°, and 125 inch diameter aperture  being similar to other AS : moisture (8.07 ± 0.5), ashes (0.10 ± 0.0), and crude protein (0.06 ± 0.00). The B. megaterium ATCC-10778. This bacterium was obtained from the strains collection of the School of Chemistry that belongs to the National Autonomous University of Mexico. The strain was spread on an agar plate with a medium that consisted of (g/L): meat-peptone broth 12.0, starch 10.0 and agar-agar 20.0. The pH of the medium was adjusted to 7.5. Plates were incubated at 37°C for 24 h. For inoculum preparation, the biomass from the agar plate was transferred to a 500 mL Erlenmeyer flask, with 50 mL of a medium at pH 7.0 that contained (g/L): starch 12.0, dextrose 10.0 and meat-peptone broth 5.0. The strain was cultivated at 37°C on a rotary shaker at 200 rpm for 24 h.CGTase was obtained using 2PO4 (2.0), K2HPO4 (1.0) MgSO4 (0.2). The medium was added with 0.5% (v/v) corn steep liquor. The medium pH was adjusted to 7.5. The fermenter was inoculated with 9.6 × 105 UFC of B. megaterium. The strain was incubated at 37°C at a constant agitation speed of 200 rpm for 96 h. Sample of 3 mL was taken every 12 h. The cells were centrifuged under cooling at 3500 × g for 15 min and in the supernatant, the CGTase activity was evaluated as well as, the content of protein and starch and pH were determined. Finally, the biomass was determined by dry weight.Biosynthesis of CGTase in SmF was carried out in a 1 L fermenter with 250 mL of sterile broth based on that used by Usharani et al. , that coAssay of biomass X = X(t) was done by using the Velhurst-Pearl or logistic equation:max is the maximal (or equilibrium) biomass level achieved when dX/dt = 0 for X > 0. The solution of Velhurst-Pearl equation is as follows:Where μ is the maximal specific growth rate and Xmax -X0)/X0, and X = X0; the initial biomass value.Where, C = (XE = Emax/t) was evaluated by using the time of Emax. YE/X is the yield of CGTase per unit of biomass produced, estimated as the relation between maximal CGTase activity (Emax) and Xmax. CGTase productivity per unit of substrate (YE/S = Emax/S).The estimation of kinetic parameters in the above equation was performed using a non-linear least square-fitting program “Solver” . The assessed kinetic parameters were: CGTase productivity  method utilized by Costa et al. . The β-CThe optimum pH and optimum temperature of CGTase were assessed according to More et al.  with somThe isolation of the CGTase was performed according to a previous method described by Gheetha and More , with miThe parameters of CGTase were assessed through a Michaelis-Menten equation and the double reciprocal plot method by Lineweaver-Burk. Km and Vmax were determined with 10–100 mg/mL of AS in 50 mM Tris-HCl buffer (pH 8) at 50°C for up to 30 min. After incubation of the mixture reaction, the cyclization activity of CGTase was measured.A. hypochondriacus L. was used to obtain β-CDs. Corn starch (CS) was also used as a positive control of the trial. The method carried out by Ibrahim et al.  substrate in glycine-NaOH buffer (pH 8) at 50°C for 12 h. The enzymatic reaction was stopped by boiling it for 10 min and after that, the reaction mixture was submerged in cold water for 10 min.The native AS isolated from grain of m et al. , with so2O/ACN/FA  (A) and MeOH/ACN/FA  (B) in an A:B ratio of 90:10, v/v. HRMS (high resolution) measurements provided by a TOF analyzer in order to enable the processing of the elemental composition of the registered ions. The percentage of starch conversion (%) was defined as the weight percentage of initial substrate converted into total CDs (g β-CD/100 g starch).The CDs produced were measured by mass spectrometry (MS). The following settings were used: electrospray ionization (ESI) in positive mode. The dry gas (nitrogen) flow rate was set at 4.0 L/min and the dry heater operated at 180°C. The capillary voltage was set at 4500 V and the end plate offset at −500 V. Collision energy varied in the range of 25–30 eV. All ESI–MS experiments were performed on a MicrOTOFQII mass spectrometer equipped with an electrospray ion source . MS data were recorded in full scan mode (from 50 to 3000 m/z). Data processing was carried out with Chromeleon 6.8. Next the samples were introduced directly to the electrospray source of the MS using an LC pump and the mobile phase at a flow rate 3.0 μL/min. The mobile phase consisted of Hp < 0.05).All experimental results were analyzed by one-way analysis of variance (ANOVA) and the Tukey's multiple comparison test  when AS was used as carbon source (μ = 0.094 ± 0.001 h−1) than that with CS (Table p < 0.05). Values of pH were very similar between cultures   Figure . The timX/S = 11.47 gX/ gS, YE/X = 9775 U/gX, respectively) than those reported when was used the CS under the same fermentation conditions. The CGTase activity reported per g of AS was three times higher (YE/S = 44602 U/g S) that the obtained per g of CS (YE/S = 11355 U/g S). This proves that AS is a good alternative carbon source to obtain a higher yield of CGTase.Table The active fraction used for the biochemical characterization of the enzyme was located between fractions number 23 and 30 obtained from the gel filtration Sephadex G-200 column Figure . These f−3/s, respectively . Thus, fermentation conditions may change CGTase yield. Additionally, researchers have been reported recent advances in heterologous expression strategies for improving CGTase production and molecular engineering approaches for enhancing the catalytic properties of CGTases for effective application  and B. firmus (80 kDa) using CS as carbon source  was reported for the one produced by Brevibacterium sp. no. 9605  in comparison to that reported by Urban et al. (The CGTase shows a Mr similar to other CGTases such as the produced in SmF by n et al.  which wan et al. . The pren et al. . Higher n et al. . The infn et al. .The amaranth starch displays a higher amylopectin content, higher dispersibility, and higher starch-granule susceptibility to amylases activity than those properties displayed by corn starch. These features can facilitate the CGTase production by a SmF as well as the synthesis of cyclodextrins when the partially purified CGTase is used in the enzymatic reaction. Therefore, amaranth starch might be a good alternative not only to obtain CGTase, but also to produce a higher α-, β-, and γ-cyclodextrins content than that afforded by corn starch. The use of CDs in the pharmaceutical and food industries is limited by high costs. Thus, many efforts have been directed to produce CDs by continuous processes in substitution of the batch process, and immobilized cells have shown higher productivity when used in continuous processes (Moriwaki et al., This work was carried out in collaboration between all authors. Author JS designed the study, contributed reagents/materials and supervised work in all its aspects. Author MA carried out trials and prepared the protocol. Author EP managed the literature searches. Authors ED and RP performed the statistical analysis and also supervised this study. Author GD followed and supervised the fermentations performed in this proyect.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.


Objectives: A method of closing a large tracheocutaneous fistula by a combination of a palatal mucosal graft with a turnover adiposal flap is presented. Methods: Mucosa of the same size as the tracheal defect was harvested from the hard palate and grafted just caudal to the fistula. After the mucosal graft had taken, a local flap containing the mucosal graft was turned over the tracheal defect facing the mucosa inward of the tracheal lumen. The defect caused by harvesting the flap was closed horizontally. Results: The fistula was closed successfully, and 1 year after the operation, the patient had no airway compromise and the operative scar was inconspicuous. Conclusions: Although the described method is a 2-stage procedure, it can be used to simply and reliably reconstruct the mucosal layer of the tracheal lumen and overlying skin. Prolonged endotracheal cannulation sometimes causes a tracheocutaneous fistula that must be closed surgically. A defect of the tracheal lumen should be replaced by the mucous membrane. A surgical procedure for closing a tracheocutaneous fistula using a local flap and palatal mucosal graft as a substitute for endotracheal lining is described. Although this is a 2-stage method, each procedure is simple and reliable. We believe the present method is feasible even for elderly or debilitated patients.a). She was constantly coughing up sputum and required tissue paper all day long. Since preoperative computed tomography showed no stenosis or obstruction of the trachea, we decided to simply cover the defect without carrying out a procedure to enlarge the trachea (b), and resurfacing the inner lumen of the trachea with mucosa to facilitate her expectorating was planned.A 78-year-old woman who had a subarachnoid hemorrhage 2 years earlier was referred to our department for closure of a tracheocutaneous fistula. She had undergone tracheostomy to support her breathing with a respirator. At the time she was referred, she had regained clear consciousness and could speak by putting her hand over the fistula. Prolonged intubation left a tracheal wall defect measuring 15 × 20 mm just above the sternal notch a. She wa trachea b, and reSkin just below the stoma measuring 15 × 20 mm was removed, and full-thickness mucosa from the hard palate of the same size was grafted on the raw surface following the skin resection. The resected skin was grafted on the donor site of the hard palate. Tie-over gauze was fixed on both grafts .After it was clear that the mucosal graft had taken, the second operation was performed 17 days after the first operation. A semicircular incision was made around the stoma with a margin of 3 mm and hinged. A rectangular flap measuring 45 × 20 mm containing the mucous graft in the middle of the flap was designed, and the skin beside the pregrafted mucosa was de-epithelialized . The flaIt took about 1 hour to carry out each operation. The fistula was closed successfully without complications associated with tracheal air or secretion leakage. The skin graft on the hard palate where the mucosal graft had been harvested had taken. Her frequent cough stopped postoperatively. Twelve months after surgery, the patient refused bronchoscopy, but computed tomographic scan showed successful reconstruction. Her neck scar was inconspicuous, and she was very satisfied with the result .2Various methods have been described for closure of tracheocutaneous fistulae. Since simple closure and end-to-end anastomosis have the risks of tracheocele, pneumopericardium, pneumothorax, and pneumomediastinum, their use is limited to patients who have a small defect with good local and general conditions.4 However, the circumference of the stoma is scar tissue, with poor blood circulation, so the length of the peristomal hinged flap should be limited to a few millimeters. Therefore, this technique was not used in the present case.A peristomal hinged turnover flap to make the inner lining of the trachea with another local flap to cover the skin defect has been reported as a useful method.5 stated that a defect of the inner lumen of the trachea is ideally resurfaced with ciliated mucous membrane. However, he had concerns regarding the reconstruction of cylindrical tracheal defects of a certain length, and it remains unclear that the principle should be applied to reconstruction of a tracheocutaneous fistula, that is, a partial defect of the trachea. Some authors have reported the procedure of closure in a tracheocutaneous fistula using mucosa with a flap.8 Shinohara et al6 treated a patient complaining of postoperative difficulty of expectorating who had undergone tracheal reconstruction with a deltopectoral fasciocutaneous flap. The inner layer of the trachea of the patient had been reconstructed with the cutaneous surface of the flap. Since they thought that dirt or sputum on the inner skin layer of the trachea might disturb the respiratory tract, they reconstructed a tracheal defect of another patient with a deltopectoral flap in which the skin island had been replaced by previously grafted palatal mucosa, and the patient had a favorable course without any complications. The watershed of the size of the defect that demands mucosal lining for successful tracheal reconstruction was unknown, but because the present patient had expectorated much sputum preoperatively, a mucosal graft together with a flap was performed. Of course, palatal mucosa, which consists of stratified squamous epithelium and underlying connective tissue, is histologically different from tracheal mucosa, which consists of ciliated epithelium and goblet cells, but it was thought that palatal mucosa could be a suitable substitute for the inner layer of the tracheal lumen.Considering the importance of the moisturizing the airway and expectoration, Belsey7 reconstructed a tracheal defect using a palatal mucoperiosteal graft. They performed a mucosal graft and overlying neck skin closure simultaneously. They used bolster suture fixation on the skin flap over the mucosal graft with a cotton ball to ensure the taking of the graft. Although their case made good progress after the operation, in the present case, there was concern that this maneuver could jeopardize the blood circulation of the skin flap with the pressure of the cotton ball, and if the mucosal graft did not take, it might become dirt and obstruct the bronchus. For this reason, the palatal mucosa was grafted prior to the flap surgery, and it was confirmed to have taken. Royer et al8 reported reconstruction with a prefabricated forearm flap combined with a buccal mucosa and conchal cartilage graft. The reason they chose the free flap for closure was the patient's history of thyroid lymphoma treated with chemotherapy. Since the present case had no history of radiation therapy, a local flap was used.Yoshimura and Nakajima7 we consider the graft on the palate valid in the present case.Defect of palatal mucosa can be epithelialized spontaneously. However, since we secured a piece of skin as a waste generated by preparation for the mucosal grafting, we utilized it for palatal resurfacing. In view of the fact that the skin graft on the palate took in a week, which might be a shorter period than that of spontaneous healing,Although the fact that the described procedure consists of 2 stages may be a shortcoming, we believe that it can be easily performed with basic surgical techniques to provide reliable physiologic reconstruction of the trachea.


H, we investigate the statistical properties of the iso-height lines of correlated surfaces in the framework of Schramm-Loewner evolution (SLE). We show numerically that in the continuum limit the external perimeter of a percolating cluster of correlated surfaces with H ∈  is statistically equivalent to SLE curves. Our results suggest that the external perimeter also retains the Markovian properties, confirmed by the absence of time correlations in the driving function and the fact that the latter is Gaussian distributed for any specific time. We also confirm that for all H the variance of the winding angle grows logarithmically with size.Motivated by the fact that many physical landscapes are characterized by long-range height-height correlations that are quantified by the Hurst exponent  H. It has been shown recently that the iso-height lines taken at the percolation threshold of a long-range correlated random surface are scale-invariant with a fractal dimension df that depends on H. What still remains elusive is whether these curves have a richer symmetry in the form of conformal invariance.Random landscapes have been used as the basis for modeling a vast range of properties of different natural systems such as the sea surface temperature, ocean depth, height of land masses above sea level and plasma vorticity fields. Generally, such surfaces are correlated and in some cases they can have long range correlations that are characterized by the Hurst exponent, 4. The interest in such curves was triggered by the seminal works of Schramm6, who combined conformal mapping with stochastic processes into a process now known as Schramm-Loewner Evolution (SLE). SLE is a one-parameter family of non-intersecting paths exhibiting conformal invariance that can be generated from Brownian motion whose diffusivity corresponds to the SLE parameter, κ. It has been conjectured, and in a few cases proven, that SLEκ is the scaling limit of a variety of discrete random processes in two-dimensional space7. Inversely, it provides us with an alternative way to validate existing conjectures regarding the dependence of critical exponents on the Hurst exponent in percolation8. Moreover, the SLEκ approach allows us to generate directly such conformally invariant curves without the need to generating correlated surfaces or simulate growth models. Taking advantage of this feature, several numerical and empirical studies of correlated random systems such as turbulent vorticity fields10, graphene sheets11, topology landscapes12, percolation in correlated surfaces13 and accessible perimeters at fixed scale14, have analyzed the corresponding two-dimensional random curves in the context of SLEκ.As in most physical systems, symmetry plays an important role in classifying and understanding the nature of these iso-height lines and the random landscapes from which they are extracted. For this reason, conformally invariant random curves extracted at critical heights of random surfaces and their fractal properties have received a lot of attention in the last decadeschordal SLE, the random continuous non-intersecting curve under study is parametrized over time such that at t = 0 it starts from the origin located on the boundary of the upper half-plane t → ∞. Although such a curve does not intersect itself, in the continuum limit it might touch itself . The union of the space inside the loops formed when the trace touches itself, together with the curve up to time t is called the hull and denoted by 15. Such a definition guarantees a simple connected domain, i.e., a domain without holes, gt(z) which maps 16. This map satisfies the Loewner differential equation,g0(z) = z and ζt is a continuum function called the driving function. Schramm6 proved that if the curves are conformal invariant and follow Markov properties, then ζt must be a Brownian motion with a single parameter κ.In what is known as SLEκ. More precisely, we investigated whether the complete perimeter of a percolating cluster obeys the SLEκ statistics properties in the continuum limit. By taking the percolation threshold iso-height lines from correlated surfaces with −1 ≤ H ≤ 08, we find that the lines indeed do follow SLE statistics. For H = −1 and H = 0 we recover the analytical results that predict κ = 6 and κ = 4, respectively. Using the relationship between κ and df demonstrated by Beffara7, we also show that the conjectured H-dependencies of the diffusivity κ and fractal dimension8, df mutually corroborate each other. Finally, we also verify the Markov property of the curves by showing that the corresponding driving functions are uncorrelated in time and that they follow Gaussian statistics.We present our study of iso-height lines of long-range correlated surfaces in the framework of chordal 14 only deals with the accessible perimeter of percolation clusters. This is not a simple difference because an approach using the accessible perimeter does not include the effects of fjords in the calculations. Moreover, the exclusion of fjords is subject to the scale used to define the accessible perimeter, a problem that is not faced by the current study. Furthermore, our study presents a consistent positive answer to the proposed problem, based both on the evaluation of κ by the driving function within the zipper-algorithm, and by the evaluation of m using the winding angle (see below).In this study we use the complete perimeter of the percolation clusters generated by random Gaussian surfaces, whereas previously published results were based either on different physical systems or different curves. For example, ref.L = 4096, by associating to each lattice site  the height h(x) = h and we use the Fourier Filtering Method (FFM)19, in order to impose long-range correlations. Furthermore, we define the Hurst exponent associated with the correlation by choosing an appropriate power spectrum S(q) in the form of a power law such that,βc = 2(H + 1)20. By multiplying a real-valued random variable u(q) in two-dimensional Fourier space by the square-root of the power spectrum and subsequently applying the inverse Fourier transform, we obtain the correlated random Gaussian surfaceu(q) is taken to be Gaussian distributed with unit variance.We generate correlated random Gaussian surfaces on square lattices, with maximum lattice size H = −1 and therefore βc = 0, the power spectrum in Eq.  that connects two opposite borders of the system. At the critical height, the fraction of occupied sites reaches the percolation threshold pc. From the percolation cluster we extract the fractal iso-height line that corresponds to the complete perimeter, which is the boundary between the percolating cluster and unoccupied sites. More precisely, the complete perimeter consists of all the lattice site edges that separate sites belonging to the percolating cluster from unoccupied sites that can be reached from the boundaries of the system without crossing the percolating cluster itself4. In the following study and analysis of the winding angle, direct SLE method and correlation time of the driving functions, we consider only the complete perimeter of the percolating cluster extracted from such correlated surfaces8.After generating the discrete random Gaussian surfaces, we use the rank method12. Although the presence of conformal invariance does not guarantee SLE, it is certainly a necessary condition. Since we are working with a discrete set of points that define the curve on the square lattice, we can consider the winding angle θi at a point zi to be the sum of all the turning angles αi along the curve, starting from a point z028. Therefore, the winding angle at a point zN is given byαi is the turning angle between two consecutive points on the curve. Curves that are conformally invariant have a probability distribution of the winding angle that is necessarily Gaussian with a variance that increases logarithmically with L so thata is a constant. Furthermore, it has been shown that for SLE curves29, the following relation holds:A simple and straight-forward necessary condition for conformal invariance is based on the statistical properties of the winding angle of the curve under studySLEκ and estimate the value of κ, we use the zipper algorithm with a vertical slit discretization31, to solve Eq. , by using the inverse of 30, its driving function can be recovered by applying the relationsωk’s are determined recursively byIn order to determine whether a curve is indeed tk are not equally distributed, we linearly interpolate the measured driving function at equally spaced time intervals.Given that even for curves with equal length and step sizes the discretized times H in the interval . In Fig. H = −1 (uncorrelated surface) and H = 0. Schrenk et al.4 made the conjecture  = 3/2 − H/3 for H ∈ . Moreover, the H-dependence was later also shown to be independent of the shape of the distribution of the random numbers, u(q), used to generate the correlated landscapes8.Our main goal is to study the properties and symmetries of the complete perimeter of the percolation cluster extracted from correlated landscapes with nterval −, 0. In Fsee Fig.  that the32 and demonstrated by Beffara7 that the SLEκ curves are fractals whose dimension, df, is related to the diffusion coefficient, κ, by the expressionIt was conjectured by Rohde and Schrammκ estimated from a random curve can be verified by comparing the value of df obtained via Eq.  are due the different Hurst exponents of the random surfaces from which the curves were extracted. The mean square displacement error N is the total number of samples of driving functions.We therefore investigate this dependence for critical site percolation interfaces of random landscapes with κ we used the function,κ, which describes the behavior of t → ∞. When t → ∞, the linear term prevails due to the condition d > e, being the non linear term only relevant in the low t range. Indeed, the second term in f(t) is sufficient to describe this localized effect for small values of t, and that it does not affect the asymptotic region. We then considered two straight lines Y(t)Max and Y(t)Min, that bound the evolution of κ, respectively , defined by:In order to confirm that a random curve is c goes to zero after a few times steps, as expected for Brownian motion. The short time correlation is associated to the discretization of the curve, i.e. due to the finite grid size. To complete the investigation of the Markov property we also calculated the distribution of the driving function for a specific time (t*), which is shown to follow a Gaussian distribution  evaluated at t* = 60. Hence, justifying our claim regarding the lack of correlation.As shown in Fig. H ≤ 0) is statistically equivalent to SLE curves. We found consistent agreements between the diffusion constant κ calculated by the zipper algorithm and the value obtained via the fractal dimension of the SLE curves7. We also proposed a new conjecture for the dependence between κ and H, in the assumed interval, on correlated random surfaces. In addition, we also showed that, in the scaling limit, the curves are Markovian in nature, in the sense that their driving functions are uncorrelated in time and Gaussian distributed at specific points in time. A practical consequence of having established that the curves under study are SLE is that we can extend the established results from SLE theory to iso-height lines that correspond to the completer perimeter of percolating clusters. Indeed, it is possible to generate an ensemble of such curves just by solving a stochastic differential equation, without the need to generate the entire landscape.Given that many systems can be viewed as long-range correlated landscapes, properties of the iso-height lines extracted from them become relevant. Our results suggest that the complete perimeter of the percolating cluster of long-range correlated landscapes (−1 ≤


Cyclodextrin production was confirmed using thin layer chromatography. Six different starch substrates, namely, soluble starch, potato starch, sago starch, corn starch, corn flour, and rice flour, were tested for CD production. Raw potato starch granules were found to be the best substrate giving 13.46 gm/L of cyclodextrins after 1 h of incubation at 60°C. Raw sago starch gave 12.96 gm/L of cyclodextrins as the second best substrate. To achieve the maximum cyclodextrin production, statistical optimization using Central Composite Design (CCD) was carried out with three parameters, namely, potato starch concentration, CGTase enzyme concentration, and incubation temperature. Cyclodextrin production of 28.22 (gm/L) was achieved with the optimized parameters suggested by the model which are CGTase 4.8 U/L, starch 150 gm/L, and temperature 55.6°C. The suggested optimized conditions showed about 15% increase in β-cyclodextrin production (28.22 gm/L) at 55.6°C as compared to 24.48 gm/L at 60°C. The degradation of raw potato starch granules by purified CGTase was also confirmed by microscopic observations.Cyclodextrin glucanotransferase  is an important member of  Cyclodextrin molecules have hydrophilic outside, which can dissolve in water, and hydrophobic nonpolar cavity, which is described as a “microheterogeneous environment” [Cyclodextrins are cyclic oligosaccharides commonly composed of six, seven, or eight D-glucose units  are produced as a result of intramolecular transglycosylation (cyclization) reaction during degradation of starch by CGTase enzyme. The CGTase is a multifunctional enzyme and catalyzes four different reactions: cyclization, disproportionation, coupling, and weak hydrolysis reaction .α-, β-, and γ-CDs is highly dependent on the nature of the enzyme used [Cyclodextrin production mainly depends on the process and incubation conditions used, while the distribution of yme used . Cyclodeyme used . The majyme used , 8. Duriyme used –11.β-Cyclodextrin was purchased from HiMedia, Mumbai, India. Soluble starch, yeast extract, and peptone were obtained from Qualigens, India. Phenolphthalein was purchased from Merck India Ltd. Finely ground corn flour and rice flour were collected from local flour mills and were sieved through fine cheese cloth to get fine powder. All other chemicals used were of analytical grade. Microbacterium terrae MTCC 8083 at IMTECH, Chandigarh, India [The CGTase producing organism used in this study was isolated from native soil in our laboratory as described by Park et al. . This nah, India .2HPO4, 0.2 g/L MgSO4·7H2O, and 10 g/L Na2CO3 (autoclaved separately) in 250 mL flasks at 30°C, 150 rpm, on rotary shaker for 72 h. After incubation, cells were removed by centrifugation and supernatant was used for enzyme purification.CGTase production was carried out using 100 mL medium containing 20 g/L soluble starch, 10 g/L yeast extract, 1.0 g/L Kβ-CD in 50 mM phosphate buffer, pH 6.0, for 30 min at 37°C with stirring. After that, it was centrifuged at 9000 ×g, 4°C for 20 min, and after centrifugation desorbed CGTase containing supernatant was carefully transferred to another tube. The elution was repeated once again with 2.0 mL of the elution buffer. The eluted fractions were pooled together and dialyzed against 50 mM Na-phosphate buffer, pH 6.0, at 8°C for 24 h with three buffer changes.Cyclodextrin glucanotransferase enzyme was purified by starch adsorption method described by Martins and Hatti-Kaul  with sliβ-cyclodextrin production were determined by phenolphthalein assay method described by Goel and Nene [μL of appropriately diluted purified enzyme was incubated with 1.0 mL of 50 mg soluble starch in sodium phosphate buffer  at 60°C for 30 min. The reaction was stopped by quickly cooling the tubes on ice. Four milliliters of working phenolphthalein solution was added, the tubes were vortexed, and the absorbance of the mixture was immediately measured at 550 nm. The working phenolphthalein solution was prepared by adding 1 mL of phenolphthalein stock (4 mM in ethanol) to 100 mL of 125 mM Na2CO3 prepared in 4% ethanol. The standard β-cyclodextrin estimation was also carried out using the same method. One enzyme unit is defined as the amount of enzyme that produced one μmole of β-cyclodextrin per minute under assay conditions.CGTase activity and and Nene  with minβ-cyclodextrin production using appropriately diluted purified enzyme. Six different starch substrates, namely, soluble starch, potato starch, sago starch, corn starch, corn flour, and rice flour, were tested for β-CD production. 10 mL of each starch (20 gm/L) in 50 mM phosphate buffer (pH 6.0) was heated in boiling water bath (10 min) for gelatinization. After cooling down to 60°C, 100 μL of appropriately diluted enzyme (4.8 U/L) was added in each flask and incubated at 60°C for β-cyclodextrin production. After 1 h and 2 h of incubation period, samples were withdrawn and β-CD production was estimated by phenolphthalein method as described above.Different starch substrates were tested for β-CD production with double CGTase concentration. A soluble starch was also kept as control for β-CD production. Reaction mixture of each starch (100 gm/L) in 10 mL phosphate buffer  and 100 μL of appropriately diluted enzyme (9.6 U/L) were prepared and incubated at 60°C for β-CD production. Samples withdrawn after 1 h and 2 h were centrifuged to remove the suspended raw starch particles and then supernatants were analysed for β-cyclodextrin production by phenolphthalein method.Preliminary experiments of different raw starch degradation using purified CGTase were successful. In subsequent set of experiments, three starch substrates, namely, potato starch, corn starch, and sago starch, were tested in raw form for β-cyclodextrin production. Their coded levels and actual values are shown in Enzymatic production of cyclodextrin depends on the reaction conditions like pH, temperature, concentration of substrate, concentration of enzyme, and incubation time. In this study, we have selected CGTase concentration (Units/L), potato starch concentration (gm/L), and temperature (°C) as the independent variables. Central Composite Design (CCD) was generated using the Design-Expert software  to test the statistical significance of these variables on Xi is the coded value of the variable, Ai is the actual value, A0 is the actual value of Ai at the centre point, and ΔA is the step change.For statistical calculations, the relation between the coded values and actual values was calculated according to the following:β-cyclodextrin production . Appropriately diluted purified enzyme was added to achieve required enzyme units in each experimental run as shown in β-cyclodextrin in gelatinized soluble starch with purified CGTase was detected using thin layer chromatography . Aliquot from the abovementioned test was spotted on the TLC plate along with glucose, standard β-cyclodextrin, and gelatinized soluble starch (without enzyme) as control. The plate was developed with a solvent system containing dioxane and 25% ammonia solution (1 : 1 v/v). The β-cyclodextrins produced were detected by spraying the plate with 50% ethanolic sulphuric acid solution followed by heating at 100°C for 10–15 min.Presence of As purified CGTase is able to degrade raw potato starch and produce cyclodextrins, we examined the degradation of these raw potato starch granules in optical microscope .Microbacterium terrae KNR 9 produced 0.9 U/mg of crude CGTase enzyme at 30°C, 150 rpm, on rotary shaker after 72 h. After incubation, cells were removed by centrifugation and supernatant was processed for enzyme purification. CGTase from Mic. terrae KNR 9 could be purified to homogeneity in a single step purification using starch adsorption method described earlier with 45.22 U/mg of specific activity, 50-fold purification, and 33% purification yield.β-CD production, sago, potato, and corn starch showed higher β-cyclodextrin production while the rest of the starch sources tested showed comparatively lower β-CD production , starch 150 gm/L, and temperature 55.6°C with the predicted response 29.67 gm/L of and Nene . Pishtiyand Nene  achievedf et al.  have repβ-cyclodextrin production was confirmed on TLC and detectable amount of β-cyclodextrin was produced from gelatinized soluble starch at 60°C in 1 h (lane C) which can be compared with β-cyclodextrin standard (lane D) on TLC plate (The LC plate . In the β-CD production was obtained using potato starch granules in raw form, microscopic observation of raw potato starch granules after degradation was carried out (As higher ried out . Confirm Microbacterium terrae KNR 9 can degrade gelatinized form as well as the raw form of all the different starch substrates tested except corn starch. Raw potato starch and sago starch were found as the most suitable raw starches for this enzyme. Thus, purified CGTase has potential for use in enzymatic production of β-cyclodextrins.Cyclodextrin glucanotransferase produced by