The thermal enthalpy decreased significantly in HMT. Modification significantly changed the pasting profiles of the native proso millet starch, and the peak viscosity, setback, and breakdown values decreased. These results clarify the mechanism of starch changes caused by thermal treatment.Proso millet starch was modified by heat–moisture treatment (HMT), autoclaving treatment (AT), and microwave treatment (MT). The effects of these treatments on the starch physicochemical, structural, and molecular properties were investigated. The amylose and resistant starch contents were increased by AT and MT, but only slightly by HMT. HMT and AT significantly increased the water‐holding capacity, to 172.66% and 191.63%, respectively. X‐ray diffractometry showed that the relative crystallinity of the HMT sample decreased by 20.88%, and the crystalline peaks disappeared from the AT and MT sample patterns. The thermal treatments decreased the proso millet starch molecular weight to 1.769 × 10 In this study, heat–moisture, autoclaving, and microwave treatments affect the physicochemical properties of proso millet starch, and microwave treatment has a greater impact on proso millet starch. HMT causes physical modifications of the starch granules with respect to size, shape, and birefringence  and water (80 ml) were mixed well. The HMT sample was heated for 30 min at 100°C in a sealed glass bottle, the AT sample was heated for 30 min at 121°C (0.1 MPa), and the MT sample was heated at a microwave power of 500 W for 10 min. The samples were then freeze‐dried, ground, and stored in plastic vials until further use.2.3Amylose/amylopectin assay with K‐AMYL 07/11  was used for amylose and amylopectin determination; the method described by Gibson, Solah, and Mccleary  was usedA K‐RSTAR kit  was used to determine the resistant starch content. The samples were hydrolyzed with α‐amylase and amyloglucosidase (provided with the kit) for 16 hr at 37°C. In all the methods, free glucose formed by enzymatic hydrolysis was quantified colorimetrically with an oxidase–peroxidase glucose reagent.2.4g, 10 min). After separation of the supernatant, the water content of the sediment was determined directly by weighing.The Water holding capacity (WHC) was determined by the method described by Yamazaki . The sam2.5g for 15 min. The supernatant was poured into an aluminum box and dried at 105°C to obtain the water‐soluble starch. The precipitate was swelled starch.Wss is the weight of soluble starch (g), Wsp is the weight of sediment paste (g), and Ws is the weight of the sample (g).The solubility and swelling power were determined by using a modified version of the leaching method reported by Adebowale, Afolabi, and Olu‐Owolabi . Starch 2.6Mw), z‐average radius of gyration (Rz), and polydispersity index (PDI) of the samples were determined by high‐performance size‐exclusion chromatography coupled with multiangle laser‐light scattering and refractive index detection (HPSEC‐MALLS‐RI) under the conditions described by Zhang, Li, Chen, and Situ  with LiBr (50 mmol/L) and left at 60°C overnight. Before injection, the sample solution was filtered through a membrane filter (5.0 μm). The mobile phase was DMSO  with LiBr (50 mmol/L) and was filtered through a 0.22‐μm membrane filter and degassed by ultrasound before use; the injection volume was 100 μl. The flow rate was 0.5 ml/min, the column was maintained at 60°C, and the nd/cd value was 0.0740 ml/g. The data were analyzed by Astra software (Wyatt Technology).The weight‐average molecular weight (and Situ  with som2.7λ = 0.15405 nm). Scanning was performed from 3° to 50° (2θ) with a step interval of 0.02° and a scanning rate of 10°/min. The relative crystallinity was calculated as the ratio of the crystalline peak area to the total diffraction area.The crystal structures of the native and modified samples were investigated by X‐ray diffractometry (XRD) (Rigaku AXS Model SmartLab) under the conditions described by Watcharatewinkul, Puttanlek, Rungsardthong, and Uttapap . RD patt2.8All Differential scanning calorimetry (DSC) data were obtained with a Q2000 instrument . The sample processing method described by Hélène et al.  was used2.9The pasting properties of the samples were determined with a Rapid Visco Analyser  by using a standard starch profile. A mixture of the sample (3.5 g) in distilled water (25 ml) was stirred at 160 rpm. The samples were held at 50°C for 1 min and then heated to 95°C at 4°C/min and then held at 50°C for 5 min. The pasting temperature, peak viscosity (PV), breakdown (BD) value, final viscosity (FV), and setback (SB) value were recorded.2.10The sample particle microstructures were examined by Scanning electron microscopy (SEM) ; the method described by Kiseleva et al.  was used2.11Sample analyses were performed in triplicate, and standard deviations were reported. A comparison of the means was ascertained by Tukey's test to be at a 5% level of significance by analysis of variance.33.1The amylose, amylopectin, and resistant starch contents of proso millet starch samples modified by three different methods are presented in Table 3.2p < .05) to 172.66%, 191.63%, and 197.13%, respectively  refers to the theoretical probability of finding a molecule at a given distance from the center. The branch length and chain‐branching mode affect the Rz value  was much lower than that of the native proso millet starch (22.68%). It has been reported that the starch crystallinity is inversely proportional to the amylose content. In this study, all three treatments increased the amylose content, which is consistent with previous results. The crystallinity is affected by crystal size, amylopectin content, amylopectin chain length, extent of interactions between double helices, and orientation of the double helices within the crystalline domains. Starch recrystallization is a complicated process, which involves conformational changes, chain alignment, crystal packing, and phase propagation  in their pasting properties compared with those of native proso millet starch. The Rapid Visco Analysis results for the HMT, AT, and MT samples were lower (p < .05), and the PV, FV, BD, and SB values were higher than those of the native proso millet starch. The PV of the native proso millet starch  was the highest (p < .05), and the PV of the HMT, AT, and MT samples was lower by varying degrees. This indicates that the interactions between starch chains slowed down, and hydrogen bond formation between chains decreased. Generally, the PV was positively related to the starch swelling power, which is consistent with the results of previous studies of swelling power. The BD values of HMT, AT, and MT decreased to 755, 1,085, and 838 cP, respectively. The BD value reflects the stability of a hot starch paste, and resistance to heat and shear. A decrease in the BD indicates that the starch grains have become stronger, which hinders swelling and rupture and gives good thermal stability after processing. Compared with that of native proso millet starch, the SB values of HMT, AT, and MT decreased to 267, 364, and 313 cP, respectively. The SB value reflects the stability and aging trend of a cold starch paste and is related to the degree of polymerization of amylose and the amylopectin structure. Amylose with a low polymerization degree and amylopectin with short outer chains can lead to cold starch paste stable retrogradation (Doutch et al., The pasting properties of the samples are summarized in Table 3.8Scanning electron microscopy was used to investigate and confirm changes in the surface morphologies of the starch gels Figure . Changes4Mw may be related to the amylose content, but there are many reasons for the molecular weight reduction, and further research is needed. HMT, AT, and MT affected the WHC, solubility, and swelling power of proso millet starch in varying degrees. A decreased swelling is desirable for some food applications such as noodle production.This study explored the effects of HMT, AT, and MT on the physicochemical properties of proso millet starch. HMT, AT, and MT significantly affected the amylose and resistant starch contents, and the fractions of amylose and resistant starch; the values were highest after MT. The decreased Heat–moisture treatment decreased the relative crystallinity compared with that of native proso millet starch; AT and MT were more effective in reducing the structural recrystallization of the starch samples. The microstructure of a gel obtained by AT had a thicker stromal wall and looser network structure than those of a HMT gel. MT gave a thinner stromal wall, and the network structure was neater and more regular. The different microstructures may affect the physicochemical properties of proso millet starch to different degrees. RVA and DSC showed that the thermal properties of the samples improved after all three treatments, the setback and BD values decreased significantly, and the cold paste and hot paste stabilities of the samples improved. Further studies are needed to investigate the potential use of proso millet starch to improve the physicochemical and functional properties while increasing the nutritional value.The authors declare that they do not have any conflict of interest.The study did not involve any human or animal testing.Written informed consent was obtained from all study participants.


A single-step ethanol production is the combination of raw cassava starch hydrolysis and fermentation. For the development of raw starch consolidated bioprocessing technologies, this research was to investigate the optimum conditions and technical procedures for the production of ethanol from raw cassava starch in a single step. It successfully resulted in high yields and productivities of all the experiments from the laboratory, the pilot, through the industrial scales. Yields of ethanol concentration are comparable with those in the commercial industries that use molasses and hydrolyzed starch as the raw materials.Saccharomyces cerevisiae  produced the maximum ethanol concentration, pmax at 81.86 g/L (10.37% v/v) with a yield coefficient, Yp/s of 0.43 g/g, a productivity or production rate, rp at 1.14 g/L/h and an efficiency, Ef of 75.29%. Scale-up experiments of the single-step ethanol production using this method, from the 5-L fermenter to the 200-L fermenter and further to the 3000-L industrial fermenter were successfully achieved with essentially good results. The values of pmax,Yp/s, rp, and Ef of the 200-L scale were at 80.85 g/L (10.25% v/v), 0.42 g/g, 1.12 g/L/h and 74.40%, respectively, and those of the 3000-L scale were at 70.74 g/L (8.97% v/v), 0.38 g/g, 0.98 g/L/h and 67.56%, respectively. Because of using raw starch, major by-products, i.e., glycerol, lactic acid, and acetic acid of all three scales were very low, in ranges of 0.940–1.140, 0.046–0.052, 0.000–0.059 (% w/v), respectively, where are less than those values in the industries.Before single-step ethanol production, studies of raw cassava starch hydrolysis by a granular starch hydrolyzing enzyme, StargenTM002, were carefully conducted. It successfully converted 80.19% (w/v) of raw cassava starch to glucose at a concentration of 176.41 g/L with a productivity at 2.45 g/L/h when it was pretreated at 60 °C for 1 h with 0.10% (v/w dry starch basis) of Distillase ASP before hydrolysis. The single-step ethanol production at 34 °C in a 5-L fermenter showed that The single-step ethanol production using the combination of raw cassava starch hydrolysis and fermentation of three fermentation scales in this study is practicable and feasible for the scale-up of industrial production of ethanol from raw starch. The “hydrolysis and fermentation” of starch to bioethanol is widely employed for the production of biofuel, pharmaceutical and cosmetic ethanol, potable alcohols, e.g., beer, whiskey, other distilled spirits, and other ethanol products. The production of fuel ethanol from starch was first introduced in the United States at the beginning of the twentieth century . BioethaStarch is the first generation 1G) feedstock that is the most abundant renewable carbon source and is more readily digestible for conversion to biofuels than cellulosic second generation (2G) feedstock. However, starchy corn gains the lowest ethanol yield per unit area of cultivation when compared to that of other crops. Bioethanol can be produced from various kinds of feedstock. However, when ethanol yields per unit area of cultivations are compared, cassava is the highest potential crop to gain the highest yield. Average yields of cassava, sugar beet, sweet potato, wheat, sugar cane, rice, sorghum, and starchy corn are about 31.25, 56.00, 30.00, 9.00, 62.50, 7.31, 6.25, and 6.00 MT/ha/year (metric ton/ha/year), respectively. Carbohydrate contents (as starch or sugar) of those crops are approximately 28.0, 14.0, 24.5, 70.0, 10.5, 80.0, 70.0, and 70.0 (% w/w), respectively. Theoretical yields of bioethanol per unit area of cultivation per annum of those crops could be 4.95, 4.44, 4.16, 3.57, 3.53, 3.31, 2.48, and 2.38 MT/ha/year (tons of ethanol/ha/year), respectively. They were calculated from a ton of starch produces 566 kg of ethanol, and a ton of sucrose sugar produces 538 kg of ethanol  was obtained from using cassava starch medium at a concentration of 20% (w/v) with 65.11% of theoretical yield (% efficiency) [S. cerevisiae strains displayed improved fermentation vigor on raw corn starch and broken rice, reaching 97% efficiency and converting 100% of the available carbon to products within 120 h in small-scale CBP fermentations on broken rice [K. marxianus SS106 in 5-L stirred tank fermenter by cold hydrolysis process was conducted. Ethanol at a concentration of 6.17% w/v with a productivity of 0.86 g/L/h was obtained [The literature review of recent research updates is addressed here. For bioethanol production based on the cold process, large companies developed novel and efficient enzymes for the saccharification of starch at lower temperatures. Genencor International Inc. (now DuPont) released STARGEN™ and Novozyme released the BPX™ for cold processes . S. cere73 g/L/h . A singliciency) . The amyken rice . Ethanolken rice . A singlobtained .To overcome the high-temperature-cooking fermentation in the industrial ethanol production from cassava starch, a single-step ethanol production process of simultaneous raw starch hydrolysis and ethanol fermentation in a single fermenter was undertaken in this study. This process is not only yeast fermentation, but also includes simultaneous hydrolyzation of raw cassava starch at a low temperature by the addition of a mixture of raw starch-digesting enzymes at the initial stage, which significantly decreases the energy consumption and the operation cost. Moreover, the low-temperature fermentations have been conducted at the pilot-plant-scale and industrial-scale productions without contamination by bacterial cells and yielded the fermentation efficiencies similar to those of the conventional fermentations. Another advantage of the single-step process is able to maintain a low concentration of glucose during fermentation, which could decrease the inhibitory effects of glucose on the enzyme and yeast activities . This reThe objective of this study is to combine both raw cassava starch hydrolysis and ethanol fermentation in a single process step by utilizing the cold enzymes which are capable of hydrolyzing raw cassava starch under fermentation conditions. This reduces the complexities of operations by a combination of raw cassava starch hydrolysis and fermentation in a single step and single fermenter. It results in saving on high energy consumption, operation cost, and time. This study aims to optimize the hydrolysis of raw cassava starch by the enzyme StargenTM002 and to develop a practicable single-step ethanol production process using raw cassava starch as the raw material at the laboratory, the pilot, and the industrial scales.–7.0 were verified for their effects on raw starch hydrolyses. Results showed that glucose concentrations and degrees of conversion were increased when pH values of starch slurry were decreased from 7.0 to 3.0. The starch hydrolysis by StargenTM002 at pH 3.0 yielded the maximum glucose concentration of 114.39 g/L with 52% (w/w) degrees of conversion. However, this experiment was concerned that the pH value below 4.0 could be a negative effect on yeast cell growth and fermentation activities. The pH is one of the most important parameters influencing yeast cell growth and fermentation activities. Low initial pH values cause chemical stress on yeast cells affecting the accumulated biomass loss, the decrease in consumption rate of sugar, the decrease in final concentration of ethanol, and the increase in final concentrations of glycerol [S. cerevisiae fermentations have been published. A pH at 4.5 gave the highest ethanol production from S. cerevisiae [Formations and degree of conversions of glucose from raw cassava starch hydrolyses by varying concentrations of the enzyme StargenTM002, temperatures, and pH values, for those optimal values are shown in Table glycerol , 29. Sevrevisiae . A pH berevisiae . Optimalrevisiae . The pH revisiae . TherefoFormations and degrees of conversion of glucose from raw cassava starch hydrolyses by enzyme StargenTM002 at the optimum conditions for 72 h are shown in Table Table Moreover, the pretreatment of raw cassava starch at the sub-gelatinization temperature of 60 °C together with Distillase ASP increased glucose formation and its degree of conversion. The highest glucose concentration of 176.41 g/L with a 80.19% (w/w) degree of conversion was successfully achieved when raw cassava starch was pretreated at 60 °C together with 0.10% (v/w ds) of Distillase ASP before the step of StargenTM002 hydrolysis. However, increases in Distillase ASP dosages to 0.20 and 0.30 (% v/w ds) for the pretreatments did not significantly increase further raw starch hydrolyses by StargenTM002.–3.0% (w/w sd) did not significantly affect raw cassava starch hydrolyses when compared with the pretreatment without urea. This result indicates that urea pretreatment does not improve raw cassava starch hydrolysis by StargenTM002. This studied result does not agree well with [On the contrary, the heat pretreatment at 60 °C together with Distillase ASP plus urea at the concentrations of 1.0ell with  who repoell with  are unclMorphological and microstructural changes of the pretreated and hydrolyzed raw cassava starch granules revealed by a scanning electron microscope (SEM) are shown in Fig. .Sub-gelatinization temperature pretreatment would allow the starch granules to swell and open up the pore on the granule surfaces  which inResults from the previous study showed uncompleted hydrolysis of raw cassava starch by StargenTM002  were conducted in the 5-L fermenters each contained 4 L of fermentation medium composed of the pretreated raw cassava starch at a concentration of 200 g/L. Enzyme StargenTM002 and S. cerevisiae inoculants  were added into the fermentation medium at the concentrations of 0.30% (v/w ds) and 0.10% (w/v), respectively. To study the effect of temperature on single-step ethanol production, fermentations were performed at 30 and 40 °C, and without temperature control . Fermenters were agitated at the design speed of 200 rpm for 72 h along with the fermentations.Single-step ethanol fermentations by Temperature is one of the most important factors that affect ethanol production. Figure The concentration of glucose in fermentation broth at the temperature below 40 °C decreased with time as the fermentation proceeded normally. In contrast to at 40 °C, glucose rapidly decreased within the first 36 h, after that it could not be utilized by yeast which consequently caused the results of final glucose accumulation and lower ethanol in the system.S. cerevisiae growth profiles of all fermentation temperatures tended to approach similar values except that at the temperature of 40 °C which was lower than those of the 30 °C and non-control temperatures, results indicate that increasing the fermentation temperature resulted in decreasing the growth and the ethanol production by the yeast. The obtained data clearly show the optimum temperature of around 34 °C at which both yeast and StargenTM002 work best together. If lower than 34 °C, the reaction rate of StargenTM002 declined, and if above this value the yeast cell growth and its activities would be inhibited.Although +–ATPs and transport systems [Temperature directly affects metabolism and growth of yeast cells. At a wormer temperature, yeast cells show a rapid decline in viability at the end of fermentation while at an excessively high temperature, enzyme and membrane functions may be disrupted resulting in the stuck fermentation . Moreove systems . High te systems . It affe systems . TherefoS. cerevisiae whilst the use of temperature at 30 °C increased the yeast activity, but reduced the hydrolytic rate of raw cassava starch. As explained above, the maximum final ethanol concentration (pmax) at 81.86 g/L (10.37% v/v) with an ethanol yield coefficient (Yp/s) of 0.43 g/g, a productivity (rp) at 1.14 g/L/h, and an efficiency (Ef) of 75.29% was obtained under the non-control fermentation temperature (34 ± 1.0 °C). This result revealed that the single-step ethanol production by S. cerevisiae at approximately 34 °C provides the best-compromised temperature that enhanced enzyme activity and promotes S. cerevisiae to produce ethanol at a high concentration.In this study, we combined the enzymatic hydrolysis of raw cassava starch and ethanol fermentation within a single stage. However, the optimum temperature at 40 °C for the hydrolysis of raw cassava starch was higher than that of the fermentation. The design of operating temperature for single-step ethanol production was very important. As mentioned before, the temperature at 40 °C was optimal for enzyme activity but it could reduce metabolism and growth of Furthermore, it was interesting to note that by-products, i.e., glycerol, lactic acid, and acetic acid were very low at maximum values of only 1.14, 0.05, and 0.00% (w/v), respectively (Table Major objectives of this section were to (i) evaluate the potential implementation of the single-step ethanol fermentations at the larger scales; (ii) check the ethanol yield and productivity of the single-step fermentations, and (iii) identify problems that were not significantly noticed at the laboratory scale. According to results in laboratory-scale fermentation, further single-step ethanol fermentations were conducted in a 200-L pilot and in a 3000-L industrial fermenter. After enzyme additions and yeast inoculations, single-step ethanol fermentations using the combination of raw cassava starch hydrolysis and fermentation were operated at the same temperature of 34 °C as that of the 5-L fermentation above for 72 h at the agitation speeds of 125 rpm for 200-L fermenter and 55 rpm for 3000-L fermenter. To maintain the designed scale-up parameter, i.e., the energy dissipation rate per unit mass or power input, pmax at 80.90 g/L (10.25% v/v) with a Yp/s of 0.42 g/g, an rp at 1.12 g/L/h and an Ef of 74.40% was achieved when the single-step ethanol fermentation from raw cassava starch has been scaled-up to the 200-L fermenter  of the 5-L laboratory fermentation. Results in the 200-L fermentation indicated that operations, conditions, and its performances at this scale-up were significantly as effective as those of results obtained in the 5-L fermentation. Furthermore, the single-step ethanol fermentation in the 3000-L industrial fermenter was also studied. In Fig. pmax at 70.74 g/L (8.97% v/v) with a Yp/s of 0.38 g/g, an rp at 0.98 g/L/h and, an Ef of 67.56%. Its performances could be observed that there were similar profiles in ethanol production and sugar utilization between both scales of fermentations  the production of ethanol from raw starch can be done in a single-step of operation without liquefaction at very high temperature, and saccharification. (ii) Consequently, complexities, times, and costs, of operations can significantly be minimized. (iii) As the raw starch is used as the substrate, major by-products, i.e., glycerol, lactic acid, and acetic acid are essentially minimized. (iv) With high concentrations, yields, productivities, and efficiencies of ethanol productions at all experimental scales, comparable with those in commercial industries that use molasses and hydrolyzed starch as raw materials, this can be implemented in the industries. (v) Using fluid dynamics for the design of impellers speeds and operations, the lysis reactor for raw starch pretreatment, and the ethanol fermenter at every scale can be designed and scaled-up to the industries.S. cerevisiae  at 34 °C in a 5-L fermenter was achieved. This ethanol was produced at the concentration of 81.86 g/L (10.37% v/v) with a yield coefficient of 0.43 g/g, a productivity at 1.14 g/L/h, and an efficiency of 75.29%. The scale-up from the 5-L laboratory fermenter to the 200-L pilot-scale, and further to the 3000-L industrial fermenter was essentially successful. There were no significant differences of those values, results, and performances between the 5-L and the 200-L fermentation scales. There were little differences of those values of the 3000-L scale comparable with the former two. These results indicated that the single-step ethanol production using the combination of raw cassava starch hydrolysis and fermentation can be scaled-up to the novel industrial production of bioethanol.The combination of raw cassava starch hydrolysis and fermentation is practicable for the single-step ethanol production. The granular starch hydrolyzing enzyme StargenTM002 showed a high ability for raw cassava starch hydrolysis. Under the optimum condition, 68.56% (w/w) of raw cassava starch was converted to glucose. Moreover, the pretreatment of raw cassava starch at 60 °C for 1 h with Distillase ASP (a blend of glucoamylase and pullulanase) greatly improved subsequent further raw starch hydrolysis by StargenTM002 which converted 80.19% (w/w) of raw cassava starch to glucose. For ethanol production from raw cassava starch, the highest ethanol concentration from single-step fermentation by Saccharomyces cerevisiae , obtained from AB Mauri , was used in this study. This yeast strain could produce the maximum ethanol yield exceeding 18% (v/v) or 12% (w/v) depending on fermentation procedures. Furthermore, it is extremely thermotolerant and it has a wide range of fermentation temperatures from 25 to 40 °C. The active dry yeast inoculants were re-hydrated in distilled water at 40 °C for 20 min prior to inoculations into the single-step ethanol fermentations.The manufactured active dry yeast, Cassava starch of the “Three Elephants” brand was obtained from Chorchiwat Industry Co., Ltd  with complements. After manufacture, freshly cassava starch was kept at a dry and cool place in the laboratory store. Moisture content and other compositions of the cassava starch in this study were totally ~ 10 ± 1.00 (% w/w) so that the carbohydrate (fermentable carbon source) is ~ 90 ± 1.00 (% w/w). Its pH was 5–7. Cassava starch chemical compositions, i.e., carbohydrate, moisture, crude fiber, ash, protein, and fat were 90.80 ± 1.22, 7.10 ± 0.10, 1.20 ± 0.00, 0.45 ± 0.22, 0.32 ± 0.01, and 0.17 ± 0.00 (% w/w), respectively .Aspergillus kawachi α-amylase expressed in Trichoderma reesei and glucoamylase from T. reesei) and (ii) Distillase ASP . Properties of these commercial enzymes are presented in Table Commercial enzymes provided by DuPont Industrial Biosciences  were utilized in this study. They were (i) StargenTM002  of raw cassava starch prepared in distilled water, were incubated in a water bath at 30 °C for 48 h, with continuous stirring at 100 rpm using an overhead stirrer. StargenTM002 was added into raw cassava starch slurries at concentrations of 0.10–0.40%  before starts of hydrolysis.For the study of temperature effect on raw cassava starch hydrolysis, starch slurries were incubated at 30–40 °C with StargenTM002 at a concentration of 0.10% (v/w ds). Effects of pH on raw cassava starch hydrolysis were verified from 3.0 to 7.0. Starch slurries at pH 3.0–4.0 were prepared in sodium acetate buffer and those at pH 5.0–7.0 were prepared in potassium phosphate buffer before hydrolyses by StargenTM002 at a concentration of 0.10% (v/w ds).A 10 L of starch slurry, containing 20% (w/v) of raw cassava starch prepared in sodium acetate buffer at pH 4.0, was hydrolyzed by StargenTM002 at a concentration of 0.30% (v/w ds) in a 15-L lysis reactor at 40 °C and agitated at 220 rpm  of native starch to release free glucose molecules . In addi4)2HPO4, HK2PO4, Na2HPO4, and MgSO4.7H2O, were also supplemented into the medium at concentrations of 0.1, 1.5, 1.8, and 3.8 g/L, respectively. After homogeneous medium mixing, StargenTM002 was added at a concentration of 0.30% (v/w ds) and inoculants of re-hydrated active dry yeast, S. cerevisiae Fali, were further inoculated into fermenters at a final concentration of 1.0 g/L (0.10% w/v). During fermentations, samples of 10 mL were withdrawn at every 12 h interval for analyses.In order to study the effect of temperatures on single-step ethanol production, fermentations were performed at different temperatures of 30, 40 °C, and without temperature control . pH values were not controlled, but undergoing with the initial value at 5.5. They were carried out using 5-L fermenters , each fit with 2 Rushton turbine impellers of the diameter, D of 0.065 m, operating at the designed agitation speed of 200 rpm, equivalent to the power input, T and 1.00 m height, H  The pilot-scale ethanol fermentation was carefully conducted using the 200-L fermenter of 0.50 m diameter, I.(2) The industrial ethanol fermentation was practically implemented using the 3000-L fermenter of 1.25 m diameter, T and 2.50 m height, H ), fit with 2 Ekato Intermig impellers of the diameter, D of 0.85 m, agitating at 55 rpm  of the pretreated raw cassava starch slurry plus 4.0% (w/v) of sugarcane molasses containing 50% (w/v) sucrose, and 0.10 g/L (NH4)2HPO4, 1.50 g/L of HK2PO4, 1.80 g/L of Na2HPO4, and 3.80 g/L of MgSO4.7H2O. When mixed well and reached the designed set-point temperature at 34 °C and the initial pH at 5.5  the enzyme StargenTM002 was added into the fermenters at a concentration of 0.30% (v/w ds) and subsequently inoculants of re-hydrated active dry yeast, S. cerevisiae Fali, were inoculated at a concentration of 0.10% (w/v). Fermentations were performed at the temperature of 34 ± 1.0 °C for 72 h at designed agitation speeds of each fermenter mentioned above. During fermentations, samples of 10 mL were withdrawn at every 12-h interval for further analyses.The fermentation mediumStudies of single-step ethanol productions at the 200-L pilot-scale and the 3000-L industrial-scale fermenters were practically based on principles of scale-up rules: (i) geometric similarity where dimensional lengths of all fermenter geometries among different fermenter scales were designed to maintain the same values; (ii) conditional similarity where all optimal conditions, i.e., temperature, pH, substrate and enzyme concentrations were imitated from the 5-L laboratory research work; (iii) operational similarity where a selected key operational method was designed. For this work, the power input  method was utilized to quantify concentrations of glucose, ethanol, glycerol, lactic acid, and acetic acid in fermentation broth samples by comparisons with standards of those known concentration values. The HPLC system  with the refractive index (RI) detector (Smartline 2300) and with the Eurokat H vertex column was used. An eluent solution of 0.01 N Hof 20 µL , 45.2SO4 solution were added, mixed well, and capped. Test tubes with mixtures were boiled in a water bath at 95 °C for 30 min. Neutralizations were done with the addition of 4 N NaOH and re-centrifugations were operated to precipitate residues. Supernatants were analyzed using the HPLC to quantify concentrations of glucose as the total sugar [.Total sugars mean all concentrations of carbon sources, i.e., raw starch plus free residual glucose of which raw starch in the mixture of fermentation broth was completely hydrolyzed by acid to release all glucose molecules, and then glucose from both sources in the same mixture was measured as the total sugar or total glucose. The total sugar of cultured samples during fermentation was analyzed by the modified sulfuric acid hydrolysis method. Samples of 1 mL in micro-tubes were centrifuged at 10× rpm for 10 min. Supernatants of 0.5 mL were transferred into 20-mL test tubes and then 2-mL volumes of 2 N Hal sugar , 45.Photographic characteristics of the pretreated and hydrolyzed raw cassava starch granules were observed under the SEM . Samples were mounted on circular aluminum stubs with carbon tape, coated with gold, and examined in SEM at an accelerating voltage of 10 kV and photographed.6H12O6 is the glucose with a molecular weight (MW) of 180.156 g/mol; H2O is the water with a MW of 18.015 g/mol, and n is the number of glucose molecules of glucose and of starch or degree of polymerization (Dp). For example, if a starch chain polymer of 1000 glucose molecules was completely hydrolyzed, where n = 1000 molecules, then substitute into the above term to obtain The degree of conversion of raw starch to glucose was calculated as the percentage of glucose released from the raw cassava starch hydrolysis using the equation:entclass1pt{minimap (g/L), and concentration of raw starch carbon substrate used, s (g/L). (i) The yield coefficient of ethanol production, Yp/s (g/g) is from Yp/sp is the ethanol produced (g/L) and Δs is the substrate used (g/L), (ii) The productivity or production rate of ethanol, rp (g/L/h) is from rs (g/L/h) is from Yp/s is from the experiment  and Y'p/s is from the theoretical yield coefficient or stoichiometry [p/s of ethanol from starch is 0.566 g/g, calculated from“a” is the glucose yield of 1.11 g from 1.00 g of starch as in Eq. (p/s) of ethanol from 1.00 g of glucose, calculated from Three kinetic parameters of ethanol productions were calculated using experimental data, i.e., cultivation time, t (h), concentration of ethanol, hiometry . The thementclass2pt{minimP (W) is calculated from n is the number of impellers of 2 for all the 5-L fermenter, 15-L lysis reactor, 200 pilot-scale fermenter, and 3000-L industrial fermenter. Po is the power number of 5 (no unit or dimensionless) for one Rushton turbine impeller of the 5-L fermenter and Po of 0.33 for one Ekato Intermig (high-efficiency) impeller of every scale, i.e., 15-L lysis reactor, 200-L and 3000-L fermenters. 3) of lysis reactor or fermenter. N is the impeller speed (rps), and D is the impeller diameter, where D of 0.065 m is for the 5-L fermenter, 0.115 m is for the 15-L lysis reactor, 0.30 m is for the 200-L fermenter, and 0.85 m is for the 3000-L fermenter.For fluid dynamics in the stirred tank bioreactor , 47, theV is the fluid volume (m3). Thus, designed impeller speeds, N, of the power input of 0.10 (W/kg) are calculated fromThe power input or energy dissipation rate per unit mass, Both these kinetic and fluid dynamic parameters from the laboratory and the pilot-scale experiment are very crucial for the scale-up of further fermentation at the industrial scale. That is the foreseen reason why impeller speeds were designed based on the power input rather than just the speed in rpm Table .Table 6Lp-value ≤ 0.05 using the Minitab version 17 software. Standard errors (± SE) were shown together with mean values as error bars in the graph. If the error bars of SE in the plots did not  appear, assuming that they were smaller than the sizes of legends.Statistical analyses of each data set from each experiment were undertaken with the one-way analysis of variance (ANOVA). Differences of treatment mean values from three replications (each experiment was done in three replicates) were compared with the Tukey’s range test method at Additional file 1.Additional file 2.


Mean −1 was ≥ 2.12 or above in males. In females, it was just above 2.00 for −1 and increased to ≥ 3.33 for −1. Conclusion: The study demonstrated the feasibility of carotid stiffness measurement in a community setting. A positive association between the risk factors and carotid artery stiffness provides evidence for the device’s use in resource-constrained settings. Clinical Impact: The device paves the way for epidemiological and clinical studies that are essential for establishing population-level nomograms for wide-spread use of carotid stiffness in clinical practice and field screening of ‘at-risk’ subjects.Objective: We investigate the field feasibility of carotid stiffness measurement using ARTSENS® Touch and report the first community-level data from India. Method: In an analytical cross-sectional survey among 1074 adults, we measured specific stiffness index ( I.Cardiovascular Diseases (CVD) is a leading cause of several fatal and nonfatal events, accounting for 31% of all deaths worldwide Altered hemodynamics, loss of arterial elastic properties, and change in arterial wall dynamics result in a range of CVD-related abnormalities. Hence, early vascular health markers measured in terms of biomechanical properties of arteries offer a unique view of the underlying disease progression Over the years, our group has developed and clinically validated an image-free alternative – ARTSENS® – for non-invasive assessment of vascular wall dynamics and stiffness. The system exploits A-mode ultrasound data with intelligent algorithms for automated measurement, thereby eliminating the need for imaging equipment/reference, expert operator, manual annotation, as well as offline analysis. A detailed discussion on the ARTSENS® technology can be found elsewhere ARTSENS® Touch  is one oBeat-by-beat diameter parameters and blood pressure (BP) values are used to evaluate the measures of carotid artery stiffness In this work, we conducted the first community-based study in India to describe the distribution of carotid artery stiffness in a general population. Such an attempt has never been reported in large cohorts owing to the lack of appropriately usable, cost-effective, and field-deployable tools for epidemiological trails. This study aimed to (1) demonstrate the field deployability and measurement reliability of the ARTSENS® Touch in a resource-constrained setting, and (2) describe relationships between the carotid stiffness and cardiometabolic risk factors in an Indian population. The study methodology, observations, results, and the outlook are presented in the following sections.II.A.The study executed as an analytical cross-sectional survey in a rural village of Tiruvallur district, Tamil Nadu, South India. The selected village is part of our institution’s field practice area; hence all the households were enumerated, and a line list was available for another ongoing cohort study. Since our primary objective was to investigate the distribution of carotid stiffness, and there exists a lack of community-level data from India to compute the sample size, all adults aged 30 years and above in the line list were considered eligible for the study. A total of 1074 subjects were recruited, and written informed consent was obtained prior to participation. Ethical approval was obtained from the National Institute of Epidemiology, Indian Council of Medical Research (NIE/IHEC/201407-02). The study was carried out in accordance with the latest revision of the Helsinki Declaration.B.All participants reported to our field setup as per preassigned slots. They were asked to fast for at least 10 hours and to abstain from dietary products known to alter stiffness (e.g. caffeinated beverages) for at least 18 hours prior to the study. Upon arrival, weight, height, waist circumference, and body-mass-index were measured. The data concerning risk factors such as age, tobacco use, alcohol consumption, current medication on hypertension and/or diabetes, and education were obtained using a structured questionnaire. Participants were asked to rest for at least 5 minutes on a comfortable chair with headrest.All individuals underwent BP measurement on the right upper arm using an automated oscillometric apparatus , the measurement was repeated after 5 minutes and averaged for analyses. Blood sample (5 mL) was collected from the antecubital vein to perform laboratory tests, including high-density lipoprotein cholesterol (HDL-C), fasting plasma glucose (FPG), and triglycerides (TGL) serum level.C.Carotid stiffness measurements were performed by either one of the two field operators who were trained at the beginning of the study period . Once the subject comfortably seated in a chair with back support, the operator recorded BP and entered it in the device. The operator then identified an approximate location of the left common carotid artery by palpation. The tip of the image-free ultrasound probe (applied with water-based ultrasound gel) was positioned near the identified location. The probe orientation was adjusted to get strong and sharp distinct echoes from both arterial walls as visually guided by the device and displayed on-screen . An on-screen progress bar fills up while capturing high-fidelity distension cycles, and the device automatically pops up results-page upon completing the measurement. All recorded data and results were saved into required file formats. Procedures were repeated after 10 – 15 minutes, and averaged values were used for analyses. The study photographs are given in Supplemental Material.D.1)Central obesity: Waist circumference ≥90 cm in males or ≥80 cm in females (for Asian Indians).2)Elevated TGL: TGL ≥150 mg/dL (1.7 mmol/L) or on drug treatment for elevated TGL.3)Reduced HDL-C: HDL-C < 40 mg/dL (1.03 mmol/L) in males or < 50 mg/dL (1.29 mmol/L) in females, or on drug treatment for reduced HDL-C.4)High BP: Systolic BP ≥130 mmHg or diastolic BP ≥80 mmHg, or on antihypertensive drug treatment.5)Elevated FPG: FPG >100 mg/dL (5.6 mmol/L) or drug treatment for elevated FPG.Clustering of three or more of the following cardiometabolic risk factors in an entity was considered as high-risk and marker of future events An individual who has smoked more than 100 cigarettes over his/her lifetime and continued to smoke at the time of the study  was considered as a current smoker. One who had quit smoking earlier was considered a former smoker E.Results are given as mean ± standard deviation or minimum-to-maximum range, unless otherwise stated. Percentage relative difference (RD) of the measurements in 2 consecutive trials was obtained as an estimate of repeatability. It was calculated as the ratio of the absolute difference between successive readings to the absolute value of their mean. Gender-specific differences in the population distribution of stiffness indices were identified by subgroup analyses. Student t-test was performed to examine the equality of two means  for each of the stiffness indices. Analysis of variance was performed to compare means of stiffness indices in different age group . Chi-square test was used to analyze the linear trend across the quintiles of stiffness indices and multiple risks. The relationship between the clustering of multiple (three or more) risk factors and ranges of each stiffness index (across quintiles) was examined separately for males and females. Logistic regression analysis was performed to compute unadjusted odds ratio (Model-1) and age-adjusted odds ratio (Model-2), with 95% confidence interval. Multiple regression analysis was used to identify the risk factors associated with stiffness indices and to develop predictive models. A p-value ≤0.05 was considered statistically significant. Analyses were conducted using PASW Statistics software .III.A.Among 1074 participants, reliable measurements (set of two) were performed on 983 (91.5%) as per protocol. Others’ data (8.5%) was excluded from the analysis due to any recent history of cardiac events or stroke (1.7%), the use of statins (0.8%), and dropout or incomplete measurements (6%) concerning practical challenges discussed in B.Individual characteristics, for a comparison between male  and female  participants, are given in C.The average carotid artery lumen diameter and distension for the first and second trial by the same operator were  vs , respectively. Nearly 80% of the diameter measurements fell within an RD ≤15%. Both the systolic and diastolic BP values yielded a group average RD < 5% , with < 4% values outside respective confidence intervals (mean ± 1.96 standard deviation).−1, and AC: 0.58 ± 0.28 mm2kPa−1. The field measurement of −1. The standard deviation of differences for 2kPa−1, respectively.The group average of stiffness indices pooled over all the subjects was D.−1 vs 5.81 ± 1.16 ms−1 (p < 0.001), and AC: 0.54 ± 0.36 mm2kPa−1 vs 0.72 ± 0.38 mm2kPa−1 (p < 0.001). The group averages of stiffness indices among females and males were As in tegories ; p < 0.0tegories , which yNote that the gradient in the mean of each stiffness index among males and females, across age groups , increas−1 , and 2kPa−1 . The corresponding measures for the subgroup free from hypertension and diabetes were −1 , and 2kPa−1 . Age-specific variations in the carotid stiffness were apparent across successive decades  in E.−1 vs 5.98 ± 1.18 ms−1 (p < 0.0001), and AC: 0.52 ± 0.25 mm2kPa−1 vs 0.55 ± 0.26 mm2kPa−1 (p = 0.34)). Males exhibited a similar trend with the corresponding measures as −1 vs 5.56 ± 1.02 ms−1 (p < 0.01), and AC: 0.67 ± 0.32 mm2kPa−1 vs 0.65 ± 0.29 mm2kPa−1 (p = 0.70). There was no difference in the proportions of multiple risks among males and females for The variation in stiffness of the carotid artery among subjects diagnosed with ≥3 risks and those with 0 – 2 risk(s) was studied separately for females (N = 550) and males (N = 393). (40 subjects were excluded due to unavailability of HDL-C data.) The prevalence of risk factors was 58.5% in female subjects and 42.7% in male subjects. Females with three or more risks exhibited significantly higher mean values of −1 (4th and 5th quintiles) was ≥ 2.12 or above in Model-1 as well as Model-2. Among females (−1 ≤ PWV−1 (3rd and 4th quintiles) was just above 2.00. The odds ratio increased to 3.45 (95% CI: 1.95 – 6.09) in Model-1 and 3.33 (1.78 – 6.25) in Model-2 for th quintile), and 3.42 (1.94 – 6.03) in Model-1 and 3.25 (1.76 – 6.02) in Model-2 for PWV−1 (5th quintile). The highest odds ratio values for Logistic regression analysis yielded a significant relationship between the clustering of three or more risks and high values of ng males , the odd females , the oddin males  and 1.77in males , respectHigh BP was the most prevalent risk factor among the study population. Different hypertension stages −1, and AC = 0.33 ± 0.16 mm2kPa−1. No significant difference was observed in −1, 8.1%, p < 0.001). Conversely, the changes in 2kPa−1, 6.1%, p >0.05). The values of −1, 19.0%, p < 0.001) and hypertensive crises groups . A significant rise in 2kPa−1, −12.1%, p < 0.01) as well as in hypertensive crises subjects .As depicted in ocument} , PWV\dococument} , or AC , their differences were marginally significant ; and 2kPa−1, −17.1%, p < 0.05). Compared with smokers of less than 5 years, the greatest changes in stiffness indices were seen in chronic smokers of more than 10 years ; and 2kPa−1, −23.4%, p < 0.01).Because gender was found to be an important determinant of arterial stiffening, the effect of smoking on the carotid stiffness was analyzed for (age-matched) males separately.  between the stiffness indices and age , enabling easy handling in the field. All the measurements were performed without failures throughout the study. Device’s icon-based custom touch-screen GUI was appreciated for improved usability. A rugged enclosure (machined nylon) offered protection from mechanical impacts during transport. As no electrical outlet sockets were available in the field, the device was operated on its 3000 mAh internal battery. A fully charged device was sufficient for a day’s study, which lasted for nearly four hours.Two sets of carotid stiffness measurement completed on 983 subjects demonstrate field usability of the device. The stiffness indices exhibited sufficient repeatability and are comparable to those reported in clinical studies conducted using ultrasound imaging systems Based on the study feedback, we have evolved the design and developed a much smaller device (ARTSENS® Pen) that takes power from a USB and operate with Microsoft Windows-based computer/tablet. This hand-held device (weight ≈ 100 g) with certified ingress protection greatly improves the portability and field-deployability. We have also integrated a custom solid ultrasound couplant into the updated probe for future studies.B.A gender-specific difference and an age-specific progression of the carotid stiffness were observed in the recruited subjects . The obsA nonlinear trend was evident in the age-specific progression of the carotid stiffness. All the stiffness indices accelerated from younger to older age-group, and later slowed in the geriatric subjects . This paRecently, efforts have made to develop age- and gender-wise normative reference for carotid stiffness indices C.Strong positive associations of in-vivo stiffness markers are summarized elsewhere It may be noted that distensibility coefficient (DC) is a well-studied marker of arterial stiffness, and has been reported to be an independent predictor of cardiovascular events and all-cause mortality Evaluation of AC is most prone to be corrupted by the error propagation as per the theoretical definition. Hence, it is least suited for use in field trials where the measurement repeatability cannot be as high as that in clinical settings. The measure of Finally, the observed high prevalence of multiple risk factors in the study population agrees with a previous community study performed in South India Further cohort studies using our device would examine whether the excess CVD events associated with clustering of cardiometabolic risk factors are partly mediated through the amplified alterations in vascular properties and endothelial dysfunction. Note also that predictive models reported herein may be attributed to the characteristics of the population under study. A multiethnic cohort study to develop generalized models is worthy for future work.D.viz. aortic PWV), thereby making it an excellent choice for large-scale assessment of the vascular stiffness.The major strength of this study is the use of a portable device to assess the carotid arterial stiffness in a resource-constrained field setting. The study demonstrated that the device enabled a reliable measurement of clinically accepted stiffness indices at a population-level. Thus, the developed device could overcome the limitations of ultrasound imaging equipment and operator expertise needed for stiffness evaluation. It also eliminates the need to access the groin region  caused dissatisfaction in participants since it was impractical to provide a precise diagnosis of their vascular health after the test by comparing their results with that of nomogram values. The study had an inherent limitation in determining the temporal relationships of carotid stiffness with risk factors due to its cross-sectional nature. In general, only association and not causality can be inferred from the data. Selection of a specific region further restricted the extension of findings to the Indian population.V.We conducted the first community-level study in an Indian rural cohort to describe the distribution of the carotid stiffness. This study established a reliable assessment of carotid stiffness indices in out-of-hospital settings using ARTSENS® Touch. It demonstrated the functionality and sufficient repeatability for potential use in population-level screening. The age-/gender-specific distribution of stiffness indices was explored. A strong relationship between the stiffness indices and cardiometabolic risk factors indicates their utility for screening patients at high-risk in clinics as well as field settings. Learnings from this study were incorporated into the instrument hardware, probe design, and algorithms to develop smaller and easy-to-use prototypes. ARTSENS® devices enable large-scale field studies to develop population-specific nomograms, thereby establishing the innate role of carotid stiffness in cardiovascular risk stratification.


Trade friction has always been a prominent feature in the current economic development of the world. Its impacts on multinational enterprises are self-evident, but its psychological effects on the multinational entrepreneurs are still unclear. In order to understand the impacts of trade friction on psychological effects of multinational legal service entrepreneurs, 305 multinational entrepreneurs were selected in this study for questionnaire survey, and Spearman’s correlation and regression models were used to analyze the correlation among economic pressure, the thought of recession, self-efficacy, and social support. The structural equation model was used to analyze the influence path of economic pressure and social support on the thought of entrepreneurial recession, as well as the influence path of multinational entrepreneurship orientation and value-chain potential on the international performance. The results show that economic pressure is significantly and positively correlated with the thought of recession and self-efficacy extremely and significantly and negatively correlated with objective support and support utilization extremely; social support will reverse the thought of entrepreneurial recession caused by the economic pressure; the indirect impact path coefficient of social support utilization in economic pressure and entrepreneurial recession is – 0.281; the indirect impact path coefficient of value-chain potential in multinational entrepreneurial motivation and international performance is – 0.424. It shows that trade friction will indirectly trigger the thought of entrepreneurial recession of entrepreneurs by reducing their economic incomes. Besides, the social support utilization can significantly regulate the relationship between the economic pressure and the thought of entrepreneurial recession. Therefore, the value-chain potential plays an intermediary role in multinational entrepreneurial motivation and international performance. In recent years, there is an upsurge of entrepreneurship in China. Nevertheless, there is a serious problem of oversupply in all fields; especially in the traditional fields, due to the increasing competitiveness in domestic market of China, it is difficult for more and more entrepreneurs to get success in the domestic market . There aWith the deepening of economic globalization, the trend of entrepreneurial strategy and international diversification strategy are considered as key factors for multinational enterprises to succeed in the global market, and related studies have shown that the trend entrepreneurial strategy has a directly positive correlation with the entrepreneurial performance of multinational enterprises . The entIn order to explore the impacts of the trend of internationalization on the psychological effects and work performance of multinational entrepreneurs, the legal service entrepreneurs of knowledge-based service companies were thus selected as the research objects. The impacts of trade friction on the psychological effects of multinational entrepreneurs and the impact mechanism of multinational entrepreneurial orientation on the international performance were explored in this study firstly. Finally, the validity and authenticity of hypotheses proposed in this study were verified through actual investigations. The results of this study aimed to lay a foundation for understanding the psychological effects of multinational entrepreneurs and promoting the international work performance of multinational enterprises.Entrepreneurship can help entrepreneurs achieve their personal ideals and promote economic development and technological innovation. Researches in entrepreneurship-related fields had been conducted from the perspectives of management, psychology, and sociology. Entrepreneurship is a significant form to achieve the social and economic development. In exploring the international entrepreneurship, it is very important to improve the performance of multinational enterprise. In order to explore the impact of strategic responsibilities of the multinational enterprises on the performance of their subsidiaries, To sum up, the multinational legal service entrepreneurs were taken as the research objects in this paper. In the form of questionnaire, the impact path of the economic pressure and social support of the trade friction on the entrepreneurial recession of entrepreneurs and the impact path of multinational entrepreneurial orientation and value-chain potential on the international performance of enterprises were analyzed. In this way, this study will lay the theoretical foundation for finding out the way to relieve from the psychological pressure of multinational entrepreneur and improving their working performance.A total of 310 Chinese entrepreneurs of legal services who started businesses in the United States, Canada, Indonesia, Brazil, and the United Kingdom were selected as the research objects, the questionnaires were distributed online from June 2019 to December 2019, and 305 questionnaires were returned, so the response rate was 98.39%. The 305 subjects were 21–38 years old, with an average age of 26.88 ± 5.52. Studies show that the risk aversion of female entrepreneurs is higher than that of male and that self-efficiency has a strong predictive effect on the entrepreneurial behavior. Therefore, the gender, age, and self-efficiency were taken as the control variables for subsequent analysis.It is believed in this study that the economic pressure brought about by the trade friction could cause the entrepreneur to experience some negative emotions . Based on the theory of learned helplessness, the theoretical model was built for effect of the economic pressure and social support caused by the trade friction on the entrepreneur’s thought of recession. The theoretical model is shown in According to a.The economic pressure could be promoted significantly by the trade friction;b.The thought of entrepreneurial recession could be promoted significantly by the economic pressure;c.The promotion of economic pressure on the thought of entrepreneurial recession could be regulated reversely by the social support;c1.The promotion of economic pressure on the thought of entrepreneurial recession could be regulated reversely by the subjective support;c2.The promotion of economic pressure on the thought of entrepreneurial recession could be regulated reversely by the objective support;c3.The promotion of economic pressure on the thought of entrepreneurial recession could be regulated reversely by the support utilization.In order to explore the correlation on the multinational entrepreneurial orientation and international performance, the theoretical model shown in Based on the above model, the below hypotheses are proposed in this study:d.The international performance could be promoted by the multinational entrepreneurial orientation;d1.The international performance could be promoted by innovation in the multinational entrepreneurial orientation;d2.The international performance could be promoted by antecedence in the multinational entrepreneurial orientation;d3.The international performance could be promoted by adventure in the multinational entrepreneurial orientation;e.The international performance could be performed by the value-chain potential;e1.The international performance could be performed by complexity of the trade;e2.The international performance could be performed by codability of the product;e3.The international performance could be performed by the supply capacity;f.The value-chain potential is the intermediary variable for multinational entrepreneurial orientation to affect the international performance.The evaluation scales used in this study were all widely used, reliable, and valid evaluation tools in line with the actual conditions.The Social Support Rating Scale (SSRS) was used by The Economic Pressure Rating Scale (EPRS)  was compThe exiting entrepreneurship assessment scale was used for recompilation and was applied to evaluate the entrepreneur’s thought of recession . Three iThe entrepreneurship self-efficacy assessment scale  was usedBased on the three dimensions of innovativeness, proactiveness, and risk taking and the “9-item semantic difference scale” , the scaThe value-chain potential was measured based on the trade complexity, product codability, and supply capacity . The finBased on the research results of other scholars , the intp < 0.05). The reliability tests of the evaluation tools were performed by using the Cronbach’s α (it was deemed as high reliability when Cronbach’s α was higher than 0.7). The principal component analysis (PCA) was used to extract the factors, whose eigenvalue was higher than 1, in the variable tables. The descriptive statistics was used for statistical analysis of the sample distribution of each variable. The correlation of each variable was analyzed with the Pearson correlation. Hypotheses proposed in this study were verified by the process of hierarchical regression. When P < 0.05, it is considered that it has a statistical significance.The statistical processing was performed by SPSS 19.0. The validity tests of the evaluation tools were performed by using the Bartlett’s spherical test and KMO value  and has an extremely significant negative correlation with the objective support and support utilization (p < 0.01). The thought of recession has an extremely significant positive correlation with the self-efficacy (p < 0.01) and has a significant negative correlation with the objective support and support utilization (p < 0.05). The self-efficacy has a significant negative correlation with the subjective support and objective support (p < 0.05) and has an extremely significant negative correlation with the support utilization (p < 0.01). The subjective support has an extremely significant negative correlation with the objective support and support utilization (p < 0.01). There is an extremely significant positive correlation between the objective support and support utilization (p < 0.01).Then, the correlation between the thought of entrepreneurial recession, the impact of entrepreneurial work performance, and other variables in this study were compared. The results are shown in p < 0.05).It could be seen from p < 0.01). b. The economic pressure has an extremely significant positive effect on the thought of entrepreneurial recession . c. The social support × economic pressure can negatively affect the thought of entrepreneurial recession, that is, the social support can regulate the thought of entrepreneurial recession caused by the economic pressure reversely . c1. and c2. The subjective support and objective support cannot regulate the thought of entrepreneurial recession caused by the economic pressure significantly . c3. The support utilization can regulate the thought of entrepreneurial recession caused by the economic pressure . d. The multinational entrepreneurial orientation has a significant positive impact on the international performance . d1. The innovation dimension in multinational entrepreneurship has an extremely significant positive impact on the international performance . d2. The proactiveness dimension in multinational entrepreneurship has a significant positive impact on the international performance . d3. The risk-taking in the multinational entrepreneurship cannot affect the international performance . e. The value-chain potential has an extremely significant positive impact on the international performance . e1. The transaction complexity has an extremely significant positive impact on the international performance . e2. The product codability cannot affect the international performance . e3. The supply capacity has an extremely significant positive effect on the international performance . In addition, it is also found in this study that the multinational entrepreneurial orientation can affect the value-chain potential significantly .The hypotheses proposed earlier in this study were analyzed by the regression analysis. It could be seen from It could be seen from The impact path of the regulated economic pressure and social support based on the trade friction on the thought of entrepreneurial recession is shown in The impact paths of the regulated multinational entrepreneurial motivation, value-chain potential, and international performance are shown in In this study, it is found that the United States, European Union, Association of Southeast Asian Nations, and Canada are main exporters of China, so the current export markets of China will continue to be the “main battlefield” of the trade friction in the future . On the The protection policies for the knowledge-based enterprises in various countries are not less than those for the manufacturing industry, and the incidents caused by trade friction for the knowledge-service enterprises entering their own countries are possible. Therefore, the legal service entrepreneurs have to bear greater economic pressure, so the trade friction may cause certain economic pressure on multinational entrepreneurs . When thIt is found that the entrepreneur’s thought of entrepreneurial recession can be affected positively by the size of economic pressure, which is similar to the results of With assistance of statistical methods combined with questionnaire surveys, regression model, and structural equation models, this study analyzed the impact mechanism of trade friction on the mentality of multinational entrepreneurs. It was found that trade friction can trigger the thought of entrepreneurial recession of entrepreneurs indirectly by reducing their income. The social support utilization can significantly regulate the relationship between economic pressure and the thought of entrepreneurial recession. The multinational entrepreneurship motivation can promote the international performance of multinational enterprises through the value-chain potential. It fails to consider other factors of the external environment  and the interactions from the personal internal factors to the thought of entrepreneurial recession and the entrepreneurial working performance. Accordingly, the quantity of samples has to be increased in the future and to take specific research in a certain area to explore the effects on external environment and the internal factors of individuals on the thought of entrepreneurial recession and entrepreneurial working performance. In conclusion, the thought of entrepreneurial recession of the entrepreneur can be reduced and the subjective initiative of the entrepreneur can be played by using the social support to the maximal extent. International performance of the enterprise can be improved by complying with the international market and improving the service ability. Results in this study can provide a basis for understanding the impact mechanism of the thought of entrepreneurial recession of entrepreneur of multinational legal service and its work performance.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by the Shandong Normal University Ethics Committee. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CX wrote the manuscript. ZZ reviewed the manuscript. Both authors contributed to the article and approved the submitted version.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.


This paper examines the effect of PBO (P-phenylene benzobisoxazole)–FRCM (Fabric Reinforced Cementitious Matrix) reinforcement on the stiffness of eccentrically compressed reinforced concrete columns. Reinforcement with FRCM consists of bonding composite meshes to the concrete substrate by means of mineral mortar. Longitudinal and/or transverse reinforcements made of PBO (P-phenylene benzobisoxazole) mesh were applied to the analyzed column specimens. When assessing the stiffness of the columns, the focus was on the effect of the composite reinforcement itself, the value and eccentricity of the longitudinal force and the decrease in the modulus of elasticity of the concrete with increasing stress intensity in the latter. Dependences between the change in the elasticity modulus of the concrete and the change in the stiffness of the tested specimens were examined. The relevant standards, providing methods of calculating the stiffness of composite columns, were used in the analysis. For columns, which were strengthened only transversely with PBO mesh, reinforcement increases their load capacity, and at the same time, the stiffness of the columns increases due to the confinement of the cross-section. The stiffness depends on the destruction of the concrete core inside its composite jacket. In the case of columns with transverse and longitudinal reinforcement, the presence of longitudinal reinforcement reduces longitudinal deformations. The columns failed at higher stiffness values in the whole range of the eccentricities. The FRCM (Fabric Reinforced Cementitious Matrix) system, in which a composite mesh is bonded to the substrate with mineral mortar, is becoming the preferred method of choice in increasing the load capacity of concrete elements or the method in repairing them. FRCM reinforcements are applied to strengthen or repair concrete and reinforced concrete elements subjected to bending, shearing and compression. Increasingly more experimental and theoretical investigations into the behavior of elements strengthened with a composite mesh on mineral mortar are reported ,5,6,7,8.The FRCM system is characterized by higher resistance to elevated temperatures than the FRP (Fiber Reinforced Polymer) system in which non-metallic composite fibers are embedded in epoxy resin ,10. An aσc in the concrete do not exceed its compressive strength f′co, the strains in the composite remain low. From the instant when σc > f′co, the transverse stresses in the composite increase, so does the PBO mesh action on the concrete core. In order to develop a general method of dimensioning columns reinforced with FRCM, it is necessary to determine the effect of this reinforcement on the change in the stiffness of such columns with increasing stresses.The knowledge about the considered subject can be extended through tests and analyses of the confined concrete elements subjected to compression ,24,25,26Tests ,18,19,20Ombres and Verre  carried Ec) with the increasing load. The value of the modulus of elasticity of the concrete (Ec) in a given stress state is highest at a stress close to zero. As the stress increases, the modulus of elasticity of the concrete decreases. For pure concrete at longitudinal stresses σc > 0.5f′co, the ratio of the concrete’s instantaneous modulus of elasticity Ec,time to its initial modulus of elasticity E0 amounts to about 0.77 . Th. Th19]. εv2,lim and εv1,lim on the more and less compressed (tensioned) sides of the cross-section, respectively.The curvatures of the specimens were calculated from Equation (7) on the basis of the measured maximum longitudinal strains εv, in the confined columns with the PBO mesh only, with horizontal layout fibers over the main direction (C_1H and C_2H), failure was observed at a comparable level of strain  were calculated from (8) on the basis of the maximum deflections wmax (The bending moments at the instant of failure (ons wmax .(8)MmaxThe bending stiffness of columns can be numerically analyzed with the use of Bernoulli’s hypothesis with or without eccentric load and additional reinforcements such as fiber materials. The additional longitudinal composite reinforcements contribute to the increasing bending stiffness directly and transverse composite reinforcements give confinement effect to increase stiffness. The axial stiffness should not be evaluated under the combination of axial force and bending moment.r), bending moment Mmax and bending stiffness EI (9), the change in stiffness was analyzed depending on the type of strengthening of the column and the stress intensity in the latter.Assuming that Bernoulli’s hypothesis is applicable in this case (plane section remains plane) and starting with the general dependence between the curvature of the specimen’s deformed axis P=0 for the load eccentricity of, respectively, 0, 16 and 32 mm. The vertical axis represents the ratio of the ultimate force to the load capacity of the axially compressed column in a given group for the load eccentricity of 0, 16 and 32 mm. The broken line marks the trend in stiffness change.The next three diagrams  show theEc,max before failure. The smaller decrease in stiffness of the reinforced concrete columns, than that resulting from the change in the elasticity modulus of the “plain concrete” itself, is evident due to the presence of the longitudinal reinforcement and the shape of the cross-section of the columns.In the case of column C_C_0 (most stressed), the stiffness at the point of failure amounts to 35% of the initial value . For theA similar trend in the change of stiffness is observed in the columns with a single layer (1H) of transverse composite reinforcement . The addEf is given in R is the radius of a circle with the circumference equals the circumference of a considering cross-section. For the considered columns with the square cross-section with the side length a:The stiffness of the composite jacket in these investigations is defined with the equivalent modulus of elasticity of the PBO–FRCM strengthening according to the following formula:One should note here that in comparison to the reference columns, the decreases in load capacity were observed for columns C_1V2H_0 and C_1V2H_16  18]. Th. Th18]. The next two diagrams  and TablMmax is the first-order moment. The slenderness ratio of the RC columns λ < λlim according to [The ductility of the columns in these investigations is defined as the ability to horizontally displace the columns, which is induced with the bending moments (eccentric load) what is presented in rding to .Ec) due to the stress destruction of the concrete core. Microcracks develop in the concrete beyond the level of stress in the column at which Poisson’s ratio ν is no longer a liner [ν quickly increases, which is due to the extensive microcracking of the concrete core. This stress level corresponds to 60–70% of the maximum (ultimate) force Pmax observed during the tests.The effect of the composite jacket in PBO–FRCM columns is closely connected with the variation in the elasticity modulus of the concrete  is clearly visible and ratio ν), due to the destruction of the concrete inside the composite jacket should be taken into account in the standards.With regard to the variability of PBO–FRCM column stiffness, the variation in the ratio of transverse strain to longitudinal strain  is a difficult and complicated task. One cannot adopt, a priori, the standard regulations dedicated to reinforced concrete or composite elements to determine the stiffness of such columns. The aim of the investigations presented in this paper was to assess the effect of the PBO–FRCM reinforcement of eccentrically compressed columns on their stiffness depending on the level of stress intensity.In the case of unstrengthened columns, the expected results were measured. As eccentricity increased, the longitudinal ultimate force decreased and the deflection of the columns increased. As the curvature of the columns increased, so did the bending moment at half of their height. Each of the columns failed at a correspondingly lower intensity of the stress in the whole cross-section, which was produced by applying longitudinal force on the more compressed side.In the case of columns C_1H and C_2H, which were strengthened only transversely, our measurements and evidence collected point to the fact that this kind of reinforcement increases their load capacity, but at the same time, the stiffness of the columns increases due to the confinement of the cross-section. In comparison with the reference (unstrengthened) specimens, the curvature would decrease at the initial eccentricities after the first and second PBO layers were laid. This was accompanied by an increase in the ultimate force and a decrease in the horizontal deflection of the columns. At eccentricities of 16 and 32 mm, the columns failed at correspondingly higher stiffness values than the reference members, owing to the use of transverse composite reinforcement. Whereas in the case of columns C_1H_0 and C_2H_0, their load capacity was reached at lower stiffness values, which indicates considerable destruction of the concrete core under axial compression.In the considered case of quadrangular columns, the tri-axial state of stress induced by the confinement of the concrete is insignificant and the addition of another layer of transverse composite reinforcement only increases the stiffness of the composite jacket.Unfortunately, in the case of columns C_1V1H and C_1V2H, their higher ductility and greater horizontal deflectability in the bending plane do not directly translate into an increase in their load capacity in comparison with the columns without longitudinal composite reinforcement. As shown in the earlier research, longitudinal PBO–FRCM reinforcement improves the ductility of eccentrically compressed columns. The presence of this reinforcement, however, reduces longitudinal deformations at which the mesh ruptures, which is disadvantageous. Thanks to the presence of longitudinal PBO reinforcement, the columns failed at higher stiffness values in the whole range of the eccentricities: 0, 16 and 32 mm.This paper investigated the effect of PBO–FRCM reinforcement on the stiffness of eccentrically compressed RC columns. The dependencies between the change in the elasticity modulus of the concrete and the change in the stiffness of the tested specimens were examined. This subject is of great practical relevance. Few such experiments are available in the literature. The analyses presented in this paper can be used to design a wider series of tests of PBO–FRCM columns with various types of reinforcement, which are subjected to eccentric compression. The results of such tests can be used to formulate a standard relation for a column stiffness reduction coefficient depending on the type of reinforcement, the longitudinal force and the cross-section’s stress intensity.