Bifidobacterium adolescentis. After intake of raw potato starch (pSt), B. adolescentis dramatically increased in every carrier of the species, accompanied by an increase in bifidobacterial metabolite acetate. In the other subjects, Eubacterium rectale and its metabolite butyrate increased, but it was suppressed in B. adolescentis carriers. A correlation analysis indicated the contribution of these species to respective metabolites. In vitro analyses of isolates of major gut bacterial species confirmed that these species are major colonizers of pSt and that B. adolescentis can colonize pSt even in the presence of the known starch granule–degrading bacterium Ruminococcus bromii. Collectively, we propose that specific binding of B. adolescentis or E. rectale to pSt selectively induces acetogenic or butyrogenic response of gut microbiota, where the former determines the response of the latter.Prediction of individualized responses is one of biggest challenges in dietary intervention to modulate human gut microbiota. Bacterial interspecies competition for dietary factors should underlie the inter-subject heterogeneity of microbial responses. Microscale localization of bacterial species around intestinal food structures could provide direct evidence for understanding this, however, little information is currently available. Here we analyzed human fecal sections and found multiple types of bacterial colonization of food structures. The most eminent one was dense and frequent colonization of starch granules by  Bifidobacterium adolescentis, although another colonizing species, Eubacterium rectale, also can bind and respond to the granules. The major gut fermentation product in each subject was determined by which of the two species responded and especially by the presence of B. adolescentis.The intestinal microbiota contributes to many aspects of host health and diseases via bioactive metabolites including short-chain fatty acids (SCFAs). Diet is a rapid and powerful tool for modulating human microbiota and their metabolism –3. A speAll human studies were conducted in accordance with the Declaration of Helsinki. The studies were scientifically and ethically reviewed and were approved by the Yakult Central Institute. Written informed consent was obtained from the participants after explanation of the study  before enrollment.Five healthy male adults  who had not taken antibiotics for at least one week prior to the day of the first sampling participated. Fecal specimens were collected twice with a 2-week interval. Subjects were asked to avoid overeating or overdrinking during the period. Contents of meals for 2 days before sampling were surveyed.Ten healthy male adults age, 29–56 years) were recruited. This experiment was designed according to a previous study  of granular starch, Maruko Foods, Saitama, Japan) at lunch time for 4 days, followed by 8 days of the post-intake period . Six fecal specimens were collected in total from each subject (two for each period). Intake of banana and additional raw Japanese yam (other than that provided by the researcher) was prohibited during all periods of this experiment. Two subjects (F and G) were recruited again after the experiment, another informed consent was obtained, and fecal material was collected once for isolation of dominant bacteria. For this sampling, food intake was not restricted.Ten healthy male adults  were recruited. After 9 days of the pre-intake period, they were asked to take 40 g/day of raw Japanese yam  for fluorescent microscopy, or using Entellan New (Merck Millipore) for observation of iodine staining without FISH and of alcian blue–PAS staining. For double staining (FISH and iodine), specimens were first stained by FISH and then by iodine, and mounted using Vectashield with DAPI. Before mounting, more than ten independent fields of view containing one or more deep-purple starch granules were randomly selected for each slide and memorized by the microscope operating software MetaMorph ; immediately after mounting, observation of stained starch and image acquisition were performed using memorized positions . Fluorescence was observed using a DM6000B microscope system . Monochrome images were acquired for each fluorescent dye and pseudocolors were assigned to make overlay images. Bright-field images for starch granule measurements were obtained under a BZX700 microscope .To confirm the specificity of starch staining, serial sections were incubated with 0.5% porcine amylase  in PBS at 37 °C for 2 h and then stained with Lugol’s solution.A macro program that selects starch granules but not solubilized starch was designed in Image-Pro Plus 6 . Representative images containing average amounts of starch granules in each specimen were analyzed by this macro to calculate the percentage of starch granule area in the field. Area outside of fecal material and area occupied by air bubbles were omitted from the calculation. Area of excreted starch granules (%) was calculated as [area of starch granules during the intake period (%)] – [that during the pre-intake period (%)].Microbial profiling based on 16 S rRNA gene sequences was performed as previously described . DNA wasSCFAs were measured as previously described . Fecal sg for 1 min to remove large debris. Aliquots of the supernatants were mounted using Vectashield with DAPI, and fluorescence of DAPI was imaged for ten independent fields of view using Leica DM6000B microscope. The cells in these images were counted using Image-Pro Plus 6.Fecal samples were washed with PBS, resuspended in PBS containing 0.1% Tween-80, sonicated and centrifuged at 100 × Fresh fecal samples or those stored at −80 °C were used. A small fraction of each sample was diluted and plated onto TOS propionate agar  supplemented with 50 μg/ml mupirocin, or BL agar  supplemented with 5% defibrinated horse blood. Plates were incubated for 1–2 days at 37 °C in an anaerobic glove box. Colonies were sorted into types by their morphology. For each type, several single colonies were isolated and subjected to taxonomic identification by 16 S rRNA gene sequencing . Approximately 1 μl of a colony was taken with an inoculating loop and suspended in 150 μl of PBS; then, 12 μl of this suspension, 80 μl of pSt suspension (1% w/v in PBS), and 88 μl of PBS were mixed by vortexing, and formation of bacteria–starch aggregates was examined. If aggregation occurred and the flocks fell immediately to the bottom of the tube making the supernatant transparent, the strain was judged positive. If no aggregation was observed, the strain was judged negative. In negative cases and in the negative control (no bacteria), visible precipitation of starch requires more than ~15 s. Strains that showed self-aggregation  were categorized as undeterminable.Starch-degrading ability was screened as follows. Strains were grown anaerobically on modified GAM agar, the culture plates were flooded with 1/2 diluted Lugol’s solution for about 30 s and then the solution was removed. Starch-degrading ability was judged from a clear zone around the colonies. When the diameter of this zone was <~1 mm or staining around the colony was reduced but was clearly visible, the strain was judged as weakly positive.Utilization of pSt was analyzed as follows. The powder of GAM Semisolid without Dextrose  was thoroughly dissolved in water, agar was removed by precipitation, and the solution was autoclaved at 115 °C for 15 min. Broths containing different carbon sources were prepared by adding 1% w/v glucose, fructose, or pSt before autoclaving. All broths were introduced into an anaerobic chamber at least 1 or 2 days before use. A separate broth was prepared by adding 1% w/v pSt into the autoclaved broth without any additional carbon source, in an anaerobic chamber just before starting culture; starch used in this broth had been UV-irradiated in advance for at least 16 h. Bacterial strains were grown anaerobically on modified GAM agar. Each colony (~1 μl) was suspended in 150 μl PBS, and 5 μl was inoculated into each broth and incubated at 37 °C in the anaerobic chamber for 3 days, and then pH of the culture was measured.Bifidobacterium adolescentis G202, Ruminococcus bromii YIT 6078 T, and R. bromii ATCC 51896 were grown anaerobically on modified GAM broth at 37 °C. Fully grown cultures of either of two R. bromii strains and G202 were inoculated into GAM broth supplemented with 0.5% pSt (pSt-GAM) at an approximate 2:1 ratio and incubated for 24 h. Alternatively, R. bromii YIT 6078 T was grown on pSt-GAM broth for one day, then G202 was inoculated, and the resultant culture was incubated for 48 h.g for 10 s, the supernatant was removed, PBS was added to the pellet and resuspended, the sample was centrifuged as above and the supernatant was discarded. The pellets were heated at 95 °C for 15 min in TE, centrifuged at 13,000 rpm for 1 min, and the supernatants were used as PCR templates. PCR was performed using Premix Ex Taq Hot Start Version (Takara) and equal amounts of three primers: 27Fmod2-notag, g-Bifid-R, and Clept866mR  were denoted as B. adolescentis in this study.Output fastq files were analyzed in QIIME2 software (version 2018.11) and its plugins . Noise as value represents the “false sign or small” (FSOS) rate, where “small” denotes an absolute LFC less than 1. In identification of significantly increased OTUs, we analyzed the data from B. adolescentis carriers  and non-carriers  separately.OTUs whose abundance increased in response to pSt intake were identified using DESeq2  with a dB. adolescentis carriers and non-carriers separately and adjusted the p value using the Benjamini–Hochberg correction [To determine whether there was a significant change in SCFA concentration from the pre-intake period to the pSt intake period, we performed a Wilcoxon signed-rank test using the R-package exactRankTests  functionrrection  for multp value between the abundance of OTUs and SCFA concentrations were calculated using the R-package Hmisc [Spearman’s rank correlation coefficient (Spearman’s ρ) and ge Hmisc  functionge Hmisc .B. adolescentis carriers and non-carriers using two-sided Welch’s t test.Excretion of starch granules (% area) was compared between B. adolescentis [Lachnospiraceae localized along a plant exodermis–like cell layer . This method has been employed in a murine study and proven useful for elucidating how specific bacteria acquire nutrients in the intestine . Stool sescentis , by usinyer Fig. , and somyer Fig. .Fig. 1MiB. adolescentis led us to speculate that the species is a key degrader, which breaks down a large fraction of starch granules, possibly providing oligosaccharides, or a key consumer, which takes up and metabolizes a large fraction of starch granules, and affects the overall response of the microbiota to the nutrient. To test this hypothesis, we next checked the response of B. adolescentis and whole-gut microbiota to intake of starch granules in ten healthy adults and evaluated how the presence or absence of the species affects the response of the microbiota , the abundance of another known starch-utilizing species, Eubacterium rectale [R. bromii [Ruminococcus torques  method, and successfully confirmed that the same conclusion as above was obtained  that were significantly correlated with the concentrations of acetic and lactic acids, and E. rectale was the most abundant among OTUs that were significantly correlated with the concentration of butyrate, suggesting that they produced a major fraction of each SCFA from pSt  among all bacterial species detected in this study, B. adolescentis most efficiently consumes intestinal pSt granules, which is facilitated by their colonization, to increase its abundance and generation of acetic and lactic acids, and that (ii) E. rectale can increase its abundance upon pSt intake, generating butyrate, but only in the absence of the dominant consumer B. adolescentis.Surprisingly, though all subjects had ers Fig. . Similarers Fig. . Howeverned Fig. . Betweeners Fig. . FurtherpSt Fig. . Insteaders Fig. . In SpeapSt Fig. . In anotria Fig. . For B. B. adolescentis and other species, especially E. rectale, we analyzed fecal sections again to distinguish colonizing and non-colonizing species (Experiment III). For this purpose, we employed another cohort, who consumed lower amounts of starch granules, to identify colonization more easily  pSt was shared by many taxa; all evaluated Bacteroidaceae species and E. rectale. However, pSt granule–binding ability was shared only by E. rectale and C. aerofaciens, and it was strain-dependent . This increase suggests that M. indica rather than butyrate producers could benefit from crossfeeding of lactate in a strongly acidic environment.It should be considered that the relationship between d States , 28, 35.sm genes . Future escentis . Meanwhia indica  in B. adB. adolescentis in utilization of starch granule is substantiated by in vitro activities of dominant species isolated from the participants. These results reinforce that B. adolescentis is highly adapted to raw granular starches and suggest that monopolization of the starch surface by adhesion promotes the selective response of the species as it limits expansion of other species, which can increase in abundance in the absence of B. adolescentis. The model is substantiated by the results of our in vitro competition assay, which suggest that B. adolescentis can take over the pSt surface from the known starch granule-degrading species, R. bromii. The acidic environment induced by B. adolescentis  may also contribute to the advantage of B. adolescentis over other species such as E. rectale [E. rectale cannot utilize raw pSt in vitro, but it did respond to the intake of raw pSt. This may be explained by the activity of host amylase or amylases from other bacteria. Because E. rectale responded to pSt in the absence (or presence below the detection limit) of R. bromii , there should be another mediator with activity similar to that of R. bromii.The advantage of  rectale . We conf rectale , 42 thatTaken together, our results provide a good example of competition for a common but specific food ingredient and hierarchical relationship among major bacteria in the normal human intestine. Our findings could provide a basis for understanding similar interspecific relationship. In situ colonization analysis brought us mechanistic understanding of inter-species hierarchy in response to a nutrient and prompted us to propose future plans for a precise control of microbiota. A similar approach may reveal competition among other species for other nutrients and contribute to our understanding and ability to use the microbiota.Supplementary notesSupplementary figuresSupplementary tables


Development of new additive manufacturing materials often requires the production of several batches of relatively large volumes in order to print and test objects. This can be difficult for many materials that are expensive or difficult to produce in large volumes on the laboratory scale. Bioprinter systems are advantageous in this regard, however, commercial systems are expensive or do not have the ability to use photopolymers. Herein, we outline a Syringe Pump Extruder and Curing System (SPECS) modification for inexpensive filament-based 3D printers which enables the use of standard bioplotter materials and photopolymers. The system is capable of using multiple syringe volumes and needle sizes that can be quickly and easily exchanged. The SPECS modification is demonstrated using a Prusa i3 mk3 fused filament fabrication printer to print several 3D objects and films using stereolithography (SLA) photopolymer resin. Geometric accuracy in the X, Y, and Z directions was ±0.1 mm using a 5 ml syringe, 22-gauge needle, and commercial SLA resin. The SPECS system could be of great benefit for laboratories pursing material development in the area of additive manufacturing. Specifications table11.1Research and development of new materials that are compatible with additive manufacturing (AM) processes have received a dramatic increase over the last decade 1.2While the system validation described herein focuses on the use of UV photopolymer resins, the SPECS modification can also be used for liquids, pastes, and gels that are commonly employed in pump-based AM systems such as bioprinters and food and paste printers. Recently, Kretzschmar et al. documented an AM system that also incorporated UV curing along with a paste extrusion process There are numerous research areas that can benefit from a 3D printer with variable volume capabilities. The SPECS modification meets the demand for an AM system for testing small batches of photopolymer build material by using a detached syringe pump which can hold a wide range of syringe volumes. The hardware provided includes 3D printed holders for 5 ml and 60 ml syringe volumes. This makes it possible to initially test small volumes of material and scale up to the printing of larger objects, if needed.1.3Extrusion-based AM systems that can use liquids, gels, resins, or pastes as feedstock are ideal for researching advanced materials in small batches 1.4−1 and a layer height of 0.2 mm showed that curing was sufficient to build 3D objects.The SPECS modification of the Prusa i3 Mk3 preserves its bed leveling function and repurposes the filament drive motor to operate a syringe pump. Similar to the open-source system developed by Pusch et al., this allows for control of the flow rate and the application of a consistent extrusion pressure with the ability to reverse flow direction 2General description of hardware. The SPECS modification consists of three parts: (1) the syringe pump, which delivers the fluid build material, (2) the extrusion head that holds the syringe tip and the bed leveling sensor, and (3) the UV lamp assembly, which is used to attach the UV lamp to the printer frame and is required to cure photopolymers during the build process.2.1The syringe pump design is based on standard laboratory syringe pump hardware in which multiple syringe sizes can be used. The current maximum size syringe that will fit into the syringe pump with its entire volume being utilized is 60 ml and the minimum syringe size is 5 ml. This allows for either high volume prints or small prints where conservation of materials is important, material batches are small, volatile materials must be used quickly and in small volumes, or material availability is low.The syringe pump is also designed for simplicity. All 3D printed parts can be printed by the Prusa i3 mk3 prior to the conversion, can be printed with no supports, and the entire pump only consists of eight 3D printed parts and minimal addition hardware. The syringe pump utilizes the existing extruder stepper motor and alterations to the slicing software allow for a fine level of adjustment for fluid flow rates.2.2The extrusion head is a simple design which preserves the original position of the automatic bed leveling sensor and extrusion location. The clamp-style design of the needle retention location makes it easy to adjust the needle height and change needle sizes as needed. Due to the high-quality construction of the Prusa i3 mk3, little to no vibration is present in the needle, even at high print velocities. The extrusion head utilizes the existing mounting holes for the stock extrusion head as well as cable routing location for the automatic bed leveling sensor.2.3The UV lamp assembly allows for the lamp to be mounted on the top cross member of the Prusa i3 mk3 without interfering with the printer’s function.2.4•The FFF printer modification employs a detached syringe pump which can be used to adapt most consumer-grade printers.•The modification preserves the bed leveling functionality of the Prusa i3 Mk3 printer allowing for accurate thin film printing and high resolution.•The existing stepper motor is used to drive the syringe pump.•The slicer software, Prusa Slicer, is open source and can be easily altered to be fully integrated with this fluid deposition system.•Syringes with different volumes and syringe tip sizes can be quickly swapped out allowing for the use wide range of materials with varying viscosities and particle additive inclusion sizes.•A multitude of needle sizes can be used for deposition.•All primary parts of the conversion can be printed with the Prusa i3 mk3 prior to the conversion.The SPECS modification is of interest to researchers developing new materials for fluid-based AM processes. Additionally, the printer allows for the unique ability to use photopolymers.Engineering drawings, renderings, and exploded diagrams of the conversion hardware are included in the table below.Design Files Summary3.1Full Assembly: An STL and CAD file of the complete assembly is provided that includes all 3D printable parts.Pump Frame – Top : The top section of the syringe pump frame.me – Top A: The toLead Screw Knob : The lead screw knob attaches to the top of the driving lead screw and allows for easy manual manipulation of the syringe pump.rew Knob B: The leLarge and Small Syringe Clamp : Clamp inserts are designed to securely hold both a 60 ml and a 5 ml standard syringe. Additional inserts for different syringe volumes may easily be printed by the user.ge Clamp C: Clamp Plunger Plate : The plunder plate is the primary translating component of the syringe pump. The plunger of the syringe is clamped to this plate with the plunger clamps and the translation of this plate extrudes or retracts the build material.er Plate D: The plPump Frame – Bottom : The bottom section of the syringe pump frame. The bottom of the pump frame also has holes to optionally mount the syringe pump directly to the Prusa i3 mk3 on the back side of the machine above the control board housing.– Bottom G: The boPlunger Clamps : Small lever clamps that secure the syringe plunger to the plunger plate and allow for retraction of the syringe.r Clamps K: Small Extruder Head : The extruder head replaces the existing extrusion head of the Prusa i3 mk3. The needle and the automatic bed leveling sensor are mounted on this component.der Head N: The exSyringe Tip Shield : The syringe tip shield prevents clogging or curing on the tip of the needle when exposed to the UV curing lamp during printing.p Shield O: The syUV Light Mount : Mounting bracket to attach the 6 W 405 nm UV lamp to the Prusa i3 mk3 top cross-member.ht Mount P: MountiSyringe Pump Stand : Optional 3D printed part that allows the syringe pump to be mounted separately from the Prusa i3 mk3.mp Stand Q: Option45WARNING: UV EYE PROTECTION AND UV SHIELDING SHOULD BE USED TO PROTECT USERS FROM UV EXPOSURE.•Syringe clamps: Two different types of syringe clamp are included for applications requiring up to 60 ml of material in a 60 ml syringe or a 5 ml syringes size for small material applications.•Syringe pump stand: Used to mount the syringe pump separately from the Prusa i3 MK3. If the syringe pump stand is not printed, the syringe pump has mounting holes for mounting on the Prusa i3 MK3 frame.•UV lamp mounting bracket: Used to attach a UV lamp to the Prusa i3 MK3 frame for UV photopolymer resin curing.Begin by 3D printing all parts included in the Mendeley repository. It is recommended that these parts are printed using PLA with an infill above 60% and a wall thickness above three to reduce the likelihood of part failure or deformation. These parts can be printed on the Prusa i3 mk3 prior to its disassembly and can be printed with no support material. The Mendeley repository includes some optional parts that can be printed if needed. These parts include:•3 mm Allen wrench•4 mm Allen wrench•5 mm box end wrench or socket•Handheld drill or drill press•4 mm drill bit•Vice or press•Hack sawTools required for assembly include:5.11.Ensure that the printer is unplugged.2.Disassemble the print head until only the bearing retention plate that capture the linear bearings remain A. Additi3.Open the control box to expose the motherboard.4.a.Part cooling fanb.Hot end fanc.Hot end wiresd.Auto bed leveling sensore.NEMA 17 extruder stepper motorDisconnect all wires that connect to the print head from the motherboard. These include the:5.Clear all removed cables from the motherboard case6.aDue to the Prusa Mk3 i3 safety features, the hot end thermistor must remain plugged into the motherboard to allow operation.bTo remove the hot end thermistor, loosen the grub screw next to the nozzle and remove the thermistor (white wires).cThe thermistor can be covered with a piece of electrical tape and the thermistor wires can be coiled and tucked into the motherboard enclosure or another safe location.dThe hot end assembly can be stored as it is not needed for this conversion.Thermistor bypass:7.Reconnect the extruder stepper motor and route cables out the bottom of the control box.8.Tidy up cables and close control box.In order to install the new extrusion head, the stock extrusion head on the Prusa i3 mk3 must be removed and some rerouting and removal of wires is required. The procedure is outlined below. For further diagrams and instructions on the disassembly of the Prusa i3 mk3, refer to the Mendeley repository for the which contains step-by-step images of the disassembly of the stock Prusa i3 mk3 and assembly of the conversion. Referencing the assembly manual for the Prusa i3 mk3 may also be useful in the disassembly process. The disassembly process is outlined below:5.21.You can now install the extrusion head onto the Prusa Mk3 i3. Locate the bed leveling sensor that was left connected to the motherboard and the disassembled extruder mount.2.On the back of the extruder mount, remove the rear plate to expose the linear bearings.3.Thread the bed leveling sensor through the cutout in the rear plate B.4.Re-attach the rear plate.5.Secure the extruder head to the front of the extruder mount using 3 M3x8mm bolts C.5.31.Begin assembly by bolting the pump Frame – Top and pump Frame – Bottom together using 7 M3 × 12 mm bolts and M3 nuts A.Fig. 4A2.Use 4 M3 × 12 mm bolts to attach the NEMA 17 stepper motor to the bottom of the pump frame B.3.If the syringe pump stand is to be used, attached the pump frame to the syringe pump stand using 4 M3 × 12 bolts and 4 M3 nuts C.4.Insert the 2 M3 × 60 mm bolts through the access holes on the back of the pump frame and thread the bolts through the plastic and gently seat them .5.Insert the two M4 threaded inserts into the two 4 mm diameter holes in the plunger plate by heating the threaded insert using a heat gun, torch, butane lighter, or soldering iron and pressing them into the plastic until the insert is flush with the top of the plunger plate. Due to print settings, the holes for the threaded inserts may be slightly undersized. Use a 4 mm drill bit to clearance the holes if necessary.6.Press the two linear bearings into the large diameter holes in the plunger plate L until t7.Use four M3 × 8 mm bolts to attach the lead screw nut into the recess in the plunger plate. (Do not over tighten these bolts).8.Install the lead screw coupler onto the NEMA 17 motor shaft and tighten the grub screw to secure the coupler to the motor.9.Install the lead screw knob onto the top of the lead screw and secure the knob using an M3 × 8 mm bolt D.10.Insert the lead screw through the access hole in the pump frame- top and thread the lead screw through the lead screw nut in the plunger plate approximately halfway E to allo11.Lower the lead screw with the plunger plate attached down into the coupler and tighten the grub screw on the coupler to secure the lead screw.12.Ensure that the plunger plate can move freely through the entire range of the lead screw by rotating the lead screw.13.Insert the two 310 mm linear rails through the top of the pump frame so that they pass through the linear bearings and seat in the recesses on the bottom of the frame E. NOTE: 14.Again, check that the plunger plate can move freely through the entire range of the lead screw.15.Use two M4 × 14 mm bolts to secure the plunger clamps to the plunger plate.16.Optional: If desired, mount syringe to the backside of the Prusa Mk3 i3 support frame, above the motherboard enclosure using 4 M3 × 12 mm bolts and nuts.17.Insert the desired syringe clamps and syringe into the upper slot in the syringe pump frame and tighten with 2 M3 wing nuts F.5.41.Attach the UV resistant tubing to the syringe using a female Luer lock hose barb connector A.Fig. 5I2.Attach the other end of the UV resistant tubing to the desired needle size using a male Luer lock hose barb connector B.3.Install the needle tip shield onto the needle by sliding it onto the needle B. NOTE: 4.Attach the UV curing lamp to the UV light mount with three M3x8mm bolts and three M3 nuts. The UV light mount can then be snapped onto the top cross member of the Prusa i3 mk3 C.5.Cut to size and attach the Teflon sheet to the build platform of the printer using the adhesive backing.5.51.Prusa Slic3r (slicing software)2.Pronterface (printer communication software if the use of an SD card is undesirable)This system uses free software for modeling of parts, slicing STL models, and communicating with the printer. Begin by downloading and installing the following software:5.5.11.Install the configuration bundle by opening Prusa Slicer and navigating to File/Import/Import Config Bundle.2.Once the configuration bundle is installed, you can select the settings profiles from the right-side drop-down menus for Print Settings, Filament Settings, and Printer Settings that match the desired printing function. The “I3 MK3 FLUID DEPOSITION CONVERSION” preset should be chosen for Printer Settings. This preset adds G-code to allow for cold extrusion. For the Print and Filament Settings, the “COLD EXTRUDE” preset provides baseline settings for 3D objects. The presets “COLD EXTRUDE THIN FILM” and “COLD EXTRUDE MESH” provide settings for printing thin solid films and mesh films optimized for 2 layers of material, respectively.A Prusa Slicer configuration bundle file is included in the Mendeley repository which will automatically make all of the adjustments outlined below to print using a 5 ml syringe with a 22-gauge needle. Note that these configurations should be treated as a baseline and adjustment to parameters inside of Prusa Slicer will be required for individual testing environments, materials used, needle sizes, and syringe sizes. Performance optimization steps are given in 5.5.21.Connect the Prusa i3 MK3 to the computer via USB.2.Select the com port that the printer is connected to in the top left corner and set the baud rate to 240,000.3.Click on connect and wait for the prompt to display “connected”4.If the prompt shows an error or unrecognizable symbols, change the baud rate and attempt to connect again.5.Once you are connected to the printer, click on “load file” and select your G-code file that Prusa Slic3r created.6.When you are ready to print, click on the print button.7.Do not close Pronterface while the printer is running.5.61.Lay the 24″ × 24″, 1/8″ Thick Laser Shielding panel on a flat surface.2.Position the two 24″ × 12″, 1/8″ Thick Laser Shielding panels on opposite sides of the 24″ × 24″, 1/8″ Thick Laser Shielding panel.3.Place three of the folding butt hinges at equal spacings at each seam between the 24″ × 24″ and 24″ × 12″ panels.4.Mark the locations of the holes in the hinges and remove.5.Drill holes in the panels at the marked locations using a 4 mm drill bit.6.Attach the hinges to the panels using the M3 × 12 mm Hex Bolts and M3 Hex Nuts.7.Fold the side panels in at a 45° angle and stand up in front of the printing system.UV eye protection and UV shielding should be used to protect users from UV exposure. If the user does not have any existing shield, the Trifold UV Shield included in these instructions is a low-cost solution.6WARNING: UV EYE PROTECTION AND UV SHIELDING SHOULD BE USED TO PROTECT USERS FROM UV EXPOSURE.6.11.Unscrew the 2 M3 wing nuts and remove the syringe and remove the female Luer lock hose barb connector, if attached.2.Fill the syringe with the required volume of fluid. It is recommended that the syringe is slightly overfilled to account for the volume of the nylon tube and needle and priming waste. NOTE: The required volume for a particular object is displayed in Prusa Slicer.3.Reattach the female Luer lock hose barb connector, place the loaded syringe into the syringe pump clamps, and gently secure with 2 M3 wing nuts. NOTE: If the wing nuts are over tightened it can deform the syringe and cause squeeze out of material or lock up the motion of the syringe.4.Raise the plunger plate using the lead screw knob so that it contacts the syringe plunger and clamp the plunger using the plunger clamps.5.Secure the syringe plunger using the plunger clamps A.6.If there are any air bubbles in the resin, let the syringe sit for several minutes until the bubbles are observed to rise towards the nozzle. Tapping the syringe with a finger can facilitate this process.7.Slowly rotate the lead screw knob until the nylon tube and needle are filled with resin and all air is ejected from the tubing and syringe needle.8.The system is now primed and ready to print.6.21.Begin by opening your desired design to be printed in Prusa Slicer.2.Select the correct presets as outlined in 3.It is recommended to print with a skirt in order to ensure that fluid is flowing cleanly and at the desired height prior to beginning to print the object.4.Export the g-code file to a known location.5.Perform a mesh bed leveling prior to printing by following the steps outlined in the Prusa i3 mk3 user manual and adjust the needle to the desired height off of the print bed.6.Connect Pronterface to the printer and load the gcode file as outlined in 7.Click print on Pronterface to initiate the print.8.Turn on the UV lamp, if applicable.9.Closely monitor the first layer of the print and use the live Z adjust function of the Prusa i3 mk3 to adjust the first layer height if needed.10.Once the print is complete, remove part from print bed and perform post-processing as specified by the build material documentation.11.Clean the syringe, tubing, and needle by removing from the system and flushing with isopropyl alcohol.7−1, 0.1 mm, and 0.2 mm, respectively, were used for 3D objects ( objects A. The reThis SPECS modified printing system is capable of producing 3D objects for a fraction of the cost of commercially available bioprinters, allowing for greater access of this manufacturing method. 3D printed parts for this conversion can be printed with the Prusa i3 mk3 prior to disassembly, eliminating the need for a second 3D printer for parts manufacturing. The conversion can be completed in hours, requires minimal hardware, and is non-destructive and reversable. With traditional SLA printers, a large amount of material must be used to fill the build tray. This system allows for a minimal material loss with only a thin film of uncured resin being lost in the cleaning process. The SPECS modification is beneficial for research and development with limited material quantities or high-cost materials.8Mendeley repository.https://doi.org/10.17632/cv9cfj39b7.1.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Further, it was indicated that the Tp and degree of retrogradation (DR%) also increase with increasing time and decreasing temperature of storage. All samples stored at −20 °C for 12 h exhibited the highest degree of retrogradation DR%. Amongst all five varieties stored at −20 °C for 12 h, Omkar exhibited the highest %DR, followed by Bili rajamudi, Khaju lahi, Dhusuri bao, and Diasang lahi. A negative correlation was also established between Tp and resistant starch content (RS%). It was also observed that the resistant starch (RS%) content increased with the increasing time and decreasing temperature of storage. A strong negative correlation was observed between RS% and non-resistant starch (NRS%). Further, rheological studies indicated that retrogradation also affects the viscosity and dynamic rheological properties of starch. In this study, it was evident that extending storage duration from 6 to 12 h and lowering temperature from 4 to −20 °C impact retrogradation of rice starch, which in turn affects the starch’s gelatinization, digestibility, and rheology. Rice starch retrograded at lower temperatures for a longer period could prove to be extremely beneficial for development of food products with better textural properties and high RS content or low glycemic index. Retrogradation is defined as the recrystallization or realignment of amylose and amylopectin chains upon cooling of gelatinization starch gels. The storage conditions such as the storage time and temperature are crucial factors that influence and govern the degree of retrogradation and in turn, affect the formation of resistant starch and alteration of thermal and rheological properties. This article investigates the effect of storage time and temperature on the properties of retrograded rice starch. Rice kernels of five different indigenous varieties, namely Diasang lahi, Khaju lahi, Dhusuri bao, Omkar, and Bili rajamudi were cooked by boiling in water and stored at 4 °C and −20 °C for 6 and 12 h, respectively. Differential scanning calorimetry (DSC) studies revealed in raw form that Bili rajamudi exhibited the highest peak gelatinization temperature (T Rice is one of the most economically indispensable crops, that contributes to the staple diet for most of the world’s population. The quality of rice-based food products essentially depends on the properties of its main component, starch . Starch Starch is usually gelatinized by heating in the presence of water, which results in the disruption of the native starch granules along with an order-to-disorder transition . On storThe time and temperature of storage are major factors affecting the process of starch retrogradation . Based oIn addition to alterations in thermal properties and RS%, retrogradation also affects the rheological properties of starch. Rheological characterization of starch includes evaluation of its flow and deformation. The rheological property of starch impacts its important applications as thickeners and binders in the food and pharmaceutical industries. Starch rheology plays a crucial role in the regulation, production, optimization, and sensory properties of starch-based food products. Rheological properties also indicate the behavior of starch as the raw materials and quality end products after processing. This study attempts to investigate the effect of time and temperature of storage on the digestibility, thermal, and rheological properties of five different varieties retrograded rice collected from the states of Assam and Karnataka, India. These rice varieties were chosen based on their apparent amylose content (AAC%). p) increases as the time of storage increases  when it is subjected to lower temperatures [p of native starch and retrograded rice samples. Since amylose is more susceptible to retrogradation than amylopectin, the influence of retrogradation on gelatinization temperatures is less pronounced for varieties belonging to the waxy group (exhibiting AAC% ≤ 10%). The very low amylose level makes the structural retrogradation less noticeable.Further, it can also be seen that as the temperature of storage decreases from room temperature to 4 °C, and −20 °C, the Teratures ,11. Henceratures . Table 1G) and enthalpy change on reheating of retrograded starch (ΔHR). ΔHG and ΔHR indicate the starch crystallinity which in turn indicates the loss of molecular order within the starch granule subjected to gelatinization [G was highest for raw rice. For the retrograded rice, ΔHR was lower for the freshly cooked rice compared to the ΔHG of raw rice. Further, with increasing time and decreasing temperature of storage, the ΔHR for stored rice was higher than the freshly cooked rice at room temperature  of rice samples with increasing time and decreasing temperature of storage was calculated and depicted in nization . It was perature . Based oHowever, as the time of storage increased from 6 h to 12 h, the gelatinized starch molecules tended to rearrange and form more ordered and stable structures leading to a higher %DR. According to a recent study, short-term retrogradation of starch often takes place within a few hours and is completely irreversible due to amylose recrystallization. However, long-term retrogradation begins after 12 h of storage and is reversible. This retrogradation is driven by amylopectin recrystallization. .In a similar study, it was reported that the peak temperature of retrograded amylose occurs between 100 to 165 °C . In anotThe alteration in the thermal properties of retrograded rice compared to native raw rice starch is beneficial for several applications in the food industry such as rice vermicelli, noodles, breakfast cereals, and parboiled rice. Determination of TS%, NRS%, and RS% of raw, freshly cooked, and retrograded rice varieties are depicted in Further, it is also observed that rice stored at −20 °C shows more RS% compared to rice stored at 4 °C. This is because at 4 °C, retrogradation occurs spontaneously as the cooked rice begins to cool, reducing its digestibility as indicated in by the increasing RS% , Table 3In a similar study, the digestibility of six indigenous Philippine rice cultivars subjected to refrigeration at 4 °C for 24 h was studied. The results indicated substantial differences in the estimated glycaemic index between the rice cultivars . For coop  binding them together . With thIt was reported that the G’ and G” values were higher for non-waxy rice and corn starches than the waxy corn and rice starches. Additionally, they observed that the addition of amylose to waxy rice and corn starches resulted in a rise in G’. Waxy corn and rice cultivars’ starches have lower G’ and G” than regular corn and rice types. Unlike regular starch, which has starch granules embedded in a continuous network of amylose, waxy starches experience the phenomena of gel formation .In a study , dynamicIn general, the tan δ values of the retrograded rice samples were lower than one, indicating the samples have a typical gel network . Lower tp and RS% as opposed to Khaju lahi and Omkar. Based on the correlation coefficients, it may be suggested that no linear relationship exists between Tp and RS% or NRS%. A correlation matrix was plotted to analyze the relationship between RS%, NRS%, Tp, and ΔH. A similar trend was observed for all 5 samples, as exhibited in p is highest for cooked rice stored at −20 °C for 12 h. Amongst samples stored at −20 °C for 12 h, Omkar exhibited the highest RS%, followed by Diasang lahi, Bili rajamudi, Khaju lahi, and Dhusuri bao. However, at −20 °C for 12 h, Bili rajamudi had the highest Tp followed by Dhusuri bao, Diasang lahi, Omkar, and Khaju lahi. Further, it was evident that all five varieties exhibited increased RS% with increased DR%. Investigating the viscosity and dynamic rheological properties of starch and starch-based foods gives a direct assessment of their processability. Food rheology contributes to giving crucial information for process design, quality assurance, sensory evaluation, and product development. It was evident that with increasing time and decreasing temperature of storage, both G’ and G” increased, whereas the tan δ was seen to be decreasing. These findings indicate that retrogradation can be used to improve the quality of rice starch-based food products by producing a higher amount of beneficial resistant starch, increasing the Tp, and improving the rheology properties. In many Asian nations, rice is a common food. For diabetic patients, it has long been thought that consumption of stale or stored rice is not preferable compared to recently prepared rice. However, the present study contradicts this idea. Due to retrogradation, some of the cooked rice’s starch becomes resistant to digestion, which prevents it from being absorbed in the small intestine. In contrast to freshly cooked rice, stale or stored rice may exhibit a decreased glycaemic response based on the increased RS%. Further, the hindrance of gelatinization also hinders the availability of starch for amylase hydrolysis. Hence, stored cooked rice is a better option for diabetic patients.This study provides useful information on how to monitor the retrogradation of starchy foods using RS%, thermal, and rheological studies. Retrogradation has a significant impact on the activities of the rice starch food business. Retrogradation is recognized to have an impact on the staleness and hardness of cooked rice. However, the current study emphasises the advantageous uses of retrogradation that may be found in a variety of rice-starch based food products, such as ready-to-eat rice products, breakfast cereals, and rice noodles/vermicelli. The current study also highlights the impact of retrogradation temperature and time on functional qualities such rheological parameters and RS%. Retrograde rice starch is, therefore, preferred for the manufacture of food products with improved texture and greater RS%. The results from this study indicate that the practical application of retrograded starch is found to be more prominently at lower temperatures and with longer storage time. Further, rice varieties with intermediate or high AAC% such as Khaju lahi are more prone to retrogradation as they show the highest %DR and can be easily used to reap the benefits of retrograded starch for various applications. RS% produced from retrograded rice starch and TFive indigenous varieties, namely Diasang lahi, Khaju lahi, Dhusuri bao, Omkar, and Bili rajamudi were collected from the paddy fields during harvest season. Diasang lahi, Khaju lahi, Dhusuri bao were collected from the state of Assam, India, whereas Omkar, and Bili rajamudi were collected from the fields of Karnataka, India. The seeds were threshed, sun-dried, de-husked, and milled using a McGill Mill No. 2. The kernels were stored at −80 °C for further experimentation . The appInitially, the rice was cooked in excess water at the minimum cooking temperature (MCT) determined previously . The freG) and there was an enthalpy change on reheating of retrograded starch (ΔHR) [Differential scanning calorimetry (DSC)  was used to investigate the gelatinization and retrogradation properties of various rice varieties. A total of 3 mg of raw rice, freshly cooked rice, and stored or retrograded rice kernels were weighed into an aluminium pan along with distilled water to obtain a ratio of 1:1 (w/w), sealed, and heated from 30 °C to 160 °C. The experiment was conducted once for each sample. The thermograms were analyzed using TA−60  software. The degree of retrogradation (%DR) was calculated based on the following formula: ch (ΔHR) .Using the RS assay kit, the resistant starch (RS) content of rice samples was calculated . Briefly, a 100 mg sample was digested in 4 mL of pancreatic amylase (10 mg/mL) with amyloglucosidase (300 U/mL) at 37 °C with continuous agitation at 200 rpm for 16 h, and then 4 mL of 100% ethanol was added with vigorous stirring. Next, centrifugation at 3000 rpm for 10 min followed. The pellet was centrifuged at 3000 rpm for 10 min while suspended in 8 mL of 50% ethanol. The non-resistant starch content of the supernatant was analysed. The pellet was resuspended in 2 M KOH for 20 min, and then 8 mL of 1.2 M sodium acetate buffer and 0.1 mL of amyloglucosidase (3300 U/mL) were added. The tubes were centrifuged at 3000 rpm for 10 min after being incubated at 50 °C for 30 min. A different set of test tubes received aliquots of 0.1 mL of supernatant, to which 3 mL of glucose oxidase/peroxidase (GOPOD) reagent was added. For 20 min, these tubes were incubated at 50 °C. At 510 nm, there was a comparison to a blank for the reagent. Based on the readings, the RS%, NRS%, and TS% were determined .−1, to determine the effect of shear rate on apparent viscosity and to describe the steady shear rheological properties of the samples, and the data was fitted to the well-known Herschel–Bulkley power law model.Briefly, using a mortar and pestle, the rice kernels were mashed into a smooth paste and immediately transferred to a parallel plate rheometer  with a diameter of 50 m diameter, and a gap size of 1 mm. The continuous shear tests were carried out at 37 °C, over a shear rate range of 0–40 sFor the study of the dynamic rheological properties, frequency sweeps were performed at 37 °C, over the angular frequency range of 0–100 rad/s at 2% strain. The mechanical spectra were obtained to record the storage modulus (G’), loss modulus (G”), and loss tangent (tanδ = G”/G’) as a function of the frequency (ω). . p  <  0.05 was used followed by Tukey’s significant difference test to compare the means of each case. Pearson correlation among the RS%, NRS%, Tp, and ΔHG/R was calculated using OriginPro2022b (Learning Edition).The mean and standard deviation of values from the resistant starch content (RS%), non-resistant starch (NRS%), and total starch (TS%) content were obtained in triplicate using a built-in function in Microsoft Excel 2013. A one-way analysis of variance (ANOVA) was used to evaluate the differences among the means of obtained values for TS%, NRS%, and RS% using Graphpad Prism 9 (Learning edition). A level of


It is highly expected to develop a simple and effective method to reinforce polyamide 6 (PA6) to enlarge its application potential. This is challenging because of frequently encountered multi-component phase separations. In this paper, we propose a novel method to solve this issue, essentially comprising two steps. Firstly, a kind of poly (amide-block-aramid) block copolymers, i.e., thermotropic liquid crystalline polymer (TLCP)-polyamide 6 (TLCP-PA6), that contains both rigid aromatic liquid crystal blocks, and flexible alkyl blocks were synthesized. It is unique in that TLCP is chemically linked with PA6, which is advantageous in excellent chemical and physical miscibility with the precursors of monomer casting polyamide 6 (MCPA6), i.e., ε-caprolactam. Secondly, such newly synthesized block copolymer TLCP-PA6 was dissolved in the melting ε-caprolactam, and followed by in situ polymerization to obtain composite polymer blends, i.e., MCPA6/TLCP-PA6. The thermodynamic, morphological, and crystalline properties of MCPA6/TLCP-PA6 can be easily manipulated by tailoring the loading ratios between TLCP-PA6 and ε-caprolactam. Especially, at the optimized condition, such MCPA6/TLCP-PA6 blends show an excellent miscibility. Systematic characterizations, including nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FT-IR), differential scanning calorimeter (DSC), and polarizing optical microscope (POM), were performed to confirm these statements. In view of these results, it is anticipated that the overall mechanical properties of such PA6-based polymer composites will be satisfactory, which should enable applications in the modern plastic industry and other emerging areas, such as wearable fabrics. Polyamide 6 (PA6) is well known as high performance engineering plastic with high strength, good fatigue resistance, and satisfactory wearing durability ,2,3,4. HAs we know, among those polymer modifiers ,13,14,15In this paper, we present a novel method to prepare well compatible PA6/TLCP composite, which shows good flexibility and scalability. In detail, the matrix-type polymer (PA6) is introduced into the main chain of TLCP as the spacer to improve the compatibility between the TLCP and the precursor of MCPA6, i.e., ε-caprolactam, which is followed by in situ copolymerization to achieve MCPA6/TLCP-PA6 composite. Firstly, a new thermotropic liquid crystalline polyamide block copolymer (TLCP-PA6) see  is desig3) was prepared according to the earlier report ) of TLCP-PA6 was measured in NMP/LiCl (5 wt. %) at a concentration of 0.1 g∙dL1H nuclear magnetic resonance (1H-NMR) spectra of TLCP-PA6 were measured by an equipment of Bruker 400 MHz, i.e., preparing sample solutions in D2SO4 and using 3-(trimethylsilyl) propanesulfonic acid sodium salt (DSS) as an internal standard.−1 were measured for these polymers by using a Nicolet 5700 spectrometer and potassium bromide (KBr)  as the window plate.Fourier-transform infrared spectroscopy (FTIR) spectra ranging from 4000 to 500 cmThe measurements of polarizing optical microscope (POM) were carried out with a polarizing microscope  equipped with a heating stage (YAZAWA) .Scanning electron microscope (SEM)  was used to characterize the cryo-fractured surfaces of all these MCPA6/TLCP-PA6 blends. Specimens were prepared as follows. Firstly, the specimens were fractured in liquid nitrogen. Secondly, the surfaces of the samples were etched with NMP for 24 h at room temperature to remove TLCP-PA6 domains and then dry thoroughly, which was followed by sputtering gold onto the etched surfaces prior to SEM examinations.−1. The samples were first heated to 260 °C and maintained for 3 min. to erase thermal history, which were followed by a cooling step at the rate of 5 °C∙min−1. The crystallization and melting thermograms were taken from the subsequent cooling and the second heating cycles. The crystallinities of the polyamide parts of those samples were determined according to the following equation:f is the melting enthalpy of MCPA6 in the sample, f is the homopolymer weight fraction in the blend and ΔHf* is the melting enthalpy of the matrix polymer of 100% crystallization (ΔHf = 230 J∙g−1). Tc,0 is the initial temperature of crystallization in the cooling scan and used for evaluating the nucleating effect [Differential scanning calorimeter (DSC) were carried out on a SCINCO S-650  equipment. All measurements were protected under nitrogen atmosphere, starting at room temperature and using a heating rate of 10 °C∙ming effect .Wide angle X-ray diffraction (WAXRD) was performed on an X-ray diffraction analyzer Bruker AXS  equipped with a rotating Cu anode generator system using Cu Kα-alpha1 (λ = 1.5406 Å) radiation. The diffraction angles (2θ) were measured ranging from 5° to 40°. The data were accumulated for 6 s at angular intervals of 2θ = 0.1°.1H NMR (1H NMR clearly support the proposed structure of TLCP-PA6 sample. The area from the absorption of the protons in the TLCP-PA6 is in the range of 1.0~9.0 ppm. In the FTIR spectrum of TLCP-PA6 (curve c in −1), N-H connected to amide II (3035 cm−1), amide I, II , and methylene (2922 cm−1), respectively.The chemical structure of TLCP-PA6 is confirmed by 1H NMR  and FTIR1H NMR . As it im = 311.4 °C). After that, it turns to isotropic at ca. 350 °C. Such a temperature is also lower than the clearing temperature of TLCP (Ti = 371.7 °C) as seen optically. These results are consistent with those observed by DSC  the compatibility of MCPA6/TLCP-PA6 blend at the phase interface is satisfactory. There is no obvious phase separation between these two components. It is believed that at least two reasons should account for it. Firstly, since the chemical structures of MCPA6 and TLCP-PA6 are similar, they intrinsically possess good physical compatibility (similarity-intermiscibility principle). Secondly, TLCP-PA6 interacts with MCPA6 during the in situ polymerization of ε-caprolactam monomers. Such chemical interactions further help to overcome the phase separation issue.−1 to the lower wavenumbers, which is between that of MCPA6 and TLCP-PA6. In addition, the stretching vibration peak of NH connected to amide II is present in the FTIR of the MCPA6/TLCP-PA6 (100 wt. %/15 wt. %) blend. It should be ascribed to the formation of the new hydrogen bonding and/or an amide reaction between TLCP-PA6 and MCPA6 [2 asymmetrical and symmetrical stretching vibrations of the MCPA6/TLCP-PA6 (100 wt. %/15 wt. %) blend are almost the same as those of the MCPA6 counterpart. However, the intensity ratio of them is different. From the above analysis, it is also suggested that the chemical interaction should occur between MCPA6 and TLCP-PA6 in the MCPA6/TLCP-PA6 blended composites.nd MCPA6 . The CH2m, ΔHf, Tc,m, Tc,0, ΔHc, and Xc(DSC) of MCPA6 in MCPA6/TLCP-PA6 blends are monotonically decreased. It is interesting to note that in the case where 30 wt. % TLCP-PA6 is added, there is no endothermic peak. While 20 wt. % or 30 wt. % TLCP-PA6 is added, the exothermic peak disappears, but both glass transition peaks appear. The Tg of those MCPA6/TLCP-PA6 blends is increased by increasing the content of TLCP-PA6. However, neither of them shows the lower Tg value, compared to pure TLCP-PA6. Combined, all of those results indicate that TLCP-PA6 greatly influences the thermal dynamic properties of those MCPA6/TLCP-PA6 blends because of its fine dispersion and its chemical interaction with MCPA6 during polymerization progress. The strong physical and chemical interaction improves the compatibility between MCPA6 and TLCP-PA6. Additionally, the finer the dispersion, the more energy is consumed in spherulite growth, i.e., the decreased Tc,0 in phenomena [c,m of MCPA6/TLCP-PA6 blends is decreased by increasing the content of TLCP-PA6. Compared to pure MCPA6, the crystalline peaks of MCPA6/TLCP-PA6 blends are broadened, which indicates that the crystallinity of MCPA6 in MCPA6/TLCP-PA6 blends is decreased and the crystallization is becoming incomplete.henomena . On the 1 and α2, correspond to the crystallographic planes of (200) and (002 + 202), respectively [2 form. When the content of TLCP-PA6 is increased to 15 wt. %, the α1 peak becomes indistinct and the α2 peak disappears. Meanwhile, the γ-form crystal is clearly present at this condition [2 peak. Combined, all of these suggest that the incorporation of TLCP-PA6 copolymer significantly influences the crystalline structure of the MCPA6 in those MCPA6/TLCP-PA6 blends. The degree of crystallinity of these blends is decreased by increasing the content of TLCP-PA6. Meanwhile, the diffraction pattern of all those blends shows an interesting feature, i.e., a small crystalline peak at 2θ = 29.4°, which is analogous to earlier reports [ectively ,34. Withondition . It is wondition . When th reports ,37. In vf, Tm, ΔHc, Tc,m, Tc,0, and Xc(DSC) of MCPA6 in MCPA6/TLCP-PA6 blends are significantly decreased by increasing the content of TLCP-PA6. In addition, introducing the moiety of TLCP-PA6 into the MCPA6 matrix is confirmed to influence the crystalline of MCPA6, as verified by WAXRD measurement. Especially, while the content of TLCP-PA6 is higher than 15 wt. %, a satisfactory γ-form crystalline of PA6 appears for these blends, which can enhance the impact strength of the composites. By contrast, MCPA6/TLCP-PA6 blends with a lower content of TLCP-PA6 merely display the well-known α1- and α2-form crystals. In short, these novel explored MCPA6/TLCP-PA6 composites are expected to be an advanced reinforcing engineering plastic material with an excellent mechanical performance. Further mechanical evaluations and practical applications are under consideration and will be reported in future work.In this study, a new kind of thermotropic liquid crystalline polyamide block copolymer (TLCP-PA6) is synthesized and fully characterized. It shows liquid crystalline (nematic phase) in a wide temperature range (290~350 °C) and is capable of fine dissolving in ε-caprolactam. Accordingly, in situ anionic polymerization of ε-caprolactam with TLCP-PA6 is successfully performed and then a series of MCPA6/TLCP-PA6 blends with different blending ratios are obtained. The results of SEM images indicate that at a low loading content of TLCP-PA6 (2.5~10 wt. %), it is well dispersed as nanoparticles in the MCPA6/TLCP-PA6 blends. While in the range of 15~20 wt. %, such nanoparticle features disappear and it turns to a homogeneous morphology without an evident two-phase boundary. While the content of TLCP-PA6 is 30 wt. %, the blends display the layered structure. In short, MCPA6/TLCP-PA6 blends with a suitable loading content of TLCP-PA6  are well compatible composites, which is due to strong physical and chemical interactions between these two components . The results of DSC measurements show that various parameters including ΔH


Moreover, due to different contents and types of monomer phenols, the HBP extracted with acetone were more effective in inhibiting starch digestion than those extracted with methanol. Ferulic acid and catechin were two key components of HBP. Further results indicated that with the increased content of ferulic acid and catechin (from 1% to 5%), they formed a more ordered structure with amylose, resulting in the lower digestibility of the complex. Collectively, this study suggested that highland barley polyphenols could effectively delay starch digestion by forming a more ordered starch crystal structure. Highland barley polyphenols can be used as functional ingredients in regulating the digestive properties of starchy foods.Slowing starch digestibility can delay or even prevent the occurrence and development of type 2 diabetes. To explore the hypoglycemic potential of highland barley polyphenols (HBP), this study investigated the structural characteristics and starch digestibility of individual or mixed HBP-starch complexes. The results showed that a V-type structure was formed in HBP-starch complexes through non-covalent bonds, resulting in a decrease in rapidly digestible starch and an increase in resistant starch. Specially, the compounding of HBP extracted by acetone significantly reduced the rapidly digestible starch content in amylose from 41.11% to 36.17% and increased the resistant starch content from 6.15% to 13.27% ( Starch is the main component of most cereals. It provides the main glycemic carbohydrates in the human diet . Starch-Polyphenols are a kind of phytochemicals with benzene rings and multiple hydroxyl chemical structures . SeveralHordeum vulgare L.) is the main crop in Tibetan areas of China. It has attracted increased attention in recent years due to its high content of dietary fiber, β-glucan, and polyphenols [Highland barley  to investigate the interaction between HBP and starch; (b) to further explore the effects of HBP on starch digestion properties; and then (c) to reveal the effects of the main monomer phenols in highland barley on the structure and digestion characteristics of amylose.Highland barley (Beiqing 3) was provided by Shanxi Dongfangliang Life Technology Co., Ltd. . Ferulic acid  and catechin (purity ≥ 98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. . Pepsin , pancreatin (P7545), invertase , and α-glucosidase  were purchased from Sigma-Aldrich . All other chemicals were of analytical grade and purchased from Beijing Banxia Technology Development Co., Ltd. .w/v). Highland barley flour was defatted using n-hexane and dried at 40 °C [The HBP was extracted by ultrasound  using an ultrasonic cleaner  with pure water, 80% methanol, 80% ethanol, and 80% acetone  complexes, ferulic acid-amylose complex, and catechin-amylose complex were prepared according to the method described by Amoako and Zheng ,18 with The total phenolic content and total flavonoid content were determined according to previously reported methods ,20. The 2+ equivalents/g of samples, and the TEAC results were described as µmol Trolox equivalents/g of samples.The ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) was measured using the total antioxidant capacity kit  according to the manufacturer’s instructions. The FRAP results were described as µmol FeFollowing Zheng , the binThe microstructure of the samples was analyzed using an SEM  according to previous methods ,22. Brie−1 by accumulating 32 scans per spectra at 4 cm−1 [The chemical bonds of the samples were determined with an FT-IR spectrometer . The samples were equilibrated at room temperature for 24 h, then mixed with KBr and pressed into thin slices before measurement. Scanning wave numbers ranged from 400 to 4000 cmt 4 cm−1 . The crystal types of the samples were characterized with an X-ray diffractometer  following Zheng et al. . The samThe thermal properties of samples were measured using a DSC  based on He’s method  with somw/v) were placed in a 50 mL test tube, incubating in a shaking water bath at 37 °C for 30 min. After the pH was adjusted to 5.5 with 0.1 mmol/L acetate buffer solution, the active mixed enzyme solution containing 0.75 mL amyloglucosidase (1200 U/mL) and 1 mL invertase (3000 U/mL) was added, shaking at 37 °C. At specified time points  during digestion, 0.2 mL of samples were taken out and mixed with 4 mL of anhydrous ethanol to deactivate the enzymes. After centrifugation at 1500× g for 5 min, the glucose contents in the supernatant were measured by a glucose assay kit .The digestibility of samples was analyzed according to the method described by Englyst et al. . Briefly0, G20, and G120 represent the content of glucose released at 0, 20, and 120 min, respectively. TS represent total starch. And 0.9 means the conversion factor of glucose from starch.The rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) contents were calculated using the following formula:p < 0.05.All data were expressed as mean ± standard deviation (mean ± SD) for at least three replicates per sample. ANOVA and Tukey’s test were performed using SPSS 17.0 . All graphs were produced using Origin 2018 . The statistical significance was set at 2·−, DPPH·, and ABTS·+ scavenging activities than the 80% ethanol. Therefore, the methanol and acetone solvents were used to extract HBP in subsequent experiments in this study.The phenolic compounds and antioxidant activities in highland barley flour extract by different solvents are shown in The binding abilities of HBP and starch are displayed in The formation of polyphenol–starch inclusion complexes can be demonstrated by various techniques including XRD, FT-IR, and so on ,28. On t−1, which belonged to the stretching vibration of -OH and the absorption of hydrogen bonds; the spectral peak near 2926 cm−1, which was assigned to the anti-symmetrical stretching vibration of −CH2 [−1, which was attributed to the amorphous region vibration [−1, mainly attributed to the vibration of ether bonds, ester bonds, and ether bonds [The FT-IR spectra of HBP-starch complexes are shown in  of −CH2 ; and theer bonds . Compareer bonds . A similer bonds .−1 in the FT-IR spectrum are related to the crystalline and amorphous regions. The ratio of 1047/1022 cm−1 peak intensities can represent the ordered structure of starch crystals [−1 were calculated and are shown in −1 of HBP-starch complexes were higher than those of amylose or HBS, indicating that a more ordered crystal structure was formed in HBP-starch complexes.The bands of 1047 and 1022 cmcrystals . Generalcrystals ,32. The XRD is an analytical technique for studying the molecular structure of crystals and has been recognized as an effective method for studying the interactions between small-molecule ligands and macromolecular receptors at the molecular level, such as polyphenol–starch complexes ,34. The In the present study, amylose and HBS exhibited strong characteristic peaks at 15.2° and 23.1° and unresolved doublet peaks at 17.2° and 18°, showing a typical A-type crystal structure . CompareThe SEM images of starch and HBP-starch complexes are shown in The starch digestibility of HBP-starch complexes extracted with different solvents is shown in Notably, the compounding of AHBP had a significant effect on the decrease of RDS content and the increase of RS content in amylose but had little effect on HBS. This might be attributed to the interaction between polyphenols and starch was more likely to be enhanced through extensive hydrogen bonding and hydrophobic interactions in starch with high amylose content . BesidesIt has been reported that different types and contents of polyphenols have different inhibitory effects on starch digestion ,43. In tThus, we further analyzed the main composition of HBP. The results showed that the main monomer phenols in highland barley were ferulic acid, catechin and p-coumaric acid . The conThe effects of different types and additions of main monomer phenol on amylose digestibility are shown in All in all, adding ferulic acid and catechin significantly reduced the RDS content and increased the RS content of amylose as expectedly. Considering the results of HBP-starch complexes, we attribute this to the complexation of ferulic acid, catechin with amylose, which might change the crystal structure of starch to form a more ordered structure, and then increase the resistance of starch to enzymatic hydrolysis . TherefoThe effects of different types and additions of main monomer phenol on thermal properties of phenol-amylose complexes were shown in Consistent with the DSC results, XRD analysis indicated an interaction between ferulic acid-amylose and catechin-amylose . AmyloseThis study investigated the structural properties and in vitro digestibility of HBP-starch complexes. HBP could form highly ordered V-type complexes with HBS and amylose through noncovalent bonds and then significantly reduce the content of RDS and increase the content of RS. In addition, the digestibility of polyphenol-amylose complexes varies with the type and content of monomer phenols. Both the ferulic acid-amylose and catechin-amylose complex effectively regulate the digestibility of amylose by altering the structure of amylose. It should be noted that other phenolic compounds interact with starch through noncovalent bonds to form complexes that change the digestive characteristics of starch.The inhibitory mechanism of HBP on starch digestion was relatively complex. This study concluded that HBP could form V-shaped complexes with starch to inhibit starch digestion. It was still difficult to attribute this inhibition to a single mechanism, however. Whether the release of HBP in the complexes and other factors affect digestive enzymes during in vitro digestion remains to be further studied. Moreover, it is necessary to conduct an in vivo study of the dose–response relationship to quantitatively control postprandial blood glucose response. Given the worldwide prevalence of T2DM, our findings provide further support for the current recommendation that phenolic compounds including HBP can be used as functional components in starch-based food systems to delay starch digestion.


In this work, the fatigue damage of CFRP uniaxial composite specimens were studied using thermal methods to determine the fatigue behavior. The aim was to evaluate the fatigue damage as a function of the number of cycles. Consequently, the damage process was studied in terms of a global indicator, considering the stiffness decay, and in terms of local parameters, considering the evolution of temperature maps acquired during the fatigue tests. A direct correlation between the damage index, corresponding to 90% of the fatigue life, and the temperature variation of the most stressed area was found. Another parameter taken into consideration was the heating rate during the application of the first thousands cycles. This parameter was proportional to the stress amplitude, making it a useful parameter since it refers to the initial part of the specimen fatigue life. Fiber-based composites, a relatively new type of material, are proving particularly interesting to the aerospace and automotive sectors due to their ability to decrease component weight, increase performance, and improve impact tolerance/crashworthiness, all factors that are important in lowering fuel emissions and increasing occupant safety. More recently, increasingly sectoral and promising applications have emerged in the field of civil engineering, such as in bridge structures and the oil extraction industry ,2. In alComposite fatigue behavior can therefore be related to stiffness and strength degradations resulting from damage accumulation. Several empirical evolution models have been developed that describe a gradual macroscopic reduction in the laminate stiffness related to the damage evolution on a microscopic scale ,20. ArafIn the present work, the study of the fatigue behavior of uniaxial carbon fiber and epoxy resin composite laminate specimens, made with resin transfer molding (RTM) technology, is presented. The innovative aspect of this study is the use of thermal methods to investigate the damage evolution in terms of stiffness decay. The goal is to evaluate the damage as the number of load cycles increases. Differently from the cited works, the damage process was studied here both in global terms, evaluating the decay of the stiffness of the composite, and in local terms, evaluating the variations of the temperature maps as a function of the number of cycles. For this purpose, fatigue tests were conducted at constant amplitude, with R = 0.1 and a load frequency equal to 10 Hz. The thermographic analysis, associated with the study of stiffness degradation, has the purpose of obtaining some parameters useful to highlight the state of localized damage in a phase preceding the appearance of an evident crack or other rapid degradation phenomena: delamination, detachment, or accentuated deformation.The experimentation was carried out on a unidirectional composite material in carbon fiber and epoxy resin (carbon–epoxy) obtained with resin transfer molding technology. Principal characteristics of the material are summarized in 0 equal to 150 mm, rectangular cross sections with thickness (t) equal to 2 mm, width (w) of 20 mm, and length of the reinforcements (Lt) equal to 120 mm :0. By doing so, it was assumed that the variation in gauge length could be approximated using the excursion of the crosshead, thus neglecting the deformation of the clamped part and, in general, of the reinforced part.During the execution of the fatigue tests, the load peaks FDuring the application of fatigue loads, the material undergoes progressive damage which often does not manifest itself with significant deformations but is linked to localized and sometimes sudden dissipation phenomena. The areas where the damage accumulates are always affected by dissipative phenomena that can be highlighted by starting from temperature maps. In order to detect these areas, a FLIR 7500 M thermal imaging camera was used, with noise-equivalent thermal sensitivity (NETD) equal to 25 mK and image resolution 320 × 256 pixels, which allowed us to observe the presence and evolution of heterogeneity in the internal part of the material as an indication of localized damage and to follow its propagation until the final break.Due to the long duration of the tests, especially those at the lowest load levels near the fatigue limit, it was necessary to limit the recording time. It was decided to record the first 20 s of each test in order to be able to capture the thermal transient and to make recordings at regular intervals for a total duration of 1 s at an acquisition frequency of 200 Hz and an integration time of 1000 µs. Taking into account that the frequency of application of the load was equal to 10 Hz, each recording of 1 s corresponded to 10 load cycles, for each of which 20 thermograms were available. The recording interval was set at 5 min for the first 2 h of testing and was thereafter variable between 5 min and 3.5 h depending on the expected duration of the test.The heat maps obtained showed the onset from the first heating cycles, localized in correspondence with the reinforcement areas; in particular on the one near the lower grip, which is the one connected with the fixed part of the test machine. This heating is due to the slipping phenomena, existing between the reinforcements and the surface of the specimen, caused by the high loads applied.a D=In this work, Equation (2) is rewritten and explained in terms of the variation of stiffness R:%.The probabilistic aspect in the study of the composite materials fatigue involves a significant dispersion in the experimental data obtained . As alreThe linear part of the stiffness trend is identified as a function of the number of cycles;0lin) and ; the first point is obtained by extrapolating the trend line up to the ordinate axis, obtaining the ideal stiffness value at cycle 0, and the second point is obtained by considering the stiffness value at 90% of the specimen’s life R90lin;The linear trend of the stiffness is traced as a function of the number of cycles through two points: , the fracture mechanisms between fibers and matrix interact with each other; as the number of cycles increases, the curve presents, in this section, a linear trend but with a less pronounced slope than in region I. It is noted that the behaviors relating to the various load levels present almost identical slopes, generating almost parallel curves;Finally, the third region (III) was identified. In it occurs the fracture of the fibers and the consequent rupture of the specimen. This phase takes place in a very short time. There is no warning regarding a possible failure of the specimen in the final part of its useful life;The progressive fatigue damage was then followed up by examining the recorded temperature maps. As expected, after a short transient, the temperature of each specimen first undergoes a slow increase, stabilizes at a steady state around a constant value, and then rapidly increases in the terminal phase.In all cases , areas wThe thermal profile was then observed along a horizontal segment at the selected hot spot to analyze the temperature trend along the width of the specimen and evaluate the increase in the damaged area and the extent of the damage. As an example, the trends for two different load levels are shown , correspBy comparing the results obtained from the thermographic analysis to those relating to damage D, a correlation was found between the maximum temperature increase for each specimen and the relative D index at 90% of the fatigue life .90% of 0.6, while next-to-unit damage causes a higher ΔT of 41 °C.In the present work, the fatigue behavior of a unidirectional composite material in carbon fiber and epoxy resin made with RTM technology was evaluated. In particular, after obtaining the Wöhler curve, the damage was evaluated in terms of stiffness variation. It was verified that the percentage change in stiffness ΔR% cannot be used as an index of damage, due to the high dispersion of the data. On the contrary, there was a direct correlation between the damage index corresponding to 90% of the fatigue life and the temperature variation of the most stressed area. A ΔT equal to 34 °C is associated with a DAnother parameter taken into consideration is the heating rate during the application of the first several thousand cycles. This parameter is proportional to the amplitude of stress and is a useful parameter since it refers to the initial part of the fatigue life. It increases by an order of magnitude from a damage of 0.6 to a damage of 0.99. Future developments of this work will concern the study of different types of FRP composites in terms of composition, glass fibers and layup. Future work will include microscopic analysis to better understand the fatigue failure mechanism of composite plates.


Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease. Patients with NIID may present with heterogeneous clinical symptoms, including episodic encephalopathy, dementia, limb weakness, cerebellar ataxia, and autonomic dysfunction. Among the NIID cases reported in China, patients often have complicated and severe manifestations. Therefore, many clinicians do not consider the disease when the patient presents with relatively minor complaints.We present the case of a 39-year-old man showing migraine-aura-like symptoms for the past 3 years. Brain magnetic resonance imaging (MRI) revealed hyperintense signals in the splenium of the corpus callosum and corticomedullary junction on diffusion-weighted imaging (DWI) over time. In addition, brain atrophy that was not concomitant with the patient’s age was detected while retrospectively reviewing the patient’s imaging results. Genetic analysis and skin biopsy confirmed a diagnosis of NIID. The patient was treated with sibelium, and the symptoms did not recur.Migraine-aura-like symptoms may be the predominant clinical presentation in young patients with NIID. Persistent high-intensity signals on DWI in the brain and early-onset brain atrophy might be clues for the diagnosis of NIID. NOTCH2NLC genes have been found to cause NIID. Although there were records of Chinese NIID patients presenting with migraine, all of the patients showed complications with severe clinical manifestations such as epilepsy and disturbance of consciousness [Neuronal intranuclear inclusion disease (NIID) is a chronic progressive neurodegenerative disease characterized by eosinophilic intranuclear inclusion bodies in the central and peripheral nervous systems and internal organs . The cliiousness , 4. HereNOTCH2NLC gene  score  was 7/10ene Fig. , which cThe differential diagnosis for increased intensity of DWI signals in the corpus callosum consists of cerebral vascular disease , vasculitis (Susac syndrome), inflammatory or autoimmune disease (multiple sclerosis), and genetic metabolic disease  . For thiSymptomatic migraine has been systematically described in a recent publication. According to the SNNOOP10 list of red flags, the patient was suspected to have mild cognitive impairment based on MMSE and MoCA scores. Side-locked symptoms were not typical in this patient, as visual deficits occurred bilaterally, and the lesion was located on both sides of the brain , 10. We NOTCH2NLC gene [SCN5A gene [NIID is considered a progressive neurodegenerative disease with infiltration of eosinophilic hyaline intranuclear inclusions in the central and peripheral nervous systems as well as visceral organs , 11–13. N5A gene .DWI showed high-intensity signals in the subcortical white matter area of the bilateral frontal lobes. A pathological study showed that the high-intensity signals on DWI and ADC at the corticomedullary junction corresponded with spongy changes in the pathology; the loss of myelin sheath and axon was also observed . Since tThe mechanisms of paroxysmal symptoms in NIID remain unclear. Of note, the vascular-related theory was a plausible explanation of the symptoms. One speculation is that cerebral hypoperfusion and vascular dysfunction contribute to the accumulation of eosinophilic inclusions, resulting in ischemic and hypoxic changes in neurons, finally leading to migraine attacks and NIID progression. Another hypothesis is that the accumulation of eosinophilic inclusions causes the development of vascular dysfunction, resulting in hypoperfusion and hypometabolism, which finally lead to migraine attacks . When reSibelium was used to relieve headache symptoms in the patient. Sibelium is a drug with flunarizine hydrochloride as its active ingredient. Flunarizine hydrochloride is a selective calcium antagonist that blocks excess calcium ion flux through transmembrane proteins into cells, prevents excessive intracellular calcium load of neurons during ischemia and hypoxia, inhibits cerebral vasospasm, and promotes cell membrane stabilization. Numerous controlled clinical studies have established the efficacy of flunarizine, which has long been used in the prophylactic management of migraines , 20.In conclusion, we have reported the case of a patient with NIID who mainly presented with migraine-aura-like symptoms, which are rare in NIID. NIID should be considered as a differential diagnosis when a patient presents with migraine aura-like symptoms, persistent high-intensity signals within the corticomedullary junction or corpus callosum on DWI, and early-onset brain atrophy. Timely gene analysis and skin biopsy are helpful for a definitive diagnosis.


Ruminiclostridium thermocellum was one of the most efficient cellulase systems in nature. However, the product of cellulose degradation by R. thermocellum is cellobiose, which leads to the feedback inhibition of cellulosome, and it limits the R. thermocellum application in the field of cellulosic biomass consolidated bioprocessing (CBP) industry. In a previous study, R. thermocellum M3, which can hydrolyze cellulosic feedstocks into monosaccharides, was isolated from horse manure. In this study, the complete genome of R. thermocellum M3 was sequenced and assembled. The genome of R. thermocellum M3 was compared with the other R. thermocellum to reveal the mechanism of cellulosic saccharification by R. thermocellum M3. In addition, we predicted the key genes for the elimination of feedback inhibition of cellobiose in R. thermocellum. The results indicated that the whole genome sequence of R. thermocellum M3 consisted of 3.6 Mb of chromosomes with a 38.9% of GC%. To be specific, eight gene islands and 271 carbohydrate-active enzyme-encoded proteins were detected. Moreover, the results of gene function annotation showed that 2,071, 2,120, and 1,246 genes were annotated into the Clusters of Orthologous Groups (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively, and most of the genes were involved in carbohydrate metabolism and enzymatic catalysis. Different from other R. thermocellum, strain M3 has three proteins related to β-glucosidase, and the cellobiose hydrolysis was enhanced by the synergy of gene BglA and BglX. Meanwhile, the GH42 family, CBM36 family, and AA8 family might participate in cellobiose degradation.The cellulosome of  Trichoderma and Aspergillus sp. are known for their potential to produce cellulases, while these fungi lack a complete cellulase system, which leads to a decrease in the catalytic efficiency of cellulase , the result indicated that the β-glucosidase activity expressed by recombinant strain was 2.3 times higher than that of the wild strain at the late logarithmic growth stage 2SO4, 1.0 g KCl, 0.2 g/L; L-cysteine, 0.5 g/L; KH2PO4, 3.0 g/L; CaCl2•2H2O, 0.025 g/L; NaCl, 1.0 g/L; Yeast, 1.5 g/L; and Avicel, 5.0 g/L. The temperature of the culture was maintained at 60°C at 120 rpm.R. thermocellum samples for whole genome sequencing analysis were cultured in MA medium for 24 h at 60°C, centrifuged for 5 min at 4°C, 12,000 × g, then the cell pellet was washed twice with normal saline (NS), and crushed with the FastPrep-24 instrument in lysing matrix B tubes  for 40 s to release the genomic DNA from cells. The extraction of genomic DNA was performed with the Tiangen bacterial DNA mini kit  according to the manufacturer’s protocol. The harvested DNA was detected using agarose gel electrophoresis and then quantified by Qubit 4.0 . The genomic sequencing was conducted by the Pacbio sequencing platform with de novo assembly  was performed with the above four databases. The prediction of carbohydrate-active enzymes was conducted with the Carbohydrate-Active enZYmes Database (The genome annotation and gene function were predicted in the Gene Ontology (GO) database, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the Clusters of Orthologous Groups (COG) database, and the Non-Redundant Protein databases (NR). The whole genome BLAST search . The GeneMarkS program was conducted to retrieve the coding genes , and genome visualization were analyzed to reveal the result of comparative genomics . The genTZ tools . Core geTZ tools . The relsoftware .R. thermocellum M3 was sequenced and analyzed with regard to the predictions of coding genes. The identified total size of the genome R. thermocellum M3 was 3,602,270 bp with 39% GC content using SPAdes, and the number of coding genes was 3,195 with an average gene length of 973.56 bp  in the aligned COG database genes, accounting for 6.62% of the COG annotations; the number of carbohydrate transport and metabolism-related genes (group G) was 130, accounting for 6.28% of the COG-annotated genes; the quantity of energy production and conversion-related genes (group C) was 107, taking up 5.17% of the COG-annotated genes; and only 16 genes fall into group Q , antibiotic resistance (CARD), and pathogen-host interactions (PHI-base) were present in mocellum . MeanwhiR. thermocellum M3, 2021 genes were annotated in the GO database  prediction obtained eight GI with an average G + C content of about 36.4%, which is slightly lower than the G + C content of the M3 genome. It is worth noting that the G + C content of GI5 (33.7%) is significantly different than that of other GI, which indicates GI5 may be an exogenous sequence by horizontal transfer. The main components of the exogenous sequence are the transposase protein and the hypothetical protein , followed by strain M3 (192 genes). The high resemblance of five R. thermocellum strains was reflected by the large proportion of core genes.To reveal the genetic and evolutionary relationships between  boxplot  revealedR. thermocellum pan-genome, the COG database was used to classify the functional genes. In the pan-genome of R. thermocellum, the most core, dispensable, and specific gene clusters fell in the metabolism category , it was found that the proteins are not only homologous but also have a good linear relationship in sequence, while R. thermocellum ATCC 27405 was related to M3 but showed a large number of inversions. At the same time, we performed a genome-linear analysis of M3 and R. thermocellum ATCC 27405 , carbohydrate binding molecules (CBMs), and glycosyltransferases (GTs), whereas a few genes were annotated in carbohydrate esterases (CEs), polysaccharide lyases (PLs), and auxiliary activities (AAs). To be specific, 271 cellulase proteins were detected in R. thermocellum M3 using dbCAN2 , GH9 (n = 17), and GH5 (n = 13) being the most abundant families. In addition, there were 75 carbohydrate-binding module (CBM) proteins, 55 glycosyl transferases (GTs) proteins, 22 carbohydrate esterases (CEs) proteins, seven polysaccharide lyases (PLs) proteins, and four auxiliary activities (AAs) proteins. It is worth noting that there were nine CBM genes and 24 GH genes that had not been identified in R. thermocellum before , BglB (Protein: PROKKA_00862), and BglX (Protein: PROKKA_01050 and PROKKA_02059). BglA encoded glycoside hydrolase family 1 (GH1) protein (β-glucosidase). BglB encoded glycoside hydrolase family 1 (GH1) protein (6-phospho-β-glucosidase). BglX (PROKKA_01050) encoded glycoside hydrolase family 3 (GH3) protein (β-glucosidase). BglX (PROKKA_02059) encoded glycoside hydrolase family 3 (GH3) protein (β-glucosidase). The result of protein sequence alignment indicated that the ratio of similarity of pairwise comparison of proteins encoded by three genes , which correlated with β-glucosidase, were lower than 28.39%. To further demonstrate the diversity among the three Bgls, we constructed phylogenetic trees of Bgls from different strains by MEGA software. Not surprisingly, the phylogenetic tree based on β-glucosidase showed that each of the three Bgls was located in one of three different clusters (R. thermocellum before.Four encoded proteins associated with cellobiose degradation were found among the cellulase-encoded proteins of strain M3 by Conserved Domain Database (CDD) analysis , includiclusters . FurtherR. thermocellum M3  about cellobiose phosphorylase coming from glycoside hydrolase family 94 (GH94) were detected in the genome of ellum M3 . Two groRuminiclostridium thermocellum, as a typical genus of thermophagic cellulosic-degrading bacteria, was a high-utility candidate in lignocellulosic biomass refinement by means of a powerful cellulosome system (BglA) was low in wild-type R. thermocellum strains  and β-glucosidase B (BglB), gene BglX (β-glucosidase) was first found in R. thermocellum in this study. In general, β-glucosidase A was sensitive to temperature and thus inactivated under the optimum temperature of R. thermocellum  may encode β-xylosidase or β-glucosidase, since R. thermocellum cannot utilize xylose as a carbon source as the previous report (BglX (Protein: PROKKA_01050) gene mainly plays a role in the hydrolysis of cellobiose in strain M3 and that the BglX (PROKKA_01050) gene encodes an enzyme with β-glucosidase activity predominantly. It is reported that the β-glucosidase encoded by BglX was a periplasmic cellulase that hydrolysis cellobiose into glucose , which leads to catalytic activity varying greatly in different regions. As a result, there are higher local cellobiose concentrations at particular sites. However, the β-glucosidase of wild type R. thermocellum was not bound to the CBM which led to the ineffective hydrolysis of local cellobiose  can be isolated or appended to a CBM. Proteins contain iron reductase domains and may generate reactive oxygen species that could contribute to the non-enzymatic degradation of cellulose chains by the generation of highly reactive hydroxyl radicals (OH•) via Fenton’s reaction , cellulose-binding modules Family (CBM), Carbohydrate Esterase Family (CE), Glycosyl Transferases Family (GT), and Glycoside Hydrolase Family (GH). The hydrolysis of cellobiose by R. thermocellum M3 was conducted by the synergy of BglA and BglX, which not only hydrolyze cellobiose but also increase the affinity of Bgl for cellulosic feedstocks, increasing catalytic activity. Meanwhile, the GH42, GH116, CBM37, and AA8 families might participate in the cellobiose degradation that released the R. thermocellum M3 from the feedback inhibition of cellobiose.The genome of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ST, MQ, LZ, SC, LiL, and LiuL contributed jointly to all aspects of the work reported in the manuscript. ST and LZ designed the experiment. MQ performed the experiments. ST, MQ, LiL, SC, and LiuL contributed to the data analysis. ST and MQ drafted the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (No. 51908200), the Heilongjiang Provincial College Youth Innovation Talent Project (No. UNPYSCT-2020028), and the Basic Scientific Research Operating Expenses (No. 2021-KYYWF-1465).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1079279/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.