Picea jezoensis and Betula ermanii, the two treeline species naturally occurring in Changbai Mountain in China, and measured the concentration of non-structural carbohydrates (NSC), soluble sugars and starch in one-year-old leaves, shoots, stems and fine roots at different elevations. We found that compared with P. jezoensis, the NSC and soluble sugars concentrations of leaves and shoots of B. ermanii were higher than those of P. jezoensis, while the starch concentration of all the tissues were lower. Moreover, the concentration of NSC, soluble sugars and starch in the leaves of B. ermanii decreased with elevation. In addition, the starch concentration of B. ermanii shoots, stems and fine roots remained at a high level regardless of whether the soluble sugars concentration decreased. Whereas the concentrations of soluble sugars and starch in one-year-old leaves, shoots and stems of P. jezoensis responded similarly changes with elevation. These findings demonstrate that compared with P. jezoensis, B. ermanii has a higher soluble sugars/starch ratio, and its shoots, stems and fine roots actively store NSC to adapt to the harsh environment, which is one of the reasons that B. ermanii can be distributed at higher altitudes.Environmental factors that drive carbon storage are often used as an explanation for alpine treeline formation. However, different tree species respond differently to environmental changes, which challenges our understanding of treeline formation and shifts. Therefore, we selected  The alpine treeline is the upper limit of mountain forest distribution and is highly sensitive to climate change and human disturbance ,2. With Elevation usually causes drastic changes in environmental factors, such as temperature, water and light, which greatly affect trees’ growth and treeline formation ,5. This Pinus sylvestris had a “drought memory effect,” using more carbon for storage, whereas Tilia platyphyllos primarily used more carbon for growth [The NSC concentrations of tree tissues can reflect the balance between carbon sources (photosynthesis) and the carbon sink (respiration and tree growth) as well as mirror tree growth in response to environmental changes . Differer growth . In addir growth . As a rer growth ,26.Betula ermanii is 2200 m a.s.l. [Picea jezoensis is 1800 m a.s.l. With climate warming, the upper limit of their distribution may shift up to varying degrees [Currently, most studies on alpine treeline formation and shifts primarily focus on the restriction of environmental factors in tree physiology ,12, wherm a.s.l. , while t degrees . Thus, uP. jezoensis and B. ermanii, the treeline species naturally occurred in Changbai Mountain, and measured the concentration of NSC, soluble sugars and starch in one-year-old leaves, shoots, stems and fine roots along with elevation. Our objectives were to determine (1) the differences in the concentrations of NSC, soluble sugars, starch and NSC storage strategies in different tissues of B. ermanii and P. jezoensis; and (2) the influence of these differences on treeline formation and the spatial distribution of tree species.In this study, we selected p < 0.05). Compared with P. jezoensis, soluble sugars concentration of leaves and shoots in B. ermanii were higher, but soluble sugars concentrations in fine roots were lower (p < 0.01). In addition, the starch concentration of P. jezoensis tissues was 1.5 times higher than that of B. ermanii . Moreover, the soluble sugars/starch ratio in B. ermanii tissues was significantly higher than that of P. jezoensis (p < 0.01).The concentrations of soluble sugars, starch, NSC and the soluble sugars/starch ratio were all significantly different in tree species, tissue types and their interactions . As for P. jezoensis, one-year-old needles NSC, soluble sugars and starch concentrations were significantly higher at the medium elevantion (1600 m a.s.l.) than at the low elevation (1300 m and 1 400 m a.s.l.) and the high elevation (1700 m and 1800 m) (p < 0.05). Shoots starch concentrations in B. ermanii were not significantly different at the various elevations .Different tissues NSC, soluble sugars and starch concentrations in evations c, whereaevations a,e. A siB. ermanii stems appeared to also remain stable at varying altitudes (p < 0.05). Therefore, except for the altitude of 1,800 m, the stems NSC concentrations in the B. ermanii did not change with elevation (p > 0.05). Similarly, the elevation did not significantly affect stems NSC, soluble sugars and starch concentrations in P. jezoensis (p > 0.05). Except for maximum value at an altitude of 2200 m a.s.l., no significant differences were observed in the concentrations of soluble sugars and NSC in fine roots of B. ermanii with elevation (p > 0.05). But fine root starch concentration in B. ermanii and NSC, soluble sugars and starch concentrations in P. jezoensis tended to increase with increasing elevation (p < 0.05).The concentrations of soluble sugars in ltitudes a, but thB. ermanii leaves (P. jezoensis stems (p > 0.05), However, the soluble sugars/starch ratio of the shoots, stems and fine roots in B. ermanii were higher at lower elevations (1800 m a.s.l.) than at higher elevations (2100 m and 2200 m a.s.l.) (p < 0.05). More specifically, a higher value of soluble sugars/starch ratio of P. jezoensis leaves and fine roots was observed at the lower elevations (1300 and 1400 m a.s.l.), but the soluble sugars/starch ratio of the shoots tended to increase with increasing elevation (p < 0.05). Overall, elevation significantly affected the soluble sugars/starch ratio among the tissues of trees, and the different tree species and tissues varied in their response to elevation.The soluble sugars/starch ratio of i leaves a and P. is stems f did notB. ermanii were significantly higher than those of P. jezoensis (p < 0.05). This is because of different adaptation strategies to high elevations. Compared to evergreen trees, deciduous hardwood leaves are more vulnerable to environmental damage, so the leaves and shoots store higher levels of NSC to enhance their resilience and new leaf growth in the second year [The concentrations of soluble sugars and NSC in the leaves and shoots of the alpine treeline species ond year . In addiond year ,19. Li eond year . By comps p < 0.0. This isond year ,29.B. ermanii was significantly higher than that of P. jezoensis and the soluble sugars/starch ratio in each tissue was > 3, while the soluble sugars/starch ratio in stems of P. jezoensis was < 3 (p < 0.05). This is also one of the physiological mechanisms that B. ermanii utilizes to adapt to the alpine environment and become a treeline species.Most studies have shown that the growth and survival of trees at high elevation are not only dependent on NSC concentrations but also require higher soluble sugars/starch ratio (soluble sugar/starch > 3) ,30. HighB. ermanii tended to decrease with increasing elevation . Therefore, by combining the changes of soluble sugars and starch concentrations in the shoots and stems with elevation, we can predict that the probability of carbon starvation in B. ermanii is lower than that in P. jezoensis under stress conditions.The shoots and stems of levation . In addilevation . In the B. ermanii, but the starch concentration increased significantly with elevation (p < 0.05). This indicates that, although the stems and fine roots of B. ermanii at high elevation increase NSC storage, they do not have a surplus of soluble sugars, but actively convert the excess soluble sugar into starch. However, the concentrations of soluble sugars and starch in the fine roots of P. jezoensis increased significantly with elevation, indicating that P. jezoensis provided enough soluble sugars for the growth of fine roots at high elevation. Thus, the increase in starch concentration in the fine roots of P. jezoensis may be a passive storage process. Furthermore, trees have self-regulatory mechanisms. When the NSC concentrations increase because of the decrease of the carbon utilization capacity, the trees will reduce the photosynthetic rate through self-regulatory to promote excess carbon utilization [B. ermanii did not decrease [B. ermanii increases NSC storage near alpine treeline [B. ermanii to the high-altitude environment and is one of the reasons it can be distributed in higher altitudes. However, whether the alpine treeline formation is due to reduced carbon utilization resulting in growth restriction must be revisited.Li et al. (2018) studied 11 treeline species around the world and found that the roots of high-altitude trees increased their starch reserves in the summer at the expense of growth, which was an active carbon storage process. In turn, when the photosynthetic products exceeded the demand, it was a passive storage process of NSC . Similarlization . Howeverdecrease . This prtreeline  due to aB. ermanii was due to the significant increase in starch concentration with increasing elevation , due to the harsh environment, the trees are sparse and dwarf forest [The research area is located in the western slope of the Changbai Mountain Nature Reserve  in Jilin Province of northeastern China. This region has a temperate continental monsoon climate. With the elevation gradient rising from 713 m to 2500 m, the annual mean temperature is from 4.9 °C to −4.8 °C, and the annual precipitation is between 800 mm to 1340 mm, with more than 60% of the precipitation concentrated in the growing season (June–September) . The soi0–1800 m . Above tf forest .B. ermanii and P. jezoensis  was cut to extract the xylem (1–2 cm). Below the alpine treeline, stem samples of P. jezoensis and B. ermanii were drilled with a growth cone with a diameter of 5 mm at a height of 1.5 m, and the xylem part 3 cm outside the core was intercepted. To avoid canopy differences, the upper, middle and lower canopy leaves, as well as shoots, were selected to represent the whole canopy. The fine roots (<0.5 cm in diameter with bark) were collected from each of the trees. To reduce the influence of daily temperature range and illumination, all samples were collected at noon and quickly transferred to 4 °C for storage [At the end of July 2019 (the growing season), six elevation gradients from high to low were selected for sampling based on the distribution of ezoensis . A total storage . After tThe growth season average temperatures (100 cm above ground) were measured with a data logger  at 1700 m, 2000 m, 2100 m, and 2200 m. Other elevation growth season average temperatures were provided by  Table 2Table 2.Soluble sugar and starch concentrations were determined using the anthrone colorimetic method . A totalThe extracted precipitate was placed in an 80 °C shaking water bath to dry the residual alcohol, and 2 mL distilled water was added. The solution was gelatinized in the boiling shaking water bath for 15 min. After being cooled to ambient temperature, 2 mL 9.2 mol/L HClO4 was added, stirred, and the starch was hydrolyzed for 15 min. A volume of 4 mL distilled water was added and centrifuged at 4000 rpm for 10 min to collect the supernatant. A volume of 2 mL 4.6 mol /L HClO4 was added to the precipitate for extraction for 15 min, and 6 mL distilled water was added. After mixing, the supernatant was centrifuged at 4000 rpm for 10 min, and all of the supernatant was collected from each centrifugation. The supernatant was mixed and brought to a volume of 50 mL in a volumetric flask to determine the starch concentration . The solB. ermanii and P. jezoensis at different elevations, and the significance level was set as α = 0.05. All the data were analyzed using SPSS 20.0 software  and drawn using Origin Pro 8.5 .Before the data were analyzed, the NSC, soluble sugar and starch data were tested for normality. Two-way ANOVAs were performed with tissue and elevation species as factors to identify the significant effects of NSC, soluble sugars, starch and the soluble sugar/starch ratio. A Tukey HSD test was used to analyze the significance difference of NSC, soluble sugars and starch concentration in different tissues of


The aim is to describe a new arthroscopically assisted Latarjet technique.We evaluated the clinical and radiological findings of 60 patients with chronic recurrent anterior gleno-humeral instability who underwent, between September 2013 and November 2014, an arthroscopically-assisted Latarjet procedure with double round endobutton fixation. Inclusion criteria were: chronic anterior recurrent instability, Instability Severity Index Score (ISIS) greater than three points, a glenoid bone defect > 15% or a Hill Sachs lesion with concomitant glenoid bone defect > 10%. During surgery the joint capsule and the anterior glenoid labrum were detached. Two drill tunnels perpendicular to the neck of the glenoid were made through a guide. An accessible pilot hole through the glenoid was created to allows the passage of guidewires for coracoid guidance and final fixation onto the anterior glenoid. Through a restricted deltopectoral access a coracoid osteotomy was made. Finally, the graft was prepared, inserted and secured using half-stitches.The mean follow-up was 32.5 months (range 24–32 months). At a mean follow-up, 56 of the 60 subjects claimed a stable shoulder without postoperative complaints, two (3.3%) had an anterior dislocation after new traumatic injury, and two (3.3%) complained of subjective instability. At the latest follow-up, four subjects complained of painful recurrent anterior instability during abduction-external rotation with apprehension. At 1 year, the graft had migrated in one patient (1.7%) and judged not healed and high positioned in another patient (1.7%). Moreover, a glenoid bony gain of 26.3% was recorded. At the latest follow-up, three patients had grade 1 according to Samilson and Prieto classification asymptomatic degenerative changes. Nerve injuries and infections were not detected. None of the 60 patients underwent revision surgery. Healing rate of the graft was 96.7%.This technique of arthroscopically assisted Latarjet combines mini-open and arthroscopic approach for improving the precision of the bony tunnels in the glenoid and coracoid placement, minimizing any potential risk of neurologic complications. It can be an option in subjects with anterior gleno-humeral instability and glenoid bone defect. Further studies should be performed to confirm our preliminary results.Trial registration number 61/int/2017Name of registry: ORSDate of registration 11.5.2017Date of enrolment of the first participant to the trial: September 2013 ‘retrospectively registered’IV The Latarjet procedure is a popular procedure to manage recurrent shoulder instability .Recently, the arthroscopic technique has also been applied to the Latarjet procedure –4. ConceIn fact, despite the excellent and reproducible results of the Latarjet procedure in terms of stability, the incidence of complications  is around 15% in open Latarjet , 8. MoreCoracoid graft malpositioning is one of the most common complications . When grIf the screw direction is not correct, the screw may be too prominent laterally and it may impact with the head of the humerus and the scapular spine damaging the joint and potentially the suprascapular nerve , 13, 14.The arthroscopic technique allows better positioning of the tunnels, whereas the open procedures have the advantages of a better preparation of the graft.To overcome these difficulties, we propose a combined mini-open and arthroscopic approach for improving the precision of the osseous tunnels in the glenoid and the coracoid placement, minimizing any potential risk of neurologic complications. The present technique takes inspiration from Boileau’s procedure for Latarjet and Taverna’s method for bone-block performed through a mini-open approach , 17.The parallel drilling of the two tunnels perpendicular to the glenoid neck can be obtained through an arthroscopic guide that aims to decrease the risk of non-union and graft resorption , 18.The mini-open preparation of the graft allows safe preparation, avoiding the risk of nerve injuries which may occur during the arthroscopic technique, because of the use of portals medial to the coracoid , 8, 19.This study aims to describe an arthroscopically assisted Latarjet, which combines a mini-open and arthroscopic approach for improving the precision of the osseous tunnels in the glenoid and the coracoid placement, minimizing any potential risk of neurologic complications for subjects with glenoid bone deficiency and anterior instability.The study is a retrospective consecutive case series.The local Ethics committee of Ospedale San Raffaele of Milan approved the present study. All participants were enrolled at “Istituto Ortopedico Galeazzi” of Milan.Between September 2013 and November 2014, 60 patients undergoing an arthroscopically-assisted Latarjet procedure with double round endobutton fixation were enrolled.Inclusion criteria were: chronic anterior recurrent instability (more than five dislocating episodes and first episode having occurred more than 3 years before), Instability Severity Index Score (ISIS)  greater The “best-fit circle” technique on preoperative Computer Tomography  with OsiBeach chair position without any traction to allow intra-operative mobilization is used. The surgeon starts performing the joint inspection; then the joint capsule and the anterior glenoid labrum are detached. At this point, the surgeon frees the capsulo-labral complex, visualizing the subscapularis tendon. The anterior osseous defect is polished through the motor-powered burr.The drill guide  is used to ensure that the two drill tunnels are perpendicular to the neck of the glenoid and parallel to each other . It is iIn this way, an accessible pilot hole through the glenoid is created, and it allows the passage of guidewires for coracoid guidance and final fixation onto the anterior glenoid. The bullets and the guide are removed at this stage, leaving the drill sleeves in place.To prepare the anterior capsular reconstruction, 3 soft anchors suture-based are placed in the anterior glenoid margin.The scope and the cannulas are then removed and, employing a restricted deltopectoral access, and the mid-glenoid portal is incorporated on the apex of the cut, as described by Young and Walch  Fig. 5)5). FolloIn the point among the superior 2/3 and the inferior 1/3, the surgeon performs a subscapularis split to expose the glena using a Fukuda retractor and Hohman retractors Fig. .Fig. 12To shuttle and transfer the coracoid process, a Suture Retriever is inserted from the posterior side of each sleeve, pushed anteriorly and fed into the prepared holes on the coracoid Fig. . The metThe posterior implants are advanced to the posterior portion of the glenoid Figs.  and 19, At this point the capulolabral complex is re-inserted on the glenoid side, recovering the suture limbs through the subscapularis split and tightening the knots of the inserted suture-based anchors. The coracoid and the conjoint tendon maintain their extra-articular stabilizer function and position Fig. . SurgicaThe arm was immobilized with an abduction pillow for the immediate 3 weeks post-surgery. After removal of the sling, passive movements were possible from the 4th week only under supervision. Active movements were allowed from the 4th week but below 90 degrees of flexion and abduction. Complete active movements were permitted after the 7th week. Strengthening exercises were started at the 15th week.The following clinical outcomes were evaluated: return to sport at pre-injury level, apprehension and relocation tests positivity, shoulder range of motion, complication or reoperation rate, Rowe score , Walch-Den face projection of the glenoid on the sagittal plane at CT scan. The surgeon drew a circle above and one below the glenoid flush with its margins using the Osirix software .At one-year follow-up, each subject was assessed through an The vertical axis of the glenoid is defined as the straight line connecting the central point of both drawn circles, while the horizontal axis as a line crossing the middle of the glenoid height and perpendicular to the vertical axis . The perA graft was considered healed when it was not malpositioned and a bone bridge could be observed between glenoid and graft on the CT. Bony gain was calculated at one-year follow-up on CT scans to evaluate bone remodelling. The original glenoid bone area (A1) and the defective area (A2) were measured to evaluate the bony healing and the effective glenoid area with defect as A3 = A1-A2. Bone graft area (A4) and the A4:A3 ratio, that represents increase of the glenoid surface by the bone block graft, were also measured Fig.  35].Fi.Fi35].On the Computer Tomography and the last X-ray control through Grashey and axillary views, the potential migration of the graft, the graft fractures, the possible bone resorption around an endobotton and the osteoarthritis grade according to Samilson and Prieto classification were evaluated by three observers . To avoiThe mean follow-up was 32.5 months (range 24–32 months). Sixty patients  were enrolled in this study . The dominant arm was affected in 47 patients (78.3%). No patients were lost to follow-up.Twenty-eight patients (46.7%) were involved in sports. Seventeen of which participated at a competitive level and eight in high-risk sports . Preoperative average ISIS  was 4.6 At a mean follow-up, 56 of the 60 subjects reported a stable shoulder without postoperative complaints, two (3.3%) had an anterior dislocation after new traumatic injury, and two (3.3%) complained of subjective instability without apprehension and recurrent anterior dislocation or subluxation. At the latest follow-up, four subject complained about painful recurrent anterior instability ) during abduction-external rotation with apprehension. No neurologic complication or infections were recorded.Most of the patients were satisfied, and 93.3% declared, if needed, that they would undergo the same procedure again.Functional and radiological outcomes are summarized in Tables The average loss of external rotation with the elbow at the side was 9.8°  and sixteen patients (26.7%) were aware of it.Twenty-three patients out of 28 came back to sport at the preinjury level (82.1%), 13 out of 17 at a competitive level.At 1 year follow up, in one of the patients affected by recurrent dislocation, the graft had migrated (1.7%). Of the remnants, one was judged not healed and high positioned (1.7%) and none dissolved.At one-year follow-up, a glenoid bony gain of 26.3% was recorded .At the latest follow-up, no signs of cartilage joint degeneration were observed in 57 patients (95.0%), and only three patients had grade 1 asymptomatic degenerative changes according to Samilson and Prieto classification . No patiNone of the 60 patients underwent revision surgery.This arthroscopically-assisted Latarjet technique combines the advantages of an open approach to harvest, prepare and handle the coracoid and the accuracy of an arthroscopically guided graft positioning and fixation. It has the advantage of avoiding dangerous portals medial to the coracoid. The use of an arthroscopic guide to perform the tunnels allows to achieve optimal graft positioning and fixation; the double pair of round-endobuttons avoids rotational instability of the graft.The site of graft positioning, according to literature, is controversial. Several studies reported the graft placement on the scapular neck, without a specific description of the site , 39; othWith the arthroscopically-assisted Latarjet, we found 3.3% of anterior redislocation after new traumatic injury, and 3.3% of subjective instability without apprehension and recurrent anterior dislocation or subluxation.Nerve injury is a dreadful complication of arthroscopic Latarjet procedure . In thisThe round-endobutton fixation and the glenoid guide provides better bony integration of the coracoid than the use of the screws through an anterior approach . CowlingThe use of the aiming device, with creation of two bony tunnels, permit the realization of two perfectly parallel tunnels with 5mm offset from the glenoid rim , 44. TheThe augmented blood flow through the bony tunnels, enhances graft perfusion and therefore probably increases bone integration of the graft . MoreoveThe Bristow-Latarjet technique has several criticisms. The potential loss of external rotation made this procedure unpopolar in some overhead throwing sportsmen –50. HoweSummarizing, this new mini-open technique allows good positioning of the tunnels and graft preparation.Weaknesses of this study are the retrospective model and the lack of a control group which precludes definitive conclusions. However, we do intend to perform a prospective randomised trial in future.Strengths of this study include that patient examination was performed by observers different from the orthopaedic surgeon; a minimal number of subjects was lost at follow-up; every surgical procedure was made by the same senior surgeon; CT scans were performed postoperatively.This technique of arthroscopically assisted Latarjet combines mini-open and arthroscopic approach to improve the precision of the bony tunnels in the glenoid and coracoid placement, minimizing any potential risk of neurologic complications. It can be an option in subjects with anterior gleno-humeral instability and glenoid bone defect. Further studies should be performed to confirm our preliminary results.


Halophytis incurva (HIEs) were characterized by the HPLC-DAD-MS analysis. The H. incurva anti-amyloidogenic role was explored by incubating both HIEs with hen egg white lysozyme (HEWL), a well-known protein model widely used in amyloid aggregation experiments. The aggregation kinetics and morphological analysis of amyloid aggregates were performed by ThT and AFM analysis, respectively, while their cytotoxicity on SH-SY5Y human neuroblastoma cells was examined by MTT assay. HIEs showed a different efficacy, probably dependent on their metabolic composition, both in inhibiting amyloid fibrillation and in obtaining short and less toxic pre-fibrillary aggregates. Overall, this work sheds light, for the first time, on a Mediterranean red seaweed as a promising renewable resource of bioactive compounds, potentially useful in preventing the formation of toxic amyloid aggregates.Neurodegenerative diseases are generally characterized by the presence of neurotoxic amyloid aggregates underlying progressive neuronal death. Since ancient times, natural compounds have been used as curative agents for human health. Amyloid research is constantly looking for safe natural molecules capable of blocking toxic amyloid aggregates’ formation. From the marine environment, seaweeds are recognized as rich reservoirs of molecules with multiple bioactivities, including the anti-amyloidogenic activity. Here, hydroalcoholic extracts of two seasonal samples of the Mediterranean red seaweed  Halopithys incurva (HIE) (Hudson) Batter  is a slow-growing, sun-adapted perennial to annual small filamentous red seaweed classified as Rhodophyta, Ceramiales, Rhodomelaceae. It is particularly abundant in the Mediterranean Sea, generally located on rocky substrates in a not very sunny area at a depth ranging from a few centimeters to about ten meters up to a maximum of twenty meters . In the past, H. incurva has been studied in terms of adaptive response to seasonal changes [H. incurva seaweed. Some studies have attributed extracts’ effective antibacterial, antiviral, antiplasmodial, antiprotozoal, antimycobacterial and strong anti-inflammatory properties to H. incurva [H. incurva has exhibited polysaccharide-mediated immunomodulatory properties [H. incurva seasonal samples on the HEWL amyloid aggregation was investigated in this work. In addition, the effect of H. incurva extracts on cytotoxicity, typically induced by HEWL fibrils, was verified on SH-SY5Y human neuroblastoma cells.Neurodegenerative diseases affect millions of people around the world with a growing prevalence along with rapidly aging populations. They are characterized by the progressive loss of neurons due to the deposition of proteins with aberrant physical–chemical properties in the brain and peripheral organs. With a relatively long course of the disease and high costs for medical care, neurodisorders have become a huge burden on families and society and a serious public health challenge . Current changes ,13, in p changes . However incurva ,16,17. Foperties  and antioperties . To the operties ,21,22. Foperties . Severaloperties ,25,26. Loperties . Moreoveoperties . In partoperties . Hence, operties . TherefoH. incurva seasonal samples allowed us to recover about 58 mg (~1.5%) and 520 mg (~13%) of dry extract, respectively from the May (m) and November (n) samples, starting from 4 g of dry seaweed . This result suggests that changes in the H. incurva growth conditions, driven by seasonal variations, have a strong influence on the composition of its secondary metabolites. Hence, seasonality is undoubtedly a relevant factor to consider when collecting the sample, since a different content of secondary metabolites may reflect differences in the seaweed bioactive properties and in their effectiveness. The phenolic and carbohydrate content of HIEs were measured, respectively, by Folin–Ciocalteau and phenol-sulfuric acid assays. As shown in The hydroalcoholic extraction method from plant material has already been proven to be an effective method for recovering a significant quantity of hydrophilic compounds . In this seaweed . The finTo investigate the HIEs’ metabolic composition, a first characterization on HIE/m and HIE/n was performed with HPLC-DAD-MS analysis. The reference wavelength of 280 nm was selected as the most appropriate for showing the profile of the extracts. 1, with a different chromophore having a maximum at 370 nm . For all the isobars, the ion clusters at 465 and 467 m/z indicated the loss of a bromine atom and the species at 401 and 403 m/z were attributable to a moiety containing a bromine atom that originated the ion at 321 m/z after the loss of 80 Da. All the mass spectra of the three isobars showed the presence of a non-halogenated fragment at m/z 211.The isotopic distribution of bromine ions allows the mass spectra of molecules containing these atoms to show diagnostic clusters . Some moed algae . The thrtructure . SeveralH. incurva extracts obtained from the two seasonal samples. This substantial difference reflects a difference in the role of the two extracts on the amyloid aggregation process as demonstrated in subsequent experiments on the formation of amyloid aggregates in the presence of HIEs.The results from HPLC-DAD-MS noticed strong difference in terms of metabolic composition between the H. incurva anti-amyloidogenic role, here proven, is the result of the synergistic action of its secondary metabolites. Although this work sheds light for the first time on the anti-amyloidogenic role of a red Mediterranean seaweed, however its composition, rich in total phenols and carbohydrates, finds analogies with other seaweeds, with similar bioactivities, widespread in the Pacific and Indian Ocean countries, such as the red seaweed G. acerosa [H. incurva as a new potential source of bioactive compounds against amyloidosis.Since amyloid aggregation is considered the initiator of events leading to neurotoxicity and clinical symptoms of amyloidosis diseases, the search for effective anti-amyloidogenic agents has become an important strategy in amyloid research . To date acerosa ,46 or th acerosa  which arTo verify whether HIEs had an effect on the HEWL aggregate-induced cytotoxicity, as well as on the reduction in aggregate load, an MTT test was performed on SH-SY5Y human neuroblastoma cells, a cell line model widely used to test amyloid aggregate-induced neurotoxicity . SpecifiH. incurva compounds. When cytotoxicity was assessed, HIEs as well as HEWL aggregates were further diluted 1:100. We emphasize that this extreme dilution of HIEs (1:5000 in cell medium) substantially excludes the contribution to neuroprotection against amyloid aggregate-driven injury of their inherent  to exalt possible important inhibitory properties against amyloid aggregation of hydrophilic rent see  antioxidDulbecco’s modified Eagle’s medium (DMEM), Ham’s F-12 nutrient mixture, fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin, 1--3,5-diphenyl formazan (MTT), α,α-Diphenyl-β-picrylhydrazyl (DPPH), 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4′′-disulfonic acid sodium salt (Ferrozine™), Thioflavin T (ThT), Folin–Ciocalteau’s reagent, hen egg white lysozyme (HEWL), gallic acid, ascorbic acid, D-glucose and all other chemicals, analytical grade and HPLC grade solvents were purchased from Sigma Aldrich-Merck . Disposable plastics were from Sarstedt .H. incurva samples were carried out by CIBM experienced personnel in May and November from the rocky substrates of the Tyrrhenian Sea  at a depth of about 2–5 m. Immediately after harvesting, H. incurva seaweed was gently washed with bi-distilled water to remove salts, epibiota and other contaminants and dried overnight at 60 °C. Then, dried seaweed was minced and resuspended with 5 mL of EtOH/H2O (70:30 v/v) per gram of dry seaweed with the aim at collecting substantially hydrophilic compounds [H. incurva hydroalcoholic extract was separated from debris and mixed with n-hexane (1:1 ratio). The hydrophilic fraction, recovered in the lower phase, was dispensed in several batches and then dried using a UnivapoTM vacuum-spin concentrator . Each batch (corresponding to 0.8 g of dry seaweed) was suspended in 2 mL of bi-distilled water prior to use. Hydroalcoholic extracts of H. incurva May and November samples were hereinafter called HIE/m and HIE/n, respectively.Field collection and identification of ompounds . The extThe total phenolic (TP) and carbohydrate (TC) content of HIEs was determined according to the Folin–Ciocalteau’s and phenol–sulfuric acid methods, respectively, described in ,53,54. TThe antioxidant and radical scavenging activities of HIEs were determined according to the ferric-reducing/antioxidant power (FRAP) assay and DPPH assay, respectively, previously described ,53,54. BH. incurva extracts from the two seasonal samples were dried, and aliquots corresponding to 0.2 g of dried seaweed were dissolved using 0.5 mL of an ethanol/water mixture pH 3.2 by HCOOH 8:2. Then, samples were centrifuged at 12,000 rpm for 4 min and the supernatant was directly injected in HPLC. Analyses were carried out using a HP 1100L liquid chromatograph equipped with a DAD detector coupled to a HP 1100 MSD mass spectrometer with an API/electrospray interface . A 150 × 3.9 mm i.d., 4um Gemini, RP18 column (Phenomenex-USA) was used for all analyses. The mobile phases were: A) water with 0.1% HCOOH and B) CH3CN. The following multi-step linear solvent gradient was used: from 0 to 5 min at 95% A; from 5 to 15 min to 75% A; then a plateau of 5 min, from 20 to 30 min to 20% A; then a plateau of 6 min; from 36 to 44 min at 5% A. The analysis was carried out for a total time of 30 min, plus an equilibration time of 10 min with flow rate 0.4 mL min−1, oven temperature 26 °C and injection volume 8 μL. The mass spectra were recorded in negative ion mode, setting the fragmentation energy between 80 and 180 V and applying the same chromatographic conditions as described previously. The mass spectrometer operating conditions were: gas temperature, 350 °C; nitrogen flow rate, 9 L min−1; nebulizer pressure, 30 psi; quadrupole temperature, 40 °C; and capillary voltage, 3500 V.The hydroalcoholic HEWL was used as a well-established model to study amyloid fibril formation ,55. A HEFor Atomic Force Microscopy (AFM) analysis a drop of sample  was vortexed and laid onto a freshly cleaved mica disc for about 2 min. Sample excess was removed by washing twice the sample mica disc with 1 mL of bi-distilled water, then the preparation was dried with a soft nitrogen flow. AFM experiments were performed in air, in non-contact mode, using a PicoSPM microscope equipped with an AAC-Mode controller . The probes were non-contact Silicon cantilevers  with 150 KHz typical resonance frequency. Scanner calibration was periodically checked by means of a reference grid  with known 3 μm pitch and 100 nm step height. At least three different areas were imaged on each sample. Typical image size was in the range 1–10 μm, at 256 × 256 pixel resolution. The images were processed and analysed by WSxM software , version 4.0 ,57.®, Manassas, VA, USA), were cultured in a 1:1 mixture of DMEM and Ham’s F12 supplemented with 2 mM L-glutamine, 100 μg/mL streptomycin, 100 U/mL penicillin and 10% FBS (complete medium), at 37 °C in a humidified atmosphere containing 5% CO2. At 90% confluence cells were appropriately propagated after trypsinization (trypsin 0.025%-EDTA 0.5 mM).Human neuroblastoma SH-SY5Y cells, obtained from American Type Culture Collection  was added into each well (100 μL/well). After 2 h incubation at 37 °C in the dark, cells were lysed with 100 μL/well of DMSO and the absorbance at 595 nm was measured using iMARK microplate reader . Data were expressed as percentage ratio compared to untreated control cells.SH-SY5Y cell viability was assessed using the MTT assay adapted to cell line used in this work . BrieflyH. incurva extractions. Linear regression analysis on calibrator compounds and the corresponding interpolations were wade with Excel . Non-linear curve fitting was performed with Origin 6.0 .All the results were expressed in terms of mean ± standard deviation on the basis of at least three different experiment from three independent H. incurva, whose metabolic composition and bioactivity were strongly influenced by seasonality. This work focused on the bioactivity of H. incurva phytocomplex to evaluate the synergistic action of its various constituents, while the structure and functionality of its secondary metabolites will be the subject of further studies. Overall, we believe that our findings can be a useful guide in relation to the screening of promising new natural anti-amyloidogenic agents in the search for innovative preventive strategies against neurodegeneration. Future investigations will aim to verify a potential in vitro disaggregating role of H. incurva on preformed amyloid fibrils and the possible cytoprotective role of the seaweed against amyloid aggregate-induced cytotoxicity, as well as its mechanism of action.The close relationship between the amyloid aggregation process and the onset of amyloidosis constantly encourages scientific research in the identification of new natural compounds capable of suppressing the formation of toxic amyloid aggregates. For the first time, our findings demonstrated the in vitro anti-amyloidogenic role of the


Dioscorea hispida tuber plant to explore their potential as renewable materials. The extraction of the Dioscorea hispida starch and Dioscorea hispida fibres was carried out and the chemical composition, physical, thermal, morphological properties, and crystallinity were studied. The chemical composition investigations revealed that the Dioscorea hispida starch (DHS) has a low moisture t (9.45%) and starch content (37.62%) compared to cassava, corn, sugar palm, and arrowroot starches. Meanwhile, the Dioscorea hispida fibres (DHF) are significantly low in hemicellulose (4.36%), cellulose (5.63%), and lignin (2.79%) compared to cassava, corn hull and sugar palm. In this investigation the chemical, physical, morphological and thermal properties of the Dioscorea hispida fibre and Dioscorea hispida starch were examined by chemical composition investigation, scanning electron microscopy (SEM), particle size distribution, thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), and Fourier transform infrared (FTIR), respectively. It was found that Dioscorea hispida waste is promising alternative biomass and sustainable material with excellent potential as a renewable filler material for food packaging applications.This study was driven by the stringent environmental legislation concerning the consumption and utilization of eco-friendly materials. Within this context, this paper aimed to examine the characteristics of starch and fibres from the  Non-renewable materials have caused critical environmental issues in many countries in the world regarding waste disposal ,2,3. ThiRecent environmental regulation has forced production industries to seek for other potential materials for the development of their product that are more environmentally friendly than synthetic materials  ,16. In rBiocomposites are changing the research activities carried out in the scope of material engineering because the number of beneficial properties proven, such as biodegradability, lightweight, low energy consumption, usability, and eco-friendly ,31,32,33Currently, biocomposites are used in various applications, e.g., automotive, aircraft, building materials, sports equipment, and food packaging . From thDioscorea hispida plants, also known as Ubi Gadong in Malaysia is a poisonous tuber plant that contains the alkaloid of dioscorine [Dioscorea sp. discovered in numerous parts around the globe, specifically in the subtropical and tropical regions [Dioscorea hispida tubers is edible after the removal of the dioscorine compound [Dioscorea hispida starch is largely based upon empirical studies that investigated its use as a crude drug for inflammation [Dioscorea hispida starch demonstrated the hydrogel created from the starch functioned as an antibacterial agent to prevent microorganism activity [oscorine . Recent  regions . Studies regions . The stacompound . It requcompound . The knoammation . Previouactivity . Additioactivity . This isactivity ,46. Geneactivity . These gactivity ,61.Dioscorea hispida plants. Tuber parts have been particularly identified and some of the recent research works published are tabulated in Dioscorea hispida plants can also generate large quantities of sustainable lignocellulosic materials every year, which can be used for the production of starch, bioplastic, and potential biocomposites to minimize environmental impact and renewability.Up to now, several studies have been carried out on various parts of Dioscorea hispida species as filler materials. In this study, the chemical, physical, morphological, and thermal properties of the Dioscorea hispida fibre and Dioscorea hispida starch were examined by Chemical Composition Investigation, Scanning Electron Microscopy (SEM), Particle Size Distribution, Thermogravimetric analysis (TGA), X-ray Powder Diffraction (XRD), and Fourier Transform Infrared (FTIR). To the best of our knowledge, no previous study had proposed Dioscorea hispida as a promising material with strong potential in the development of biodegradable films. In comparison with other natural fibres and starch, the objective of this work was to characterize novel starch and fibre from Dioscorea hispida tuber as renewable materials for food packaging applications. It must be noted that the Dioscorea hispida tubers used in this study were chemically treated to remove the toxin.According to the literature, limited studies have been conducted on the investigation of the native starch and fibre solid wastes from Dioscorea hispida was collected from a local farm M Abd Halim Enterprise in Kuala Terengganu, Malaysia. Dioscorea hispida starch and fibres were extracted from the fresh tubers. All samples were characterized in powder form.The native Dioscorea hispida starch and fibre from the Dioscorea hispida tuber was conducted with a number of processes  at a temperature of 60 °C. The fibres were shredded, sieved through a 300 µm mesh sieve  and characterized prior to application as filler in the Dioscorea hispida starch biocomposites.The extraction of rocesses . InitialDioscorea hispida starch. The procedures used to study acid detergent fibre (ADF), neutral detergent fibre (NDF), ash, lignin (LIG), hemicellulose, and cellulose of Dioscorea hispida fibres were adapted from [Dioscorea hispida fibres. The most practised procedure was used for evaluating the amount of fibre constituents, such as hemicellulose, cellulose, and lignin. The amount of hemicellulose and cellulose were determined by using Equations (1) and (2), respectively.The analysis was conducted to investigate the moisture, ash, starch, crude protein, energy, and carbohydrate contents of ted from . The ADFm = mass (g), V = volume (cm3)The density was determined using an AccuPyc II 1340 pycnometer gas  intrusion using helium gas flow. The samples underwent oven drying at a temperature of 105 °C for 24 h to eliminate the moisture content within the fibres and starch. Then, the samples were placed inside a desiccator to remove water traces from the dried sample before putting inside the pycnometer. Samples’ densities were calculated from measurements at a temperature of 27 °C using Equation (3).i and after, Wf the heating to evaluate the moisture content using Equation (4)The moisture content investigation was performed with fibres and starch samples. The samples were heated in an oven at 105 °C for 24 h. The samples were weighed before, WThe instrument Mastersizer 2000 E Ver. 5.52  was used to identify the particle size distribution for all samples through a built-in Q-space powder feeder. Prior to distribution analysis, the particle size for the tested samples was investigated with a 1000 μm sieve.The instrument scanning electron microscope  was used to determine the samples’ surface morphology. The samples were coated with a layer of gold and 20 kV voltage was passed through at high vacuum condition to produce an electron beam. The electrons were connected with the sample atoms and produced signals giving a report on the surface topography, by generating images of high resolution.The thermogravimetric analyser  was applied to analyse the thermal behaviour of the samples. The samples with the range of mass 5–10 mg were placed in platinum crucibles and the temperature was raised to 600 °C from room temperature under a heating rate of 10 °C/min in a nitrogen atmosphere with flow rate, 50 mL/min.−1 to 400 cm−1 with 4 cm−1 of spectral resolution. The sample preparation was conducted using the potassium bromide (KBr) disc method, and the scan per specimen used was 16 scans.The infrared spectrometer  was used to obtain the FTIR spectrum for samples at frequencies over a wide spectral range, 4000 cmθ) at scattering speed of 0.02 (θ) s−1. The operating current and voltage were set to 35 mA and 40 kV, respectively.XRD analysis was conducted using a 2500 X-ray diffractometer  with an angular range from 5° to 60°  and starch had low moisture contents of 9.45% and 37.62%, respectively compared to cassava, corn, sugar palm, and arrowroot starches. Meanwhile, the Dioscorea hispida fibres (DHF) were significantly low in hemicellulose (4.36%), cellulose (5.63%), and lignin (2.79%) compared to cassava, corn hull, and sugar palm. The composition of these components could affect the characteristics of thermoplastic starch-based film composites, including the water barrier properties [f plants . Howeveroperties .Dioscorea hispida starch was relatively high due to the presence of phosphate groups. In terms of metals, the ash of native starches consists mainly of potassium, calcium, sodium, and magnesium [Dioscorea hispida tuber composition, even though they differed according to genetic, ecological, and agronomic factors [In addition, the ash content in the agnesium . Carbohy factors ,67. This factors . Since tDioscorea hispida starch showed slightly higher density, 1.74 g/cm3 compared to other natural starches shown in The most important factor in the materials selection process is the molecular weight of the materials since it could affect the performance of the products . MoreoveDioscorea hispida fibre is the amount of cellulose, hemicellulose, and lignin in the fibre. The results of this study were compared to other studies’ findings, as tabulated in Dioscorea hispida fibre were considerably low compared to other fibres. Furthermore, the lignin content (2.79%) in the Dioscorea hispida fibre was comparatively lower than the other samples’ lignin contents, as shown in Dioscorea hispida tubers. Apart from that, Dioscorea hispida fibre density (1.47 g/cm3) values were smaller compared to sugar palm fibres but higher than cassava and corn hull fibres [The critical finding for l fibres ,70.Dioscorea hispida fibres was relatively low compared to the other samples. The current study found that the relative amounts of different compounds discovered in natural fibres varied with each plant, including different parts of the same plant [Ibrahim et al.  reportedme plant . Generalme plant . Previoume plant . Similarme plant ,47.Fibres are assumed to be natural source composites mainly consisting of holocellulose  and lignin, with low sugar, starch, protein, extractives, and ash content . The incDioscorea hispida starch and Dioscorea hispida fibre samples. It was apparent that considerable weight loss occurred up to ~100 °C in all of the samples as shown in Dioscorea hispida starch and Dioscorea hispida fibre. For example, Dioscorea hispida starch sample’s onset thermal degradation temperature was about ~41.3 °C, while for samples of Dioscorea hispida fibre was ~40.7 °C. It might be due to the amount of moisture content of the samples  than Dioscorea hispida fibre, as presented in Dioscorea hispida starch and Dioscorea hispida fibre. The migration of volatile materials occurred as a result of the movement of water from the lower interior part of the starch and fibres to a better water potential region, at the surfaces of Dioscorea hispida starch and Dioscorea hispida fibre, as the water molecules on the surface of samples were evaporated. Hence, this movement of water indirectly moved the volatile materials, leaving them on the Dioscorea hispida starch and Dioscorea hispida fibre surfaces [Thermal stability is frequently assessed by identifying the thermal decomposition’s onset temperature.  samples . The evasurfaces .Dioscorea hispida starch and 203.9 °C for Dioscorea hispida fibre. The results showed that the initial heat tolerance of Dioscorea hispida starch was considerably greater than Dioscorea hispida fibre. It was revealed that when temperatures reached 140 °C, the inorganic materials and lignin of Dioscorea hispida fibre were degraded after the thermal decomposition of hemicellulose, cellulose, and volatiles [Dioscorea hispida starch began with the decay of the water-soluble amylopectin at the onset temperature and continued until achieving a weight loss of approximately 66.1%. The highest rate of thermal degradation of Dioscorea hispida starch occurred at about 310 °C and left 20.6% residue of ash. These results were in good agreement with the findings in previous works [Nevertheless, the temperatures of the first thermal decomposition were about 260.4 °C for onset temperature of olatiles . The firus works ,70.Dioscorea hispida fibre was consistent with the degradation of hemicelluloses. The remarkable weight loss was due to the degradation of significant elements of lignocellulose, including hemicellulose, cellulose, and finally, lignin, which normally degrades at the temperature of higher than 140 °C [Dioscorea hispida fibre, similar to other lignocellulosic material decompositions [Dioscorea hispida starch and Dioscorea hispida fibre samples were found to be in the range of 20% to 25% at a higher temperature (550 °C)  . This waperature . Hemicelbranches . These rDioscorea hispida starch and Dioscorea hispida fibre samples are displayed in Dioscorea hispida starch appeared as a polyhedral shape obtained from the raw starch isolated from the Dioscorea hispida tubers with a range of size from 1.8 µm to 3.5 µm. The shape was different from other Dioscorea sp. due to the dissimilarity in biological sources and the environments of plant growth [Dioscorea sp. starch and found that Dioscorea alata had three different shapes; triangular, ellipsoid, and polyhedral [Dioscorea opposita starch such as cake-shaped, oval, and ground granules. Jiang et al. [Dioscorea hispida fibre sample showed the presence of a polyhedral shape and exhibited individualized irregular granules with a clear smooth surface and no evidence of any fissure. The fibres were much bigger than Dioscorea hispida starch granules in this study with a range of size from 3.1 µm to 5.1 µm.SEM images of t growth . Previoulyhedral . In addig et al.  publisheg et al. . The DioDioscorea hispida samples showed a single mode distribution profile for their sizes. The graph was analyzed to approximate the gradation size range of the Dioscorea hispida fibre and Dioscorea hispida starch particles. According to the refractive guideline for the sample taken from the Malvern Instrument Mastersizer 2000, the refractive index value was denoted as 1.334. Thus, the particle size distribution of Dioscorea hispida fibre and Dioscorea hispida starch are presented in Dioscorea hispida fibre particles had dimensions of less than 18 µm and the majority sizes of the particles were 333 µm. Meanwhile, 10% of Dioscorea hispida starch particles had less than 3 µm and 877 µm for the majority sizes of the particles. However, the findings of the current study were unparalleled with the previous research of corn starch [The particle size distribution of n starch ,61. ThesThe results indicated that the mean particle size of starch powder for the volume-weighted mean was 167 µm. The mean particle diameter demonstrated crystallinity pattern and degree of hardness inside the starch granules . Recent Dioscorea hispida fibre sample presented a larger particle size compared to Dioscorea hispida starch due to the morphological structure related to the samples. Nonetheless, the findings of this study strengthened the idea that it can convey an excellent dispersion behaviour that is significant in reinforcing composites.Moreover, the particle size distribution of the fibres indicated that the volume-weighted mean was 344 µm. The Dioscorea hispida starch and fibre waste are portrayed in −1, the second stage was between 800 cm−1 to 1500 cm−1, followed by the third stage within the C–H stretch area (2800 cm−1 to 3000 cm−1). The final stage showed intense peaks indicating the O–H groups’ presence in every filler part associated with the hydroxyl group discovered in hemicellulose, cellulose, and lignin [Dioscorea hispida starch and Dioscorea hispida fibre showed behaviour patterns below 800 cm−1 in the region related to the vibrations of the glucose pyranose unit [−1 in the FTIR spectrum of Dioscorea hispida fibre was mainly ascribed to the C=O stretching vibration of the ester linkage of the carboxylic group of ferulic and p-coumaric acids of hemicellulose and lignin [−1 were shown as aromatic skeletal vibrations of the operative lignin group in DHF, while the band observed at 1635 cm−1 corresponded to lignin composition. These findings were supported by determining the chemical composition of Dioscorea hispida fibres as shown in −1 indicated the structural polymer stretching of the aromatic groups existing in the lignin form. From −1 reflected the stretching of the C–H and O–H groups, correspondingly. Peaks found in the 3300–3000 cm−1 region were attributed to the adsorbed water. The absorbance bands around 856, 930, 996, 1077, 1149, and 1242 cm−1 were related to the C–O stretching, C–H rocking vibrations, and C–O–C asymmetric valence vibration, respectively, and these peaks indicated cellulose in the carbohydrates [Dioscorea hispida starch appeared to have complex spectra in the second region with outstanding overlapping originating from the monomer glucose units C–O vibrational stretching. This result might explain that the presence of the C–O–H bending mode was due to the band appearance at 1078 cm−1, while the coupling modes of C–C and C–O stretching exhibited the peak at 1148 cm−1. However, the peak at 1339 cm−1 of the infrared wavenumbers indicated CH2 bending modes [−1 was related to the water fragment vibrations in the amorphous region of the starch granules, while another possible explanation for this was the emergence of broad infrared bands. The intense peaks at 3262 cm−1 meant the presence of O–H groups in Dioscorea hispida starch. A possible explanation for this might be the Dioscorea hispida starch that was very sensitive to water molecules due to the presence of hydroxyl groups. It can be noted that the fourth vibration in the region of the O–H stretch hydroxyl groups resulted in the formation of the last band [The FTIR spectra for raw d lignin . Thus, tose unit . The pead lignin . It can hydrates . The Diong modes . In addiDioscorea hispida starch and Dioscorea hispida fibre samples. The fibres characterization was performed via the XRD diffractograms. Dioscorea hispida fibres are primarily composed of cellulose, hemicellulose, and lignin, while the lignin is amorphous having a benzene ring with cellulose existing in a crystalline form in nature [Dioscorea hispida starch possessed 27.5% crystallinity index, which was lower than Dioscorea hispida fibres of 39.0%. This was probably due to the difference in the samples’ contents, which was referred to the investigations of the chemical composition. The crystallinity index of Dioscorea hispida starch and Dioscorea hispida fibre supported the agreement of Ibrahim et al. [Dioscorea hispida fibres and Dioscorea hispida starch samples. This might be attributed to the contents of lignin, cellulose, hemicellulose, moisture, and amylose of the fibres [Dioscorea sp. which agreed on the crystallinity of the starches of Dioscorea hispida starch [XRD Analysis is frequently used to investigate the percentage of crystallinity and the structure as for the samples in this study. n nature . Based om et al.  which ree fibres ,74. The a starch . Therefoa starch . Accordia starch , the modDioscorea hispida starch and Dioscorea hispida fibres were successfully extracted from Dioscorea hispida tubers after removing the dioscorine compound in the tubers. The chemical composition investigations, particle size distribution, scanning electron microscopy (SEM), X-ray powder diffraction (XRD), thermogravimetric analysis (TGA), and Fourier transform infrared (FT-IR) of DHS and DHF samples were explored, and their chemical composition, thermal properties, and morphologies were compared. The chemical composition investigations revealed that Dioscorea hispida starch has low moisture (9.45%) and starch content (37.62%) compared to cassava, corn, sugar palm, and arrowroot starches. Meanwhile, the Dioscorea hispida fibres (DHF) are significantly low in hemicellulose (4.36%), cellulose (5.63%), and lignin (2.79%) compared to cassava, corn hull, and sugar palm Thermogravimetric analysis (TGA) showed that the Dioscorea hispida starch maximum decomposition temperature was 309.7 °C, and for Dioscorea hispida fibres, it was 315.4 °C. The particle size determination and SEM analysis results also contributed to the excellent thermal stability of Dioscorea hispida starch and Dioscorea hispida fibres. The crystallinity index of Dioscorea hispida starch was 27.5%, which was lower than that of Dioscorea hispida fibres of 39.0%. This experiment revealed that Dioscorea hispida waste could be promising alternative biomass and sustainable material with excellent potential as a renewable filler material for food packaging applications. Tests with other geographical origins of Dioscorea hispida tubers should be conducted in a future study to determine the best characteristics for the production of biodegradable packaging films.


The main applications of ionic liquids in chemistry and material research on one of the most important natural polymers—starch—are presented in this review. A brief characterization of ionic liquids and the advantages and disadvantages of using them in the modification and processing of polysaccharides is presented. The latest reports on the use of various ionic liquids as solvents or co-solvents; as media for synthesizing starch derivatives in oxidation, etherification, esterification, and transesterification, with particular emphasis on biocatalyzed reactions; and as plasticizers or compatibilizers in the processing of starch-based polymers have been investigated. The current trends, possibilities, and limitations of using this type of compound for the production of functional starch-based materials are presented. One of the main problems in the modern synthesis or modification of various types of materials is the use of solvents and reaction media that generate a number of threats, particularly environmental ones. These threats result from the source and synthesis of the solvent, its functional properties, and, finally, its disposal . This ha4−, NO3, PF6−, SO42−, and imidazolium or pyrimidinium cations, could also be synthesized [HCOO]), 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]Me2PO4)) as solvents and p-TsOH as a catalyst. The process was carried out at 80 °C using microwave heating. The depolymerization extent and dissolution time of the starch were specific to each IL. The authors demonstrated that barley starch immersed in the tested ILs was dissolved and depolymerized into water-soluble oligomers (1000–2000 Da). The reaction time was shorter for ILs with a smaller cation . A larger cation can cause steric hindrance due to the longer alkyl chain in some ILs  and slow down the reaction.The effect of [BMIM]Cl on the dissolution and degradation of starch for its modification was studied by Kärkkäinen et al. . The disIn other studies, [BMIM]Cl was also involved in the preparation of biocomposites with thermoplastic starch . The preILs are also used for the preparation of thermoplastic starch (TPS). Sankri et al. used [BMIM]Cl to obtain TPS . The IL l-lactide) (starch-g-PLLA) demonstrated good adhesion between the two components, evidenced by SEM observations. Although the grafting efficiency of PLLA reached 30%, the chains of the grafted PLLA were relatively short, as calculated according to a standard curve created by the FTIR method. Due to the high dissolubility of starch and chemical inertness, [AMIM]Cl was employed as a reaction medium for the preparation of a starch-based macro-initiator using atom transfer radical polymerization (ATRP) [4], pyrrolidinium chloride [H-NMP][Cl], and morpholinium bisulfate [H-Mor][HSO4]) as acid catalysts to prepare biodegradable polycaprolactone by ring-opening polymerization Cl, can be used for grafting starch. In one study, 1-allyl-3-methylimidazolium chloride was found to be a promising solvent in the ring-opening graft polymerization of L-lactide onto starch chains . The obtn (ATRP) . The autn (ATRP) . The gran (ATRP) . Abdolmarization . The res2O ratio on the behaviors of both these starch types. The effects of the IL were found to be dependent on the starch source and structure. Gelatinization and dissolution of starch occurred competitively and synergistically when the [EMIM]OAc/H2O ratio was increased. Differential scanning calorimetry (DSC) results showed changes in the transition of starch from a single endotherm to an exotherm/endotherm and then to a single exotherm with the increase in IL. Excellent solvent properties of maize starch were confirmed by its low gelatinization temperature, which was only 44.4 °C in [EMIM]OAc/H2O. In another study, [EMIM]OAc was used as the solvent for a corn starch and cellulose mixture [Lu et al. examined [EMIM]OAc as a solvent for maize and potato starch . They in mixture . The autIn recent studies, the effectiveness of [EMIM]OAc as a plasticizer in the preparation of starch-based films has been tested ,62,70. TIn the case of the synthesis of starch carbonyl derivatives in ILs, corn, potato, and cassava starches have primarily been described. Only a few successful attempts to etherify starch are currently known. One of them involved the use of [BMIM]Cl in the reaction of corn starch with glycidyltrimethyl ammonium chloride (GTAC), catalyzed by sodium hydroxide . The pro5PMo10V2O40) as precursors. A combination of POMs with choline chloride exhibited the same level of effectiveness as traditional catalysts such as Fe3SO4. Moreover, an IL-based catalyst for starch oxidation (such as POMs) has been found to be thermoregulated and ecofriendly.The application of ILs in the oxidation of starch is not common . Only poThe use of ILs in the esterification of starch with acid chlorides and anhydrides as well as short and long-chain carboxylic acids is much more common and has attracted great interest . The eff4 and [BMIM]OAc NO3) Cl, but the reactions had to be carried out at much higher temperatures Br, [C16-3-C16 IMBr2], and [C16-12-C16IM]Br2, which exhibited surfactant properties, were used in this reaction. The significant increase in the efficiency of biocatalyzed starch esterification and achievement of a high DS (2.75) was explained not only by the use of non-toxic ILs but also by more efficient microwave heating. Thus, the authors presented a highly efficient and environmentally friendly method of obtaining hydrophobic and thermoplastic starch derivatives in the presence of ILs. In 2019, Żarski et al. again used immobilized lipase from T. lanuginosus and [BMIM]Cl to esterify potato starch OAc . During ried out . HoweverMIM]NO3) . This tiMIM]NO3) . The syneratures . The epoeratures . Similareratures . This tinown ILs . Lipase o starch . This tio starch  was explo starch . Potato o starch  have beeo starch , and funo starch  characteThe most commonly used ILs for the modification of starch to improve its properties are 1-butyl-3-methylimidazolium chloride [BMIM]Cl, 1-allyl-3-methylimidazolium chloride, and 1-ethyl-3-methylimidazolium acetate [EMIM]Ac. They are used in many applications, including the dissolution and gelatinization of starch. In addition, they are used as solvents in the ring-opening graft polymerization of the other polymers onto starch chains. Another process that successfully uses ILs is the preparation of thermoplastic starch, wherein they play the role of a plasticizer. ILs have better plasticizing properties for starch than the most popular plasticizer, which is glycerol. In particular, ILs are often used as solvents in the preparation of starch modified with acid anhydrides or short- and long-chain acids. The obtained modified starches have hydrophobic properties, which are more desirable in industries such as packaging. In recent years, there have been several reports on the preparation of hydrophobic starch derivatives by enzymatically catalyzed esterification with the use of ILs. ILs usually constitute the reaction medium or are used for the pretreatment of the starch (gelatinization). The starch esters obtained in biocatalyzed reactions in the presence of ILs are characterized by a high DS, which can be additionally increased by using non-ionic surfactants. Biodegradability, non-phytotoxicity, hydrophobicity, and improved mechanical properties are the features of films extruded from starch esters obtained in the enzymatically catalyzed reaction of starch with fatty acids such as oleic acid and hydrolysates of pure and waste rapeseed oil. The combination of biocatalysis and ILs is highly advantageous from an economic point of view (higher process efficiency), and it is also pro-ecological (shorter time and lower temperature of process).


The curve has begun to flatten and many of us have returned to the office and the operating theater. For many of us, this is likely the longest time in our careers without seeing patients, performing aesthetic procedures, and traveling to meetings. We are glad this is now in our rearview mirror and we can return to work as normal or should we say what we hope will be a temporary new normal?Aesthetic Surgery Journal offered during the lockdown. Through the contributions of many of our lifelong peers and our editorial team, both ASJ and ASJ Open Forum quickly became a go-to resource during COVID-19.We are proud of the educational efforts the ASJ to choose the topics and speakers and we called upon our Section Editors. They rose to the challenge and did not disappoint with hour-long talks followed by copious Q&A sessions that in many cases lasted an additional hour. Dr. Cheng moderated all of these sessions with her own unique style and acumen, curating the questions with assistance from our colleague Dr. William J. Knaus . She even scrubbed out during 1 session to moderate, which is testimony to her passion and commitment to educating the next generations. In some form or fashion, the educational opportunity conceived by Dr. Cheng will continue after COVID and we at ASJ intend to explore options for furthering her efforts in the future.We must give credit where it is due. Dr. Angela Cheng, Associate Professor of Surgery in the Division of Plastic and Reconstructive Surgery at Emory University School of Medicine, conceived of the idea of “Virtual Grand Rounds” so that residents and fellows would not miss out on learning opportunities during the quarantine. Her brilliant and original idea was to host Zoom meetings 3 times per week, with 2 sessions featuring reconstructive topics and 1 session featuring aesthetic topics. She graciously agreed to allow those of us at ASJ Virtual Grand Rounds, our Executive Editor Phaedra Cress conceived of ASJ GEMS —Zoom meetings featuring experts and targeting the international community. As international submissions continue to soar, we know there is so much we can teach and learn from the global community. ASJ GEMS tackled topics ranging from eyelid complications to managing vaginal rejuvenation patients, plastic surgery practices after COVID-19, and body contouring after massive weight loss. Engaging with our international aesthetic colleagues from all 7 continents brought the presentations and discussions to a new and also gave us the opportunity to promote our new open-access journal, ASJ Open Forum, which is free to read for everyone. Allergan graciously agreed to support several of the ASJ Virtual Grand Rounds sessions, and we would like to offer our sincere thanks for promoting ASJ’s educational efforts during this challenging time.On the heels of the success of ASJ Virtual Grand Rounds and ASJ GEMS sessions were recorded and are available on Radar Resource for journal subscribers and The Aesthetic Society members. We know these videos will provide valuable knowledge for many years to come.Many of the ASJ Journal Club also continues to thrive with record attendance at the May session that featured authors such as Dr. Frank Lista, ASJ Breast Section Co-Editor, Dr. Bradley Calobrace, and our new lead Next Generation Editor, Dr. Ryan Austin. These sessions are available on the ASJ website and our YouTube channel. They will continue to be held on the first Tuesday of each month at 8:00 pm EDT (Eastern Daylight Time). The https://www.surgery.org/private/webinars.Finally, we salute The Aesthetic Society who convened a task force chaired by Dr. James Fernau making formal recommendations on reopening the office and resuming elective procedures. They have also held weekly webinars on COVID-19 and have made those recordings available free to all. The task force recommendations and webinars are available here: https://academic.oup.com/asj/pages/practice-management-thematic-issue and includes practice management articles by leading aesthetic surgeons, such as Dr. Jeffrey Kenkel, Dr. Guy Massry, Dr. James Zins, Dr. Jamil Ahmad, Dr. Greg Evans, and many others.In addition to these virtual learning resources, we thank Dr. Tom Fiala who served as Guest Editor for a thematic issue entitled: “Practice Management and Getting Back to Work After a Pandemic” that can be read here ASJ and ASJ Open Forum and we wish you all continued safety and good health. We know we will meet again “face-to-face” at an upcoming conference and we welcome that opportunity.Thank you for continuing to support Aesthetic Surgery Journal and Aesthetic Surgery Journal Open Forum. The articles are identical and either citation can be used when citing this article.This article has been co-published with permission in the The authors received no financial support for the research, authorship, and publication of this article.


The relationship between in vitro and in vivo starch digestion kinetics was studied in portal vein catheterised pigs fed breads varying in dietary fibre (DF) content and composition. The breads were a low DF white wheat bread, two high DF whole grain rye breads without and with whole kernels and two experimental breads with added arabinoxylan or oat β-glucan concentrates, respectively. In vitro, samples were collected at 0, 5, 10, 15, 30, 60, 120 and 180 min and the cumulative hydrolysis curve for starch was modelled, whereas the in vivo cumulative absorption models for starch were based on samples taken every 15 min up to 60 min and then every 30 min up to 240 min. The starch hydrolysis rate in vitro (0.07 to 0.16%/min) was far higher than the rate of glucose appearance in vivo (0.017 to 0.023% absorbed starch/min). However, the ranking of the breads was the same in vitro and in vivo and there was a strong relationship between the kinetic parameters. Bread is a stable carbohydrate rich food consumed in various forms around the world . In Denmark, bread accounts for ~17% of dietary energy intake . It is kd-glucan (referred to as β-glucan), which has been approved by the Food and Drug Administration in the US [The presence of DF in cereals, either occurring naturally or added as concentrates, can influence the glycaemic response due to its physicochemical properties in the gastrointestinal tract. Soluble DF may form viscous solutions or gels in the stomach that delay gastric emptying and physically inhibit the rate of digestion and absorption of starch in the small intestine,  therebyn the US  and the n the US  for healn the US ,12, simin the US ,13.The glycaemic response is related to the rate of starch digestion in the intestine and the GI values of cereal products have been positively and negatively related to rapidly available glucose and slowly available glucose, respectively, determined in vitro ,15. In tThe breads studied were kept frozen from a previous in vivo kinetic study with portal vein catheterised pigs . They co2SO4 for 1 h was used instead of 1 M H2SO4 for 2 h for the NSP analysis. The content of non-digestible carbohydrates (NDC) was determined by direct acid hydrolysis without starch removal and alcohol precipitation and NDC calculated by the subtraction of the starch content. The content of low molecular weight ((LMW) NDC) was calculated asThe breads were freeze-dried and ground to a particle size less than 0.5 mm to make the samples homogenous for chemical analyses. All analyses were performed in duplicate. Dry matter (DM) was determined by drying to a constant weight at 103 °C for 20 h, ash was analysed by the AOAC method 923.03; AOAC) , protein.03; AOACThe content of resistant starch (RS) in the breads was calculated asA modification of the in vitro starch digestion assay of Vangsøe et al.  and Engl2  were prepared in duplicates, except the blank standard which was prepared in singular.All the reactions and sample preparations were performed in Greiner 50 mL centrifuge tubes . Before the analysis, the frozen beads (stored at −20 °C) were partially defrosted and ground  for 20 s at the same speed and constant rhythm. The in vitro analyses were performed in triplicates by weighing 800 mg breads (245–360 mg of starch) into the centrifuge tubes and 50 mg guar gum  to ensure similar viscosity in all the samples. Potato starch  was used as a reference. Glucose standards , dissolved in 0.1 M sodium acetate buffer  with 4 mM CaCl2. To hydrolyse the starch to glucose, 200 µL of amyloglucosidase  diluted in 5 mL of water was added to each sample and incubated in a water bath at 70 °C for 30 min. After the 30 min, the samples were placed in boiling water for 10 min, diluted in 35 mL of water and centrifuged at 2980× g for 10 min. The timed aliquots in the 66% ethanol solution were centrifuged at 2980× g for 10 min before glucose determination using a glucose oxidase kit  after adding 260 µL of GOD-POD to 25 µL of the sample in a microplate and incubated for 20 min in a 40 °C oven before measuring in a plate-reader  at 510 nm.All samples received 5 glass balls to enhance agitation and provide grinding action. Initially, the samples were incubated horizontally in a 10 mL Pepsin solution  for 30 min in a water bath at 37 °C to mimic gastric digestion. After that, 10 mL of 0.25 M sodium acetate buffer was added to neutralize the acidity, and the samples were incubated for 10 min under the same conditions. In an arranged scheme for precise timing in the collection points, an enzyme mix containing Pancreatin , amyloglucosidase  and Invertase  was added as described by Kasprzak et al. [g for 10 min and glucose was determined in a coupled reaction with NADP+ as described by Larsson and Bengtsson [The content of free glucose was determined separately, by weighting duplicated samples of 500 mg into a centrifuge tube, adding 25 mL of acetate buffer (0.1 M at pH 5.0) and incubating for 60 min in a water bath at 65 °C. After cooling to room temperature, the samples were centrifuged at 2980× engtsson .The in vivo data were obtained from the study of Christensen et al.  where tht is the glucose released at time t, C0 is the glucose released at time 0, and TS is total starch. The hydrolysis of starch in the time frames 0–5 min, 5–10 min and 10–15 min were calculated by taking the cumulative starch hydrolysed at the end of the periods, at 5, 10 and 15 min, and subtracting the cumulated starch at the start, 0, 5 and 10 min, respectively.The proportion of hydrolysed starch (%) over time in vitro was obtained from the released glucose multiplied by a factor of 0.9 to convert to anhydro sugars:p is the concentration of glucose in the portal vein, Ca is the concentration of glucose in the mesenteric artery, F is the blood flow in the portal vein at time period dt, Q is the cumulative amount of starch absorbed from t0 to t300 and TS is the total starch.The absorbed starch in vivo was calculated based on absorbed glucose to the portal vein, as described by  and convi is the effect of diet  and εij describes the random error. Levels of significance were reported as being significant when p < 0.05.The data from the in vitro studies were analysed by a one-way analysis of variance model for each time point of the hydrolysis curve and for the generated variable:i is the effect of diet , βj the effect of the pig and εijk describes the random error. Levels of significance were reported as being significant when p < 0.05.The data from the in vivo study were analysed by a two-way analysis of variance model for each time point of the hydrolysis curve and for the generated variable:The relationship between the cumulated hydrolysis of starch in vitro and time was described by a mechanistic growth model:The relationship between the cumulated hydrolysis of starch in vivo and time was described by a Gomperts 3P model:k values obtained in vitro and in vivo was analysed by a linear regression model:i is the slope and εij describes the random error.The relationship between the ®, Version 13.2, .All statistical analyses were carried out using JMPThe WWB bread had the highest total starch content (711 g/kg DM), whereas the content was lower in all high DF breads (514–612 g/kg DM) . The breT = 0 was significantly different (p < 0.05) between breads, being highest for AXB, followed by WWB, WRB and lowest for WRBK and BGB; the last two breads were not significantly different (R2 > 0.93) following first order kinetics. The rate of hydrolysis differed between the breads up to 30 min with the highest hydrolysis rate for WWB and the lowest for BGB, and with the other breads in between , followed by WRB (10.4% starch/min), WRBK (8.7% starch/min) and finally AXB and BGB (7.4–8.5% starch/min) . From 5 2R ˃ 0.98). The cumulative starch hydrolysis after 15 min was highest for WWB and the lowest was from WRB and WRBK, with AXB and BGB in between (p < 0.05). At 30 min and onwards, there was no significant difference between the hydrolysis rates between the breads but the WWB continued to give the highest numeric values and WRBK and WRB the lowest ones. The cumulated starch hydrolysis approached the asymptotic values (57–72%) at approximately 240 min for all breads and with no difference between the breads.The cumulative portal glucose appearance expressed in units of hydrolysed starch per 100 g of dry starch is shown in k values in vitro varied from 0.0744 to 0.1595% hydrolysis/min with significant differences between WWB and BGB  and when the data were combined with the data from the previous study of Vangsøe et al. [k values was r = 0.71 (p < 0.05)  .WM β-glucan [The current study investigated the in vitro starch digestion kinetics of breads varying in total DF and the proportion between soluble and insoluble DF. Compared to the previous study of Vangsøe et al. , samplesβ-glucan  resultedβ-glucan . Due to β-glucan . Arabinoβ-glucan ,30 also β-glucan . In vivoβ-glucan  and glucβ-glucan . In humaβ-glucan , possiblThe WRB also resulted in a lower rate of hydrolysis the first 5 min of incubation, which corroborates with Juntunen et al. , who fouThe inclusion of intact kernels into breads have been shown as an efficient way of regulating the rate of starch hydrolysis because the insoluble fibrous network surrounds starch, and thereby forms a physical barrier to α-amylases and limits the degree of starch gelatinization ,34. In tk values of the raw diets in the study of Vangsøe et al. [k values in vitro and in vivo obtained not only with the breads of the current study but also with the raw diets of the study of Vangsøe et al. [The in vivo cumulated absorption of glucose found in this and other studies ,35,36 are et al. ; the hige et al.  is encouFor all diets, the in vivo starch digestion displayed lower digestion asymptotic values than in vitro and slightly lower than the values reported in the study of Vangsøe et al. . The lowk values obtained in vitro and in vivo, not only with breads in this study but also when including data from a previous study, indicating that the in vitro model can provide valuable information about the digestion rate in the gastrointestinal tract of value not only in a pig model but rather in general.The in vitro method ranked the breads in a similar manner as in vivo but the rate of glucose release was much faster in vitro than in vivo in portal vein catheterised pigs. Although the in vivo model is more complex than the in vitro model used, it is encouraging that there is a strong correlation between the


Yarrowia lipolitica yeast production. Various acid concentrations and various roasting temperatures were used to determine effects of process conditions on ester properties, including the number of acid residues attached to starch chains, starch susceptibility to amylolysis, and thermal characteristics of starch phase transitions. Study results demonstrated the effect of both the composition and the dose of the fermentation broth and of roasting temperature of starch on the number of acid residues attached to starch chains. Citric acid was more susceptible to esterification with starch (DS = 5.65%) compared to the α-ketoglutaric acid (DS = 0.12%). In the case of the latter, a higher degree of substitution was determined in the esters produced at higher roasting temperatures. The lowest digestibility (RS = 20%) was demonstrated for the starch esters with the highest degree of substitution with citric acid, whereas all starch esters showed decreased values of the thermal characteristics of pasting.Potato starch was esterified with carboxylic acids contained in the fermentation broth from  Starch modification through esterification involves starch chain reactions with inorganic and organic acids, salts of inorganic acids, as well as anhydrides or chlorides of organic acids. During the esterification process, acid groups linked by ester bonds with carbon atoms at positions 2, 3, and 6 of the anhydroglucose rings attach to the molecules of amylose and amylopectin . ProductApart from ample descriptions of starch ester synthesis upon starch modification with homogenous reagents, the available literature provides some reports on starch esterification with mixtures of acids. Šubarić et al.  and AčkaYarrowia lipolityca yeast production. This broth contains various naturally-synthesized acids whose ratios differ depending on yeast strain applied, substrate used for biomass production, and culture conditions [Ample research works indicate the feasibility of starch modification using various acids and methods. Most of them concern the use of pure reagents as esterifying agents, whereas only few describe starch modification with naturally-synthesized acids derived from natural sources, like agri-food waste, but not isolated from them. An interesting method of starch modification involves the use of the fermentation broth from nditions ,18,19,20Yarrowia lipolityca are capable of absorbing carbon from agri-industrial wastes [Yeast of the species l wastes  and of cl wastes ,18,19,20l wastes . The feaThe possibility of starch modification with a mixture of organic acids contained in a fermentation broth was described in our previous paper . HoweverYarrowia lipolitica yeast production and temperature of starch roasting on selected properties of starch esters produced by starch chain esterification with carboxylic acids from the broth.This study aimed to determine the effect of the composition and dose of the fermentation broth left after Yarrowia lipolitica yeast. Experiments were carried out with two culture broths having various concentrations of organic acids, which were obtained from the Department of Food Biotechnology and Microbiology of the Wrocław University of Life Sciences. Their composition was as follows:Potato starch produced by PEPEES Niechlów in 2018 was modified with various doses of a mixture of carboxylic acids from the fermentation broth of Broth A—α-ketoglutaric acid 60 g/L; citric acid 18 g/L; pyruvic acid 1 g/L.Broth B—α-ketoglutaric acid 49 g/L; citric acid 46 g/L; pyruvic acid 1.5 g/L.Starch was modified according to the method for starch citrate production described by Kapelko-Żeberska et al.  and ZdybSeries I: Native potato starch was mixed with the culture broth. Broth doses were adjusted so as to obtain 15, 20, 25, 30, 35, and 40 g of the acids from the broth in 100 g starch dry matter. The mixtures were air-dried at 40 °C for 24 h, then cooled and ground. Next, the mixtures were dried, ground, and roasted in a convective dryer  at 130 °C for 3 h. Afterward, the roasted samples were rinsed with portions of 60% ethyl alcohol  at an alcohol to sample ratio of 4:1 to remove the excess of the reagent. Then, the samples were rinsed ten times, poured with 60% ethanol and left covered at a room temperature for 24 h. The rinsing cycle was repeated three times. The blank sample was prepared acc. to the analogous procedure but without broth addition.Series II: Starch preparations were produced as in series I, but the culture broth added contained 20 g of acid per 100 g of starch, and the dried mixtures were roasted at various temperatures: 120, 130, 140, or 150 °C.The degree of starch substitution (DS) was expressed as percentage content (g/100 g) of acid residues in the modified starch preparation and determined according to the method described by Zdybel et al. .−1 were used. Compounds were identified based on chromatograms of pure chemical standards [Two grams of the starch preparation tested were mixed with 100 mL of 0.5 M NaOH  and left at room temperature for 24 h with continuous stirring. After this time, 250 mL of concentrated ethyl alcohol  were added and the mixture was left again at room temperature for 24 h. The solution was filtered and concentrated to a volume of 50 mL. The content of acid residues esterified with starch was determined by HPLC . A Carbohydrate H + column coupled with a UV detector (λ = 210 nm) and a refractometric detector at 65 °C with a liquid phase (25 nM trifluoroacetic acid) flow rate of 0.6 mL mintandards ,27. The The phase transition characteristics were performed with a DSC 822 differential scanning calorimeter . Ten milligram dry weight of the modified starch preparations were weighed into a 100-µL aluminum crucible. Then, 30 mg of water were added and the crucible was covered with a lid. The samples thus prepared were conditioned at room temperature for 30 min and then placed in a calorimetric chamber at 25 °C and heated to 100 °C at a heating rate of 4 °C/min. The temperatures of onset, maximum, and end of the phase transformation, and the specific heat of phase transformation were read from the thermal curve obtained. The analysis was carried out in triplicate ,25.An aqueous suspension of the modified starch preparations (0.72 g of preparation per 100 mL of solution) was prepared, which was diluted twice with an acetate buffer (pH = 4.3). The mixtures were then placed in a 37 °C water bath, and enzymes (α-amylases and glucoamylases) were added . The amount of enzymes was adjusted to ensure complete saccharification after 20 min of hydrolysis of the native starch paste. Samples were incubated at 37 °C for 20 and 120 min. Afterward, a 1-mL sample was taken in which the enzymes were deactivated by placing the sample in a water bath at 100 °C. After cooling, the glucose content was determined using a glucose assay kit , which contains glucose oxidase and peroxidase. The concentration of the resulting colored complex was measured spectrophotometrically  at 500 nm and compared with a standard curve. The determined amount of glucose was used to calculate the percentage of rapidly digestible starch (RDS), slowly digestible starch (SDS), and amylolysis-resistant starch (RS) in the starch preparations. The analysis was carried out in triplicate .Study results were subjected to a statistical analysis using Statistica 13.1 software package . Two-way analysis of variance was conducted. The significance of differences between mean values was determined with Duncan’s test at a significance level of α = 0.05.A similar dependency was noted while analyzing starch substitution with α-ketoglutaric acid. Only the 20 g dose of acid mixture added to starch caused an increase in α-ketoglutaric acid content compared to the dose of 15 g. The successive increase in the dose of acids had no effect on the amount of α-ketoglutaric attached to starch chains.The analysis of the roasting temperature on starch substitution degree demonstrated the temperature of 130 °C to be optimal in the samples with a higher content if citric acid. The successive increase in roasting temperature had no effect on the amount of acid attached. In the case of the broth with a lower citric acid content, the roasting temperature of 140 °C resulted in a small increase in starch DS compared to the samples roasted at 130 °C. However, the successive temperature increase failed to ensure a statistically significant increase of the degree of substitution. As reported by other authors, the long-term roasting at a high temperature can decrease reaction dynamics due to the degradation of citric acid , therefoNone of the produced preparations contained pyruvic acid, despite its presence in the fermentation broth .The roasted starch has small amounts of resistant starch (RS) and slowly-digestible starch (SDS), which was confirmed in works of other authors . The reaThe analysis of the effect of roasting temperature on digestibility of starch esters produced with broth A  revealedIn the case of starch esters produced at various roasting temperatures , both thThe study demonstrated the effect of both the composition and dose of culture broth as well as of roasting temperature on the number of acid residues attached to starch chains. Citric acid was more susceptible to the esterification with starch compared to the α-ketoglutaric acid. At acid doses below 35%, the degree of starch substitution with citric acid increased along with increasing amounts of acids used. In turn the degree of starch substitution with α-ketoglutaric acid showed no changes at acids doses above 20%. In the case of α-ketoglutaric acid, a higher degree of starch substitution was determined in the preparations produced at the higher roasting temperatures. Lower digestibility was found for the preparations having the highest degree of substitution with citric acid. All starch esters produced showed decreased values of the thermal characteristics of pasting.