Vigna unguiculata), chickpeas (Cicer arietinum) and kidney beans (Phaseolus vulgaris). Texture analysis and in vitro oral-gastro-intestinal digestion of each legume was combined with kinetic modelling to explore the rate and extent of their changes observed during hydrothermal processing. All three legumes showed rapid initial texture decay in the first 30 min of processing. Chickpeas showed the fastest rate of texture degradation with processing duration, whereas texture degradation of kidney bean was slower but reached the lowest hardness value among all beans when processed up to 120 min. The rate of starch and protein digestion increased with prolonged processing duration, whilst showing an inverse relationship with texture values. The extent of starch digestion continually increased with processing duration for all three legumes, whereas the extent of protein digestion decreased after 60 min in cowpeas. This study systematically demonstrated how choosing different processing times can modulate the rate of texture degradation, starch and protein digestion in legumes. The findings of this study can aid consumers and manufacturers on optimal processing to achieve the desired texture or modulate starch and protein digestibility.Legumes are a vital candidate in the fight for food security as a sustainable and nutritious food source. The current study systematically investigated the effects of hydrothermal processing of varying durations (15–120 min) on the texture, starch and protein digestibility of cowpeas ( Vigna unguiculata), chickpeas (Cicer arietinum) and kidney beans (Phaseolus vulgaris).Legumes are a sustainable source of (plant-based) protein, carbohydrate, fibre and minerals. Legumes are consumed as a staple food in developing countries due to their economical cost and long  shelf life . Legume Despite the health and sustainability benefits, legumes are not as widely consumed in developed countries. This is due to a combination of factors, such as the involved process in their cooking, the potential of gastrointestinal discomfort from gas, undesirable odour  and geneIn an attempt to maximise the utilisation of legume seeds, hydrothermal processing  is the most widely adopted technique in domestic household cooking as well as in the food industry to be sold as canned products to consumers for convenience purpose. Hydrothermal processing softens a legume’s texture, so it is palatable for consumption. Moreover, reduction and/or inactivation of antinutrients has been known effective with hydrothermal processing . DespiteThere is an emerging interest in utilising a kinetic approach in describing the starch and protein digestibility of foods ,19,20,21ct = 15–120 min) on three types of legumes, namely cowpeas (Vigna unguiculata), chickpeas (Cicer arietinum) and kidney beans (Phaseolus vulgaris). The three legumes were chosen based on three criteria: (i) each legume is widely cultivated in many regions of the world; (ii) each legume has a distinct macronutrient composition; and (iii) each legume was of different shape, colour and size. Hydrothermal processing was chosen because it is the most common and widely available processing technique to consumers regardless of socio-economic status. In this study, legumes processed at different hydrothermal processing duration were digested in an in vitro oral-gastric-intestinal process and the hydrolysis of starch and protein was followed as a function of digestion time (up to 6 h). The softening of legume texture along the hydrothermal processing duration was also measured.The objective of the current study was to investigate the effects of hydrothermal processing duration (Vigna unguiculata), chickpeas (Cicer arietinum) and kidney beans (Phaseolus vulgaris) were purchased from a local market in Dunedin  in August 2017. Deformed, discoloured and damaged seeds were discarded and excluded from the experiment. The seeds were then vacuum-packed in opaque aluminium bags and stored in the dark at 4 °C until processing and analysis. Starch, protein, lipid and moisture content of each legume  for 16 h at 20 °C in an incubator , after which the soaking water was discarded. The seed’s moisture content increased from 9.13% to 58.24% during soaking due to water uptake overnight. The soaked legume was aliquoted (~100 g) and transferred into six individual stainless-steel sieve cages . A portion (~100 g) of the soaked legumes was set aside to represent an unprocessed state (ct = 0 min). Two large stainless-steel hydrothermal processing pots (10 L) and induction hot plates  were used in this experiment, where three cages were assigned to each of them . The legumes were boiled (~95 °C) in excess distilled water (≥1:20 (w/v) seed-to-water ratio). At each hydrothermal processing time point, the specified cage was removed from the pot and immersed in ambient tap water (~20 °C) to stop the thermal treatment whilst minimising starch retrogradation. The three legumes were then divided into two lots. The first lot was subjected to texture analysis and in vitro digestion within the same day (see Legume seeds (500 g) were soaked in distilled water at a seed-to-water ratio of 1:5  was monitored using a thermocouple  and recorded every 5 s using a data logger . The time taken for the interior of the seed to reach 90 °C was approximately 30 min .Texture analysis of unprocessed, soaked, and boiled legumes were conducted using a compression test, as per Zhang et al. , with moTwo textural parameters were determined from each of the obtained texture profiles. Hardness is defined as the maximum force achieved during the compression test, represented in Newtons (N) . CohesivAt the end of each compression test, the disintegrated legumes were removed from the platform and probe using a clean spatula. The disintegrated samples were expected to mimic the way boiled legumes undergo a size reduction in the oral phase before being swallowed, and hence, were found to be suitable to be utilised for the subsequent in vitro digestion . The disMechanically disintegrated legumes obtained from the compression test  were immThe in vitro starch and protein digestibility kinetics of compressed legumes were conducted according to the harmonised INFOGEST static in vitro digestion procedure . The conw/v) trichloroacetic acid  to stop protein hydrolysis [Samples of digesta were withdrawn from the digestion vessels at different intervals throughout the 6 h-long in vitro digestion procedure to determine the degree of starch and protein digestibility. In the gastric phase, digesta were withdrawn at 0, 60 and 120 min for starch digestibility analysis, with additional time points at 30 and 90 min for protein digestibility. In the intestinal phase, digesta were withdrawn at 0, 20, 60, 90, 120, 180 and 240 min for both starch and protein digestibility analysis. Digesta allocated to starch digestibility analysis (0.5 mL each) were immediately subjected to a heat shock treatment at 100 °C for 10 min to stop activity of digestive enzymes [drolysis . SamplesThe total starch content in boiled and uncooked legumes was quantified using the Megazyme Total Starch Assay Kit  . The Megg for 10 min at 4 °C  and supernatant was collected. D-glucose within the supernatant was measured according to the kit’s instructions. The amount of D-glucose released was converted by a factor of 0.9 (=162/180) into starch equivalents [Legume starch digestibility was approximated through measurement of the D-glucose present in the aqueous phase of the digesta when hydrolysed using the Megazyme D-glucose Assay Kit . Digesta were gently thawed overnight at 4 °C and then centrifuged at 1090× ivalents . This ino-phthalaldehyde (OPA) spectrophotometric assay based on the work of Lassé et al. [g for 20 min. For each digestion time studied, 26.7 µL of supernatant was withdrawn and placed into a 96-well microplate . An OPA solution (200 µL) containing 6 mM o-phthalaldehyde , 50 mM sodium tetraborate , 1.0% (w/v) sodium dodecyl sulfate , 2.0% (v/v) methanol  and 0.2% (v/v) β-mercaptoethanol , prepared and used within 2 h of the assay, was added to the microplate. This resulted in a digesta-to-OPA solution ratio of 2:15. The microplate was placed inside a plate reader  and the reaction was allowed to proceed with agitation for 30 s, and then without agitation for 90 s. Finally, the absorbance of the coloured solution was read at 340 nm. Solutions of L-serine standards  were used to prepare a standard curve, from which the hydrolysis of proteins and peptides within the digesta were calculated. Protein digestibility was then expressed as mg L-serine equivalent per g of legume sample dry weight. The digesta obtained at each time point were analysed in four replicates.Legume protein digestibility was measured using an é et al. . Digestact = 0). The converted experimental data were fitted to the model using SAS  via a nonlinear regression procedure using the Marquardt maxiter fitting method from SAS. Two kinetic parameters (k expressed as × 10−2/min and fT expressed as a percentage) were estimated:ct (min); fT indicates the proportional (%) texture properties of legumes at prolonged hydrothermal processing time; T0 represents the initial texture properties of uncooked legumes ; and k indicates the rate constant (×10−2/min) of texture changes/degradation due to hydrothermal processing. The standard error of the estimated kinetic parameters from the model was generated as part of the output from SAS.The change in texture of the legumes at varying hydrothermal processing times was described with a first-order fractional conversion model, see Equation (2). To allow a fair comparison of the changes in the texture parameters as a function of hydrothermal processing time between the three types of legumes, the actual value of the texture parameters’ cohesiveness and hardness was converted and expressed as the percentage of the texture parameter with respect to the soaked bean  were estimated:dt (min); xC indicates the asymptotic amount of digested starch (replace subscript x as s) or protein (replace subscript x as p) at prolonged small intestinal digestion; C0 represents the amount of digested starch or protein at the start of small intestinal digestion (dt = 0 min); and xk indicates the rate constant (×10−3/min) of starch (replace subscript x as s) or protein (replace subscript x as p) digestion. The standard error of estimated kinetic parameters model was generated as part of the output from SAS.During the 4 h-long in vitro small intestinal digestion phase, the changes in the starch and protein digested for each legume sample, boiled at varying durations, were also described using a first-order fractional conversion kinetic model, see Equation (3), using SAS software. Two kinetic parameters , and a visual examination of the residual  and parity plots:m is the sum of observations; modelSSQ is the sum of squares of the regression model; totalSSQ is the sum of squares of total observations; and j is the number of parameters estimated by the model.The fitting quality of the experimental data obtained from texture analysis and starch and protein digestion to the first-order fractional conversion model, see Equations (2) and (3), was evaluated by calculating the corrected Rxk:LC denotes the amount of starch or protein of a boiled legume sample being digested in vitro at time dt (min); C0 represents the amount of digested starch or protein at the start of small intestinal digestion (dt = 0 min); and xk indicates the rate constant (×10−3/min) of starch (replace subscript x as s) or protein (replace subscript x as p) digestion.For certain cooked legume samples exhibiting a linear starch and/or protein digestogram rather than following a first-order fractional conversion behaviour, a linear regression was used instead to estimate ct and changes in starch and protein digestibility during gastric digestion were compared by one-way ANOVA for significant difference (p = 0.05) , followed by Tukey’s post hoc test.Changes in the texture parameters as a function of Examination and comparison of the visual texture profile can give a rapid insight into the texture of legumes. According to During hydrothermal processing, the texture profile of all three legumes experienced two clear trends, illustrated in Phaseolus vulgaris after hydrothermal processing (95 °C) in excess of demineralised water. A good fit was found when using a first-order fractional conversion kinetic model to describe the texture changes of legumes as a function of processing time (Vigna subterranean (L.) Verdc.) during hydrothermal processing [From the texture profile curve, the cohesiveness and hardness of each uncooked and cooked legume were calculated. In all three legumes, cohesiveness and hardness showed a sharp decrease during the first 15 min of hydrothermal processing, with a sustained decrease until 45 min of hydrothermal processing in cowpeas and chickpeas, and up to 90 min for kidney beans . Hydrothing time . A firstocessing .k and texture of legumes at prolonged hydrothermal processing time; fT), estimated using the first-order fractional conversion kinetic model, see Equation (2), to describe the texture degradation for the three types of legumes during hydrothermal processing are presented in k for cohesiveness and hardness was the highest in chickpeas, followed by cowpeas and kidney beans. In other words, the degradation rate (k) of the texture of cowpeas and chickpeas in terms of cohesiveness and hardness was approximately 1.78–1.94 and 2.13–2.55-fold higher than that of kidney bean, respectively (k) for cohesiveness and hardness may be due to species-specific differences in the middle lamella and cell composition. Another factor that may have contributed to kidney beans’ texture retention is its larger size and homogeneity, compared to cowpeas’ smaller size and chickpeas’ irregular seed surface  during hydrothermal processing was higher than the rate of hardness degradation , the cohesiveness for cowpeas, chickpeas and kidney beans dropped to 12.29, 12.29 and 7.61% of the initial value, respectively  were calculated from the initial texture as detailed in ct ≤ 60 min) than cowpeas or chickpeas.At the end of hydrothermal processing , a typical condition in which legume seeds can harden and take a long hydrothermal processing time (>2–5 h) to soften . The impThe amount of digested starch (%) of uncooked, soaked and hydrothermal processed legume samples at varying durations, during the 2 h-long in vitro gastric digestion is presented in dt = 0 min), it was found that the amount of digested starch in cowpeas, chickpeas and kidney beans increased from 5.98% up to 12.55%, 6.56% to 10.59% and 2.89% to 5.77%, respectively, with increasing hydrothermal processing time between 15 and 120 min , a slight increase in the amount of digested starch was observed in hydrothermally processed cowpeas (up to 8.5%) and chickpeas (up to 1.2%), whereas for kidney beans, changes were very small overall (less than 1%) compared to when dt = 0 min (start of gastric digestion). A considerable increase in the amount of digested starch during the gastric phase is not expected since the only active hydrolytic enzyme present in the digestion vessel was pepsin, as oral α-amylase was previously deactivated by dropping the pH of the digestion mixture to 3. Any increase in the amount of digested starch during the gastric phase in cowpeas and chickpeas may be due to pepsin weakening the encapsulation of the storage protein-encapsulated starch granules. This phenomenon has been illustrated in common beans, wherein hydrolysis of storage protein  was illustrated to enhance starch digestion in vitro [At the end of the 2 h in vitro gastric digestion . This is because the starch gelatinisation associated with boiled legumes had not occurred for the U and S samples, thus limiting the susceptibility of the starch granules to enzymatic hydrolysis . In addidt) before slowing down and reaching a plateau. This is expected as the amount of (gelatinised) starch available to be hydrolysed by amylase (present in pancreatin) becomes rate-limiting. For legumes that were processed at lower hydrothermal processing times, however, it was found that their starch digestograms tended to exhibit a slow linear increase in the amount of hydrolysed starch. To quantitatively describe the starch digestion behaviour of the three legumes, a first-order fractional conversion kinetic model was applied to estimate the rate (sk) and the extent (sC) of starch digestion, see Equation (3). In cases where the samples were observed to not follow a first-order fractional conversion behaviour (in conjunction with a large standard error of the estimated kinetic parameter), a simple linear regression was utilised instead, see Equation (5). In this study, the changes in the starch digestibility of all cooked cowpea samples as a function of dt fitted well with the fractional conversion kinetic model. However, simple linear regression model was applied on chickpeas samples with a ct of 90 min or less, and on kidney beans samples with a ct of 60 min or less. The estimated kinetic parameters sk and sC for all boiled legumes are summarised in sk) increases as a function of ct  for all legumes, suggesting an enhanced enzyme–substrate interaction between α-amylase and starch present in legumes that have undergone a longer hydrothermal processing. A higher estimated sk value indicates that more starch is being hydrolysed by α-amylase per minute at intestinal phase, and in the case of legumes subjected to hydrothermal processing, the starch gelatinised and would have increased their susceptibility to hydrolysis by α-amylase [sk value was observed in kidney beans, increasing from 0.43 × 10−3/min when cooked for 15 min up to 6.93 × 10−3/min when cooked for 90 min, presenting a 16-fold increase in the starch digestion rate (sk).Based on -amylase . TherefoPhaseolus vulgaris) as a function of ct. Expectedly, their estimated sk also increased at prolonged ct, increasing from 0.65 to 1.30 × 10−3/min during ct of 30 min and 120 min, respectively. This is logical, as legume cotyledons naturally have encapsulated starch (and protein), restricting contact of digestive enzymes to their substrates. The rigid cell structure, especially of the cell wall, is a physical barrier to the diffusion of digestive enzymes into the cell, limiting hydrolysis of starch granules in vitro [sk), likely through process-induced cell wall permeability.Another explanation for the increase in the rate of starch digestion is likely through hydrothermally induced cell wall permeabilisation , which cin vitro  and in vin vitro ,48. Therdt = 120 min). These samples also have overlapping predicted sC values, falling within 67.94% to 69.95%  and achieved a similar final amount of starch being hydrolysed at the end of small intestinal digestion. If sC had been able to be predicted for chickpeas and kidney beans, perhaps a similar behaviour might have been observed near their respective gelatinisation temperature.An interesting finding with respect to boiled cowpeas was observed, where COW45, COW60 and COW90 samples appeared to share similar proportions of digested starch (between 65.29% and 66.90%) at the end of the small intestinal digestion  at lower ct (probably due to incomplete starch gelatinisation), the concentration of starch (substrate) remains essentially constant throughout the experimental time frame. Hence, a zero-order model is used to describe the behaviour of chickpea and kidney bean samples, subjected to a shorter c,t during in vitro small intestinal digestion. In agreement with the study of Rovalino-Córdova, Fogliano and Capuano [ct as the concentration of available starch becomes rate-limiting. In other words, the experimental time frame only allows observation of the initial phase (linear part) of the expected starch digestogram. Of course, the in vitro small intestinal digestion was not investigated beyond 4 h in this study because the transit time for digesta passing through the small intestinal ileum is limited and usually occur within 4 h in the human body [In contrast to boiled cowpea samples, the starch digestibility of chickpeas and kidney beans could only be observed to follow a first-order fractional conversion behaviour when they were cooked longer, i.e., 120 min for chickpea and above 90 min for kidney beans. At lower gression a. A line Capuano , a lineaman body .sC) in cowpeas increased from 59.19% to 75.61% from when hydrothermal processing time (ct) was increased from 15 to 120 min. In kidney beans, the estimated sC can be seen increased from 56.37% to 66.92% when increasing ct from 90 to 120 min. Lastly, even though sC was not predicted for shorter hydrothermal processing time, chickpeas had the highest sC, attained at 94.09%, after 2 h of hydrothermal processing. The result suggested that a greater extent of starch digestion (sC) could be associated with higher hydrothermal processing time. A higher sC indicates that proportionally more of the starch granules within the legume cell was able to be accessed by α-amylase, which could be attributed to two major factors. First, hydrothermally induced cell wall degradation could have contributed to the increased sC at the end of 4 h-long small intestinal in vitro digestion. Secondly, hydrothermal processing could promote conformational changes (from protein denaturation) in storage proteins within the cell’s cytoplasmic matrix, thus degrading the encapsulating barrier between α-amylase and starch granules. This would explain an increase in the estimated sC for legumes that had been boiled longer.As illustrated in Bringing together the findings, hydrothermal processing not only increases the rate of starch digestion of the studied legumes, but also the extent of digestion. The main contributing effect is most likely starch gelatinisation and induced cell permeability. Furthermore, while the general trend is the same, each legume, though containing a similar starch content, exhibited different responses to hydrothermal processing. Therefore, it was demonstrated that there are different nutritional implications for different legumes hydrothermally processed for the same duration.ct) nor gastric digestion time (data not shown). The amount of L-serine equivalent (mg) released in the whole digesta per g dry weight was found to be no more than 11.35 mg for all legume type regardless of the duration hydrothermal processing (ct) and whether they were unprocessed or soaked. This may be due to pepsin’s enzyme specificity; as Rovalino-Córdova et al. (2019) reported, pepsin is capable of hydrolysing only ~5% of kidney bean protein even after 6 h of in vitro gastric digestion. Therefore, it is possible that the pepsin utilised in this investigation does not effectively hydrolyse the storage protein present in kidney beans, and thus, the storage protein continued to encapsulate starch (negatively affecting starch hydrolysis). Perhaps the poor specificity of pepsin towards protein in kidney beans is also extended to cowpeas and chickpeas. As such, under the experimental timeframe of 2 h gastric (pepsin) digestion, no discernible protein digestion could be observed. Thus, this section will only discuss results from the small intestinal phase of the in vitro digestion.During the gastric phase of the in vitro digestion, there was no apparent trend in protein digestibility as a function of hydrothermal processing (pk) and extent (pC) of protein digestion, see Equation (3). Again, where the first-order fractional conversion model is unable to predict a kinetic parameter, a linear regression was utilised where appropriate. The estimated kinetic parameters pk and pC for all boiled legumes are summarised in During the in vitro small intestinal digestion, the protein digestogram of boiled cowpea samples exhibited a fast release of digested protein during the first 60 min of the in vitro small intestinal digestion, followed by a gradual plateau b. Converct) has a positive impact on the rate (pk) of protein digestion in mostly all legumes . Another contributing factor may be that the proteolytic enzymes present in pancreatin also experienced enhanced diffusion through the cell wall, thus promoting the enzyme–substrate interaction [pk) and extent (pC) of protein digestion is likely attributed by protein denaturation [The results showed that hydrothermal processing duration  were very comparable for the samples boiled between 15 to 60 min, ranging between 6.09 and 8.72 × 10−3/min. This indicates that hydrothermal processing, ct of up to 60 min, did not appeared to affect pk to a considerable extent. This apparent lack of effect may be related to the presence of antinutrients and their incomplete inactivation within a short hydrothermal processing time. Indeed, it was demonstrated that approximately 45 min was required for the internal temperature of the seeds to reach 95 °C . This difference may be due to an interspecies difference, or the difference in the type and amount of antinutrients in each legume. Chickpeas have been reported to contain lower levels of antinutrients, such as protease inhibitors, compared to other species of legume [In contrast to the larger increase in f legume ,40.pC) at long digestion time (dt = ∞) could only be estimated for cowpeas with the first-order fractional conversion model; hence, the discussion will focus on cowpeas. At the end of the 4 h-long small intestinal digestion, cowpeas showed an increasing trend from an initial 33.25 mg L-serine equivalents released/g observed in COW15, increased to 46.04 mg/g in COW60, before showing a decreasing trend to 39.99 mg/g and 35.99 mg/g as observed in COW90 and COW120, respectively. The initially increasing pC trend observed in COW15, COW45 and COW60 is likely due to a combination of hydrothermally induced protein denaturation and the previously discussed inactivation/removal of antinutrients. The work of Naidoo, Gerrano and Mellem [pC compared to other legume types.The estimated extent of protein digestion . This decrease may be due to heat-induced modifications such as protein insolubilisation and the creation of protein cross-links, as the partially unfolded peptides resulting from hydrothermal denaturation can subsequently aggregate to form complexes, which are resistant to enzymatic proteolytic attack [ct of 60 min for cowpeas.The observed increase in the estimated c attack , who obssk and pk, respectively) of legumes boiled at different durations are discussed in this section.In this study, it was evidenced that hydrothermal processing can cause microstructural changes, leading to loss of legume cohesiveness and hardness, as shown in sk) , a small change of texture in the legumes due to hydrothermal processing could result in a considerable increase of estimated sk. For example, the estimated sk for cowpeas hydrothermally processed within 15 and 30 min fall within a similar magnitude between 6.2 and 8.8 × 10−3/min  owing to solubilisation of the pectin-rich middle lamella [One of the key finding was that a decrease in cohesiveness and hardness of legumes due to prolonged hydrothermal processing could pose a major impact on the estimated rate of starch digestion (ks) a,b. The  lamella ,62.pk) and legume cohesiveness and hardness  tend to exhibit a lower estimated pk (~4–7 × 10−3/min). For boiled samples with moderate texture degradation , these samples were predicted with modest pk values (7–8 × 10−3/min). Lastly, boiled samples with severe texture degradation  exhibited a faster estimated rate of protein digestion (pk > 8 × 10−3/min). In legumes, an overall lower texture parameter as an effect of hydrothermal processing appears to positively impact the estimated pk. Again, this may be due to an increased surface area for enzyme action, due to increased cell separation.Likewise, an exponential relationship was observed between the estimated protein digestion rate  and protein (pk) digestion  and protein (pk) digestion. The nutritional implication is to simply enjoy legumes as a source of starch and protein; however, it is not critical to boil them until their texture is severely degraded. Conversely, to benefit from a slow, sustained starch and protein digestion, the duration of hydrothermal processing can be tailored to result in an appropriate texture for different legume types.The duration of hydrothermal processing required for cowpeas, chickpeas and kidney beans to achieve a softened texture, without severe texture degradation, is likely to be different for each legume. For example, cowpeas and chickpeas to be processed for 30 to 45 min, and kidney beans are required to be processed for 45 to 60 min for them to attain similar hardness within the range of 20% to 25% of their soaked seed. According to Gwala et al. , legumesigestion , for alligestion . Finallyduration . As suchct) and during small intestinal digestion (dt), where the texture degradation of kidney beans was found to be slower than in cowpeas and chickpeas during processing, whereas the estimated rate of starch (sk) and protein (pk) digestion increased with increasing ct, but at different rates depending on the legumes. The estimated extent of starch digestion (sC) at the end of the 4 h in vitro small intestinal phase also increased with samples subjected to increased ct. In cowpeas, the estimated extent of protein digestion (pC) at the end of 4 h in vitro small intestinal phase decreased for samples with prolonged processing time (ct > 60). Future studies could explore the phenomenon potentially occurring during hydrothermal processing of legumes  to gain a mechanistic understanding. Our finding has created valuable insight into how legumes’ texture, as well as starch and protein digestion, may be affected by inter-species differences. In addition, a link between texture and the rate of nutrient digestion was observed. Overall, the starch digestion rate increased, as legumes lose their hardness and cohesiveness when boiled. In the practical perspective, this information could aid household and commercial processes to process legumes to a certain texture level to achieve the desired digestion characteristics, be that to maximise or modulate starch and/or protein digestion.The systematic approach of studying legumes processed for varying duration has never been studied before, and the use of a suitable kinetic approach to describe the texture degradation, starch and protein digestibility is presented for the first time on cowpeas and kidney beans. The kinetic approach demonstrated how each legume responded differently to processing (


This editorial is part of an article series published by the Academy for Multidisciplinary Neurotraumatology (AMN) neurotrauma includes Acquired Brain Injury (ABI) and Spinal Cord Injury (SCI). While injuries to the brain can cause many problems, including troubles with attention, planning, and interaction, the primary result of spinal cord injury remains paralysis [The umbrella term aralysis . Traumataralysis . Some riaralysis . Moreovearalysis . The advHomo Habilis, Australopithecus, Homo erectus) as well as Homo sapiens being traced back to the Paleolithic, when most of the head trauma was caused by animals or injuries [trephination-the oldest documented surgical procedure performed by humans-on over 1500 skulls  worldwide  into the neurocranium”, with most procedures including one or two holes [Interestingly, the practice is still used to treat chronic subdural hematoma (SDH) . Trephinwo holes . Howeverwo holes . Some ofwo holes , 8, 10. wo holes , 8, 10. wo holes . Althougwo holes , 9, 10. wo holes . Sometimwo holes  were usewo holes .The data from Neolithic reveals insightful patterns in neurotrauma, including equal affection of men and women, the rarity of sharp-force trauma , a higher frequency of antemortem trauma in earlier periods, increasing size of the lesion with time, as well as a more common occurrence of healed lesions in the northern Mesopotamia . The disthe first written documentation of brain injuries, the first known surgical treatise, as well as the first medical document in history ,  history  [3, 5, 1The papyrus also showcased the symptoms and the diagnostic procedure in patients with TBI, along with the therapeutic principles and prognostic detailed in specific cases, including mentions of spinal cord injuries with categorization , 14. AmoTreatments included applying fresh meat, honey, or grease on the wound, dressing the wound for those without skull fractures, and positioning the patients upright. The cases’ presentation is well-structured and follows a logical order, representing a possible guideline for the management of combating head injuries . The papThe document mentions four sections  and a classification of fractures into 1) splits, 2) smashes, 3) compound comminuted, and 4) comminuted and depressed . It was standing on the shoulders of giants!While neurotramatology has made significant progress throughout the centuries, its history provides a fascinating insight into the medical landscape of our ancestors. The broad gaps between our eras highlight how humanity has exploited the understanding gained over time to kindle and forge scientific and technological progress – modern neurotraumatology is certainly


Journal of Materials Science: Materials in Medicine (2021) 32:20Correction to: 10.1007/s10856-021-06491-0 published online 27 February 2021The authors agree to withdraw the Fig. Supporting Information-2


International Journal of Neonatal Screening (IJNS) has solidified its position as the preferred platform to publish the scientific output of the members of the International Society of Neonatal screening (ISNS) and professionals in fields related to neonatal screening. With the recent achievements of becoming a journal enlisted in PubMed Central (PMC), we have now achieved a place among scientific journals to be taken seriously. Below, we want to look back on the route IJNS has taken to get there, and our planned scheme for a road ahead.Since our inaugural issue in 2015, the IJNS can look back on six years of history. Founded in 2015, we could not have envisioned or foreseen whether our journal was going to be a success. Attempts in the past had not been fully successful, and so success was not guaranteed. For instance, would we be able to attract sufficient contributions for a viable scientific journal?Secondly, we had to build up relationships with a new publisher, in a world that was relatively new to us. While open access has become one of the standards in scientific publishing, it was relatively new in 2013–2014. Would we be able to build up a readership, and become a constant in neonatal screening, if authors were to pay a fee for publication? Especially in a world where thousands of journals are available, and relatively few with an established reputation and high impact factor, many others were trying to find a niche in the increasingly competitive world of scientific publishing.What would be the right format and how could we find and choose the right partner for publication? These were the questions we had in 2014, and most of the details were published in our first two editorials [For the first four years, we were able to waive all publication costs, which may have been encouraging for our first authors to send in their papers. We were able to advertise at various congresses, especially those of the ISNS and the Association of Public Health Laboratories (APHL), and that may have contributed to making a name for our journal.Slowly but steadily, we were able to generate a steady number of contributions. Even within the first few years, we were able to reach some milestones. In December 2018, we became Scopus-enlisted, receiving our first CiteScore™ in May 2019, and also in May 2019, we became an ESCI (Web of Science)-enlisted journal.IJNS were already listed in PubMed, mostly because these studies were funded by the NIH, or similar funding bodies, enabling PMC-enlistment. With the growing realization that we were establishing a sustainable journal, a second ambition was to be permanently and systematically enlisted in PubMed. In December 2017, we sent a first application to become PMC-enlisted to the National Library of Medicine (NLM).Here, the knowledge and guidance of our publisher, MDPI, should be mentioned prominently. Our colleagues at MDPI knew the proper channels for this application and guided the editorial board through the various procedures.At the end of 2017, 13 papers of This first application failed, but the April 2018 review report of the NLM gave us insight into what was needed to improve. We have since then continuously worked to improve the quality of reviews , and we added three associated editors to our board.IJNS passed the evaluation of the NLM for PMC indexing, which means all the papers published since 2018 in our journal will be indexed in PMC. As a result, if you type “Int J Neonatal Screen” into the search field of the PubMed website, you will find the 209 hits that comprise the 2018–2020 volumes, and 13 hits from former volumes (2015–2017).A second attempt to obtain PMC indexing was more successful. In September 2020, IJNS has steadily increased, and so too has the number of published papers  for publication in IJNS.After this, the management of the journal worked on expanding the editorial board and the board of reviewers. IJNS also depended on the fact that regional associations for neonatal screening were prepared to affiliate with IJNS. Currently, the Association of Public Health Laboratories (APHL), ISNS, the German Society for Neonatal Screening (DGNS) and the Japanese Society for Neonatal Screening (JSNS) are affiliated with IJNS . We intend to invite more regional associations in the months and years ahead.The success of IJNS are easier to find and read, and are thus obviously cited more frequently. Hopefully, Web of Science will grant IJNS its first Impact Factor in the near future as well. Since IJNS is the most prominent journal and platform for original papers in our field, and holds the most recent reports on many subjects, we expect that the best references for your upcoming papers can be found in IJNS.Now, we are at a new cross-roads of our journal. With the PMC enlistment, articles in IJNS, and with the growing success of the journal, new APCs were negotiated. The ISNS has tried to keep these as low as possible, and MDPI was generous enough to keep the APC at a modest price . Current information on the APC can be found on the website of IJNS [IJNS as their preferred platform to publish original scientific results, opinions, technical reports, and everything in between. We also hope that with PubMed enlistment and possibly an Impact Factor, publication in IJNS will contribute to your H-index, and add to the value of the scientific output of your research groups.ISNS is the licensee for  of IJNS . We hopeIJNS. We hope that you, the readership of IJNS, will remain faithful to the cause and mission of our journal and of the society. We appreciate your support and will try to remain the best platform for the dissemination of all scientific work related to neonatal screening.We are very enthusiastic about the development of the journal. It serves to disseminate the high quality work of our members, the members of affiliated societies and, increasingly, the non-affiliated authors who find their way to


Agriophyllum squarrosum) was subjected to heat‐moisture treatment (HMT) at different moisture contents . With MC≤20%, a higher MC resulted in increases in the starch orders  with unchanged granule morphology. Nonetheless, a further elevated MC (>20%) gradually destroyed the granule morphology and starch orders. Also, HMT gradually vanished the lamellar structure as MC increased during HMT. These structural evolutions in HMT‐modified starch resulted in greater thermal stability, higher pasting temperature, lower pasting viscosity and weakened digestibility. Particularly, HMT applied directly in sand rice starch at 20% MC obtained the highest amount of SDS and RS (23.6%), which was 2.2‐fold higher than that of native starch. Therefore, the small granule sand rice starch can be modulated by HMT through controlled MC to expand their application range in food production.A small granule starch from sand rice  during HMT increased starch orders with unaltered granule morphology, and HMT‐20% produced the highest amount of SDS and RS in starch. The sand rice starch can be modulated by HMT through controlled MC. The ɑ‐amylase from the hog pancreas (50 U/mg) was obtained from Sigma‐Aldrich (China) Co., Ltd. The amyloglucosidase (200 U/ml) was purchased from Megazyme International Ireland Ltd. Glucose oxidase‐peroxidase assay kit was bought from the Applygen Technologies Inc. All other chemicals of analytical grade were obtained from Sinopharm Chemical Reagent Co., Ltd.Sand rice  was applied to evaluate the granule morphology at an accelerating voltage of 10.0 kV. Before observation, every starch sample was spread onto circular metal stubs and sputtered with gold in a sputter coater .2.5−1 with a resolution of 4 cm−1. The spectrum was smoothed, baseline corrected and deconvoluted by the OMNIC software (Thermo Scientific). The peak intensity at the wavenumber of 1,047 cm−1 was divided by that at 1,022 cm−1 to calculate the intensity ratio R1047/1022.The ATR‐FTIR analysis was conducted using an IS10 FTIR spectrometer (Thermo Electron Corporation) assembled with an attenuated total reflection accessory and a deuterated triglycine sulphate. Each sample was scanned for 32 times from 1,200 to 800 cm2.6θ) at 40 kV and 4 mA, with a step size of 3° per min. The relative crystallinity was quantitatively estimated by comparing area under the crystalline peaks with the total area of the crystalline and amorphous regions.The powder method of XRD was applied to starch samples using a Bruker D8‐advance X‐ray diffractometer (Bruker AXS Inc.)  and scattering angle (θ) was calculated according to a formula q=4πsinθ/λ  was operated at 0.6 mA and 50 kV, using Cu‐Kα radiation with a wavelength (λ) of 0.154 nm as X‐ray source. The determination of starch samples (ca. 60% MC) were made between 0.2° and 3.2° for 15 min, and further processed with DIFFRAC2.8o), peak temperature (Tp), conclusion temperature (Tc), gelatinization enthalpy (ΔH), and temperature range (Tc‐To).The thermal properties of starch samples were investigated with a DSC 8,500 Thermal Analytical System . The distilled water (6 μl) was added into starch (3.0 mg) in an aluminum pan. This mixture was then immediately sealed and equilibrated at 4℃ for 24 h. Each sealed sample was heated form 20℃ to 120℃ at a rate of 10℃/min, taking the onset temperature  with paddle rotating 160 rpm at a starch concentration of 6% . The starch slurry was firstly held at 50℃ for 1 min, heated from 30℃ to 95℃ at a rate of 5.4 ℃/min, held at 95℃ for 5 min, cooled from 95℃ to 30℃ at the same rate, and then maintained at 50℃ for 1 min. Recorded attributes were the peak viscosity (PV), breakdown (BD), final viscosity (FV), setback (SB), and peak temperature (PT).2.10Englyst  of HMT‐modified starch was lowered than in native starch due to the firm conformation modification. The bands at 1,047 and 1,022 cm−1 are associated with ordered/crystalline and amorphous regions in the external region of the starch granule  of intensity at 1,047 cm−1 to that at 1,022 cm−1 was applied to quantify the degree of order in starch , and a double peak at 2θ (17.2° and 18.0°), pointing out that sand rice starch is an A‐type starch. The HMT‐modified starches still had the similar XRD patterns, indicating that the A‐type crystalline form was retained when exposed to HMT. Previously studies have demonstrated that the crystalline type of buckwheat, cassava and sorghum starch could hardly be changed through HMT  of the native and HMT‐modified starches are presented in Table To, Tp, Tc and Tc‐To of starch increased after HMT, while the △H significantly decreased, and all these changes were correlated well with the MC during HMT. These results are in agreement with the previous studies on buckwheat starch and rice starch  of crystallites are controlled indirectly by the surrounding amorphous region in the starch granule , simultaneous increases in short‐range and long‐range orders with slightly modified morphology can be seen. Nonetheless, when a higher MC (≥25%) was used, HMT might promote the starch structure disruption rather than rearrangement (shown by significantly decreased order structures). Also, HMT gradually damaged the starch lamellar structure as the moisture level increased. These structural evolutions were positively related to the transformation of RDS into SDS/RS upon HMT modification. It was proposed that the newly formed starch orders had relatively high perfection with less flaws, higher stability and greater resistance to enzyme hydrolysis  improved the molecular order structure  but unchanged the granule morphology; further elevated MC over 20% led to a gradual disruption of starch orders and granule structure. Meanwhile, HMT gradually vanished the lamellar structure with the elevated MC during HMT. Compared with native starch, HMT‐modified starch had higher pasting temperature, lower paste viscosity and weakened digestibility. Particularly, HMT‐20% gained the highest amount of SDS and RS at 23.6%. Generally, MC played a crucial role in controlling the structure and properties of the HMT‐modified sand rice starch for practical application. These results here help us in well understanding the effects of HMT modification from the structural view, which thus are beneficial for the production of sand rice starch products with tailored digestibility and pasting properties.The authors have no conflict of interest to declare.


The combination of ABO incompatibility and DRWR incompatibility was not an independent risk factor for biopsy-proven graft rejection ; however, it was an independent risk factor for pneumonia . The mortality rate due to infection was higher among patients with both ABO incompatibility and DRWR incompatibility than among patients with neither factor or with either factor alone. The combination of ABO incompatibility and DRWR incompatibility was an independent risk factor for graft and patient survival after LDKT, whereas neither factor alone significantly affected graft or patient survival. Thus, donor-recipient weight matching should be cautiously considered in LDKT with ABO incompatibility.The effect of donor-recipient weight mismatch is not well established in ABO-incompatible living donor kidney transplantation (LDKT). A total of 2584 LDKT patients in the Korean Organ Transplantation Registry were classified into four groups according to the presence or absence of ABO incompatibility and donor-recipient weight mismatch (donor-to-recipient weight ratio (DRWR) < 0.8). In a multivariable Cox analysis, the combination of ABO incompatibility and DRWR incompatibility ( Kidney transplantation (KT) is the best treatment for most patients with end-stage renal disease . Amidst Nephron mass is an important determinant of the fate and function of the kidney , as insun = 209), crossmatch-positive LDKT (n = 252), or lack of data (n = 5). The remaining 2584 LDKTs included 1941 ABOc LDKTs and 643 ABOi LDKTs. We defined DRWRi as a DRWR less than 0.8, which was near the cutoff for the 25th percentile among all LDKTs in the dataset; we defined DRWR compatibility (DRWRc) as a DRWR greater than 0.8 (n = 1570), ABOc-DRWRi (n = 371), ABOi-DRWRc (n = 519), and ABOi-DRWRi  who underwent KT in Korea between May 2014 and December 2018. Details of the KOTRY are described elsewhere [than 0.8 . For ourn = 124; .We retrieved data on the demographics of the recipients and donors, underlying kidney disease, donor-specific antibody (DSA) positivity, and induction agent. Serum creatinine was measured at the time of discharge, at 6 and 12 months, and annually thereafter. The estimated glomerular filtration rate (eGFR) was calculated from the serum creatinine level using the Modification of Diet in Renal Disease equation , which aThe causes of graft failure and patient death were recorded. We defined graft failure as a need for return to dialysis or re-transplantation. Data from patients who died with a functioning graft were censored in the analysis of graft failure. The first event of biopsy-proven rejection (BPAR) was recorded for each patient. The causative pathogens and infection sites were recorded for infections that required re-admission to the hospital after KT. The primary end point of the study was death-censored graft survival. Secondary end points were patient survival, BPAR, and infection.p-value was <0.10 in a univariable Cox analysis. A multivariable model for patient survival was established by stepwise regression to ensure statistical significance with limited number of events. The results were presented as hazard ratios (HRs) with 95% confidence intervals (CIs). All analyses were performed using standard software . p < 0.05 was considered statistically significant.Categorical variables were compared by Chi-square test or Fisher’s exact test and presented as the number (proportion). Continuous variables were compared by one-way analysis of variance (ANOVA) and presented as the mean ± standard deviation. Survival curves were generated using the Kaplan–Meier method and compared by log-rank test. Cox regression analysis was performed for outcomes adjusted with covariates for which the The demographics of the study sample are shown in 2) and ABOi-DRWRi (57.8 mL/min/1.73 m2) groups than in the ABOc-DRWRc (75.3 mL/min/1.73 m2) and ABOi-DRWRc (75.2 mL/min/1.73 m2) groups. This trend was maintained throughout the study period. The full results are provided in n = 23, 0.9%), the detailed causes of which are presented in p = 0.063, p = 0.028), together with other factors such as age and the number of HLA mismatches. The full results of the Cox regression analyses of death-censored graft survival are provided in During a mean follow-up of 36.2 ± 15.7 months, graft loss occurred in 50 (1.9%) patients. Rejection was the most common cause of graft loss , followed by unknown causes  and cardiovascular disease . The detailed causes of death are presented in p < 0.001, p = 0.008), along with other factors such as cardiovascular disease, unrelated donor, and XM-DSA+ (Thirty-one (1.2%) patients died during the study period. Infection was the most common cause of death , ABOi , and DRWRi  were not independent risk factors for BPAR , as were ABOi with DRWRc  and ABOi alone .We compared the incidences of infections that required hospitalization within 1 year after KT  and founAlthough the outcomes of ABOi LDKT have been inconsistent among different studies, patients who undergo ABOi LDKT have better long-term survival than those who wait for an extended period of time for an ABOc transplant from a deceased donor . FurtherABOi alone was not an independent risk factor for death-censored graft survival in our population, in contrast to the findings of a recent meta-analysis . One reap = 0.074), which could be due to the relatively small population and the short-to-medium duration of follow-up. Regardless, our results showed that DRWRi had a greater effect on graft survival in ABOi LDKT than in ABOc LDKT.Among the ABOi LDKT patients in our study, only those with DRWRi had lower death-censored graft survival rates compared with ABOc LDKT patients. Donor-recipient size mismatch is a well-known risk factor in KT ,27; howeIn the Kaplan–Meier analysis, patients without BPAR events during follow-up had equal graft survival regardless of ABOi or DRWRi. In contrast, among patients who experienced BPAR, the combination of ABOi and DRWRi was associated with lower death-censored graft survival, although the difference was not significant, probably because of the small sample size. The low graft survival after ABOi-DRWRi LDKT among patients who experienced BPAR might be due to low functional reserves in relatively small renal allografts, which were well demonstrated in a previous study . More poInterestingly, ABOi-DRWRi was the independent risk factor for patient survival while ABOi alone was not a significant risk factor in this study. High recipient BMI, which is characteristic of DRWRi, might affect patient survival; however, DRWRi alone did not significantly increase the risk of mortality. In fact, recent studies in the United States found that recipient BMI was not an independent risk factor for patient survival after KT . A possiOur study had some limitations. Because of the short-to-medium duration of follow-up, our results do not represent the long-term consequences of ABOi and DRWRi. The small sample size of the ABOi-DRWRi group was another limitation, which resulted in large confidence intervals of hazard for some outcomes. Additionally, detailed information, including the strength of anti-blood group antibody and desensitization protocols, was not included in the risk factor analysis because of data unavailability in the registry. Furthermore, potential confounders not controlled by the multivariable analysis because of the small number of events could be present, although we attempted to optimize the adjusted model to obtain statistical relevance.The combination of ABOi and DRWRi was an independent risk factor for graft and patient survival after LDKT, whereas neither factor alone significantly affected graft or patient survival. Thus, donor-recipient weight matching should be considered when selecting donors for ABOi LDKT.


Microcystis sp. bloom in February 2020 , which completely disappeared at the end of the following COVID-19 lockdown, when almost all urban activities were suspended. Due to potential harmfulness of cyanoHABs, crude extracts from the “winter bloom” were evaluated for their cytotoxicity in two different human cell lines, namely normal dermal fibroblasts (NHDF) and breast adenocarcinoma cells (MCF-7). The chloroform extract was shown to exert the highest cytotoxic activity, which has been correlated to the presence of cyanotoxins, i.e., microcystins, micropeptins, anabaenopeptins, and aeruginopeptins, detected by molecular networking analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data.Cyanobacteria are ubiquitous photosynthetic microorganisms considered as important contributors to the formation of Earth’s atmosphere and to the process of nitrogen fixation. However, they are also frequently associated with toxic blooms, named cyanobacterial harmful algal blooms (cyanoHABs). This paper reports on an unusual out-of-season cyanoHAB and its dynamics during the COVID-19 pandemic, in Lake Avernus, South Italy. Fast detection strategy (FDS) was used to assess this phenomenon, through the integration of satellite imagery and biomolecular investigation of the environmental samples. Data obtained unveiled a widespread  Campi Flegrei), 2.5 mi west of Pozzuoli, in Campania, southern Italy (Lake Avernus (Lago d’Averno) is a volcanic lake located in the area known as Phlegrean Fields  are triggered by an influx of nutrients, which can occur—for example—through urbanization or agricultural activities  as well Planktothrix rubescens and identified anabaenopeptin B and anabaenopeptin F from samples collected between March and June 2007 as the major toxins on the basis of mass spectrometry data.Lake Avernus experienced a cyanobacterial bloom in 2008 3MC-FR) 3MC-FR) , two bel]3MC-FR) , MP-T1 3MC-FR) ), and ot]3MC-FR)  and aeru]3MC-FR) . Nodes iOrganic extracts from the cyanobacterial bloom were assessed for their cytotoxicity in two different human cell lines, using dermal fibroblasts (NHDF) as a normal cell model and MCF-7 breast adenocarcinoma cells as a tumor cell model . Cell prn-butanol extracts from the cyanobacterial bloom were evaluated individually at three different concentrations  for 48 h. The chloroform extract resulted to be the most toxic towards both NHDF and MCF-7 cells within the first 24 h of exposure. Indeed, the chloroform fraction was shown to elicit a significant reduction (approximately 60%) in the slope of the growth curve at the highest dose tested, with a concomitant increase in cell doubling time, as compared to the relevant controls treated with 0.5% DMSO vehicle , sunlight for photosynthesis, and water pH . The bloThe temporal course of the bloom was monitored and showed the highest peak at the turn of the COVID-19 lockdown in Italy, which started on 6 March 2020, and a sudden stop at the end of the lockdown, suggesting a relationship with the concomitant variation of anthropogenic activities. This can be explained in that the stop of all the agricultural, commercial, and recreational activities on the lake suddenly eliminated the continuous supply of nutrients to lake, normally derived from incorrect behaviors and also from the simple washout of fertilizers to the lake, for example.The early detection, identification, and monitoring of the environmental problem was successfully achieved through FDS ,3. FDS iThe bloom was detected early using data acquired by the Sentinel 2 satellite. The NDVI index together with information coming from the mini-drone allowed us to localize the bloom and start the collection’s campaign.Microcystis sp. As Microcystis is known as a toxin-producing strain, a preliminary metagenomic screen of the bloom sample was carried out for detection of microcystin biosynthetic genes. PCR amplification of a mcyB fragment suggested the presence of the microcystin synthetase gene cluster in the environmental DNA sample. However, PCR detection of this mcy fragment is not the proof for the production of the encoded metabolite, in that, in some cases, biosynthetic genes remain phenotypically cryptic unless exposed to specific environmental conditions , [M+K-H], [M+Mg-2H], [M+NH3], [M-Na+NH4], and  adducts were filtered out by setting the maximum relative height at 100%. Peaks without associated MS/MS spectrum were finally filtered out from the peak list. Clustered data were then exported to .mgf file for GNPS, while chromatographic data including retention times, peak areas, and peak heights were exported to a .csv file. A molecular network [Peak alignment was performed using the Join aligner algorithm , as previously reported . NHDF anmcyB primers FAA (5′-CTATGTTATTTATACATCAGG-3′) and RAA (5′-CTCAGCTTAACTTGATTATC-3′), as previously reported [mcyB gene when the amplification product appeared before the end of the exponential phase of the PCR.Furthermore, 250 ng of environmental DNA was amplified by PCR in an Applied Bio-System StepOne Plus Real-Time Detector system (Thermo Fisher Scientific) with the fluorescent double-stranded DNA-binding dye SYBR Green (Thermo Fisher Scientific). PCR screening of metagenomic DNA was performed by using the 16S-ITS rRNA cyanobacterial specific primers CYA359F (5′-GGGGAATYTTCCGCAATGGG-3′) and 373R (5′-CTAACCACCTGAGCTAAT-3′), and the specific reported . Primers


Clostridium thermocellum offers a single step microbial platform for production of biofuels and biochemicals via simultaneous solubilization of carbohydrates from lignocellulosic biomass and conversion to products. Here, solubilization of cell wall cellulosic, hemicellulosic, and pectic polysaccharides in the liquor and solid residues generated during CBP of poplar biomass by C. thermocellum was analyzed.Terrestrial plant biomass is the primary renewable carbon feedstock for enabling transition to a sustainable bioeconomy. Consolidated bioprocessing (CBP) by the cellulolytic thermophile C. thermocellum DSM1313 fermentation platform was 5.8, 10.3, and 13.7% of milled non-pretreated poplar after 24, 48, and 120 h, respectively. These results demonstrate solubilization of 24% cellulose and 17% non-cellulosic sugars after 120 h, consistent with prior reports. The net solubilization of non-cellulosic sugars by C. thermocellum (after correcting for the uninoculated control fermentations) was 13 to 36% of arabinose (Ara), xylose (Xyl), galactose , mannose (Man), and glucose (Glc); and 15% and 3% of fucose and glucuronic acid, respectively. No rhamnose was solubilized and 71% of the galacturonic acid  was solubilized. These results indicate that C. thermocellum may be selective for the types and/or rate of solubilization of the non-cellulosic wall polymers. Xyl, Man, and Glc were found to accumulate in the fermentation liquor at levels greater than in uninoculated control fermentations, whereas Ara and Gal did not accumulate, suggesting that C. thermocellum solubilizes both hemicelluloses and pectins but utilizes them differently. After five days of fermentation, the relative amount of Rha in the solid residues increased 21% indicating that the Rha-containing polymer rhamnogalacturonan I (RG-I) was not effectively solubilized by C. thermocellum CBP, a result confirmed by immunoassays. Comparison of the sugars in the liquor versus solid residue showed that C. thermocellum solubilized hemicellulosic xylan and mannan, but did not fully utilize them, solubilized and appeared to utilize pectic homogalacturonan, and did not solubilize RG-I.The total amount of biomass solubilized in the C. thermocellum did not solubilize RG-I. These results support the hypothesis that this pectic glycan may be one barrier for efficient solubilization of poplar by C. thermocellum.The significant relative increase in RG-I in poplar solid residues following CBP indicates that The online version contains supplementary material available at 10.1186/s13068-022-02119-9. We are at an inflection point in which a resource depletion, fossil fuel-based economy must be replaced by a sustainable carbon-negative bioeconomy , 2. ThisThe bulk of cell wall biomass in dedicated cellulosic feedstock is comprised of cellulose microfibrils, hemicelluloses, and lignin; with lesser amounts of pectin and glycoproteins –7. The pWhile transportation fuel for light duty vehicles is expected to be replaced by electric energy, liquid fuel will be required into the foreseeable future for aviation and marine transportation. Thus, efforts to produce liquid fuel from biomass remain critical. Multiple biomass processing and fermentation strategies to obtain C5 and C6 sugars from lignocellulosic biomass have been developed, including thermal, chemical, biochemical and microbial processes . HoweverClostridium thermocellum, which is one of the most effective crystalline cellulose utilizing microbes found in nature [C. thermocellum offers distinct process advantages, including reduced risk of microbial contamination and increased solubilization of biomass polymers due to high growth temperatures, facile integration into existing industrial processing schemes due to reduced cooling requirements, and the production of highly efficient cellulosomes which provide unique solubilization properties on crystalline cellulose [C. thermocellum DSM1313 can hydrolyze all three major polysaccharide classes of cell wall polymers, cellulose, hemicellulose  and pectin; the latter being a major component of the “glue” that holds the cells of plant biomass together [C. thermocellum for the production of biofuels include the inability of native strains to utilize C5 sugars and a low tolerance to ethanol [A leading microbial candidate for CBP is the anaerobic Gram-positive thermophile, n nature . CBP usiellulose . C. thertogether –11. Limi ethanol , althougC. thermocellum is very efficient at hydrolyzing and utilizing cellulose, and produces enzymes that solubilize the other two classes of non-cellulosic cell wall polysaccharides, hemicellulose and pectin, it has been reported that C. thermocellum cannot utilize the non-cellulosic polymers for growth [C. thermocellum can hydrolyze xylan, the most abundant non-cellulosic polysaccharide in grasses and hardwood feedstocks, but cannot utilize xylose or arabinose for growth. There are fewer published studies on the metabolism of pectin by C. thermocellum and those that are available are contradictory. Aburaya and coworkers [C. thermocellum cannot grow on pectin, whereas Spinnler and coworkers [C. thermocellum could not grow on monogalacturonic acid, a major glycosyl residue of pectin, it did grow well on polygalacturonic acid and sugar beet pectin. These contradictory results leave open the question as to whether or not C. thermocellum utilizes pectin.Although r growth . Numerouoworkers  reportedoworkers  showed tC. thermocellum is highly effective in solubilizing pure crystalline cellulose, with reports of up to 100% solubilization of Avicel™ [C. thermocellum is more difficult due to the heterogeneous nature of these polymers and their homo- and hetero-associations. The degrees of solubilization of biomass by C. thermocellum depends on the type of biomass, severity of the pretreatment, and methods used to quantify carbohydrate yields. For example, total carbohydrate fractional yields of 20–31% and 32–45% have been reported for C. thermocellum solubilization of milled non-pretreated poplar and switchgrass biomass, respectively [C. thermocellum in solubilizing and utilizing pectin during CBP is important for understanding and potentially improving the CBP process. Avicel™ . The solectively . Pretreaectively . HoweverC. thermocellum DSM1313. Determination of cellulosic and non-cellulosic sugar mass yields in both the fermentation broth and solid residues during CBP by C. thermocellum was carried out to identify potential cell wall structural and architectural changes and limitations to solubilization of non-pretreated poplar biomass. The results of this study provide information about non-cellulosic sugars and likely corresponding polymers that are not effectively solubilized from milled, non-pretreated poplar by C. thermocellum. This study also provides a foundation for comparisons of the efficacy of C. thermocellum solubilization of biomass from feedstock mutants and variants with modified cell wall and yield properties; as well as the effects of different biomass pretreatment strategies on the ability of C. thermocellum to solubilize biomass at different biomass loadings.The goal of this study was to better understand the solubilization of cellulose and non-cellulosic sugars from milled, non-pretreated Center for Bioenergy Innovation (CBI) reference poplar  during CC. thermocellum DSM1313 is shown in Additional file C. thermocellum resulted in total solids solubilization of 5.8%, 10.3%, and 13.7% after 24, 48, and 120 h, respectively, while control reactors without the microbe  resulted in 4.5%, 4.6% and 6.3% total solids solubilization at the same time points  was subtracted, the results showed that C. thermocellum CBP solubilized 1.3%, 5.7% and 7.4% of the poplar biomass after 24, 48, and 120 h, respectively.A schematic of the microbial fermentation and analysis procedure used to evaluate the solubilization of poplar biomass by C. thermocellum solubilized biomass over the course of the fermentations. Since the amount of dry mass recovered from the liquor of the fermentation controls and the CBP fermentations was greater than the amount of total solids solubilized from the biomass  from the solid residue. The CBP and fermentation control liquor samples were lyophilized for five days and the dry mass was recorded. There was a significant increase (16–23%) in the dry mass recovered from the CBP liquor compared to the controls at 24, 48, and 120 h, respectively  derivatization and GC–MS. This method specifically quantifies the non-cellulosic sugar composition [C. thermocellum fermentations compared to the controls including 41–267% more xylose (Xyl), 35–80% more mannose (Man), and 19% to 35% more glucose (Glc) over the 120 h fermentation  were detected in the liquid fractions.In order to determine which components of the poplar biomass were solubilized by position . NotablyC. thermocellum fermentations. However, their levels were significantly less in the liquor from the CBP fermentations compared to the liquor from the fermentation controls, being 36–57% less for Ara, 24–49% less for Rha, and 32–46% less for Gal at 48 h and 120 h). The reduction in the levels of these sugars in the liquor of the C. thermocellum fermentations compared to the control fermentations, suggested that C. thermocellum assimilated these sugars. Since the levels of these sugars were greater in the control liquor than in the CPB liquor, it was not possible from these data alone to conclude whether or not C. thermocellum had solubilized these sugars from the biomass, or rather they had all resulted from the process conditions. To determine how much sugar C. thermocellum was able to solubilize from the biomass, the solid residues obtained during the fermentations were also evaluated.Interestingly, arabinose (Ara), rhamnose (Rha), and galactose  were also detected in the liquor of both the control and C. thermocellum CBP, alcohol-insoluble residue (AIR) was produced from the post-fermentation biomass (solid residues) remaining after CBP and in the fermentation controls. The resulting AIR, which represents cell walls, was de-starched for 48 h with α-amylase and the resulting de-starched AIR was analyzed for cellulose, lignin and non-cellulosic sugars. The yields of total AIR per gram of solid residue from the fermentation controls and the C. thermocellum CBP fermentations after the first 24 h were comparable  and syringyl (S) lignin subunits and the lignin S/G ratio were measured by pyrolysis molecular beam mass spectrometry (py-MBMS) [To evaluate the amount and composition of lignin in the post-fermentation solid residues of poplar wood, the amount of total lignin, guaiacyl (G), py-MBMS) . The totpy-MBMS) , a diffeC. thermocellum increased on average 25%, 24%, and 13%, respectively, compared to the fermentation controls , leading to a greater weight percentage of total lignin in the residues.Analysis of the lignin content of the CBI reference poplar biomass versus the lignin content in the solid residues of the 24 h, 48 h, and 120 h CBP fermentation controls revealed an unexpected reduction in the amount of total, S, and G lignin, suggesting that some lignin-containing wall material is solubilized during the process used for fermentation. The percentage of total, S, and G lignin content of the recovered CBP solid residues following fermentation of the CBI reference poplar by C. thermocellum CBP fermentation of poplar wood has been previously described [C. thermocellum is associated with lignin that has a higher G/S ratio, which was preferentially solubilized along with the glycans. There were no major changes in the C5:C6 ratio of poplar solid residues in the control or C. thermocellum CBP fermentations  in CBP solid residue compared to those of controls  based on the Saeman hydrolysis method, which corresponded to a 21% reduction after subtracting the fermentation controls  by GC–MS of TMS-derivatized methyl glycosides and subtracting this value from the total cellulosic plus non-cellulosic glucose as obtained by Saeman 1945) hydrolysis of cell wall material , 22 foll hydrolysC. thermocellum CBP, the glycosyl residue composition of CBI reference poplar solid residues after 24, 48, and 120 h of microbial hydrolysis was measured and compared to those of the solid residues in the fermentation controls  has been reported for other high S/G woody phenotypes [C. thermocellum. Conversely, there was a significant trend for increased Rha (12–21%) in the CBP solid residues compared to the fermentation controls, suggesting that Rha-containing polymers in the wall  were not being solubilized and thus were found to proportionally increase in the final residue recovered. Although there was a small reduction in the total non-cellulosic sugars in the solid residue from the fermentation control, the 10% to 18% greater reduction in total non-cellulosic sugars in the solid residues following CBP fermentation by C. thermocellum indicates that the microbe solubilizes at least some of the non-cellulosic wall polymers including xylans and homogalacturonan  may be a key determinant limiting solubilization of poplar biomass during CBP fermentation by C. thermocellum.A comparison of the amount of lignin, cellulose, and non-cellulosic sugars in the solid residues recovered over the 120 h fermentation Fig.  shows thlum Fig. A, C. Simion Fig. B. The neC. thermocellum fermentation and 120-h fermentation control. Since the solid residues could not be bound to the ELISA plate, they were sequentially extracted with 50 mM ammonium oxalate and 4 M KOH, solvents known extract non-cellulosic polysaccharides [C. thermocelllum fermentation  to measure the relative amount of RG-I in the starting biomass and in the solid residues of the 120-h on Figs. , 7, we hon Figs. A, confiron Figs. C. Furtheon Figs. D. These on Figs.  and showC. thermocellum secretes an extensive array of glycoside hydrolases with more than 70 types of cellulosomal proteins capable of hydrolyzing different types of lignocellulosic biomass [C. thermocellum by evaluating the starting biomass, the post-fermentation liquors, and solid residues recovered during the CBP process. Our goal was to quantify the amount of cellulosic and non-cellulosic sugars solubilized during the CBP process to provide information about which polymers in woody biomass C. thermocellum solubilizes and to identify potential limiting factors that could inform the development of an economically viable consolidated bioprocess. biomass –32. Howe biomass  by C. thC. thermocellum can efficiently solubilize crystalline cellulose, with reports of 95% and 93% solubilization of Avicel™ at low (5 g L−1) [−1) [Populus biomass at low  solids loading and the solubilization was affected by the composition of the biomass with 12% solubilization of low S/G lignin poplar (20 g L−1 DW) and 20% solubilization of high S/G lignin poplar (20 g L−1 DW) [C. thermocellum is affected by biomass loading, with total cellulose solubilization decreasing from 76 to 58% and 63% to 37% as switchgrass loadings were increased from 9.2 to 92 g L−1 and 10 to 50 g L−1 (dry weight), respectively, although Avicel™ was completely consumed at all loadings [−1 solids loading after 5 days of fermentation   and high−1) [−1)  solids l L−1 DW) . The amoloadings , 36. In loadings , 37.thermocellum are expressed and mostly arranged on multi-enzyme structures known as cellulosomes and it appears that the microbe can regulate the enzymes on the cellulosomes based on the substrate and growth conditions [.C. thermocellum hydrolysis of cellulose in poplar biomass was reported to result in solubilization of 17–18% of the glucan in recovered post-fermentation solid residues compared to the controls [C. thermocellum on P. deltoides [thermocellum of cellulose in poplar biomass results in 75–79% of the cellulose being retained in the poplar solid residues after 5 days of fermentation.The plant biomass-solubilizing enzymes produced by C. nditions –32, 38.controls . Anothereltoides . In the eltoides , 23. TheC. thermocellum CBP to solubilize cellulose in poplar biomass. The CBI reference poplar has a mutation in the 5-enolpyruvylshikimate-2-phosphate (EPSP) synthase gene and is among multiple low recalcitrance poplar natural variants affected in lignin biosynthesis with high S/G ratios that have been positively correlated with glucan solubilization [p-hydroxyphenyl (H) subunits compared to the fermentation control solid residues  [C. thermocellum is able to break down xylan, it is unable to ferment hemicellulose solubilized from the lignocellulosic biomass [C. thermocellum CBP fermentation are significantly greater than the amounts in the liquor of the fermentation controls. Specifically, the liquor during C. thermocellum CBP fermentation contained 41–267% more xylose ; 35% to 80% more mannose ; and 19–35% more non-cellulosic glucose   –45, xyloY, XynZ) , mannanaY, XynZ) , 47, andY, XynZ)  and a chY, XynZ) ) the latY, XynZ) , 50, sug biomass , 51. Acclar wood , 52. Thelization , 53.C. thermocellum grown on plant biomass, as well as the hydrolysis of Avicel™ [C. thermocellum CBP liquor  [C. thermocellum for biomass solubilization. The cell wall sugar composition analyses reported here show the largest reduction of GalA (56–71%) content compared to the other non-cellulosic sugars in the CBP biomass residues compared to the fermentation controls  in poplar wood as a carbon source, as previously reported for sugar beet [C. thermocellum fermentation compared to the control fermentations, suggesting that C. thermocellum was not efficiently solubilizing RG-I. This was confirmed by ELISA analysis of the solid residues identifying an increased relative amount of RG-I in the solid residues following CBP fermentation. There was no change in the amount of arabinose and galactose, sugars abundant in the side branches of RG-I.and PL9)  and a reand PL9) ). RGLf hand PL9) , indicatof GalA 5–71% contgar beet . SurprisC. thermocellum hydrolysis of poplar biomass removed only 17% more of the total non-cellulosic sugars after 5 days of fermentation, indicating that the residual poplar wood still retained the bulk of the non-cellulosic wall polysaccharides  in poplar [Compared to the fermentation control,  in wood . Yang ann poplar , confirmC. thermocellum a promising CBP microbe for production of fuels and chemicals from lignocellulosic biomass. The microbe by itself; however, is not able to achieve effective solubilization of all the cellulosic and non-cellulosic polysaccharides from some biomass feedstocks. The low percentage solubilization of the untreated woody biomass is a bottleneck. We previously reported that pectin contributes to biomass recalcitrance and saccharification yield although it is present in relatively low abundance in woody biomass [ bescii resulted in reduced growth of the mutated microbe on Arabidopsis, switchgrass, and poplar biomass, supporting a role for pectin in biomass recalcitrance [C. thermocellum is unable to effectively degrade the RG-I component of pectin, making RG-I a potential target for improving the ability of C. thermocellum to effectively solubilize woody biomass.The cellulosomal cellulolytic activities and non-cellulosic xylanases and pectinolytic activities makes  biomass , 61–63. citrance . The prePopulus trichocarpa genotype GW-9947 was selected as the woody reference biomass for CBI (The Center for Bioenergy Innovation). GW-9947 is an elite natural variant from the BESC (BioEnergy Science Center) top line candidates with high ethanol yields, low lignin, and high S/G lignin [G lignin . Trees wC. thermocellum strain DSM1313. Cultures were grown in 1 × MTC (Medium for Thermophilic Clostridia) solution. The MTC stock solutions were diluted with H2O and the desired carbon source to reach 1 × concentration. The medium was prepared as described in Holwerda and coworkers [Fermentations were carried out using oworkers  with the2, 10% CO2, and 5% H2 until the bioreactors for carrying out the fermentations were sterilized. This 2 × stock was then aseptically added to the autoclaved reactors already containing water and CBI reference poplar under a sterilized biological safety cabinet to bring the total volume to a 1 × MTC concentration.For bioreactor preparation, a 2 × MTC solution was prepared and filter sterilized without the carbon source. This solution was placed in an anaerobic chamber under 85% N600 of approximately 0.6 before transferring at 10% v/v to the 500-mL Avicel™ seed reactors. Avicel™ seed reactors were grown for approximately 24 h before transferring at 10% v/v to the CBI poplar containing reactors. Avicel™ seed cultures were monitored for ideal transferring phase by following total base addition (2 M KOH) and visual confirmation of a healthy culture under a microscope.Serum bottles were prepared by making a 1 × MTC stock that included cellobiose and allowing the solution to reach anaerobic conditions in the anaerobic chamber described above. The medium was then transferred to the desired serum bottle size inside the chamber, capped, and stored at 4 °C until ready for use. Serum bottle seeds were allowed to proliferate to an OD2 was sparged through the reactor headspace to maintain anaerobic conditions. All reactors were inoculated with a 10% (v/v) inoculum of DSM1313 C. thermocellum from a common seed culture containing 5 g/L Avicel™. At 24, 48, and 120 h, two reactors each were shut down and harvested  was loaded into each reactor to reach a glucan loading of 5 g/L (0.5% w/v). The reactor, biomass, and water were autoclaved for 30 min at 121 °C. MTC media components were then added post-autoclaving as described above. Cultures were controlled at pH 7.0 with 2 N HCl and 2 N KOH as the acid and base, respectively. Temperature was maintained at 60 °C, agitation at 150 rpm minimum to keep biomass from accumulating on the bottom of the reactor, and 50 ccm of NTo separate the undigested solids from the liquor, the reactor contents were split into 250-mL bottles and centrifuged at 10,000 RPM for 15 min in a Sorvall RC 6 Plus with FiberliteTM F14-6 × 250y fixed angle rotor. The supernatant was removed by pipetting, frozen for storage, and this fraction was referred to as ‘liquor’. The solids residue was washed with distilled water at a volume consistent with the liquor removed and centrifuged again under the same conditions. The wash fraction was removed by pipetting and the residual solids were dried for 5 days at 60 °C. The dried biomass was weighed, and the total solids solubilization was calculated by dividing the total recovered biomass after fermentation by the initial biomass loaded into the reactors at the start of fermentation.Specifically, the following calculations were used:A;Mass of polymer solubilized during control fermentation (without microbe) = A = mass (mg) of sugar polymer in starting biomass before CBP minus the mass of solids in 24 h, 48 h, and 120 h fermentation control;C. thermocellum) = B;Mass of polymer solubilized during the CBP process  of polymer in starting biomass before CBP minus the mass of solids in 24 h, 48 h, and 120 h CBP fermentations);C. thermocellum = B − A;Mass of polymer solubilized by 2O and two acetone washes. The AIR samples were then kept in fume hood for 72 h to dry. Glycosyl residue composition analysis of the AIR (~ 2 mg) and liquor (100–300 µg) was by GC–MS of trimethylsilyl (TMS) derivatization of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis as previously described [Alcohol insoluble residue (AIR) from solid residues post-fermentation was prepared as previously described , 17. Priescribed , 66. Bri2SO4 a borosilicate glass screw-top test tube for 1 h with vortexing every 15 min at room temperature. Samples were diluted to 1 M H2SO4 and heated to 121 °C for 1 h. Cooled samples were neutralized with 0.25 M Ba(OH)2. Supernatants were collected after centrifugation at 3600×g for 20 min and dried down by lyophilization. The quantification of glucose in the Saeman hydrolysis sample was by the GC–MS of TMS-derivatized methyl glycosides as described above [One method to determine cellulose content was to first quantify the total glucose content of the sample by Saeman hydrolysis of the cellulose in the sample , 22 folled above . This vaO-methyl alditol (MA) derivatives of the monosaccharides produced from the sample as described previously by Black and coworkers [4, and re-permethylated as before. The resulting MAs were analyzed on an Agilent 7890A GC interfaced to a 5975C MSD ; separation was performed on a 30 m Supelco Equity-1 bonded phase fused silica capillary column. The value of Glc obtained from the MA method represented the total Glc content of the sample. The Glc attributed to cellulose was calculated by subtracting the non-cellulosic Glc content as determined by TMS GC–MS (described above) from the total Glc content to give the cellulose content.Glycosyl residue composition analysis was performed by GC–MS of the per-oworkers . PermethThe Complex Carbohydrate Research Center (CCRC) high-throughput pyrolysis molecular beam mass spectrometry (MBMS) method  was usedg for 15 min at room temperature and the supernatant removed and named as the ammonium oxalate (AO) extract. The residual pellet was subsequently washed three times with the same volume of deionized water, centrifuged, and the supernatant discarded. The pellet was suspended in 20 mL of 4 M KOH with 1% (w/v) sodium borohydride and incubated at 25 °C for 24 h with constant shaking at 100 rpm. After incubation, the suspension was centrifuged at 4000g for 15 min and the collected supernatant labeled as 4 M KOH extract and stored at 4 °C. The 4 M KOH fraction was neutralized using glacial acetic acid. Both the AO and 4 M KOH fractions were dialyzed against six changes of deionized water at room temperature for 72 h and lyophilized.Sequential fractionations of AIR from post-fermentation solid residues were carried out to isolate fractions enriched for different types of cell wall components as previously described , 62. For2SO4 was added to each tube, followed by rapid vortexing to provide uniform mixing. The solutions were incubated for 20 min at room temperature in a fume hood. After incubation, 250 μL of each reaction mixture was added into the wells of a Costar 3598 ELISA plate and the absorbance was measured at 490 nm in a microplate reader (Bio-Rad). Standard curves were measured in triplicate with d-glucose as a standard to calculate the total sugar in the AO and 4 M KOH wall extracts.Both the AO and 4 M KOH cell wall extracts were dissolved in deionized water at a concentration of 200 µg/mL. The amount of total sugar in both extracts was determined by the phenol–sulfuric acid assay as previously described , 69. In  °C as described previously [. The plates were blocked with 200 µL of 1% (w/v) instant nonfat dry milk in 0.1 M Tris-buffered saline  for 1 h at room temperature. The aspiration and wash steps were performed using an ELx405 microplate washer (Bio-Tek Instruments). Blocking agent was removed by aspiration, and 50 µL of primary monoclonal antibodies (mAbs) were added to each well and incubated for 1 h at room temperature. The aspiration of the primary antibodies was completed, and each well was washed three times with 300 µL of 0.1% (w/v) instant nonfat dry milk in Tris-buffered saline (wash buffer). After washing, 50 µL of secondary antibody, peroxidase-conjugated anti-mouse IgG whole molecule goat antibodies , was added to each well and incubated at room temperature for 1 h. Wells were washed five times with 300 µL of wash buffer and 50 µL of TMB  substrate solution was added to each well. After 20 min, the reaction was stopped by adding 50 µL of 0.5 N sulfuric acid. The net OD values of the color formation in the each well of the ELISA plates were measured at 450 nm and a background reading at 655 nm was measured and subtracted using a model 680 microplate reader (Bio-Rad). The primary antibodies used were the anti-RG-I CCRC-M14, CCRC-M35 and CCRC-M72 [The ammonium oxalate and 4 M KOH extract samples were applied (50 µL of 20 µg/mL) to 96-well plates and dried overnight at 37eviously . The plaCCRC-M72 , 27 and CCRC-M72 –28.Additional file 1: Fig. S1. Schematic workflow of C. thermocellum consolidated bioprocessing (CBP) of milled un-pretreated poplar biomass. The specific amounts of biomass and water (200 mL) in fermentation vessels were autoclaved, after which 250 mL of filter sterilized 2 × MTC medium stock was added. CBP reactors were inoculated with 50 mL C. thermocellum culture from a common Avicel™-grown seed culture. Uninoculated controls (50 mL medium added instead) were included to account for non-biological solids solubilization. All fermentations were carried out at 60ºC and pH 7.0 with titration using 2 M KOH and with agitation at a minimum of 150 RPM to maintain solids suspended within the reactor. Sacrificial reactors were harvested in duplicate after 24, 48, and 120 h of fermentation, and the post-fermentation residual solids were separated from the fermentation liquor via centrifugation followed by drying of the solids at 60℃ for five days.Additional file 2: Fig. S2. Total solids solubilization of CBI reference poplar biomass by C. thermocellum over 5 days at 60 °C. Solubilization profiles were generated by comparing post-fermentation dry weights to initial biomass loading (5.68 g). Mean ± standard deviation, n = 4. Statistical analysis was with one-way ANOVA followed by Fisher’s least significant difference method *P < 0.05, **P < 0.01Additional file 3: Fig. S3. Glycosyl residue composition by trimethylsilyl (TMS) derivatization and GC–MS of sugars recovered in the liquor from the fermentations at the indicated times, as described in Fig. Additional file 4: Fig. S4. Mass of AIR extracted per gram solid residue recovered after CBP of poplar biomass. n = 4, Statistical analysis was by one-way ANOVA followed by Fisher’s least significant difference method; *P < 0.05.Additional file 5: Fig. S5. Total lignin, S, G, and H lignin of poplar biomass after CBP bioconversion with C. thermocellum. (A) Total lignin, (B) S lignin, (C) G lignin, and (D) H lignin content. Data are presented as % AIR prepared from solid residue after CBP. *P < 0.05, **P ≤ 0.01 significant level (one-way ANOVA followed by Fisher’s least significant difference method).Additional file 6: Fig. S6. Data used to determine cellulose content of AIR from CBI reference poplar solid residues recovered over 120 h fermentation by C. thermocellum and from fermentation controls. (A-C) Cellulose content was estimated as the difference of (A) total cellulosic and non-cellulosic glucose of AIR pretreated by Saeman hydrolysis minus (B) the content of non-cellulosic glucose of AIR not treated by Saeman hydrolysis as detected by TMS derivatization and GC–MS. (C) The difference of glucose content between A and B is recognized as cellulose content and also presented in Fig. D) total cellulosic and non-cellulosic glucose of AIR pretreated by methylated alditol method minus (E) the content of non-cellulosic glucose of AIR not treated by Methylated Alditol method as detected by TMS derivatization and GC–MS. (F) Cellulose content, the difference in glucose content between D and E, measured by Methylated Alditol method and also presented in Fig. Additional file 7: Fig. S7. Glycosyl residue composition of AIR  from solid residue recovered after CBP fermentation by trimethylsilyl (TMS) derivatization and GC–MS analysis. The amounts of sugar are represented as average of mol% (A), and mass (µg/mg AIR) of total monosaccharides in the solid residue (B). Mean ± SD of two biological and two technical replicates (n = 4). Stars indicate values that are different at * P ≤ 0.05, ** P ≤ 0.001 significant level.


There are numerous arthroscopic techniques available to address anterior shoulder instability. Complications are various, and in pursuit of new treatment options, an alternative arthroscopic technique with less potential for complications has been developed. The novel subscapular sling with a semitendinosus graft provides both dynamic and static stability. This procedure uses a semitendinosus graft as a sling around the upper two-thirds of the subscapular tendon, attached to the anterior glenoid rim. The sling phenomenon present in the Latarjet procedure was the basis of the development. The efficacy of the subscapular sling procedure has been verified in biomechanical studies and further investigated in a clinical pilot study. The procedure can be performed without altering the anatomy of nearby structures such as the coracoid process, the conjoined tendon, and the axillary and musculocutaneous nerves. The authors propose the arthroscopic subscapular sling procedure as an alternative to existing surgical treatment options for recurrent anterior shoulder instability. The subscapular sling procedure. This procedure uses a semitendinosus graft as a sling around the upper two-thirds of the subscapular tendon, attached to the anterior glenoid rim. The reconstruction of the labrum and the sling provide both static and dynamic stabilization of the shoulder. There are many treatment options available, depending on the structures injured.The surgical procedure is illustrated in A diagnostic arthroscopy is performed after an external examination of the shoulder stability. The rotator cuff and in particular the subscapular tendon are inspected to rule out injuries. Furthermore, the amount of glenoid bone loss, on/off-track Hill–Sachs lesions, and labral and ligament ears are assessed .Fig 3ArtThe ST tendon is identified through a small skin incision on the proximal medial tibia and harvested with a corkscrew tendon stripper. The graft is doubled, whip stitched, and the length is measured. Additional sutures are placed at both ends of the graft to ease the advancement into the joint and around the subscapular tendon before fixation . Before Remnants of the anterior labrum are removed from the 2- to 5-o'clock position using different rasps, shaver, and radiofrequency coblation. The anterior glenoid must be cleared of soft tissue to support ingrowth of the ST graft. The anterior portion of the subscapular tendon is cleared, and the axillary nerve is identified and kept at a distance from the portals and instruments during surgery. All instrumentation is performed lateral to the conjoined tendon, thus also preserving the musculocutaneous nerve, which is not visualized. The lower anterolateral portal is created lateral to the conjoined tendon from anterior to posterior direction using an aiming guide, cannulated needle, guidewire, switching stick, and halfpipe instruments with the shoulder in 20° of external rotation . The aimAfter the correct placement of the aiming guide, a long, cannulated needle is advanced into the joint and a switching stick slid carefully over it  and 9. DA banana knife or a coblation device is passed over the halfpipe and used to make a slit in the longitudinal direction between the lower and middle third of the subscapular tendon. This is performed with a neutral rotation of the shoulder and cutting from medial to lateral under visual control. A short switching stick or blunt round instrument is advanced on the anterior wall of the subscapular tendon through the anterosuperior portal down to the halfpipe to facilitate the placement of a pulling suture (#2) and subsequently the ST-graft  and 11. It is of immense importance to keep the shoulder in at least 20° of internal rotation at this point of the procedure to avoid impingement of the graft between the humeral head and the subscapular tendon. An external rotation would hinder the gliding and correct placement of the ST graft. FiberTak Soft anchors  are placed from the 3- to 5-o’clock position to fixate the graft to the anterior glenoid rim to replace the lost anterior labrum  and 16. To prevent a reduction in external rotation and abduction, the arm is at this point of the procedure held in 30° of abduction and 20° of external rotation to prevent the sling being too tight. The remaining graft is cut and removed from the joint. The graft fixation is assessed and the shoulder advanced carefully into the 90/90 position to judge the stability and containment of the joint. The joint is flushed, and the portals and camera are removed. The small incisions are sutured, and the shoulder is placed in a supportive sling for 4 weeks. Patients are recommended a postoperative rehabilitation program instructed by a physiotherapist. Pendulum and limited rotational exercises are started on the first postoperative day.This paper presents the surgical technique of the novel subscapular ST sling. The sling supports the subscapular tendon and pulls it in a posterior direction to prevent an anterior dislocation. The ST graft replaces the lost anterior labrum and has, together with the subscapular tendon sling, both a static and dynamic stabilizing effect on the shoulder .Fig 21Sc,,,This procedure is an alternative option to other arthroscopic stabilizing procedures to treat anterior shoulder instability. After a Bankart stabilization, one third of the patients  experience residual dislocation or do not return to their preinjury sporting level.,,Bone grafting procedures may change the anatomy of the shoulder. They are performed by both open surgery and arthroscopically.,The drawbacks of this procedure include the use of an ST autograft, but allografts may also be an option. There are reports of reduction in flexion strength at the donor site, but sequalae are otherwise limited to postoperative pain and loss of sensation around the donor site.The graft-sling and the subscapular tendon can eventually lose firmness and strength and subsequently reduce the stability of the shoulder. Firm attachment and ingrowth of the graft on the anterior glenoid rim are essential to achieve a negative joint pressure and suction effect. These effects may diminish if the healing is disturbed by poor graft quality and/or bone quality. The tightness of the sling is important to assess during surgery. An overconstrained sling would hinder the external rotation and may also, in the long run, induce arthritis because of the excessive pressure on the anterior part of the glenohumeral joint.