N‐lactoyl‐leucine and N‐lactoyl‐isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N‐lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.Increasing demands for protein‐based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed‐batch processes.  N‐lactoyl amino acids in concentrated feeds enables the development of next generation fed‐batch bioprocesses used to produce monoclonal antibodies or recombinant proteins.The replacement of leucine and isoleucine by  Five CHO cell lines producing three different IgGs and two fusion proteins were used in this study. Cell line 1 is a recombinant CHOK1 GS clone expressing mAb1, whereas Cell line 2 is a CHODG44 clone expressing mAb2. Cell lines 3, 4, and 5 are CHOZN clones expressing mAb3 and two different fusion proteins and are named CHOZN 023, CHOZN 034, and CHOZN 5, respectively.2.2The maximum solubility of Lac‐Ile and Lac‐Leu in water was assessed at different temperatures through the preparation of a saturated solution. The pH value of this solution was recorded. After sedimentation, the supernatant was transferred to a new vial, the aqueous solvent was evaporated using infrared , the residual mass was determined, and final solubility was calculated in g compound per kg of water.To investigate the solubility of Lac‐Ile and Lac‐Leu in a highly concentrated cell culture feed, a customized dry powder media corresponding to Cellvento® 4Feed lacking Ile and Leu was produced (Merck). Increasing amounts of Lac‐Ile and Lac‐Leu were added to the liquid feed containing already a total of 125 g/L nutrients at pH 7.0 until saturation was observed. After each addition, the feed was agitated for 10 min, and osmolality , pH and turbidity  were measured.To determine the maximum solubility that can be achieved for a feed upon the replacement of Leu and Ile with Lac‐Leu and Lac‐Ile respectively, a Cellvento® 4Feed backbone lacking Leu and Ile was supplemented with Lac‐Leu and Lac‐Ile and compared with the original Cellvento® 4Feed formulation containing Leu and Ile. Both formulations were reconstituted at 230 g/L and 265 g/L, respectively. After 15 min, the pH was adjusted to 7.0 using NaOH and the solution was mixed another 30 min. Increasing amounts of water were added until a clear solution was obtained with a turbidity value below 5 NTU (measured using Turb® 550 IR). Samples were taken before and after each of the dilution steps to measure turbidity.2.3To monitor the stability of Lac‐AA in the complex feed mixture, a targeted liquid chromatography‐mass spectrometry (LC–MS) method was developed. Lac‐Leu and Lac‐Ile concentrations from 100 μM to 100 mM in Cellvento® 4Feed lacking Ile and Leu at pH 7 were measured to determine the linear range of the method. The final linear range was defined between 10 and 400 pmol of both Lac‐AA injected on the column. Samples were injected on a Xselect HSS T3 column  heated at 40°C. The elution was performed using a gradient between buffer A (20 mM ammonium formate/0.1% formic acid) and buffer B (methanol) using a flow rate of 300 μl/min. Briefly, the gradient started with 0.1% of B for 2 min, then increased linearly from 0.1% B to 20% B at 4 min, followed by 30% B at 6 min, 80% B at 8 min, and 100% B at 8.5 min. A plateau at 100% B was maintained until 9.5 min before returning to 0.1% B until 12 min. The MS detection was performed using an Impact II (Bruker) running in ESI negative mode with an end plate offset of 500 V and a capillary voltage of 3500 V. The nebulizer was set to 1.4 bar and the dry gas was set to 9 L/min at a temperature of 250°C. The mass range was set from 20 to 1000 Da at a spectral rate of 5 Hz. Internal calibration was performed using a sodium formate solution. For the stability study, the cell culture feed (Cellvento® 4Feed lacking Ile/Leu) supplemented with Lac‐AA was reconstituted, the pH was adjusted to 7.0 and the solution was stored for 3 months at either 4°C or room temperature (RT), light protected. Every week, samples were collected and stored at −20°C for further analytics including LC–MS. The signal for Lac‐Leu and Lac‐Ile was considered stable if the measured concentrations over time did not decrease beyond 10% (standard error of the method).2.4l‐glutamine supplementation. In contrast, CHODG44 cells required supplementation with 6 mM l‐glutamine in the medium. Regarding feeds, Cellvento® 4Feed and EX‐CELL® Advanced feed were lacking Ile and Leu to allow individual supplementation or exchanges with Lac‐Leu and Lac‐Ile (equimolar concentrations or decreasing concentrations going from 100% to 50% the equimolar concentration). Fed‐batch processes were performed in 50 ml spin tubes with vented cap (TPP) at 37°C, 5% CO2, 80% humidity and a rotation speed of 320 rpm. For Cellvento® 4Feed, 3% (v/v) of the total volume was added on Day 3, 5, 10, 12, and 14 and 6% (v/v) was added on Day 7. For EX‐CELL® Advanced based processes, 5% (v/v) of EX‐CELL® Advanced™ Feed was added on Day 3, 5, 7, 10, 12, and 14. The glucose level was maintained by adding a specific amount of a 400 g/L glucose stock solution on demand to up to 6 g/L during the week and up to 12 g/L before the weekend. Cell counts and viability were measured using a Vi‐CELL™XR 2.04 cell counter (Beckman Coulter). The seeding density was 2 × 105 cells/ml for CHOK1 GS and CHODG44 and 3 × 105 cells/ml for CHOZN cells in 30 ml starting volume. Experimental conditions were performed with four biological replicates. Spent media analysis including glucose, IgG, lactate dehydrogenase, ammonia, iron, lactate, phosphate, and pyruvate was performed with the bioprocess analyzer Cedex Bio HT (Roche) after centrifugation of the sample for 2.5 min at 5000 rpm (2541 g).CHOK1 GS and CHODG44 cells were cultivated in the Cellvento® 4CHO medium, whereas CHOZN 023, CHOZN 5, and CHOZN 034 were cultivated in EX‐CELL® Advanced™ CHO Fed‐batch Medium (Merck/Sigma Aldrich). Both media are chemically defined and contain essential components for optimal cell growth and protein production. CHOK1 GS and CHOZN cells were transfected with a plasmid containing the Glutamine Synthetase (GS) gene and thus did not require For bioreactor experiments using the CHOK1 GS clone, DasGIP 1.2 L glass bioreactors were used, pH was controlled at 6.95 ± 0.15, and dissolved oxygen (DO) was controlled at 50% air saturation by sparging with air and pure oxygen gas via an open pipe sparger. Temperature and agitation were set to 36.7 C and 140 rpm, respectively. The control condition was fed with classical Cellvento® 4Feed (containing Ile and Leu) at 1x (same feeding regime as above). The Lac‐AA containing feed was designed based on the small‐scale dose finding experiment (70% Lac‐Leu and Lac‐Ile) and was concentrated 2x allowing to reduce the volume of feed added to the bioreactor by a factor 2. For the concentrated process, 1.5% (v/v) of feed was added on Day 3, 5, 10, 12, and 14 and 3% (v/v) on Day 7. To see the potential benefit of a process using highly concentrated feeds, the starting volume of the Lac‐AA bioreactors was adapted to maximize the bioreactor capacity and the process started with a 12% higher volume compared to the control condition (same seeding density).Amino acid analysis was performed by Ultra‐Performance Liquid Chromatography (UPLC) with an AccQ‐Tag Ultra C18 column (Waters). To detect AA using an UV detector, samples were derivatized with the AccQ‐Tag Ultra Derivatization Kit (Waters). Quantification was performed using external calibration curves according to the manufacturer protocol. Lac‐AA were quantified using the LC‐MS method described in paragraph 2.3 and using an isotopically labeled standard .2.5Antibodies and fusion proteins were purified from the cell culture supernatant using protein A PhyTips® (PhyNexus Inc.). Glycosylation patterns were analyzed either by capillary gel electrophoresis with laser‐induced fluorescence (CGE‐LIF) or by UPLC coupled to a mass spectrometer as described elsewhere  as indicated in the manufacturer instructions and stored at −20°C. Protein concentrations were determined using Pierce™ BCA Protein Assay Kit . Lysates were diluted with 10 mM Tris HCl (pH 7.4) to obtain protein concentrations of 37.5, 75, 150, and 300 μg/ml and mixed with 25 mM Lac‐Leu or Lac‐Ile in 10 mM Tris⋅HCl and 50 mM NaCl (pH 7.4) buffer. Samples were collected at different time points after incubation at 20°C or 37°C. The reaction was stopped by incubation at 100°C for 10 min and kept at −20°C before performing the lactate measurement using the lactate fluorescent assay kit  or amino acid analysis using the AccQ‐Tag method described above.2. The reactions were stopped by incubation at 100°C for 10 min and kept at −20°C before performing lactate and AA measurements.Recombinant human CNDP2  was incubated (37°C) with 10 mM Lac‐Leu or Lac‐Ile in 25 mM Tris⋅HCl (pH 7.4), containing 0.1 mM MnCl2.72.7.17 cells per 100 µl and incubated for 10 min at RT. The lysis mix was centrifuged at 15,000 g (4°C) for 5 min, and the supernatants were transferred in 100 µl aliquots to 1.5 ml Eppendorf tubes and stored at −20°C.Cell pellets from a fed‐batch experiment using either the control feed, or a feed containing Lac‐Leu and Lac‐Ile, were collected at Day 10 by centrifugation at 270 g for 5 min. The cell pellet was resuspended in Cytobuster (Merck) at a concentration of 1 × 102.7.2Proteins were precipitated using a TCA/acetone protocol. Briefly, 100 µl lysate were mixed with 20% TCA/80% acetone/10 mM DTT in a 1:1 ratio (v:v) and incubated at −20°C for 2 h. Then, pellets were resuspended with 40–80 µl of 8 M urea, 25 mM Tris‐HCl, pH 8 and then buffer exchanged in 100 mM Tris‐HCl, pH 8 using Zeba spin desalting column (Thermo Fisher Scientific). Protein quantification was performed using NanoDrop (Thermo Fisher Scientific).2.7.32, and 2 µl of trypsin Gold at 0.5 µg/µl (Promega) were successively added. After an incubation at 37°C for 15 h, enzymatic digestion was stopped by adding 1 µl of 100% formic acid. A centrifugation step  was performed to remove degraded surfactant and 50 µl of supernatant were transferred in a LC vial containing 50 µl of 4% ACN/0.2% TFA.Samples were digested using trypsin before nanoLC‐MS/MS analysis. Briefly, 30 µg of intracellular proteins were denatured and reduced by adding 1 µl of both 1% ProteaseMAX (Promega) and 250 mM DTT, respectively, followed by an incubation at 56°C for 60 min in a thermomixer . After cooling down the samples at RT, free cysteines were alkylated by adding 2 µl of 250 mM iodoacetamide. After an incubation at RT for 45 min in the dark, excess of iodoacetamide was quenched by adding 1 µl of 250 mM DTT. 61 µl of 100 mM Tris HCl, pH 8, 1 µl of 1 M CaCl2.7.41 µg of tryptic digest was injected on a nanoRSLC3000 (Thermo Fisher Scientific) coupled to QToF Impact II equipped with a Captive spray (Bruker). For the LC, an Acclaim PepMap trap column (100 µm × 2 cm) was used together with a nanoEase M/Z Peptide CSH C18 column . The column was heated at 40°C, two solvents were used  at a flow rate of 300 nl/min. The gradient was raised stepwise from 5% B to 95% B according to the following gradient . The mass spectrometer was run in positive mode for a m/z range from 150 to 2200 with lock mass calibration (m/z 1221.9906). The capillary voltage was set to 1100 V, the MS spectra rate was 2 Hz whereas the MSMS scan level was 8 Hz (2500 cts); 32 Hz (25000 cts).2.7.5Cricetulus griseus UniprotKB proteome database . First, data (.mgf) were converted by Progenesis QI for Proteomics software (Waters) before loading into PEAKS Xpro software. Database search was performed using the following parameters: peptide tolerance of 10 ppm, fragment ion tolerance of 0.05 Da, trypsin enzyme (specific), two missed cleavages allowed, carbamidomethylation of cysteine as a fixed modification, methionine oxidation, and asparagine/glutamine deamidation as variable modifications, allowing a maximum of two variable modifications per peptide. Only proteins identified with at least one unique peptide (FDR < 1% at the peptide level) were validated.Protein identification was performed using PEAKS Xpro software (BSI Informatics) by matching acquired peptide tandem mass spectra against the 2.7.6q‐value < 0.01 were considered as statistically significant. All proteins were plotted in a volcano plot according to their Log2(fold change) and −Log10 using GraphPad prism 7.0 (GraphPad Software Inc.).Label‐free MS quantification was performed using Progenesis QI for Proteomics software. Raw data were loaded as centroided data  without additional calibration . “All proteins” method was used for normalization and only proteins quantified with at least three unique peptides were considered. Only proteins with a fold change > 2 and a 33.1N‐lactoyl‐amino acids, Lac‐Leu, and Lac‐Ile, was determined in water using saturated solutions and residual mass determination at 4, 20, 25, and 37°C. As shown in Table To determine whether Lac‐AA present a benefit compared to canonical AA, the solubility of Leu, Ile, and their respective N‐lactoyl counterparts allows to increase by 54% the total concentration of a complex CCM formulation.Further experiments were designed to determine the maximum concentration that can be reached for a complex CCM formulation when Leu and Ile are replaced by their respective lactoyl derivatives. Data for Cellvento® 4Feed (containing Ile and Leu) indicate that this formulation is insoluble at already 1.2× . In contrast, if Leu and Ile are replaced with their equimolar concentration of Lac‐Leu and Lac‐Ile, the formulation is soluble until 200 g/L which indicates that the replacement of Leu and Ile by their respective 3.2Limited reports are available about the stability of Lac‐AA. Therefore, a new targeted LC‐MS method was developed to monitor the stability of Lac‐AA in complex feed mixtures. The method proved to be linear between 10 pmol and 400 pmol of Lac‐AA injected on the column and was used to monitor the stability of Lac‐Leu and Lac‐Ile in Cellvento® 4Feed lacking Ile and Leu and stored for 3 months, light protected, at either 4°C or RT. Results Figure  indicate3.3To understand whether Lac‐AA derivatives can replace Ile and Leu in feeds, small scale fed‐batch experiments were performed in spin tubes with five distinct CHO cell clones. The positive control feed contained Ile and Leu and was compared to three different test feeds: the first feed contained Lac‐Leu and Ile, the second contained Lac‐Ile and Leu, and the third feed contained Lac‐Leu and Lac‐Ile. Equimolar ratios of Lac‐Leu and Lac‐Ile were added to the feed to match the concentrations of the canonical AA. The negative control feed was lacking Leu and Ile and was used to monitor the overall Leu and Ile consumption from the basal growth medium.Results shown in Figure 3 values was observed when both AA were replaced simultaneously by Lac‐AA. The negative control showed undetectable levels of ammonia between Day 7 and Day 12, followed by a NH3 release from dying/dead cells  and was concentrated two times compared with the control condition. Consequently, two times less volume was added on the respective feeding days, thus reducing the diluting effect of the feed on the product. To maximize the bioreactor utilization using a concentrated feed, the Lac‐AA process started with a 12% higher volume compared to the control condition. Since the same seeding density was used, this lead to a higher absolute amount of cells inoculated at Day 0 in the concentrated process. Results Figure  indicate3.7To investigate whether the release of canonical AA from Lac‐AA is mediated by enzymes, both modified AA were incubated with cell lysates from CHOK1 GS clone and the release of Leu, Ile, and lactate was monitored over time. Results indicate that the release of both lactate and canonical AA was linear with time (not shown) and was dependent on the protein concentration of the lysate Figure . The relN‐lactoyl‐phenylalanine when incubated with its precursors phenylalanine and lactate. Since this enzyme is reported as a nonspecific dipeptidase, we hypothesized that it may also cleave Lac‐Leu and Lac‐Ile. In fact, release of lactate and respective free AA , suggesting no major change at the protein level upon usage of Lac‐AA. Altogether, these results suggest that Lac‐AA are very likely cleaved by intracellular enzymes such as CNDP2, a dipeptidase proved to be expressed by CHO cells. CNDP2 was able to cleave Lac‐AA in vitro as shown in Figure Finally, to confirm that CNDP2 is expressed by CHO cells, a proteomic characterization of a CHO GS cell lysate was performed. A sample collected on Day 10 from the control process was compared to a sample collected on the same day from the Lac‐AA process. About 1179 proteins were quantified with at least three unique peptides Table . CNDP2 w4This study sought to investigate the possibility to replace Leu and Ile in CCM formulations by their respective Lac‐AA counterparts, Lac‐Leu and Lac‐Ile. Lac‐AA are relatively new molecules, discovered by the food industry at the end of the 1990s, and especially known for their taste properties in cheese . Gregor Wille, Yannik Rey and Markus Fischer developed the synthesis process for lactoyl‐amino acids and produced them at scale; Aline Zimmer designed, supervised the study, and reviewed the data and the manuscript. All authors have approved the final article.Supporting information.Click here for additional data file.


Prior research shows that employment programs for individuals with autism spectrum disorder (ASD) fail to address ASD as a heterogeneous disorder and focus on specific ASD traits associated with difficulty in obtaining and maintaining employment. This study provides descriptive evidence that self-perceptions of self-determination improve in young adults with ASD who participate in a residential program that promotes Wehmeyer and Schalock’s essential characteristics of self-determined behavior: behavioral autonomy, self-regulated behavior, acting in a psychologically empowered manner, and self-realization. Qualitative surveys were administered to 60 participants (17-28 years old) on perceptions of self-determination, confidence in independent living skills, and program effectiveness regarding case management and sustainable employment. One-sided t-tests using pre- and post-program responses were assessed. Post- versus pre-program means were significantly higher in participants feeling confident to live alone (p = 0.0059). Findings suggest that programs adopting self-determined behavior may be more effective in increasing self-confidence for individuals with ASD. However, these findings warrant long-term analysis to assess the continuity of program success and sustained employment. Autism spectrum disorder (ASD) is a lifelong neurological condition characterized by deficits in social communication and restricted, repetitive sensory-motor behaviors . ASD chaThere are many employment programs and interventions currently available for individuals with ASD. These programs focus on employment preparation to teach necessary social, communication, and vocational skills and job maintenance through behavior and task management. The interventions obtain employment preparation and job maintenance by utilizing video modeling, role-playing, audio coaching, behavior skills training, job site, and simulation training. However, systematic analyses on the efficacy and success of these programs ,10 show Self-determined behavior is defined as the combination of skills, knowledge, and beliefs, which empowers an individual to participate in goal-directed, self-regulated, autonomous behavior . WehmeyeThe primary aim of this study is to provide descriptive evidence that self-perceptions of self-determination in young adults with ASD are malleable in a short period of time through participation in an urban residential summer camp program. Secondary aims include identifying demographic characteristics of study participants and how the addition of individual case managers affects young adults with ASD in developing meaningful employment.The present study was conducted as a pretest-posttest research design using surveys to evaluate a summer camp program for young adults with ASD. The residential program was created by a non-profit organization in 2015 to address the lack of employment opportunities for young adults on the autism spectrum leaving school and entering adulthood . The resParticipantsParticipants were referred by a state Division of Vocational Rehabilitation office, and those interested in participating in the summer program submitted an online application. Minimum eligibility criteria for the program included (1) a documented history of ASD, intellectual or developmental disability by a state Division Vocational Rehabilitation office; (2) minimum age of 17 years; (3) ability to meet personal needs without assistance ; (4) ability to self-administer any necessary medication; (5) no disruptive behavior difficulties or violent tendencies; (6) no history of running or leaving school/home unsupervised; (7) ability to participate in multiple 50-90-minute classes daily; (8) ability to navigate campus independently with guidance from counselors ; (9) ability to live in a group dorm setting and compromise with roommates; (10) interested in learning steps to employment, social skills, postsecondary education opportunities, and independent living; and (11) participation in all activities and classes. Applications were reviewed by summer program staff, and 64 participants were identified and accepted in the study. Participants and family members attended an in-person orientation where all details of the summer program and the informed consent were explained. All participants  were required to give written informed consent. The study was approved by the residential university’s Institutional Review Board (1409572-3). Of the 64 participants, two were excluded due to attrition, and two were excluded due to missing post-program survey completion. The final analytical dataset consisted of 60 participants.ProceduresThe intervention was divided into three 12-day sessions with roughly 20 participants, two case managers, and program staff in each session. Participants resided at a large undergraduate institution during the entire program duration under the supervision of program staff. Each participant was matched with a case manager. Case managers were qualified social workers with graduate degrees in special education and previous experience in clinical or rehabilitation settings. Program staff members were individuals who were required to complete a 20-hour online certification course that prepared them for interaction with young adults with ASD. The participants were split evenly such that each of the two case managers had a caseload of no more than 12 individuals for each session. The case managers were expected to have direct contact with the participants at least five times throughout the session and assessed the participants’ ability to live and function independently and their employability skills. National metrics, including the Holland Code and Career Index, were utilized in case manager assessments. Case managers were also expected to have monthly follow-ups with each of their assigned participants after the successful completion of the residential program.Table Behavioral autonomy was established by hosting participants in a large urban undergraduate institution without direct familial support. Participants were still under supervision by program staff members but were encouraged to embrace their independence from family members and were taught how to self-navigate daily life. Participants were also given options for different social activities to choose from on their own and incorporate into their schedules.Self-regulated behavior was established by giving participants individual daily responsibilities. Responsibilities were related to personal hygiene, dressing and clothing care, and time management. Participants also engaged in problem-solving activities to learn stepwise approaches to problems to apply in scenarios after program completion.Acting in a psychologically empowered manner was established through instruction in executive functioning from case managers, qualified special education instructors, and autism specialists. Lessons were taught by the way of modeling, simulation, and guided practice for participants to learn how to select and successfully monitor behaviors that facilitate the attainment of their chosen goals. Case managers specifically taught participants about specific laws and adult services that could benefit them in finding and maintaining meaningful employment.Self-realization was established through reflection and team-building activities where participants were able to consider beneficial and non-beneficial aspects of their behavior and the consequences of their behavior. Participants also met with case managers to monitor their progress and reflect on areas of improvement.MeasuresParticipants were administered program surveys that included questions focused on participant self-determination, confidence in independent living skills, and program effectiveness in terms of case management and meaningful employment. Survey questions were developed by the summer program staff with oversight by faculty and the Director of Evaluation and Assessment at an urban medical school. Survey questions included the following: (1) I feel confident enough to live alone, (2) I have learned the professional skills needed to get a job, (3) I feel comfortable meeting with/talking to my case manager, (4) I have gained one or more ideas to use in my everyday life, (5) I feel confident in succeeding at my job, and (6) I feel confident in achieving my career plan goals. Surveys were administered via Google Forms at baseline and upon completion of the summer program. Responses were recorded on a five-point Likert scale .Statistical analysisA one-sided t-test using pre- and post-program survey responses was assessed. The authors chose to do a whole group analysis rather than a paired pre-/post-analysis because not all participants completed the program to provide post-program survey responses, which rendered the data unsuitable for paired analysis. Microsoft Excel version 16.37  was used for all statistical analyses. A one-sided p-value < 0.05 was considered statistically significant.Descriptive characteristicsThe descriptive characteristics of the study participants are listed in Table Program survey responsesResults of the one-sided, two-tailed t-test using pre- and post-program survey responses are listed in Table This study evaluated the immediate effects of a residential summer camp program in a large urban undergraduate institution. The program promoted the essential characteristics of self-determined behavior through independent living and employability skills in young adults with ASD in hopes of developing sustainable careers. The major finding in this diverse cohort of young adults with ASD was that participation in the residential summer camp program was associated with increased confidence for independent living immediately after program completion. Participants also felt greater comfort meeting and talking with case managers, although mean differences did not show significance.The residential summer camp program used in this study identified and incorporated missing aspects from other vocational programs for young adults with ASD and integrated qualities necessary for adequate employment training, such as those seen in Project SEARCH with ASD supports . SystemaWhile many of the current vocational programs lacked descriptive characteristics of the participants , this stLimitations and future researchThere are limitations to this study that should be considered. First, the small sample size limited the analysis to a one-sided, two-tailed t-test. While this statistical test was chosen for attrition reasons, survey results were limited to whole group intra-mean differences. There was also no control group to establish inter-mean differences and to assess whether an increase in independent living skills would have occurred regardless of attending the summer camp program. The extensive eligibility criteria of this program limited participants to those at a higher functioning level on the ASD. Although the strict admission criteria limited generalizability to all individuals with ASD, there is still a need for vocational programs for higher functioning ASD adults . Lastly,Study results suggest that residential vocational programs for young adults with ASD who incorporate characteristics of self-determination provide short-term benefits in independent living skills. Future studies, including further follow-up and evaluation of this study cohort, are needed to obtain objective data on job placement and sustained employment rates. Additional qualitative metrics, such as interviews and focus groups, can further explain study findings and identify which aspects of the summer camp program correspond to better employment outcomes. The additional qualitative metrics can also help to elucidate specific needs of female young adults with ASD as it pertains to independent living, self-determination, and employment. Overall, future studies could utilize larger sample sizes to investigate the effects of similar residential programs across broader, more diverse populations of young adults with ASD. This residential summer program provides a unique opportunity for young adults with ASD to become self-sufficient and live independently, with the goal of these skills translating to better employment opportunities and increased job performance. The program offers a simulation for participants to practice employment skills before entering the workforce where consequences for mistakes are often much higher. The results of the study suggest that residential programs incorporating characteristics of self-determination offer young adults with ASD the skills to live independently. Future studies should expand on the long-term effects of residential programs and incorporate greater sample sizes. Increased representation of a broader spectrum of ASD persons based on race, gender, and ethnicity is also needed.


Introduction: The need for caregiver respite is well-documented for the care of persons with IDD. Social Assistive Robotics (SAR) offer promise in addressing the need for caregiver respite through ‘complementary caregiving’ activities that promote engagement and learning opportunities for a care recipient (CR) with IDD. This study explored the acceptability and usefulness of a SAR caregiver respite program responsive to feedback from both the CRs and their older family caregivers (age 55+). Methods: Caregiver-CR dyads (N =11) were recruited. A mixed methods research design was deployed in three phases: Phase I with four focus groups to inform the program design; Phase II for program demonstration and evaluation with pre- and post-surveys; and Phase III with post-program interviews for feedback and suggestions. Results: Quantitative data analysis in Phase II showed both caregivers and their CRs scored favorably the social presence of the robot (Pepper) and social engagement with Pepper. Caregivers also reported positive perceptions of Pepper in terms of anthropomorphism, animation, likeability, intelligence, and safety. Content analysis from Phase III interviews suggested that the SAR may offer physical/emotional respite to caregivers by providing companionship/friendship as well as promoting independence, safety/monitoring, and interactive engagement with children. Discussion: SAR has potential in providing respite for older family caregiver demonstrated by the levels of CR engagement and learning with Pepper. Future studies need a longer program design and larger sample size to test the feasibility and efficacy of the intervention.


Breast cancer is the most common malignancy among Iranian women and worldwide. Lymphatic vessel invasion (LVI) was found to be an independent prognostic factor in many carcinomas, including invasive carcinoma of the breast. The aim of this study was to compare the hematoxylin and eosin (H&E) staining method and use of the immunohistochemical (IHC) marker ofD2-40, for evaluation of LVI in breast carcinoma specimens.The study was conducted on 50 patients undergone surgery between the years 2010 and 2015 in Rohani Hospital, Babol, Iran with invasive carcinoma of the breast with Census sampling method. LVI was assessed by two surgical pathologists, using H&E- stained sections and two IHC markers, i.e., D2-40 and CD31.P=0.004). LVI was detected in 25 (50%) patients by H&E and in 14 (28%) patients by D2-40. Twelve out of 25 patients with positive LVI in H&E were confirmed by D2-40 and 2 out of 25 patients with negative lymphatic vessel in H&E. Only one case showed weak staining of CD31 proving LVI. These results showed a significant difference between the H&E staining and D2-40 IHC study for LVI detection (The D2-40 IHC marker is helpful in the diagnosis and confirmation of LVI in invasive carcinoma of the breast. CD31 is not suitable for the evaluation of lymphatic vessels. Breast cancer is the most common malignancy in women worldwide, particularly in Iran. Breast cancer is the second leading cause of cancer death in developed countries 15.4%) after lung cancer, and it is the fifth leading cause of cancer death in Iran like any other part of the world. The five-year overall survival rate is estimated to be 72% in women and 60% in men. This survival rate is significantly affected by the stage and number of positive lymph nodes  of variable tumors. The importance of LVI in breast cancer was first described by Teel in 1964  or -posiIn the past, hematoxylin and eosin (H&E) staining was the only method for the evaluation of LVI. It was somehow problematic due to fibrosis and fixation artifact, which resulted in false positivity . Therefo D2-40 is a monoclonal antibody detecting podoplanin, mucin-type transmembrane glycoproteins on the endothelium of lymphatic vessels .This marker has been used by many studies in variable malignancies , 12, 13.The aim of our study was to compare the H&E method and use of the IHC marker of D2-40, for evaluation of LVI in breast carcinoma specimens.th edition.The study was conducted on patients with invasive carcinoma of the breast who had undergone surgery between the years 2010 and 2015 in Rohani Hospital, Babol, Iran, and their formalin-fixed paraffin blocks of the tumor were available. The inclusion criteria were having invasive carcinoma with available paraffin block which included a rim of normal breast tissue in tumor margin. Those cases that diagnosed as carcinoma in situ or presented with extensive tumor necrosis in all slides were excluded from the study. Clinicopathological data, including age, gender, tumor stage, and grade, were retrieved from standard reports, which were prepared according to the American Joint Committee on Cancer (AJCC), 7Immunohistochemistry2O2 in dark humid condition. After that, they were washed in TBS for five minutes. Sections were incubated with primary antibody for 60 minutes at room temperature and with secondary antibody for 30 minutes. Sites of binding were detected by a 10-minute incubation with diaminobenzidine (DAB). To highlight lymphatic and blood vessels, two 3-4 µm thick sections were prepared from each block (one block per case), and IHC staining for D2-40 and CD31  was performed. Sections were dewaxed at 60°C in an oven for about one hour, and then they were put in xylol and rehydrated through a descending concentration of ethanol. For antigen retrieval, sections were microwaved for 15 minutes in ethylenediaminetetraacetic acid (EDTA) buffer (pH=9). Sections were left at room temperature for 15 minutes to cool down. They were washed in tris-buffered saline (TBS) for five minutes and incubated in 3% Ht tests were performed for statistical analysis.IHC and archived H&E slides were reviewed by two pathologists, not knowing the pathology report. Data were imported in SPSS 20 ; chi-square, Fisher’s exact, and The current study was conducted on 50 female patients with invasive carcinoma of the breast, including 47 ductal and 3 lobular carcinomas, who had undergone a mastectomy. The mean age was 47.40±12.35 years ranging from 19 to 74. The clinicopathologic characteristics of the tumors are shown in Two cases in the negative H&E group had LVI in D2-40 stained sections . These cases were the result of the presence of tumor emboli that filled the lumen entirely P=0.004). The H&E method had 0.85 sensitivity and specificity of 0.64. The difference between these two methods was significant  and D2-40 were used. There was only one CD31 positive case in the D2-40 positive group and none in the D2-40 negative group. Consequently, the interfering role of blood vessel invasion was absent in this study, and the observed significant difference could be trusted. The results of another study using D2-40 and CD31 are in agreement with our findings. It introduced D2-40 as a useful marker for lymphatic invasion and CD31 for blood vessel invasion . et al. (et al. (In the present study, 28% of the cases had LVI detected by D2-40. These results were consistent with those other similar studies. In a study conducted by Gujam et al. , 35% of  (et al.  reportedet al. (A quick survey through literature revealed that D2-40 has been announced by numerous studies as a helpful marker in LVI detection . He et aet al.  used thiIn contrast, a study conducted on 124 cases of stage I lung adenocarcinoma showed no prognostic effect for LVI detected by D2-40 staining method. In other words, patients with LVI detected by D2-40 did not have a significantly poorer outcome compared to LVI negative cases .et al. stated that compared to the H&E staining, the utilization of D2-40 for LVI assessment increased the positive rate significantly in colon cancer (In addition, this marker has been used by many studies performed on other malignant neoplasms, in which the same results were achieved. Lai n cancer . Anothern cancer . A studyn cancer . In the present study, the results showed no significant correlation between the cancer stage or tumor grade and LVI detected by D2-40; however, in a study conducted on 303 patients with node-negative invasive breast carcinoma, LVI invasion detected by D2-40 correlated significantly with the tumor size and histologic grade . This diet al. suggested using myoepithelial markers, such as p63 and D2-40 simultaneously, to distinguish tumor emboli from in situ carcinoma (Another error which could be encountered in our study was the positive staining of the myoepithelial cell layer in D2-40 IHC method, which can cause false positivity in cases of in situ carcinoma. However, we assessed the areas of invasive carcinoma without in situ component to solve this problem. In addition, the pattern of staining in lymph vessels is strong continuous membranous, and in the myoepithelial cell layer is weak-to-moderate with granular pattern . Rabban arcinoma . In our In conclusion, the use of the D2-40 IHC marker is helpful in the diagnosis and confirmation of LVI in invasive carcinoma of the breast. CD31 is not helpful in the identification of lymphatic invasion.


Brunei Darussalam commenced its living-related renal transplant program in 2013, with subsequent attainment of independent local capacity and proficiency in 2019. The preliminary outcome from the program has already begun to shape the national nephrology landscape with a 36% increment in transplant rate and mitigation of commercialized transplantations. The blueprint for the program was first laid out in 2010 and thereupon executed in four phases. The first phase involved the gathering of evidence to support the establishment of the national program, through researches investigating feasibility, public opinion, quality of life, graft survival, and cost-effectiveness. The second phase focused on laying the foundation of the program through grooming of local expertise, implementation of legal-ethical frameworks, religious legitimization, and propagation of awareness. The third phase worked on facilitating experiential exposure and strengthening local infrastructure through the upgrading of facilities and the introduction of subsidiary services. The fourth phase was implemented in Brunei in 2013 when foreign personnel worked together with the local team to perform the transplants. Between 2013 and 2019, ten kidney transplants were performed, with two being done in 2018 and three in 2019. We hope to inspire other similar countries to develop their own self-sustainable and independent local program. Despite having the smallest population in Asia , Brunei Literature on establishing a national transplant program was scarce, especially in the settings of a small, Muslim, and Asian country. Due to the country's small population, there have always been reservations about a program's viability and sustainability due to insufficient demands and expertise retention. Funding and support from experienced international associations were less forthcoming because of the affluent economic status of the country and the presumptive conviction that existing pecuniary resolve could prevail over logistical and expertise restrictions. Small countries also have to grapple with the lack of a recognized training program and the “brain drain” phenomenon with suitable foreign-trained doctors seeking work opportunities overseas . Other sNot many clinicians have the privilege and experience to start a transplant program in a country, and the task is not helped by the paucity of data available in the literature. We would, therefore, like to share our experience of setting up a national program in a previously transplant naïve country, especially focusing on overcoming issues like the lack of expertise and facilities and religiocultural and political-legislative challenges. We will also describe a case series of our patients and present the impact of the program on our services.This case series described in detail the ten transplant cases that were performed in the country. The objectives of the study were to share our experience with phases of the establishment of a kidney transplant program and share our results of the first cases done. The duration of the study was from 2013 to the end of 2019 during which 10 kidney transplants were done. Personal patients' data were obtained through personal accounts from clinicians and recorded data in hospital computerized information system. Data collected included patient demographics and data pertaining to the operations, hospital stays, and outpatient follow-ups. Patient demographics included gender, age, the relationship between patients, year of operation, etiology, medication, blood group, crossmatch results, blood results, and radiographic findings. The operation, inpatient, and follow-up data were also collected. These results are presented in Tables 1p > 0.05), and a visual inspection of the histogram was done for age, operation time, and warm ischemia time. These data are presented in Tables Data for the country were obtained from the Brunei Dialysis and Transplant Registry  and unpuThe journey started with a vision of a universally accessible local transplant program that caters to the needs of all residents of the country. Previously all transplant patients (citizens of the country only) had the option of being sent overseas for transplantations if there was a compatible donor. The patient would typically stay abroad for 3–6 months. The absence of a local program meant that many eligible patients have forfeited transplant opportunities because they had not been able to take time away from family and work. Additionally, permanent and temporary residents of the country (which comprised 30% of the population⁵) were not entitled to have government-funded treatment. The vision of an independent program was only able to come to fruition when a local team took the initiative to instigate a pioneering project to start a local transplant program ten years ago.From the outset, the project was divided into four phases. Each phase was meticulously planned, and specific objectives and targets were established and evaluated at regular intervals. The phases are described in The team wanted to answer several key questions that were specific to the needs of the country. A study in 2010 involving 300 participants (approximately 0.1% of the country's eligible population) demonstrated that 78.7% were willing to donate their kidneys if the need arose. 59.7% wanted to have a local program in the country . AnotherPhase 2 was concerned with assembling and empowering a core local team to spearhead the project on various fronts, especially to address the multitude of nonclinical issues including ethics, law, religion, and policy.To assemble a local team, we enlisted three experienced local surgeons—a general surgeon, urologist, and vascular surgeon and deliberately fragmented the transplant surgery into different parts to cater to their existing expertise. The intentions were to have the urologist retrieving the donor's kidney and performing the ureteric anastomosis, the vascular surgeon operating the vascular anastomosis, and the general surgeon anchoring the entire procedure and preparing the transplant vascular bed. As the three surgeons were very experienced, it was felt that they could acquire the required skills with less exposure and training than a freshly trained surgeon performing an entire unfragmented procedure. Numerous studies have shown that experienced general surgeons and urologists can have safe transplant outcomes with limited training exposure –22. AddiThe team worked with the lawyers from the Attorney General Office in drafting the National Transplant Act, which primarily focused on the prohibition and penalization of organ trafficking and commercialization. Enactment of the Transplant Act is anticipated to take a few years as it required sanctioning from parliamentary offices. Interim measures through strict policy regulations with emphasis on compliance to the Istanbul Declaration of Organ Trafficking and Transplant Tourism (DOI)  were necNumerous models for international transplant partnerships were studied and scrutinized. The International Society of Nephrology and The Transplantation Society sisters transplant program created new transplant centers and developed existing kidney transplant programs in developing countries like Guatemala, Palestine, Myanmar, Tanzania, and Armenia . These cThis phase was concerned with getting the team experienced and the local infrastructure and manpower ready for the incipient program. We started sending patients with our medical team to our allied foreign center in 2002 to obtain experiential exposure in surgical, medical, and nursing training. The team stayed with the patient for the duration of the hospital stay abroad until the patient was deemed fit for discharge to Brunei.With a focus on strengthening local facilities, particular attention was given to environmental infection aversion strategies and ventilation engineering controls to prevent transmission of infections. Through guidance from our infection control team, we were able to introduce purpose-built ward facilities and procure consumables to enable the proper use of disinfectants and cleaners and maintenance of medical equipment. We also established new services to the laboratory in preparation for the program like calcineurin inhibitor assays, nuclear medicine scan, bone density scan, and new medications into our formulary. For services that we cannot implement, we sent tests to overseas laboratories in Singapore and Malaysia for crossmatching , viral assay, and everolimus assay.We took into account the varied contributions of members of the interdisciplinary team and their potential impact on transplant outcomes and enhancing living kidney donations. Special efforts were made to conciliate their roles and instill an ethos of shared program ownership. Allied healthcare professionals were given special emphasis in their training through MOH-endorsed workshops, designed to help them integrate and identify with the vision of the team and to facilitate their involvement during the workup and follow-up phases.The first transplant in Brunei was performed on 21/11/2013. A transplant team comprising a physician, a surgeon, and two nurses were flown in from Malaysia and Australia to guide and assist with the procedures. By the third transplant, we had acquired self-sufficiency in many areas and, only the Australian surgeon continued to come to the country at that point. The initial few years of the program were hampered by inadequate staffing and lukewarm interest from the public. As we progressed with a few successful stories (that we had deliberately played out in the press), the public began to register more interest. By the tenth transplant, the team was deemed to have acquired full proficiency in independently performing the transplants and running the program.Throughout the course of several transplants, we were able to gradually improve and introduce new services to the program , throughOur recipient stayed in the hospital with a mean stay of 15 days which is higher than 6–12 reported in the literature . This maThe challenges that we faced were unique and covered many nonclinical areas that were not normally encountered by established centers. We had to overcome technical, religious, cultural, political, and legislative barriers to inaugurate our national program. As stated from the outset, the core principles of the program were hinged around the ideals of equity, quality, sustainability, and morality; henceforth, the pace of the fledging program can only be determined by the slow disentanglement of existing limitations to enable the successful fulfillment of these virtues. It has been a truly enriching and gratifying journey, and we hope that our experience can inspire and benefit other new centers, especially in small and developing countries.


To evaluate the progress and challenges of a hearing screening program as well as review the incidence of pediatric hearing loss in grade school children participating in this program.Medical students from the University of Ottawa established iHear, a grade school hearing assessment program that uses novel tablet audiometry. Over 3 years, children in grades 1 and 2 were assessed and those found to have abnormal results on iHear assessment were then referred to audiology for formal testing, and to otolaryngology if needed.From 2014 to 2017, 753 children aged 5–9 years old were assessed for hearing loss. Mean age of participants was 6.7 years, 51.9% of whom were female. Of the children assessed, 86 (11.4%) had abnormal results and 6 (0.8%) had inconsistent results, necessitating 92 referrals for assessment by a professional audiologist. Of the 65 participants who completed secondary audiologic assessment, 54 (83.1%) were normal and 11 (16.9%) had a definitive hearing loss or abnormal tympanometry. A total of 32 children were lost to follow-up. A total of 118 medical students were involved in the iHear program.Hearing loss in grade school populations continues to go undetected across Canada. Programs such as iHear demonstrate that gaps in the provision of hearing assessment can be filled effectively by medical students equipped with tablet audiometry. Medical student exposure to audiology and otolaryngology increased through the iHear program. Over 34 million children are affected by disabling hearing loss (HL) worldwide . WithoutThe American Academy of Paediatrics recommends that “objective screenings for hearing impairment should be performed periodically on all infants and children” . DespiteHistorically, childhood HL screening programs have been limited by access to audiology services . SoundprTo address the lack of grade school hearing assessments in Ontario, medical students from the University of Ottawa have developed iHear, a grade school hearing assessment program. The program uses tablet audiometry to screen children in grades 1 and 2 for hearing impairment in the Ottawa region . ChildreThis paper aims to evaluate the iHear hearing assessment program and to report on the incidence of paediatric HL in grade school children participating in this program.This study was approved in full by the Children’s Hospital of Eastern Ontario Research Ethics Board (protocol 14/170X), the University of Ottawa’s Office of Research Ethics and Integrity (protocol A02-15–01), the Ottawa-Carleton Research and Evaluation Advisory Committee, and involved schoolboards.iHear technicians were recruited annually from 1st and 2nd year cohorts of the University of Ottawa Medical Undergraduate program. Prior to participation on any school visit, the medical students were required to attend a full 2-h training session in which they were familiarized with consequences of HL, hearing anatomy, causes of HL, otoscopy, and audiometry, and given practical training with otoscopes and the tablet audiometer. Alternatively, students could complete AE100: Audiometry Essentials, an online training module developed to provide a foundational understanding of audiometry and hearing assessment, and then attend a 1-h practical training session with otoscopes and tablet audiometers. Training was provided by an Otolaryngologist as well as medical student iHear executive members who had extensive involvement in the program and additional training.Schools in the Ottawa region were contacted annually to arrange school visits. During each visit, children in grades 1 and 2 underwent a hearing assessment using SHOEBOX® Audiometry, an iPad-based mobile audiometer . Graphs were produced using GraphPad Prism software .Between September 2014 and May 2017, the Ottawa iHear program recruited and assessed 753 grade school children for HL in the Ottawa region  had abnormal results and 6 (0.8%) had inconsistent results, necessitating 92 referrals for assessment by a professional audiologist (Fig. 1.4% had In the first 3 years of the iHear program, 9 training sessions were held and 118 medical students were trained as iHear technicians Table . ThirteeOf the 753 children assessed by iHear, 11.4% had abnormal results (Fig. Of those referred to audiology for evaluation, 11 of the 65 who completed assessment had a definitive HL Fig. . As suchFollowing iHear referral, consultation with otolaryngology was completed in 6 cases with a mean time between iHear and otolaryngology assessment of 299.8 days Table . AccountDeveloped from the ground up by medical student volunteers, the iHear program has achieved great popularity at the University of Ottawa Faculty of Medicine. Over the first 3 years of the program 118 medical students have been involved as iHear technicians Table . This voThe iHear program exists through collaboration with the University of Ottawa Interprofessional Rehabilitation Clinic and Otolaryngologists at the Children’s Hospital of Eastern Ontario. Unfortunately, despite the best efforts of these organizations and the iHear executives, one of the largest issues faced by the program was the number of children LTFU. The most common reason for a child to be LTFU was due to lack of reliable contact information. Similar issues with follow-up were experienced by the Toronto-based Gift of Sight and Sound program, with specific barriers identified by interviewing participant’s parents: Lack of medical insurance coverage, lack of accessible transportation, inability for parents to take time off from work, and lack of confidence in navigating the health care system due to cultural and language barriers . To decrIn summary, the Ottawa iHear program has employed novel mobile audiometry technology in assessing 753 children over 3 years, identifying otherwise undetected cases of HL and connecting affected individuals with the healthcare resources they require. While the assessed cohort represents only a small proportion of children within the Ottawa area, it reflects a much larger need for grade school hearing assessments locally, provincially, and nationally. To that end, the iHear program has expanded to medical school campuses at the University of Saskatchewan, McGill University, and Dalhousie University. While expansion of the iHear program will provide access to hearing assessments for more children across Canada, the program as it stands is inherently limited to cities associated with medical school campuses.In conclusion, hearing loss in grade school populations continues to go undetected across Canada. Programs such as iHear demonstrate that gaps in hearing assessment service provision can be filled effectively by local technicians equipped with novel tablet play audiometry.


Custodial grandfamilies often face challenges, such as psychological distress, parenting burden, and grandchildren showing internalizing and externalizing behaviors. Given their circumstances, effective training and education is critical to provide a supportive family environment for both custodial grandparents and custodial grandchildren. The aim of this study was to develop, pilot and evaluate a psychosocial intervention for custodial grandfamilies. The program, interACT, was implemented virtually due to restrictions related to COVID-19. It is an intervention for custodial grandparents (CGPs) and custodial grandchildren (CGC) to improve psychological wellbeing and life skills. Participant eligibility was determined by grandfamilies having Iowa residency and legal guardianship or custody of an 8-12 year old grandchild. The program utilizes the Acceptance and Commitment Therapy (ACT) framework, focusing on psychological flexibility, acceptance, and psychosocial resilience for CGPs and CGC. The program used techniques such as mindfulness, self-compassion, and decision-making skills. During the pilot program stage, participants were divided into CGPs and CGC groups, and completed self-paced weekly 45 minute long modules through a program website. To enhance online session experience, Extension staff facilitated hour-long Zoom calls to discuss module contents and foster peer connection for both groups. Findings will be available at the conclusion of the pilot program. We expect to find increased psychological well-being and improved life skills for both groups right after the implementation of the ACT. Findings and limitations will be discussed with practical implication for program implementation via virtual delivery for the current custodial families. Future studies could extend current curriculum to other populations.


Live Long Walk Strong is a rehabilitation program that produces large clinically meaningful improvements in mobility when implemented as an outpatient program for older adults. We adapted Live Long Walk Strong for the post-acute nursing home setting within the Veterans Health Administration as a clinical demonstration project. The adapted version includes novel elements and bridges the inpatient stay and three months post-discharge. The inpatient phase focuses on maximizing functional recovery and includes activities focused on timing and coordination of gait, leg strength and power, and trunk muscle endurance. The care transition and virtual  post-discharge phase focuses on case management and engagement in physical activity programs. Coaching and behavior change are a consistent focus throughout the program. To date, 13 Veterans  have completed the inpatient phase, and of those Veterans, six have completed the entire program, five are still active, one was lost to follow-up, and one was rehospitalized. The program demonstrates feasibility, 91% of all inpatient sessions and 81% of all post-discharge sessions were completed. Regarding preliminary efficacy, 83% of Veterans who completed the program exceeded the minimal detectable change score (4 points) on the Activity Measure for Post-Acute Care (AM-PAC) Mobility scale from program enrollment to completion . Based on findings from this clinical demonstration project, the program is feasible. However, future research is needed to further examine the program’s impact on mobility and other outcomes important to older Veterans receiving post-acute nursing home care.


The CBEs fabricated by blending the POP- and SOS-rich fats in a weight ratio of 40:60 contained 63.7 w/w% total symmetric monounsaturated triacylglycerols, including 22.0 w/w% POP, 8.6 w/w% palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol, 33.1 w/w% SOS, and 1.3 w/w% DAGs, which was not substantially different from the DAG content in cocoa butter (1.1 w/w%). Based on the solid-fat content results, it was concluded that, when these CBEs were used for chocolate manufacture, they blended with cocoa butter at levels up to 40 w/w%, without distinctively altering the hardness and melting behavior of cocoa butter.Herein, we prepared 1,3-dipalmitoyl-2-oleoyl glycerol (POP)-rich fats with reduced levels of diacylglycerols (DAGs), adversely affecting the tempering of chocolate, via two-step hexane fractionation of palm stearin. DAG content in the as-prepared fats was lower than that in POP-rich fats obtained by previously reported conventional two-step acetone fractionation. Cocoa butter equivalents (CBEs) were fabricated by blending the as-prepared fats with 1,3-distearoyl-2-oleoyl glycerol (SOS)-rich fats obtained by hexane fractionation of degummed shea butter. POP-rich fats achieved under the best conditions for the fractionation of palm stearin had a significantly lower DAG content (1.6  The elevated level of DAGs was attributed to the concentration of DAGs in the liquid phase during the first step of the two-step acetone fractionation.In our previous studies ,8, we den-hexane, with polarity lower than that of acetone, was used as a solvent for fractionation, it concentrated more polar lipids than TAGs, such as DAG, monoacylglycerols, and fatty acids (FAs), in the solid phase rather than in the liquid phase [n-hexane can be an appropriate solvent for the two-step fractionation of palm stearin to fabricate POP-rich fats and the resultant CBEs containing reduced levels of DAGs compared to those in the products reported in our previous studies [n-hexane is the most widely used organic solvent in the edible oil extraction industry. Nevertheless, it has not been commonly used as the solvent for the fractionation of edible fats and oils. In South Korea, unlike acetone, the use of n-hexane is not legally permitted in the fractionation process. The aim of this study was to prepare POP-rich fats with reduced levels of DAGs by two-step hexane fractionation of palm stearin and to produce CBEs by blending these POP-rich fats with SOS-rich fats acquired by hexane fractionation of degummed shea butter. The DAG content in the as-prepared POP-rich fats was lower than that in the POP-rich fats produced by the previously reported two-step acetone fractionation [When id phase ,10. This studies ,8. SOS-r studies ,12. Howeionation . The besw/w% total SMUTs , 15.2 w/w% PPP, and 3.0 w/w% DAGs was used as the starting material for fractionation. Note that the contents of SMUTs in the lipid samples investigated in this study included the contents of their positional isomers, such as PPO  for POP, PSO  and SPO  for POS, and SSO  for SOS, as isomeric TAGs could not be separated by the high-performance liquid chromatography (HPLC)–evaporative light scattering detector (ELSD) system employed herein. The effects of temperature, volume-to-weight ratio of hexane-to-palm stearin, and crystallization time on the yields of the fractions and the contents of DAGs, POP, and PPP in the fractions were investigated, and the optimal conditions were determined.Herein, two-step hexane fractionation of palm stearin was performed to prepare POP-rich fats with lower levels of DAGs than those in the POP-rich fats prepared by previously reported two-step acetone fractionation of palm stearin . The firw/w% when the temperature was increased from −10 to 0 °C. In the temperature range 0–15 °C, no considerable difference was noticed in the yields of the liquid fraction (64.6–69.7 w/w%). w/w%) of the liquid fractions obtained at temperatures ranging from −5 to 15 °C were significantly lower than those of the liquid fractions (2.8–3.9 w/w%) produced at temperatures outside the range from −5 to 15 °C. The DAG contents (5.2–7.6 w/w%) of the liquid fractions acquired at temperatures in the range from −10 to 5 °C did not show a substantial difference; however, they were considerably lower than those of the liquid fractions (8.9–12.3 w/w%) obtained at 10–25 °C. These results suggest that the solubility of DAG in hexane significantly increases at temperatures above ~10 °C and fractionation should be performed at temperatures below ~10 °C to attain a liquid fraction with a lower content of DAGs. The maximum POP content (33.6 w/w%) was achieved at 5 °C. Thus, the best temperature for the first fractionation step was 5 °C. A possible approach to fabricate cost-effective CBE products by using targeted POP-rich fats obtained from palm stearin is to reduce the amount of hexane employed in the fractionation of palm stearin. To determine the most suitable volume-to-weight ratio of hexane-to-palm stearin for the first fractionation step, the process was performed, using different volume-to-weight ratios of hexane-to-palm stearin  at 5 °C for a crystallization time of 8 h. The yield of the liquid fraction obtained from palm stearin as a function of the volume-to-weight ratio of hexane-to-palm stearin is shown in w/w%) was not significantly different from that of the liquid fraction obtained at a ratio of 6 (61.9 w/w%), it was substantially lower than that of the liquid fraction achieved at a ratio of 8 (64.7 w/w%). The yield of the liquid fraction acquired at a ratio of 2 (47.7 w/w%) was considerably lower than those of the liquid fractions obtained at the ratios of 4–8. The contents of DAGs, PPP, POP, and total SMUTs in the liquid fraction as a function of the volume-to-weight ratio of hexane-to-palm stearin are shown in w/w%) of the liquid fractions attained at the ratios of 2–6. However, the PPP content of the liquid fraction achieved at a ratio of 4 (1.4 w/w%) was substantially lower than that of the liquid fraction acquired at a ratio of 8 (2.1 w/w%). There was no significant difference between the contents of DAGs (3.5–5.2 w/w%), POP (31.6–34.4 w/w%), and total SMUTs (41.6–42.7 w/w%) of all liquid fractions obtained at any specific ratio. Therefore, the most appropriate volume-to-weight ratio of hexane-to-palm stearin for the first fractionation step was 4.Temperature is the most important factor for solvent fractionation of edible fats because the separation of higher-melting and lower-melting TAGs occurs based on the difference in their melting degrees and solubility at a specific temperature . To detew/w%) of the liquid fractions during 1–16 h of crystallization. However, based on the two-tailed Student’s t-test results, the yield of the liquid fraction reached a constant value after 6 h of crystallization, and the yield of the liquid fraction acquired at 6 h (60.1 w/w%) of crystallization was considerably higher than that of the liquid fraction achieved at 4 h (58.4 w/w%) of crystallization. No substantial difference was noticed between the DAG (3.4–4.3 w/w%), PPP (1.4–1.6 w/w%), POP (30.8–33.2 w/w%), and total SMUT (42.0–43.8 w/w%) contents of all liquid fractions attained at different crystallization times, except for the fraction obtained at 16 h of crystallization  significantly decreased compared to that of the solid fraction attained at −5 °C (39.7 w/w%). The contents of DAGs, PPP, POP, and total SMUTs in the solid fraction as a function of temperature are shown in w/w%) in the solid fraction achieved at −5 °C was maximum; however, the difference between the yields of the solid fractions produced at −10 °C (44.9 w/w%) and 5 °C (46.8 w/w%) was not statistically significant. The contents of DAGs (2.8–4.1 w/w%) in the solid fractions obtained at temperatures ranging from −20 to −5 °C were not considerably different; nevertheless, the DAG content was significantly and remarkably high in the solid fraction attained at 0 °C (11.1 w/w%) owing to reduction in the content of higher-melting SMUTs (such as POS) than that of POP. The POS content was in the range 12.3–13.6 w/w% in the solid fraction obtained at temperatures ranging from −20 to −5 °C, whereas it decreased to 5.6 w/w% in the solid fraction achieved at 0 °C. Thus, −5 °C was determined to be the best temperature for the second fractionation step.To determine the best temperature for the second fractionation step, the process was conducted by using a volume-to-weight ratio of hexane-to-PS-LF of 4 at desired temperatures  for a crystallization time of 24 h. w/w%) of the solid fractions acquired at a crystallization time of 12–24 h. w/w%) was achieved at 12 h of crystallization time. The DAG contents (2.4–3.4 w/w%) of the solid fractions did not considerably vary at all the crystallization times investigated herein. Thus, the best crystallization time for the second fractionation step was 12 h. Finally, PS-LF-SF was attained under the best conditions  established for the second fractionation step.To discover the best crystallization time for the second fractionation step, crystallization was performed for 1–24 h, using a volume-to-weight ratio of hexane-to-PS-LF of 4 at −5 °C. w/w%) used in the small-scale fractionation was performed to prepare fractions including PS-LF-SF; moreover, the DAG contents of the fractions were compared with those of the counterpart fractions acquired by the previously reported two-step acetone fractionation of the same quantity of palm stearin [w/w%) of DAGs than that in the counterpart fraction (DAG content = 4.9 w/w%) produced by the first acetone fractionation step. These phenomena were ascribed to the higher concentration of DAGs in PS-SF produced along with PS-LF during the first hexane fractionation step (DAG content = 3.1 w/w%) than that in PS-SF obtained by the first acetone fractionation step (DAG content <0.05 w/w%); this indicated that DAGs were rarely removed from PS-LF during the first acetone fractionation step. These results are in accordance with those reported in previous studies [w/w%) of DAGs was acquired from PS-LF by the second hexane fractionation step, whose DAG level was lower than that in the counterpart fraction (DAG content = 4.6 w/w%) obtained by the second acetone fractionation step. However, PS-LF-LF achieved along with PS-LF-SF by the second hexane fractionation step also contained significantly lower level of DAGs (DAG content = 7.0 w/w%) than that in the counterpart fraction (DAG content = 14.2 w/w%) produced by the second acetone fractionation step. These results suggested that unlike the case of the first fractionation step, no distinct tendency to concentrate DAG toward the solid fraction was observed during the second hexane fractionation step compared with the case of the second acetone fractionation step. These phenomena were attributed to the crystallization of a considerable portion of DAGs at 4 °C during the second acetone fractionation step compared to that in the case of the first acetone fractionation step at 17 °C. Therefore, these findings imply that the two-step hexane fractionation method proposed in this study is a more effective approach for the preparation of POP-rich fats containing a lower level of DAGs from palm stearin than the previously reported two-step acetone fractionation procedure.To investigate whether the two-step hexane fractionation method established in this study was effective in reducing the level of DAGs in PS-LF-SF, large-scale fractionation using ten times  the quantity of palm stearin  [w/w% total SMUTs  and 1.8 w/w% DAGs was used as the starting material for fractionation. Best temperature and crystallization time for the fractionation of degummed shea butter were determined by examining the effects of temperature and crystallization time on the yields of the fractions and the contents of DAG and SOS in the fractions. Unlike the case of the fractionation of palm stearin for the preparation of PS-LF-SF, the best volume-to-weight ratio of hexane-to-degummed shea butter was not determined herein. Instead, the volume-to-weight ratio of hexane-to-degummed shea butter was fixed at 8, which was the same as the volume-to-weight ratio of hexane-to-shea butter used for degumming. Therefore, SOS-rich fats were directly acquired from the degummed shea butter existing in the liquid phase after degumming; nevertheless, herein, hexane was removed after degumming to establish the best temperature and crystallization time conditions for the fractionation of degummed shea butter.Hexane fractionation of the shea butter acquired after degumming was performed to prepare SOS-rich fats. Herein, the term “degumming” refers to the removal of gummy unsaponifiable substances  rather than the removal of phosphatides, which are typical gums present in common edible fats and oils. Shea butter contains high levels of unsaponifiable substances (5–10 w/w%) ,14. For w/w%) . Furtherw/w%) acquired at temperatures ranging from −20 to −5 °C. However, the yield of the solid fraction obtained at 0 °C (7.5 w/w%) was substantially lower than that of the solid fraction attained at −5 °C (40.3 w/w%). Degummed shea butter did not crystallize at temperatures >0 °C. No considerable difference was observed between the SOS (52.6–54.2 w/w%) and DAG (1.2–1.8 w/w%) contents of the solid fractions achieved at temperatures ranging from −20 to −5 °C  for a crystallization time of 24 h. to −5 °C B. Therefw/w%) (w/w%) than those of the other fractions (52.3–54.9 w/w%); in contrast, no considerable difference was found in the DAG contents of all the fractions (1.3–1.8 w/w%)  C. Among .8 w/w%) D. Thus, w/w%) and DAGs (1.2–1.6 w/w%) (data not shown here). The melting temperature ranges  of the blends containing larger quantities of shea stearin were lower (w/w%). The melting temperature range of all the blends, including the 40:60 blend (10.5–30.8 °C), was significantly lower than that of cocoa butter (11.4–31.5 °C), except for the 30:70 blend (10.7–33.2 °C). Unlike the other four blends showing melting events consisting of two peaks, the 30:70 blend had an additional small peak at approximately 30 °C. This different melting profile of the 30:70 blend from those of the other blends caused the higher melting completion temperature of this blend than that of cocoa butter, which might result in incomplete melting of this blend at a temperature where the cocoa butter is completely melted. To establish the best blending ratio of PS-LF-SF and shea stearin for the preparation of CBEs, five different types of PS-LF-SF–shea stearin blends with PS-LF-SF:shea stearin weight ratios of 70:30, 60:40, 50:50, 40:60, and 30:70 were fabricated, and their differential scanning calorimetry (DSC) melting and crystallization profiles were compared with those of cocoa butter. The range of the blending ratios of PS-LF-SF and shea stearin was determined based on the results of preliminary examinations. The blends contained similar levels of total SMUTs (62.1–63.7 re lower A. The low/w%) was not significantly different from that of cocoa butter (1.1 w/w%). Kang et al. [w/w%; analyzed by GC) than that of the cocoa butter  used in their study. These results suggest that via two-step hexane fractionation of palm stearin, low-DAG POP-rich fats can be produced from palm stearin and subsequently utilized to fabricate CBEs containing a similar level of DAG to cocoa butter. The CBEs comprised 22.0 w/w% POP, 8.6 w/w% POS, and 33.1 w/w% SOS, with a total SMUT content of 63.7 w/w%. The total SMUT content of the CBEs was substantially lower than that of cocoa butter (75.9 w/w%). The presence of large amounts of TAGs, such as PPP and tristearin (SSS) with higher melting points than those of SMUTs causes incomplete melting of chocolate products [w/w%) was significantly higher than that in cocoa butter (2.3 w/w). However, no considerable difference was found between the SSS contents of the CBEs (3.0 w/w%) and cocoa butter (2.5 w/w%). Because of the relatively lower content of SMUTs and higher content of SSS in CBEs than those in cocoa butter, it was expected that the CBEs prepared in this study would not satisfy the theoretical physical properties of CBEs, i.e., the ability to blend with cocoa butter in any proportion without altering the physical properties of cocoa butter.DAG contents and TAG compositions of the CBEs prepared by using the 40:60 PS-LF-S–shea stearin blend were compared with those of cocoa butter . The DAGg et al.  preparedproducts . Althougw/w% for 16:0 and 35.1 w/w% for 18:0) than those in cocoa butter (26.0 w/w% for 16:0 and 35.9 w/w% for 18:0), whereas the content of oleic acid (18:1n-9) in the CBEs (35.5 w/w%) was considerably higher than that in cocoa butter (32.8 w/w%). In both CBEs and cocoa butter, as expected, 16:0 and 18:0 were predominantly located at the sn-1,3 positions, whereas 18:1n-9 mainly existed at the sn-2 position. The tendency for this specific positional distribution of FAs was less pronounced in CBEs than that in cocoa butter. In other words, in CBEs, the contents of 16:0 (30.7 w/w%) and 18:0 (49.6 w/w%) at the sn-1,3 positions were ~3.9 and ~8.3 times higher those at the sn-2 position (7.8 w/w% for 16:0 and 6.0 w/w% for 18:0), respectively. In contrast, the contents of 16:0 (38.2 w/w%) and 18:0 (52.8 w/w%) at the sn-1,3 position of cocoa butter were ~22.5 and ~25.1 times higher than those at the sn-2 position (1.7 w/w% for 16:0 and 2.1 w/w% for 18:0), respectively. These results suggest the possibility of a more prevalent presence of SMUT positional isomers in CBEs than in cocoa butter.Total FA composition and positional composition of FAs in CBEs and cocoa butter are provided in −1 for the 95:5 blend and 54.2 J g−1 for the 10:90 blend), which were substantially lower than that of cocoa butter (68.0 J g−1) (data not shown here). Compared to the DSC melting profile of cocoa butter, the DSC melting profile of the cocoa butter–CBE blends had a shoulder peak at temperatures lower than those at which the main peak was found, and the intensity of this shoulder peak was higher for the blends containing larger quantities of CBEs. Melting and crystallization properties of cocoa butter and cocoa butter–CBE blends with different cocoa butter:CBE weight ratios of 95:5, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 10:90 were compared, using DSC thermal profiles. The melting temperature ranges of the cocoa butter–CBE blends containing larger quantities of CBEs were significantly lower than that of cocoa butter A. Moreovw/w% CBEs showed similar changes to that of cocoa butter in the temperature range examined herein  of chocolate products . The SFCd herein A. In oth20–30 °C B. These n-hexane (>99.5%), acetone (>99.3%), acetonitrile (>99.9%), and dichloromethane (>99.9%) were procured from J.T. Baker . Silica gel–coated glass plates  for thin-layer chromatography (TLC) were purchased from Merck KGaA . Pancreatic lipase was acquired from Sigma Chemical Co. . Triolein , 1,2-dioleoyl-3-palmitoyl-rac-glycerol , POP (≥99%), and PPP (≥99%), used as TAG standards, were also purchased from Sigma Chemical Co. All other reagents used herein were HPLC-grade.Spanish cocoa butter was purchased from Sunin Co. . Palm stearin and shea butter were obtained from Ottogi Co.  and Korea Similac , respectively. HPLC-grade 2 flush to obtain a liquid fraction. The liquid fraction achieved under the best conditions for the first fractionation step was designated as PS-LF. Optimal temperature, volume-to-weight ratio of hexane-to-palm stearin, and crystallization time were established by considering the yields of the fractions and the contents of DAGs, POP, and PPP in the fractions. Second fractionation step, i.e., fractionation of PS-LF, was conducted in the same manner as the first fractionation step. PS-LF (5 g) was crystallized in 20 mL hexane under stirring at 300 rpm at a desired temperature (ranging from −20 to 0 °C) for a desired time period (0–24 h). The solid fraction acquired under the best conditions for the second fractionation step was designated as PS-LF-SF. Optimal temperature and crystallization time were determined by considering the yields of the fractions and the contents of DAG and POP in the fractions. Large-scale fractionation of palm stearin (50 g) was performed in a flat-bottom glass vessel (11 cm × 10 cm i.d.) under the best conditions established for the abovementioned small-scale process to fabricate CBEs by blending POP-rich fats with the SOS-rich fats obtained by the fractionation of shea butter. For the second step of large-scale fractionation, PS-LF dissolved in hexane was used as the starting material.Herein, two-step hexane fractionation of palm stearin was performed in a flat-bottom glass vessel (8 cm × 3.5 cm i.d.) equipped with a water jacket for temperature control to prepare POP-rich fats  was crystallized in hexane (40 mL) under stirring at 300 rpm at a desired temperature (ranging from −20 to 0 °C) for a desired time period (0–24 h). The solid fraction attained under the best conditions for fractionation was denoted as shea stearin. The best temperature and crystallization time were determined by considering the yields of the fractions and the content of SOS in the fractions. Large-scale fractionation of degummed shea butter (50 g) was conducted in a flat-bottom glass vessel (11 cm × 10 cm i.d.) under the optimal conditions established for the abovementioned small-scale fractionation to obtain shea stearin in sufficient quantity for blending with PS-LF-SF for the preparation of CBEs.Fractionation of the shea butter achieved after degumming was conducted to acquire shea stearin, which is rich in SOS B. DegummPS-LF-SF and shea stearin were blended in PS-LF-SF:shea stearin weight ratios of 70:30, 60:40, 50:50, 40:60, and 30:70. The best blending ratio (40:60) of PS-LF-SF and shea stearin was determined by comparing the DSC melting and crystallization profiles of the blends with those of cocoa butter. CBEs were prepared by blending PS-LF-SF with shea stearin in the best blending ratio, and were stored at −20 °C prior to analysis. Cocoa butter was blended with the CBEs in cocoa butter:CBE weight ratios of 95: 5, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 10:90. The resulting cocoa butter–CBE blend samples were stored at −20 °C prior to analysis.−1. Injector and detector temperatures were maintained at 225 and 285 °C, respectively. Oven temperature was initially set at 100 °C for 4 min, increased to 240 °C at a rate of 3 °C min−1, and finally maintained at 240 °C for 17 min. FAMEs were identified by comparing their retention times with those of the standards, and their relative contents were calculated as w/w%. Compositions of the FAs at the sn-2 and sn-1,3 positions of TAG in lipid samples were determined according to a previously reported method [w/v% sodium cholate solution, and 0.2 mL of 2.2 w/v% calcium chloride solution. After thorough vortexing to emulsify the samples, 20 mg pancreatic lipase was added to the resulting samples. The mixtures were incubated in a water bath at 40 °C for 2 min and vigorously vortexed for 30 s, followed by the addition of 5 mL of 6 M hydrochloric acid. Anhydrous diethyl ether (15 mL × 2) was added to the abovementioned mixtures followed by vortexing. The upper diethyl ether layer containing pancreatic lipase hydrolysates was collected and passed through an anhydrous sodium sulfate column. Then, the hydrolysates were placed on a silica gel G TLC plate  and developed with hexane/diethyl ether/acetic acid . The plate was dried in air and sprayed with 0.2 w/v% 2,7-dichlorofluorescein in methanol. The band corresponding to sn-2 monoacylglycerols was scraped from the plates, methylated, and analyzed by GC as previously reported to determine the composition of the FAs at the sn-2 position. The composition of FAs at the sn-1,3 position was evaluated by using the following Equation (1):Total FAs composition of the lipid samples was analyzed according to a previously reported method . Lipid sd method . Lipid s® 3300, Alltech Associates Inc., Deerfield, IL, USA) was operated at a drift tube temperature of 70 °C, a gain setting of 4, and a N2 gas flow rate of 2.5 L min−1. Separation of TAGs was performed by using a Develosil C30 column  and a mobile phase consisting of (A) acetonitrile and (B) dichloromethane at a flow rate of 1 L min−1. Lipid samples (1 mg) were dissolved in 4 mL dichloromethane and filtered through a 0.45 μm hydrophobic PTFE membrane , and the resultant solution was injected into the column for analysis. Herein, twelve TAGs were detected in the samples and identified by comparing their retention times with those of the corresponding standards  analyzed in this study or according to previously reported results [w/w% of the total contents of TAGs and DAGs found in the samples. Compositions of TAGs in the lipid samples were examined by using an HPLC system coupled with an ELSD, according to a previously reported method  with min results ,17. Eachw/w% of the total contents of TAGs and DAGs found in the samples.DAGs present in the lipid samples were separated by HPLC–ELSD as abovementioned in −1 as a reference standard. Liquid N2 was used as a coolant. The flow of the coolant through the circulating chamber attached to the DSC system was controlled by Intracooler 2P . Lipid samples (6–8 mg) were tightly sealed in a 50 μL Al pan, and an empty sealed pan was employed as a reference. These lipid samples were rapidly heated from room temperature to 80 °C and maintained at this temperature for 10 min before being cooled to −60 °C, at a rate of 10 °C min−1, to achieve the crystallization profile. After maintaining the samples at −60 °C for 30 min, the resulting samples were heated to 80 °C, at a rate of 5 °C/min, to obtain the melting profile. The profiles were analyzed by the software provided with the DSC system .DSC melting and crystallization temperatures and enthalpies of the lipid samples were evaluated by using a DSC 4000  according to AOCS Official Method Cj 1–94 . Normal SFC of the lipid samples was determined according to the AOCS official method Cd 16b-93 . Nucleart-test or one-way analysis of variance, followed by Duncan’s multiple range test. Differences were considered significant if the p-value was less than 0.05.Statistical analysis was conducted using IBM SPSS Statistics (version 25) software . All data are presented as the mean ± standard deviation. The difference between lipid samples was assessed by using a two-tailed Student’s w/w% CBEs could be added to cocoa butter to attain the cocoa butter–CBE blend without significantly altering the physical properties (such as hardness and melting behavior) of cocoa butter for chocolate manufacture. The limit in the addition quantity of CBEs to cocoa butter was owing to the relatively lower level of SMUTs and a relatively higher level of SSS in CBEs than those in cocoa butter. Finally, this study contains limitations. The contents of individual TAG species in the CBEs reported by this study included the contents of their positional isomers, because isomeric TAGs could not be separated by the HPLC–ELSD system used in this study. Furthermore, although the existence of some more TAG species, such as stearoyl-oleoyl-linolein (SOL), is theoretically possible in the CBEs, they were not detected by using the HPLC–ELSD system. Further investigation on the separation and identification of TAGs in the CBEs by using mass spectrometry–based methods would be required to obtain the accurate TAG composition of the CBEs.In this study, CBEs were prepared by blending POP-rich fats obtained by two-step hexane fractionation of palm stearin and SOS-rich fats acquired by hexane fractionation of degummed shea butter. This study demonstrated that the two-step hexane fractionation method is a more effective approach for the preparation of POP-rich fats containing a lower level of DAGs from palm stearin than the previously reported two-step acetone fractionation procedure. We established the best conditions  for the fractionation of palm stearin to achieve desired levels of chemical constituents , affecting the physical and textural properties of chocolate, in the resultant POP-rich fats. Similarly, for the fractionation of degummed shea butter, we determined the best conditions  to prepare SOS-rich fats containing desired levels of DAGs and SOS. Accordingly, the CBEs fabricated in this study also contained comparable levels of DAGs to that of cocoa butter. Based on the SFC results, we concluded that up to 40