The impact of mulberry leaf extract at three different levels on reducing the glycemic index of white bread

In this study, the influences of mulberry leaf extract (MLE) addition on the physicochemical properties including the specific volume, texture and sensory features of white bread (WB) were evaluated by the sensory analysis technology. A double-blind, randomised, repeat-measure design was used to study the impact of MLE addition on the postprandial blood glucose response as well as the satiety index of WB. Results showed that the addition of MLE showed no significant effects on the physicochemical properties of WB except for the slight changes of color and bitterness. The addition of MLE significantly reduced the total blood glucose rise after ingestion of WB over 120 minutes, and reduced the GI value of WB in a dose-effect relationship. When the concentration of MLE reached 1.5 g per 100 g available carbohydrate, the GI value of WB could be reduced from 77 to 43. This study provides important information in terms of the appropriateness of MLE when added to more complex real food, the dose-dependent relationship could supply a reference for the application of MLE.

Twelve subjects were randomly selected from 30 subjects to participate the GI tests of the experiment groups, and 18 subjects participated in the test of control group.The investigator followed the specified randomization procedures of the protocol and ensured that the randomization code is broken only in accordance with the requirements of the protocol.

Ethics
The ethical approval for the study was obtained at the Chinese Academy of Medical Sciences and Beijing Union Medical College Hospital Ethics Review Committee (protocol code: HS-1763, first issued: November 27, 2018, latest updated on September 27, 2021).
10. Standard operation process The test was carried out in strict accordance with ISO 26642:2010 standard.In this experiment, blood was taken from the fingertip of a disposable blood sampling needle.
[Each GI test cycle] ①Set up participant's testing area with labels, test record sheet, alcohol swabs, lancets, finger prick device and timer.②Record weight of each participant with shoes off upon arrival for the test each time.③Fill out top section of blood glucose test record and double check for accuracy.④Label tube with -5, 0 labels containing participant's initials, date and test food.The participants' identity will be subsequently decoded for sample analysis.⑤Ensure participant is using own personalised blood-taking supplies and is familiar with safety procedures as outlined in consent form.⑥Wipe participants finger with alcohol pad, then prick finger for fasting samples.Note: The capillary blood samples will be collected by trained research staff or phlebotomist.⑦Collect 350 µL of blood in test tube and mix blood with small amount of anti-coagulant at the bottom of the tube by rolling between hands.If glucose strips are used, blood is directly dropped on the strip and the values read immediately.⑧Then the second fasting sample is taken at 0 minute and blood is collected accordingly.⑱Participants will be reimbursed for their time participating in the study.If a participant withdraws from the study or becomes ineligible and are withdrawn by the investigator prior to completing the full study, they will be compensated for portions of the study completed to that point.If this occurs, the participant will be replaced in order to maintain a minimum of twelve ( 12) participants with complete data for statistical validity.The randomization sequence for the replacement will be the same as the participant who drops out from the study.Note: A wash out period of three (3) days (72 hours) is required between two different tests.11.Statistical analysis methods All statistical analyses were performed using SAS version 9.4.The blood glucose data were calculated from the point values of all the time spots of every participant, and the means and standard deviation (SD) were calculated for the normality test.All data were processed and compared using origin 2021 and Graph Prism 7.0.Data were analyzed by one-way ANOVA and paired t-test and differences with values of p<0.01 or p<0.05 were considered significantly differences.
Blood collection: fingertip-prick blood sample collection.②Calculation of GI The GI was calculated with the reference glucose whose IAUC set as 100 (white bread is set as 71)，the GI value of the product is: Imean=Test food IAUC/glucose IAUC×100 ③Satiety evaluation The VAS scale was used for the comparison of satiety.Subjects' satiety change were record at 8 time points (0 min, 15 min, 30 min, 45 min, 60 min, 90 min and 120 min) within 2 hours after eating, and the data were collected and processed.
12. Publishing plan This manuscript is planned to be submitted and published in 2023.
sure participant consumes test food or reference food and water within 5 to 10 minutes.However, if the food cannot be consumed in 10 minutes, the participant should try to consume the entire food, if possible, even if it takes longer than 10 minutes.Take as long as required to consume the entire test food or reference food.Participant may have to stop eating to take the 10 minute finger prick and continue eating.⑫Recordtime taken to eat entire meal on the test record sheet.⑬Prick finger, collect blood, roll tube, label tube, place in fridge.Continue this step for 30, 45, 60, 90, and 120 minutes, ensuring all tubes are properly labeled.If glucose strips are used, no need to collect blood, but can be analysed immediately.⑭Used needles are thrown into the biohazard collection container.Any other contaminated waste is also disposed of in the appropriate container.⑮Ensure test record is complete and accurate.⑯Direct participants to snack area if they want a snack before leaving.⑰Disinfect tables and kitchen counters with 70% ethanol solution.
Taking 2 samples within 5 minutes is acceptable.⑨Servereference food/test food to the participant according to the study protocol with 250 mL of water ⑩Start timer with the first bite of test meal.Timers should be set at count down or count up to 2 hours.⑪Make