Glycemic Index, Starch, and Protein Digestibility in Tempeh Gembus Cookies

Diabetes mellitus is directly related to diet and lifestyle. Control of blood glucose levels is needed to reduce the risk of complications, and one way is to choose foods with a low glycemic index. Cookies made from tempeh gembus/tempeh gembus our are expected to be eaten as a snack and are safe for people with diabetes. e aim of this research was to analyze glycemic index (GI), glycemic load (GL), dietary ber, in vitro starch, and protein digestibility of cookies with tempeh gembus our substitution. Completely randomized design research with one primary factor used cookies with variations of 0%, 25%, and 50% tempeh gembus our substitution. GI was calculated using the Incremental Area Under the Blood Glucose Response Curve (IAUC) method. Dietary ber concentration analysis was done by enzymatic methods. e starch and protein digestion rates were calculated using the in vitro method. GI, GL, dietary ber, starch digestion rate, and protein digestion rate data were analyzed with descriptive methods. Cookies with lowest GI (47.01± 11.08%) and GL (6.90± 1.63) were found in cookies with 50% tempeh gembus our substitution. e highest dietary ber content (24.61± 0.41%), digestibility of starch (48.07± 0.01%), and protein (20.27± 0.43%) cookies were found in cookies with 50% tempeh gembus our substitution. e higher tempeh gembus our substitution produced low GI and GL while its dietary ber, in vitro starch, and protein digestibility were highest.


Introduction
Diabetes mellitus (DM) is a group of metabolic disorders characterized by hyperglycemia which is caused by impaired insulin secretion or/and insulin performance disorder. Hyperglycemia is a medical condition when the blood glucose level increases above normal levels [1]. e World Health Organization (WHO) predicted there would be a prevalence increase of DM in Indonesia, from 8.  [2].
Snacks can help to stabilize blood glucose levels. One of the best strategies of meal schedule adjustment to help control blood glucose levels is choosing food with low glycemic index (GI). Foods with low GI do not increase blood glucose quickly, so their consumption overcomes insulin sensitivity and is useful in blood glucose management [3]. Glycemic load (GL) will give more thorough information about food consumption e ects on the blood glucose rate and provide a better estimate of the amount of carbohydrates in food.
Dietary ber can a ect the blood glucose concentration [4]. Foods with high dietary ber have a high contribution towards low GI level. Fiber will slow down the food movement in the digestive tract and slow down the enzyme activity, so that the digestive process, especially starch will get slowed down and the blood glucose response will be slower. erefore, the GI rate will be lower [5]. High protein rate will stimulate insulin secretion, so the glucose in blood will not become excessive and will be controlled [6]. Protein quality in food is determined by the level of amino acids that have complete sequence, suitable composition for the body's need, and a high protein digestion rate. Low starch digestion rate means lower starch hydrolysis by digestive enzymes in a certain period of time. erefore, blood glucose level will not increase drastically after the food is digested and metabolized by the body. Foods with the high starch digestion rate will generate high GI levels.
Tempeh gembus is a popular traditional food consumed by low-income citizens. Tempeh gembus is made from tofu dregs and goes through a fermentation process by Rhizopus oligosporus [7]. A previous study showed that tempeh gembus has health benefits including antioxidants [8], antimicrobial activity [9], ability to reduce the inflammatory reaction [10][11][12][13][14][15], and reduce LDL-C and total cholesterol. Tempeh gembus contains 50% less energy compared to regular tempeh, 3 times the concentration of dietary fiber, and has complete amino acids composition [16,17].
One way to increase snack variation for diabetics is the utilization of high-fiber, low-glycemic index tempeh gembus in the form of snack products such as crackers (kerupuk) and cookies [18]. Cookies are chosen to be healthy snacks because they can be stored for a long period of time, are easy to carry, and are acceptable to people of all ages and socioeconomic levels.
Snacks for diabetics are not yet readily available in Indonesia, so the authors are interested in researching about foods such as cookies with high-fiber, high-protein, and low-GI ingredients that can be consumed as healthy snacks and are safe for diabetics. is research aimed to know the level of the GI, GL, dietary fiber, starch, and protein digestion rate of tempeh gembus cookies.

Materials and Methods
Dietary fiber, starch digestion rate, and protein digestion rate were measured in the Laboratorium Chemix, Yogyakarta. GI and GL examinations were performed in the Universitas Diponegoro, Indonesia.
is research used a completely randomized study design with one primary factor. Tempeh gembus flour substitutions that were used were 0%, 25%, and 50%. ree repetitions were done for each treatment. e following table contains the cookies' formulation with tempeh gembus flour substitution (Table 1). e baking process of the cookies started with making the tempeh gembus flour. Tempeh gembus was thinly sliced and dried at 110°C in an oven for 30 minutes. Dried tempeh gembus was ground with a blender and then sifted with a sieve. Other ingredients for the cookies are 40 g margarine, 30 g skim milk, 20 g stevia sugar, 0.1 g baking powder, 10 g corn starch, and 15 g egg yolk. Cookies were made by homogenizing margarine and stevia sugar with a mixer, adding egg yolk, then adding the rest of ingredients, and mixing until combined completely. e data collected from this research are as follows: GI, GL, dietary fiber, starch digestion rate, and protein digestion rate. e Incremental Area Under the Blood Glucose Response Curve (IAUC) method was used for the GI calculation.
GI testing was carried out on 28 subjects with the following inclusion criteria: man or woman between 18-30 years of age, normal body mass index (BMI) (18.5-22.9 kg/m 2 ), normal blood glucose concentration when fasting (70-100 mg/dl), nonsmokers and nondrinkers, no history of DM disease in family history, and completed the informed consent from stating the willingness to be a research subject. Exclusion criteria were as follows: using medicine and supplements during intervention, not present at the sample taking time, and/or sick during intervention. e day before intervention, the subject was required to fast (except water) for 10 hours from 22:00 to 08:00 until the next day. en, the subject's capillary blood was taken to calculate the blood glucose concentration when fasting. Next, the subject was asked to consume test food (pure glucose) and tempeh gembus flour cookies with the 0%, 25%, and 50% rate that contain 50 grams of available carbohydrate. e blood glucose level was tested every 30 minutes (30, 60, 90, and 120 minutes mark) after consuming the test food every two hours. e GI test was performed using a glucometer from the EasyTouch GCU brand. Dietary fiber rate analysis was carried out with enzymatic methods. e starch and protein digestion rates were measured with in vitro methods.
is research received ethical clearance from the appropriate institutional review board with no. 625/EC/FK-UNDIPXI/2018. e collected data were tested descriptively to describe the cookies' glycemic index rate, glycemic load, dietary fiber, starch digestion rate, and protein digestion rate.

Subjects' Characteristics.
ere were 28 female subjects who signed the informed consent form with age range between 18-22 years old, BMI between 18.5-22.9 kg/m 2 , and normal FBI (70-100 mg/dl). Research subjects' characteristics are shown in Table 2. Table 2 shows that subjects' average age is 20 years with an average BMI of 20.9 kg/m 2 and average FBG of 83.4 mg/ dl.

Cookies' Weight
Stipulation per Subject. Food tested for their glycemic index rate was cookies with P 0 (0%), P 1 (25%), and P 2 (50%) tempeh gembus flour substitution while the 2 Journal of Food Quality standard food used for comparison was pure glucose. Each food contained 50 g of available carbohydrate known by the amount of total sugar and starch from the food [19]. e cookies' weights for each subject are P 0 (95.45 g), P 1 (89.97 g), and P 2 (85.18 g), where each weight has the equivalent of 50 g carbohydrate. Food with standard pure glucose that was given was 50 g powder dissolved in 240 ml of water. Cookies' weight stipulation per subject is shown in Table 3. Table 4 shows that the cookies' GI rate with tempeh gembus flour substitution in P 0 (68.67) falls under medium category while P 1 and P 2 tempeh gembus flour substitutions fall under low category. GI rates are shown in Table 4. Table 5 shows that the cookies' GL level with P 0 (8.99), P 1 (7.45), and P 2 (6.90) tempeh gembus flour substitutions fall under the low category. GL rates are shown in Table 5.

Discussion
Subjects that were used in this research are suitable according to the inclusion criteria. After being tested with ANOVA analysis, age data, BMI, and FBG showed there were no significant differences, so subjects were considered homogeneous. GI testing was performed using pure glucose as standard food and cookies with 0%, 25%, and 50% tempeh gembus flour substitution as test food. All of the foods that were tested are equivalent to 50 g carbohydrate, determined by the rate of their available carbohydrate. Glycemic index (GI) is a way to give a picture of the relations between carbohydrates in food with responses in blood glucose levels. Food that has low GI can optimize glycemic control in diabetics by slowing down the carbohydrate absorption [20]. GI is classified in three categories, which are low GI (<55), medium GI (55-75), and high GI (>70). Cookies with 50% tempeh gembus flour substitution have the lowest GI, which is 47.01%. GI rate results show that the higher the tempeh gembus flour substitution, the lower its GI.
Factors that affect a certain food's GI are dietary fiber, starch digestion rate, fat and protein content, and the preparation [21]. Low GI rate may be caused by tempeh gembus, the main ingredient in this research. Tempeh gembus contains 50% of the energy level of regular tempeh, has higher protein and fat content, and has 3 times the concentration of dietary fiber [16]. Fiber affects food's GI related with its role as a physical inhibitor in the digestive process. Fiber consumption in sufficient amounts will benefit the blood glucose management [22]. It corresponds with research that stated the increase in dietary fiber consumption is related to better GI control [23].
Protein content in tempeh gembus can also affect the low GI rate. High protein content can stimulate insulin secretion, so the glucose in blood is controlled and does not become excessive. e concentration of protein in food will decrease the amount of glucose that is being taken out from systemic circulation, thereby decreasing the food GI rate [24]. Low-GI food will get digested and turned to glucose gradually, and as a result, the highest peak of blood glucose concentration will be low. is means the increase in glucose rate fluctuation is relatively low. On the contrary, high-GI food will get digested and turned into glucose quickly. erefore, to control the blood glucose concentration, diabetics are suggested to consume food with low GI [25].
GI gives information about the speed of carbohydrate transformation to blood glucose, but it does not give information about the amount of carbohydrates and the impact that certain foods have on blood glucose level. Glycemic load (GL) can give information about the effect that food consumption has on the elevation of blood glucose level. e purpose of low-GI food consumption is to decrease GL. GL is used to evaluate the effect of carbohydrate consumption with food GI taken into account [21]. Food GL is classified into three categories: low GL (<10), medium GL (11)(12)(13)(14)(15)(16)(17)(18)(19), and high GL (>20) [3]. Cookies with 50% tempeh gembus flour substitution have the lowest GL rate, at 6.90%. e GL rate result shows that the higher the tempeh gembus flour substitution, the lower GI in cookies. is is caused by the low number of available carbohydrates. Food with low GI and GL will produce low blood glucose response after being consumed [26]. is corresponds to the previous study which stated consuming food with low-GI and GL rates causes slow elevation of blood glucose levels [27].
Dietary fiber concentration in cookies with tempeh gembus flour substitution is higher than that in control treatment cookies. Cookies with 50% tempeh gembus flour substitution have higher dietary fiber, which is 24.61%. Dietary fiber concentration increase is caused by the increasing of tempeh gembus flour substitution. is is supported by the previous study which stated that the low wheat flour percentage in the cookie formula decreases the dietary fiber concentration [28]. e suggested fiber intake for diabetics is 25 g/day [29]. Fiber concentration in every P 2 cookies is 6.15 g, obtained from cookies' weight on one portion (25 g) per 100 g and then multiplied by the fiber Starch digestion is the level of easiness of certain starchy foods to get hydrolyzed by starch enzymes and broken down to simpler units of nutrition. Tempeh gembus flour substitution cookies' starch digestion rate is higher than the control treatment cookies' starch digestion rate. e higher the tempeh gembus flour substitution, the higher the starch digestion rate.
Starch digestibility is determined by several factors, such as amylose and amylopectin, fiber, along with the preparation process [30]. Based on enzymatic hydrolytic mechanism, amylose can be hydrolyzed with one enzyme which is α-amylase while amylopectin because of its branching chain will be hydrolyzed by more than one enzyme such as α -amylase and then continued by α (1-6) glucosidase. Amylopectin molecule weight is higher than amylose. Based on this consideration, amylopectin needs more time to get digested than amylose [31]. Products with low amylose are estimated to have higher starch digestion rate [32]. Dietary fiber also affects starch digestibility. Food with high dietary fiber will be more difficult to digest or have low digestion rate. Dietary fiber increases mixed food viscosity in the intestines, so it will slow the enzyme and food (starch) interaction. High-fiber food also increases stomach distention which is related to greater feeling of fullness and less appetite. is will also decrease the nutrition absorption of the food by the body [30].
Starch digestion rate elevation is suspected to come from the preparation process, which is one of the factors that influence starch digestibility. In this research, milling or grinding process was done during the tempeh gembus flour production. e grinding process made the food structure to be smooth, so the food will get digested and absorbed easily. Particle size also affects the starch gelatinization process, where smaller starch particle size will make it more easily digested by enzymes so that starch digestion and absorption is fast. While the starch digestion rate of tempeh gembus flour cookies increased, that digestion rate was still categorized as low and cookies with tempeh gembus flour substitute also had high dietary fiber.
Protein digestibility is a term for digestive enzyme (protease) performance to hydrolyze a protein to amino acids. An easily digestible protein shows that the amount of amino acids that gets absorbed and used by the body is high. e protein digestion rate of cookies with tempeh gembus flour substitute is higher than that of the control treatment cookies. e higher the substitution of tempeh gembus flour, the higher the protein digestion rate. High protein digestion rate means that protein can get well hydrolyzed into amino acids, so the amount of amino acids that can get absorbed and used by the body is high, while the low-protein digestion rate means protein is hard to get hydrolyzed to amino acids   Available carbohydrate per serving � weight per serving(g)/100 and g × available carbohydrate. * * GL � GI × available carbohydate per serving/100. * * * Category: low GL (<10), medium GL (11)(12)(13)(14)(15)(16)(17)(18)(19), and high GL (>20). and that causes a low amount of amino acids to get absorbed and used by the body because most of it will get passed through the body with feces. e increase in the protein digestion rate is caused by the preparation process.
is process can influence products' protein digestion rate. e authors did the drying process two times, when creating the tempeh gembus flour and making the cookies. ese drying and baking steps in the preparation process can increase the protein digestion rate because they denature the antinutritional protein (antiprotease). e drying process also changes the width and length of the protein. e relationship between width and length of the protein with the digestion rate is that the lower the protein size, the wider its surface; therefore, its digestion rate can increase because the protein hydrolysis process by the protease enzyme will be easier. e drying treatment can widen the protein's surface. is happens because the drying process will extract the water from protein and make the protein surface wider than before because the protein particle becomes smaller when going through the drying process, and so the protein digestion rate will be higher [33]. e increase in protein digestion rate means that more protein can get digested by the body. High protein may stimulate insulin secretion through the Gβ5-RGS protein complex, so the GI can be low [6]. e nutrition content per snack serving is usually 10-15% of daily calorie intake and can be consumed 2-3 times a day, which means the total calories of snacks for diabetics is 200 kcal in 25 grams of cookies. e carbohydrate intake amount that is recommended for diabetics is 55% of daily calorie intake, so the amount of daily carbohydrate intake in snacks is 27.5 grams. Based on the results of this research, the available carbohydrate per serving of P 2 cookies is 14.68 grams; therefore, to fulfill the daily carbohydrate intake for snacks, the amount of P 2 cookies needed is 46.83 g.

Conclusions
Cookies that have the lowest GI and GL (47.01% and 6.90%) are cookies with 50% tempeh gembus flour substitute. e higher the tempeh gembus flour substitution, the lower its GI and GL rates. Cookies with 50% tempeh gembus flour substitute have higher dietary fiber, starch digestion, and protein digestion rates. e available carbohydrate testing method needs to be performed with more thorough and standardized methods such as the anthrone-sulfuric method while using a UV-visible spectrophotometer instrument to get a more valid test result. e recommended cookies are cookies with 50% tempeh gembus flour substitute that have lower GI and GL rates.
Data Availability e cookies' formulation, subject's characteristics, cookies' weight stipulation per subject, cookies' glycemic index, cookies' glycemic load, and cookies' dietary fiber, starch digestion, and protein digestion rate data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.