Modification of the carbohydrate profile of oatmeal cookies

. Excessive sugar consumption worldwide has become an increasing problem in recent years. Excessive sugar consumption can lead to serious diseases such as diabetes and obesity. Confectionery products make a significant contribution to this problem. The carbohydrates in the formulation of traditional confectionery products have a high glycemic index. Modification of the ingredient composition of confectionery products by replacing sugar, which is conventionally used in the formulation of confectionery products, with other ingredients should help to reduce the glycemic index of these products. The purpose of the study is to develop the prescription composition and manufacturing technology for a flour confectionery product in the form of oatmeal cookies with a modified carbohydrate profile. The objects under study are a control sample of oatmeal cookies (a prototype of the developed product with an unmodified carbohydrate profile) and experimental samples of oatmeal cookies (products with modified carbohydrate profiles). The modification of carbohydrate profiles consists in removing sugar and introducing ingredients that have no hyperglycemic effect – sugar substitute trehalose or isomalt and sweetener stevioside. Organoleptic and physicochemical parameters of the developed cookies are determined using standard methods. The study demonstrates the possibility of using the method of biotesting with Tetrahymena pyriformis infusoria on oatmeal cookies with sugar and sugar substitutes. Biotesting is carried out based on analyzing the condition, behavioral characteristics, and growth of the culture population in the studied samples of cookies. The findings indicate a positive effect of the developed oatmeal cookies with modified carbohydrate profiles on living cells.


Introduction
Excessive sugar consumption in the world has become increasingly urgent in recent years.Excessive sugar consumption contributes to serious diseases such as diabetes mellitus, obesity, atherosclerosis, and tooth decay.A significant contribution to this problem is made by flour confectionery products.The most promising solution to this problem is to replace sugar in the formulation of flour confectionery products with other ingredients.
The aim of the study is to develop the prescription composition and manufacturing technology for a flour confectionery product in the form of oatmeal cookies with a modified carbohydrate profile.
The carbohydrates included in the recipe of traditional flour confectionery products in pure form, i.e., glucose, fructose, sucrose, maltose, and lactose, or as part of the ingredients have a high glycemic index.Thus, modification of the ingredient composition of flour confectionery products by replacing sugar should decrease the glycemic index of these products [1].
Of practical interest in the development of recipes for flour confectionery products with a reduced glycemic index are sugar substitutes that are similar to sucrose in their physical, chemical, and technological properties and do not have a negative impact on the rheological, physical, chemical, and organoleptic properties of the finished product [2].
In recent years, sugar substitutes such as isomalt and trehalose have attracted increasing interest.In this study, they are included as sugar substitutes in the formulation of the product under development.
Isomalt (isomaltitol) is obtained through a two-step process of sugar beet processing: by enzymatic processing of sucrose into isomaltulose (palatinose), followed by catalytic hydrogenation.Isomalt is less soluble in water than sucrose, more heat stable, has low hygroscopicity, and is resistant to enzymatic and acid hydrolysis [2].
Isomalt has a caloric value and sweetness lower than sucrose, does not cause a significant rise in blood glucose levels, and does not require insulin for its assimilation, which allows it to be used as an ingredient in the preparation of dietary food products with reduced energy value and diabetic products [3].
Trehalose, a natural disaccharide built from two identical α-D-glucopyranose residues, protects proteins and cell membranes from inactivation or denaturation caused by a variety of stressful conditions including drying, dehydration, heat, cold, oxidation, heat shock, hypoxia, and prolonged lack of oxygen.Trehalose increases autophagy, protecting cells from various stressors, acts as a modifier of abnormal protein accumulation in human neurodegenerative diseases, and has a favorable effect on the endothelium of blood vessels and on metabolic processes, including in type 2 diabetes -and this is only a fraction of the positive effects trehalose has on the human body [8].
Trehalose is abundant in nature and is found in a wide variety of organisms, from bacteria to invertebrates.The largest amounts of trehalose are concentrated in the spores of mycelial fungi and in yeast [9].
Large-scale industrial production of this disaccharide is carried out using biotechnological methods based on the enzymatic transformation of starch.
Trehalose has a very low hygroscopicity and a high glass transition temperature, which increases the stability of taste and aroma, color, and moisture content of food products with trehalose, as well as their shelf life.
Trehalose maintains blood glucose levels as effectively as products with added glucose, with a lower plasma insulin response, and blocks glucose from reaching the liver.Trehalose is non-toxic and is well tolerated even in high doses.In flour confectionery products, trehalose prevents starch retrogradation much more effectively than other sugars, thus increasing the stability of products and delaying their staling [3,10].
Furthermore, trehalose is only slightly fermentable in the oral cavity, making it safe for dental health.
Trehalose, as well as isomalt, provides pure sweetness, the intensity of which is about two times less than the sweetness of sucrose.This necessitates the use of a natural sweetening , 020 (2024) BIO Web of Conferences MSNBAS2023 https://doi.org/10.1051/bioconf/2024820203939 82 agent in the formulation of the product under development.One ingredient that provides a flavor profile characteristic of sucrose is stevioside.
Stevioside is a glycoside from the extract of the stevia plant, has an average sweetness factor of 300, and contains no calories.Stevioside is suitable for use as a sweetener in all food products, as it is highly resistant to heat and acids, does not ferment, and does not yellow when heated [11].
An experimental study of the effects of stevioside conducted by the World Health Organization has proven that it has no genotoxic, mutagenic, or carcinogenic properties.An additional advantage of this substance is the lack of harmful effects on tooth enamel.
Stevioside prevents the development of hypoglycemic and hyperglycemic states in diabetic patients.In addition, studies have reported that stevioside shows some evidence of pharmacologic effects in patients with arterial hypertension or type 2 diabetes mellitus [11].
To control the quality of new products, it is necessary to develop simple and effective methods of analysis to assess the safety of both the sweeteners themselves and the variety of products using them.
One of the indirect methods of determining the complex effects of a substance on human beings is biotesting using living organisms -higher animals or alternative models, such as protozoa.Biotesting is a procedure for establishing the toxicity of the environment using living objects that signal danger regardless of which substances and in what combination cause changes in essential functions.For the assessment of long-term exposure to low concentrations of active substances, the test response can be the death of experimental populations of monocultures over a given time, changes in motility, reproduction rate, and death of organisms.
Studies on the adequacy of biotests for various research objects have found the most universal test organisms to be protozoa (Tetrahymena infusoria, Paramecia, Stylonychia), which are highly sensitive to a wide range of toxicants [12].
Infusoria occupy a prominent place among the test organisms used in biotesting.They are very convenient objects for research, and the obtained results have a high correlation coefficient with the data of similar studies on animals [13].
Infusoria-based biotesting has enabled a comparative characterization of culinary products enriched with protein from different fish of the Pacific basin in terms of biological value [14], identification of heat treatment regimens that ensure the greatest preservation of antioxidant activity of cow's milk [15], and comparative assessment of the quality of clarified reconstituted apple juice of industrial production [16].
In the study, we explore the possibility of using the biotesting method using the infusoria Tetrahymena pyriformis to analyze the properties of cookies with sugar and sugar substitutes.

Methods
The objects under study include a control sample of oatmeal cookies (a prototype of the developed product with an unmodified carbohydrate profile) and experimental samples of oatmeal cookies (products with modified carbohydrate profiles).
The sugar substitutes used in the recipes of experimental cookie samples are trehalose (TORGSNAB LLC) -a white free flow powder, isomalt (Cargill, Inc., usa) -a white, odorless crystalline substance with a clean, sweet taste and low hygroscopicity, and , 020 (2024) BIO Web of Conferences MSNBAS2023 https://doi.org/10.1051/bioconf/2024820203939 82 stevioside (Ecotopia LLC) -an odorless white powder with a characteristic sweetish taste and a sweetness coefficient of 250 units.

Determination of organoleptic and physicochemical parameters
Organoleptic quality parameters were determined by inspection of a combined sample of products.
Alkalinity was determined by titration of the product filtrate with a sulfuric acid solution in the presence of bromothymol blue until the appearance of yellow coloring.Moisture content was assessed by drying a sample of the product at 130°C and calculating the loss of mass in relation to the mass of the analyzed sample before drying.Wettability was measured by the ratio of the product's mass after soaking to the mass of the dry product.

Biotesting
Biotesting of cookies was performed on the infusoria T. pyriformis.The culture was grown using the method on a sterile yeast-peptone base nutrient medium prepared with distilled water and containing (%): 0.5 glucose, 2 bacteriological peptone, 0.1 yeast extract, and 0.1 sea salt.The cultivation of infusoria was carried out in a cold thermostat at 20±1°C by reinoculation with an inoculation loop every 4 days.
Cookies were added to 2 ml of the medium with the culture in the amount of 0.04 g, which was found to be optimal in a preliminary study.The condition of the culture was assessed after different periods of contact time with the product, from 24 to 48 hours.The evaluation used a 5-point scale, in which the maximum number of points corresponded to the conventional behavior of infusoria, without deviations.The reduction of the score corresponded to the intensity of the decrease in activity, speed, and character of movement.The lowest score corresponded to the absolute death of infusoria.Observations of infusoria were carried out at 100x magnification after 24 and 48 hours.
To determine the number of infusoria, cell counting was performed after 48 hours of cultivation in the large square of the Fuchs-Rosenthal chamber.The culture in the medium was mixed to obtain a homogeneous suspension and 1 cm 3 of the culture was pipetted into a separate container, where 10 µl of iodine solution was added to kill the protozoa.After 2 minutes, the contents were mixed, and the suspension was added to the counting chamber.Infusoria cells were counted in 16 large squares at 100x magnification.All tests were performed in three repetitions.

Results and discussion
To determine the influence of the selected ingredients on the quality of the finished product, we developed prescription compositions and produced a control sample of oatmeal cookies with sugar and experimental samples of cookies with modified carbohydrate profiles (Table 1).The technological scheme for preparing the cookies with modified carbohydrate profiles is as follows.Trehalose/isomalt are mixed with hot water for their crystals to dissolve completely in 2-3 minutes, then egg powder and plasticized butter are added, and the obtained emulsion is mixed for 1.5-2 minutes.Into the finished emulsion, pre-boiled oat flour (with hot water at a temperature of 90-95°C) and salt are added, and the mixture is stirred for 2-4 minutes.Next, wheat flour with a chemical baking powder and vanillin are introduced.The batter is mixed until ready.To ensure a homogeneous plastic, not smearing structure of finished products, the batter allowed to rest for 30 min in a refrigerator.After resting, the batter gains a plastic and not smearing consistency and is easily molded with shaped metal molds.The cookies are baked in an electric oven at 180°C, sugar-based cookies -for 10 minutes and trehalose-and isomalt-based -for 13 minutes.After cooling, the products were assessed by organoleptic and physicochemical parameters.
Experimental cookie samples with modified carbohydrate profiles had a pleasant taste and aroma, uniform and well-developed porosity, and a dense crumbly structure and were more brittle when bitten compared to the control sample.
The assessment of the alkalinity of the cookie samples shows that this parameter virtually does not change when sugar is replaced with trehalose or isomalt.However, the wettability and moisture of the cookies based on sugar substitutes are increased (Table 2).Analysis of the chemical composition of the experimental samples reveals that 100 g of trehalose-based cookies contain 7.2 g protein, 23.0 g fat, and 49.0 g carbohydrates; energy value -432 kcal/1,807 kJ; 100 g of isomalt-based cookies have 7.3 g protein, 23.0 g fat, and 50.0 g carbohydrates; energy value -373 kcal/1,629 kJ, which is 60 kcal/183 kJ lower than the control sample.
A test was performed to investigate the effect of sugar and sugar substitute cookies on the condition and behavioral characteristics of T. pyriformis (Table 3).As indicated by the obtained results (Table 3), the introduction of cookies into the habitat of T. pyriformis hardly reduces the activity of the culture for up to 24 hours.After 48 hours, the best condition of T. pyriformis, almost identical to the behavior in the control, is observed in the presence of sugar cookies.The media with the addition of sugar substitutes are slightly less favorable.
The introduction of cookies into the habitat of T. pyriformis reduces the activity and intensity of the development of the culture as compared to the control (Table 4).The number of cells in samples with cookies is 2-4 times lower than the control, although the addition of cookies in the amount of 0.04 g per 200 µl of medium does not kill the infusoria.The comparative assessment of the results shows that after 48 hours of cultivation, the highest cell count is observed in the presence of sugar oatmeal cookies and a somewhat lesser number is recorded in the sample with trehalose cookies.The least favorable environment for protozoa is that with isomalt-based oatmeal cookies.However, T. pyriformis does develop even in the presence of this sweetener, albeit somewhat less intensively.The conducted study demonstrates that the considered sugar substitutes are relatively safe compared with traditional sugar.Biotesting indicates that the chosen sucrose substitutes are close to the traditional form of sucrose in their effects on T. pyriformis and, presumably, on the human body.

Conclusions
The conducted research resulted in the modification of the carbohydrate profile of oatmeal cookies consisting in the exclusion of sugar from their prescription composition and the introduction of ingredients that cause no hyperglycemic effect -sugar substitutes trehalose or isomalt and sweetener stevioside.The technology to produce a new type of cookies was developed, and the parameters of its production process were elaborated.The possibility of using the method of biotesting of oatmeal cookies with sugar and sweeteners using T. pyriformis infusoria was explored.It was established that the biotesting method can be utilized for a comparative assessment of the quality and safety of cookies.The positive effect of the developed oatmeal cookies with modified carbohydrate profiles on , 020 (2024) BIO Web of Conferences MSNBAS2023 https://doi.org/10.1051/bioconf/2024820203939 82 living cells is demonstrated.Cookies with sugar substitutes (isomalt/trehalose) do not decrease the metabolic activity of protozoa, the integral measure of which is the dynamics of culture growth in the sample of the assessed object.

Table 2 .
Physicochemical parameters of the developed oatmeal cookies

Table 3 .
Point estimation of T. pyriformis in the presence of oatmeal cookies with sugar and sugar substitutes

Table 4 .
Results of biotesting of cookies using T. pyriformis after 48 hours of cultivation