MINISTRY OF EDUCATION AND TRAININGHO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION GRADUATION THESIS MAJOR: FOOD TECHNOLOGY INSTRUCTOR: TRINH KHANH SON TRAN HOANG VU TRAN HUONG NG
Urgency and reason for forming the topic
Previous studies
The glycemic index (GI) is gaining prominence in nutritional literature, prompting increased research into the health benefits of resistant starch (RS) Studies are focusing on how RS consumption influences physiological factors, including blood sugar regulation, lipid metabolism, and body weight management Table 1.1 presents findings from various studies examining the physiological effects of resistant starch on experimental animals.
No Article name Author Year Main content
1 The impact of a low glycemic index diet on the metabolism of glucose and lipids, microbial composition, short-chain fatty acid levels, immune response, and levels of
FGF-19 and CYP7A1 in type 2 diabetic mice was studied
Jian-bo Luo, Xiao-juan Xie, Xiao-yu Zhou, Feng Yang, Jun-hui Li, Zheng Li, Liang Zhou, Yong He, Jia Zhou, Li-hong Niu, Min Fu
A recent study in 2024 examined the effects of a low glycemic index (GI) diet on a mouse model of type 2 diabetes, which was induced by a high-fat diet and streptozotocin The findings revealed that the low GI diet significantly lowered neutral fat and glucose levels while enhancing insulin and C-peptide levels Furthermore, it positively influenced the gut microbiota composition and increased the levels of beneficial short-chain fatty acids (Luo et al., 2024).
2 Sweet potato extract reduced obesity caused by a high-fat diet in C57BL/6J
2022 Sweet potatoes are widely consumed as a nutritious and healthy vegetable containing
2 mice, but not by pancreatic lipase inhibition
A study conducted by Bingna Pan, Xifeng Yin, Yilin You, Zhixuan Song, Dan Li, and Dejian Huang investigated the health-promoting bioactive components of white sweet potato extract (SPE) The research specifically focused on the beneficial effects of SPE on obese individuals who followed a high-fat diet (HFD) (Liu et al., 2022).
3 The beneficial effects of purple yam starch
(Dioscorea alata L.) on high blood fat levels in mice fed a high-fat diet were studied
Fengping An, Qun Huang, Lei Chen, Hongbo Song
In a 2019 study, researchers explored the impact of resistant starch (RS) derived from purple yam (Dioscorea alata L.) on lipid metabolism and gut microbiota in obese mice induced by a high-fat diet The findings revealed that high-dose RS (HR) treatment was more effective than low-dose RS (LR) in managing body weight and fat mass, while also enhancing high-density lipoprotein cholesterol (HDL-C) levels and lowering triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels (Li et al., 2019b).
In Vietnam, resistant starch supporting health improvement and diabetes treatment is a new concept that has gained attention In recent years, some studies have identified the impact of
Resistant starch, found in foods like rice, cereals, and wheat, plays a significant role in health by offering insights into its benefits for diabetes management and metabolic disorders Recent studies highlight its potential applications in both the food and medical industries, emphasizing the importance of understanding resistant starch for improved health outcomes.
No Article name Author Year Main content
1 In vitro digestibility and in vivo glucose response of native and physically modified rice starches varying amylose contents
Pham Van Hung, Huynh Thi Chau, Nguyen Thi Lan Phi
In a 2015 study, researchers investigated the in vitro digestibility and in vivo glucose tolerance in rats using various natural and physically modified rice starches with different amylose contents The starch samples consisted of five types of natural rice starch and flour, which underwent moist heat and tempering treatments The findings indicated that these treatments significantly lowered the glycemic index (GI) value of the rice starch (Van Hung et al., 2016a).
2 Determination of acute and semi- chronic toxicity of wheat starch acetate in Swiss mice
In a study conducted by Dung Tran in 2020, the acute and semi-chronic toxicity of wheat starch acetate was evaluated on Swiss white mice Over an 8-week monitoring period, various health parameters were assessed, including skin condition, hair quality, eye health, and the functioning of the digestive, respiratory, motor, and nervous systems, along with weight changes and hematological and biochemical indicators Macroscopic examinations were also performed to gather comprehensive results.
4 microscopic liver organs , kidney Then came to the conclusion that the starch sample was not toxic on test mice (Dũng et al., 2020)
Limitations of previous research
Recent studies indicate promising outcomes in the production of low glycemic index (GI) starch, particularly from purple yam, in in vitro models However, the effectiveness of these products in living organisms (in vivo) remains underexplored Factors that yield positive results in theoretical in vitro experiments may not translate similarly in vivo Consequently, our focus is on "The impact of low glycemic index starch on reducing overweight, obesity, and type 2 diabetes in experimental animals."
Aims
In our study, we evaluated the impacts of two control diets on mice: a standard diet (ND) and a high-fat diet (HF) We compared these with four experimental diets enriched with purple yam starch: standard and high-fat diets supplemented with 50% raw purple yam starch (ND:A and HF:A), and standard and high-fat diets enhanced with 70% low glycemic index purple yam starch (ND:HR0 and HF:HR0) This comparison aimed to assess the effects of these dietary modifications against the control diets.
Research contents
The study investigates the impact of a low glycemic index (GI) starch diet on experimental animals by assessing various physiological factors Key evaluations include changes in body weight and energy expenditure, blood lipid levels, fasting blood sugar, and glucose tolerance Additionally, the research examines behavior and motor skills through locomotion and rotarod tests, as well as histological analyses of liver, kidney, adipose, and muscle tissues.
Practical and theoretical implications
Purple yam is widely cultivated across Vietnam, particularly thriving in the Southwestern provinces, which yield the highest production However, despite the abundant harvest, its culinary applications remain relatively limited.
Yam is commonly used for making soup, but its potential extends to starch production aimed at low glycemic index (GI) products The glycemic index is crucial for selecting foods and designing diets for individuals at risk of obesity and type 2 diabetes, as low GI foods positively impact blood sugar control This study investigates the effects of low GI yam starch on reducing overweight, obesity, and type 2 diabetes through physiological, behavioral, and motor assessments of experimental animals The findings aim to support the application of yam starch in the food industry for its therapeutic benefits in addressing nutrition-related diseases.
Objectives and limitations
Research subjects: yam starch (Dioscorea alata) and male albino mice (Mus musculus var
In February 2024, purple yams, weighing 2-3 kg per tuber, were sourced from the Thu Duc wholesale market Additionally, male albino mice, aged 6 weeks and weighing approximately 16±1g, were acquired from the Pasteur Institute in Ho Chi Minh City.
Scope and limitations of the study: only evaluating the physiological, behavioral, and motor status of experimental mice on various starch samples.
Research content
LITERATURE REVIEW
Oveview about starch
Starch is a crucial dietary component for humans and many animals, alongside protein and fat This polysaccharide carbohydrate consists of a blend of amylose and amylopectin, with ratios typically ranging from 20:80 to 30:70, depending on the starch type It can be sourced from grains like corn and wheat, as well as from roots and tubers such as cassava, potatoes, and yams Starch is derived from diverse plants, each exhibiting unique physical and chemical properties.
Natural starch often falls short in industrial applications due to its inadequate quality, particularly in terms of heat resistance, gel formation, retrogradation, and freeze-thaw stability (Waterschoot et al., 2015) To address these limitations, various modification techniques are employed to improve starch's functional properties according to specific processing and product needs These modifications can be achieved through physical methods such as steeping and heat-moisture treatment, chemical methods including etherification and cross-linking, enzymatic processes, genetic modifications, or a combination of these approaches (Bensaad et al., 2022).
Starch modification
Chemical modification of starch through the addition of functional groups significantly enhances its physicochemical properties This alteration impacts the behavior of natural granular starch during processes such as gelatinization, pasting, and retrogradation Key factors influencing the outcomes of starch modification include the starch source, experimental conditions (such as reactant concentration, pH, and catalyst presence), types of substituent groups, and the degree of substitution (DS1) or molar substitution (MS2) These modifications can lead to various derivatives, including etherification, esterification, cross-linking, starch grafting, acid or enzyme hydrolysis, and starch oxidation, ultimately resulting in tailored functionalities for diverse applications (Masina et al., 2016).
Physical transformation methods involve altering the shape and structure of starch through factors like moisture, temperature, pressure, pH, and various forms of radiation These methods significantly impact the starch's surface size, properties, solubility index, and functional characteristics, including water absorption, swelling, adhesion, and gel formation The resulting modifications directly affect the quality, selectivity, and applicability of starch in industrial, pharmaceutical, and nutritional applications Numerous studies have explored these transformations, highlighting techniques such as heat treatment, ultraviolet and gamma ray irradiation, microwave treatment, and high-pressure methods (Nawaz et al., 2020).
Enzyme methods play a crucial role in altering starch structures in food through three main processes: hydrolysis of α-1,4 or α-1,6 glycosidic bonds, disproportionation via glucan branch transfer, and branching through the formation of α-1,6 glycosidic bonds By utilizing a single enzyme or a combination of multiple enzymes in catalytic reactions, these methods lead to significant chemical modifications in starch, resulting in changes to its molecular weight and overall properties.
8 distribution of chain lengths, and the ratio of amylose to amylopectin that is produced (Lakshmi Krithika & Ratnamala, 2019)
The digestion fractions in starch
Table 2 1 Classification of starch (Lehmann & Robin, 2007)
Digestion time (in vitro/in the human body)
Within 20 minutes; mouth and small intestine
From 20 to 120 minutes later; small intestine
After 120 minutes; not digested in the small intestine, primarily active in the large intestine
Natural sticky corn starch, sorghum, and other legumes
Boiled hot potatoes: 65 Boiled sorghum: 28 Raw potato starch: 75
Source of rapid energy supply
Source of slow, sustainable energy, and blood sugar levels maintained
Impacts gut health (e.g prebiotic, fermentation into butyrate with hypothesized anti- cancer effects)
Structure Mainly indefinite Amorphous/crystalline
The digestion of starch is significantly affected by its source, granule structure, degree of isolation, and processing methods Starches with higher amylose content are generally more challenging to digest compared to those with higher amylopectin content Starch can be categorized into resistant starch (RS), rapidly digestible starch (RDS), and slowly digestible starch (SDS) RDS is quickly absorbed in the small intestine, leading to a rapid spike in blood sugar levels, which can cause harm to cells and organs due to sudden fluctuations In contrast, RS is not digested in the upper digestive tract; instead, it is fermented by colonic microorganisms, resulting in the production of beneficial short-chain fatty acids.
SDS (slow-digesting starch) offers a steady energy source for the body by slowly releasing glucose in the small intestine, which helps maintain low initial blood sugar levels This gradual digestion process not only provides stability but also extends energy availability over a longer duration compared to rapidly digested starch Additionally, it supplies essential fatty acids and beneficial butyrate, contributing to overall health (Aller et al., 2011)
Starch fractions are categorized based on their digestibility into three types: Rapidly Digestible Starch (RDS), which measures the glucose released after 20 minutes of digestion; Slowly Digestible Starch (SDS), representing glucose released between 20 and 120 minutes; and Resistant Starch (RS), defined as the total starch minus the glucose released within 120 minutes of hydrolysis in the large intestine (Lehmann & Robin, 2007).
Glycemic index of food (eGI)
The Glycemic Index (GI) measures the ability of a carbohydrate-rich food to raise blood glucose levels compared to a standard food (like white bread or glucose) Foods with a higher
The glycemic index (GI) significantly influences blood sugar fluctuations following food consumption, with these variations not directly tied to calorie intake or portion sizes Instead, they are determined by the rate at which the body metabolizes and absorbs particular foods (Chen et al., 2010).
The Glycemic Index (GI) is determined by measuring the area under the blood glucose response curve, with glucose and white bread set as the standard at a GI of 100 Foods are categorized based on their impact on blood sugar levels: high GI (GI > 70), medium GI (GI 55 - 69), and low GI (GI < 55).
Consuming high glycemic index (GI) carbohydrates can elevate blood sugar and insulin levels, increasing the risk of heart disease, type 2 diabetes, and obesity (C Brand-Miller et al., 2005) To mitigate these risks, dietary guidelines suggest incorporating low GI foods (GI < 55) to help prevent chronic diseases (Romão et al., 2021) Low GI foods are increasingly utilized in meal planning for diabetic patients, individuals with high blood lipids, and those at risk of heart disease (Chen et al., 2010) Furthermore, a low GI diet is effective in lowering total cholesterol and LDL levels, which are significant risk factors for heart disease.
Some diseases are related to diet
The World Health Organization (WHO) identifies overweight and obesity as excessive fat accumulation that can lead to significant health risks A Body Mass Index (BMI) over 25 indicates overweight, while a BMI over 30 classifies an individual as obese Elevated BMI is a major risk factor for various non-communicable diseases, including cardiovascular diseases—particularly heart disease and stroke, which are the leading causes of death in 2012—along with diabetes, severe musculoskeletal disorders like osteoarthritis, and several cancers, including endometrial, breast, ovarian, prostate, liver, gallbladder, kidney, and colon cancers (WHO, 2021).
Childhood obesity significantly heightens the risk of various health issues in adulthood, including obesity, premature death, and disabilities Obese children are particularly vulnerable to respiratory problems, high blood pressure, early heart disease, insulin resistance, bone fractures, and psychological challenges Both overweight and obesity are preventable non-communicable diseases, with the environment and supportive communities playing a vital role in encouraging healthier dietary choices and regular physical activity Implementing evidence-based policies universally can make healthy options simple, accessible, and affordable, particularly for low to moderate-income individuals; for instance, taxing sugary beverages could be an effective strategy The food industry also has a critical responsibility to promote healthy diets by reducing unhealthy ingredients in processed foods, offering nutritious options at fair prices, limiting advertisements for unhealthy products aimed at children and teens, and ensuring access to healthy food and physical activity opportunities in the workplace.
Type 2 diabetes (T2D), formerly known as non-insulin dependent diabetes mellitus or adult- onset diabetes, is a metabolic disorder characterized by high blood sugar levels due to insulin resistance and relative insulin deficiency Typical symptoms of T2D include frequent urination, excessive thirst, fatigue, and weight loss The main causes of this disease include genetic factors,
A sedentary lifestyle, unhealthy diet, and obesity significantly contribute to the risk of developing type 2 diabetes (T2D), with environmental toxins also playing a role Family history of T2D further increases this risk The disease occurs when muscle, fat, and liver cells fail to absorb glucose, leading to insulin resistance and elevated blood sugar levels Unlike type 1 diabetes, this resistance is due to cell receptors not responding properly to insulin, rather than a lack of insulin production The World Health Organization defines T2D by specific blood sugar levels, and early prevention is possible through proper nutrition and regular exercise Recent research has linked obesity and T2D to insulin resistance in the brain Additionally, the glycemic index (GI) of foods is crucial, as high GI foods can spike blood sugar levels, while low GI foods help maintain stable levels Therefore, choosing low GI foods and managing diet are essential for controlling and preventing T2D.
Overview of in vivo experiments
2.6.1 Regulations during conducting in vivo experiments (3R Principles)
In vivo experiments must prioritize the generation of meaningful, high-quality data while adhering to the 3Rs concept—reducing, refining, and replacing animal use—as established by Russell and Burch in 1959.
To reduce the number of animals used in experiments while maintaining scientifically valid results, it is essential to enhance experimental design through statistical analysis and data optimization This can be accomplished by sharing animals across different experiments and conducting studies on groups of animals under uniform conditions, utilizing advanced technologies and methodologies.
The principle of substitution, known as "replace," encompasses both absolute and relative replacement methods (Cheluvappa et al., 2017) Absolute replacement refers to the use of alternatives such as tissue cultures, computer models, or cell lines in place of live animals Whole replacement specifically involves utilizing cells and tissues, including liver, kidney, brain, and heart, sourced from previously deceased animals that are preserved outside the body.
12 appropriate conditions, ranging from days to months or even years, to serve research purposes, thereby reducing the use of live animals and avoiding wasting the valuable resources animals provide
Refinement involves employing techniques to lessen pain and distress in animals, while also aiming to minimize anxiety and ensure their psychological and physical well-being This approach helps to mitigate factors that could compromise the validity of experimental outcomes (Cheluvappa et al., 2017).
2.6.2 Factors related to experimental procedures on mice a) Diet and body composition
A high-fat diet is commonly employed to alter body composition, particularly to increase fat mass and induce insulin resistance in mice It is essential to implement a controlled supplementary high-fat diet when there are variations in nutrient sources Careful consideration of the diet's impact on body composition is crucial, as differences in body weight and composition can significantly affect glucose metabolism and the outcomes of metabolic exchange tests, influencing the interpretation of results (Ayala et al., 2010).
Researchers have found that using anesthetized mice to test stress reduction leads to elevated blood sugar levels and altered blood flow and heart rate, resulting in non-physiological data on glucose metabolism To obtain accurate measurements, it is essential to conduct experiments on awake mice, as there are alternative methods available to reduce stress without the use of anesthesia (Ayala et al.).
Blood sampling methods in research are influenced by the operator's skills, the specific tests being conducted, and the required blood volume In awake mice, blood is typically collected from the tail tip or via a surgically implanted arterial catheter Utilizing an arterial catheter allows for stress-free access to blood vessels during experiments, which is crucial for metabolic exchange tests that demand larger blood volumes However, this invasive procedure poses risks, such as increased mortality rates post-surgery and potential complications like stroke, which can compromise the study's outcomes.
Arterial blood sampling is not universally adopted as a standard method for blood collection across all laboratories Alternatively, retro-orbital bleeding can be utilized, but it typically necessitates anesthesia and is more suitable for obtaining a single sample rather than for frequent or repetitive sampling (Ayala et al., 2010).
The breeding cage, measuring 63.4×23.9×42.7 cm, features a lid and multiple perforations for optimal mouse respiration Regular cage changes are essential to maintain cleanliness and dryness, with frequency influenced by factors such as the mice's physiological state, cage density, and experimental design For instance, diabetic mice necessitate daily cage changes, particularly in the evening when they are most active (Sharp & Villano, 2012) Additionally, the surrounding light and time of day play a crucial role in the care and management of the mice.
Mice, being nocturnal animals, are often housed in environments with a controlled light-dark cycle, typically set to 12 hours of light and 12 hours of darkness, which can be adjusted for specific experimental requirements This setup facilitates metabolic experiments at various times of the day, aligning with the mice's peak activity during the dark cycle, thus enhancing the convenience for researchers (Sharp & Villano, 2012) Additionally, maintaining appropriate temperature and humidity levels is crucial for optimal experimental conditions.
The optimal temperature to mimic human metabolic response in mice is 30°C and humidity is always kept above 70%(Keijer et al., 2019) d) Drinking water and vitamins
The automatic drinking system provides enough water for mice The temperature, relative humidity of the environment, and moisture of the feed determine the water needs of the mouse
Ensuring that pets have access to clean, drinkable water is crucial for their health Mice, in particular, require a significant amount of water, consuming daily between 10% to 15% of their body weight At a temperature of 22°C, mice's water intake can surpass their food consumption by approximately 20%, while at 30°C, their water consumption may double that of their food intake (Sharp & Villano, 2012).
Vitamins are categorized into two groups: fat-soluble vitamins, which include A, D, E, and K, and water-soluble vitamins, such as B6, B12, biotin, choline, folate, niacin, pantothenic acid, riboflavin, and thiamine In a study conducted by Sharp and Villano (2012), Thai hamster vitamins were administered in two drops to the drinking water of mice.
Figure 2 3 Thai Hamster vitamins (Vitamin Nước - Hamster Kingdom, 2024)
MATERIAL AND METHODS
Materials
Purple yam, sourced from the Thủ Đức agricultural market located at 141 National Highway 1A in Tam Binh Ward, Thu Duc City, Ho Chi Minh City, is a key ingredient in various formulations Additionally, Impact Whey Protein from My Protein in the USA is utilized, along with sodium carboxymethyl cellulose (CMC) and sodium hydroxide (NaOH), both from China Hydrochloric acid (HCl), 3,5-dinitrosalicylic acid (DNS), and the α-amylase enzyme (AHA-400 from Angek Yeast Co., China) are also included in the process Other components such as sodium azide (NaN3), monosodium orthophosphate (NaH2PO4), and disodium hydrogen phosphate (Na2HSO4), along with Panceratin enzyme (P7545 from MilliporeSigma, Germany), contribute to the overall formulation.
Obtaining raw purple yam starch method
The process of obtaining raw purple yam starch begins with cleaning the yams to eliminate soil and dirt After this, the yams are peeled and diced, resulting in 0.88 kg of usable purple yam flesh from every 1 kg of the raw tuber.
To extract colorless starch from purple yam, the flesh is soaked in a 0.2% NaOH solution at a ratio of 1:9, blended, and sieved through a mesh of 100 (φ = 0.150 mm) to create a slurry This slurry is then soaked again in the same NaOH solution to eliminate impurities like color, protein, and other unwanted compounds, ensuring the starch remains clear The starch is subsequently filtered through the same sieve to isolate it During the soaking process, color compounds, proteins, pectin, and phenols dissolve in the solution, while fibers float to the surface for removal The NaOH solution is refreshed every four hours, with the total soaking time for the purple yam starch slurry reaching 120 hours.
To determine if the soaking process has effectively removed protein from purple yam starch, the soaking solution is tested using ninhydrin reagent This reagent reacts with any remaining protein, producing a purple-blue complex when heated The testing procedure involves adding 1 mL of the soaking solution to a test tube, followed by a few drops of a ninhydrin solution (prepared from 0.2 g ninhydrin and 10 mL absolute alcohol), and heating it in a water bath for 10 minutes If a purple color persists, it indicates that protein remains, necessitating further soaking in a 0.2% NaOH solution until the solution becomes colorless, after which neutralization can occur.
The purple yam starch initially has a pH of approximately 11.6 To aid in the neutralization process, distilled water is utilized to wash away sodium hydroxide (NaOH) in a ratio of 1:9, promoting starch settling Following this, a 0.1N hydrochloric acid (HCl) solution is applied to neutralize the starch precipitate After the neutralization is complete, the starch undergoes multiple rounds of washing with distilled water to ensure thorough salt removal.
To determine if all the salt has been removed from the starch solution, add 1 mL of the solution to a few drops of AgNO3 If a white precipitate of AgCl forms, further washing of the starch with distilled water is required If no precipitate appears, air-dry the starch at 50°C, then grind and sieve it through a mesh of 100 (0.150 mm) The yield of settled starch precipitate is 0.38 kg for every 1 kg of purple yam.
The starch, after drying, achieves a moisture content below 10% and is stored in sealed packaging in a dry place until used for further studies
From 1 kg of initial purple yam material, after processing, 0.11 kg of purple yam starch is obtained The starch yield (H) is calculated using the following formula:
𝑚1∗ 100% (3.1) With m2 is mass (kg) of starch after drying; m1 is mass (kg) of raw yam after peeling
Hydrolysis and retrogradation starch method
Figure 3 2 Combined hydrolysis and retrogradation method Raw purple yam starch is gelatinized by boiling (95°C, 30 minutes) in a 3% starch solution
To prepare a starch gel, 12 grams of starch is mixed with 400 mL of distilled water and sterilized at 121°C for 15 minutes to achieve complete gelatinization After cooling, 24 mL of 0.5 N HCl solution is added to hydrolyze 100 mL of the starch gel, which is subsequently neutralized with 1.1 N NaOH solution at the zero-hour mark.
The starch gel is maintained at a consistent pH of 7 and subjected to filtration through a 500 mesh sieve (0.025mm) after various time intervals of 4, 10, and 18 hours This process separates the mixture into two distinct components: a liquid part and a residue part that contains the starch.
After the initial reaction volume is reached, the starch is filtered and cooled at 4°C for 18 hours, followed by cooling at 30°C for 6 hours, completing two cycles over 24 hours Retrogradation occurs over two days, and after this process, the swollen starch is spread thinly and dried convectively at 50°C for 24 hours until the moisture content is below 10% The final product is then ground and sieved through a 100 mesh sieve (φ = 0.125 mm).
After the combined hydrolysis and retrogradation process, 3% swollen starch (12g starch /400 mL distilled water) yields 11.47 g of starch The recovery efficiency is 95.6%.
Method for determining the degree of hydrolysis and in vitro glycemic index (eGI)
The degree of hydrolysis and the eGI index of starch were assessed following the method outlined by Arp et al (2020) An enzyme solution was prepared by mixing 0.1 mL of α-amylase enzyme (AHA-400, activity 40U/g from Angek Yeast Co., LTD) with 100 mL of sodium phosphate buffer at pH 6.9, supplemented with 0.05 g of NaN3 This enzyme solution, at a concentration of 10U/mL, was then placed in a thermostatic bath (Cleaver Scientific LTD – SWB-20L-3, UK) set to 37°C to ensure temperature stabilization prior to use The starch sample was accurately weighed to the nearest 0.5 g and prepared for analysis.
To initiate the experiment, prepare 25 mL of a pH 6.9 buffer and transfer it to a temperature-controlled bath set at 37°C, stirring at 40% speed for 10 minutes Following this, add 5 mL of enzyme solution to the starch suspension while maintaining the stirring temperature The reaction will be monitored over a duration of 0 to 480 minutes, with samples of 1000 µL taken at specific time intervals: immediately after enzyme addition, and at 10, 20, 30, 60, 90, 120, 150, 180, 210, 240, and 270 minutes.
300, 330, 360, 390, 420, 450 and 480 minutes The reaction solution was added to 1 mL of DNS solution (1 g DNS, 30 g sodium potassium tartrate, 1.6 g NaOH) ) Then, boil in water for 5 minutes
The sugar content reduction was assessed using a maltose standard curve at a wavelength of 530 nm The collected data facilitated the construction of a hydrolysis curve, which was then analyzed according to the regression equation established by Gorii et al (1997).
In which, C is the amount of reducing sugar released upon hydrolysis at a certain time t; C∞ is the digestion limit (value of C at t∞); and k is a constant rate
For comparison, the hydrolysis curve of the crude starch sample was also performed Raw starch needs to be completely gelatinized (water bath for 15 minutes and autoclave at 121 o C for
Prior to testing, a 10-minute interval was observed (Gorii et al., 1997) The glycemic index (GI) was subsequently estimated using a published method, which involves integrating each hydrolysis curve to determine the area under the curve The estimated effective glycemic index (eGI) is then calculated as a mathematical ratio of the area of each starch to that of the corresponding raw powder, designated as 100% (eGI0).
Measuring in vivo digestive index method
The digestibility of yam starch was assessed using a modified method by Englyst et al (1992) and Sang et al (2007) To prepare the enzyme solution, 0.5 g of pancreatin (P7545, 8×USP/g from Sigma–Aldrich) was dissolved in 6 mL of distilled water and stirred for 10 minutes After centrifuging the solution at 1500 rcf for 15 minutes, 5 mL of the supernatant was mixed with 0.9 mL of distilled water and 0.1 mL of amyloglucosidase (AMG 300L, activity 300 U/ml from Novozymes, Denmark) This enzyme mixture was then incubated in a thermostatic bath (Memmert - WNB14, Goettingen, Germany) at 37.5°C for 10 minutes.
A precise starch sample weighing 0.06 g was placed in a 7.5 mL centrifuge tube containing three glass beads Following this, 1.5 mL of sodium acetate buffer (0.1 M, pH 5.2) was added, and the tube was incubated in a water bath at 37.5°C for 10 minutes Afterward, 1.5 mL of enzyme solution was introduced, and the mixture was shaken continuously for a reaction period of 10 to 240 minutes To inactivate the enzyme, 1 mL of 1N NaOH was added, and the sample was centrifuged at 1500 rcf for 10 minutes to collect the supernatant (Sang et al., 2007).
To determine glucose concentration in the supernatant using the DNS method (Lorenz Miller, 1959), a glucose standard curve is employed Reducing sugars (RDS) and soluble sugars (SDS) are quantified based on the sugar content produced after 10 and 240 minutes of enzyme-catalyzed starch hydrolysis Resistant starch (RS) is defined as the undigested residue remaining after 240 minutes.
Resource equation
Before initiating in vivo experiments, it is crucial to establish the appropriate number of experimental animals, ensuring adherence to the 3R principle, which emphasizes minimizing animal use while obtaining scientifically valid results (Mohammad et al., 2012) According to Arifin et al (2007), a widely accepted method for determining sample size in animal research is essential for achieving reliable outcomes.
"resource equation" method In this method, the acceptable degrees of freedom (DF) range from 10-20
For 1-way ANOVA, DF is determined using the formula:
With N is the total number of experimental animals; k is the total number of experimental groups; n is the number of experimental animals per group From (3.3) it can be deduced:
Based on the acceptable range of DF (DFmin = 10 and DFmax = 20) to determine the minimum and maximum number of animals for each group as follows:
The calculation of 4 + 1 = 6 indicates that a sample size of 5 animals per group is ideal for ensuring reliable results while adhering to the 3R principle in in vivo testing.
In vivo experiment diagram
Figure 3 3 Schematic diagram of semi-permanent in vivo experiments
Six-week-old male Mus musculus var Albino mice were fed one of the diets (ND:A, HF:A,
ND:HR0, HF:HR0) continuously for 8 weeks Mice were examined for behavioral changes at weeks 0 and 8 Glucose tolerance test and terminal blood collection were performed at week 8
Table 3 1 Coding table of experimental mouse groups
ND Control group (Standard Diet)
ND:A The group used a standard diet to replace 50% by carbohydrates with gelatinized yam starch
ND:HR0 The group used a standard diet in the diet to replace 70% by carbohydrates with 0h hydrolyzed yam starch that was retrograded in 2 cycles
HF The group used a 70% fat diet
HF:A The group used a 70% fat diet in the diet to replace 50% by carbohydrates with gelatinized yam starch
HF:HRO The group used a 70% fat diet in the diet to replace 70% by carbohydrates with
0h hydrolyzed yam starch that was retrograded in 2 cycles.
Food rations
Table 3 2 Diet composition of groups
Ingredients (g) ND ND:A ND:HR0 HF HF:A HF:HR0
Note: 3.4 are numbers corresponding to the appendix numbers presented at the end of the article
Table 3 3 Nutritional composition (g/100g) of the diet of the experimental groups
In this trial, diets were categorized into two types: a regular diet (ND) and a high-fat diet (HF) The study utilized Fullvit JP70, a commercial food developed by Jolly Pet Products based in Ohio, USA, as the standard diet for comparison.
23 ration (ND) The nutritional content of ND is similar to the conventional AIN 93 diet given to laboratory animals(Reeves et al., 1993)
Groups used standard food rations consisting of 2 batches as follows:
Lot 1 (ND:A): used standard diet with 50% carbohydrate source being gelatinized yam starch;
Lot 2 (ND:HR0): used standard diet with 70% carbohydrate source being 0-hour hydrolyzed yam starch retrograded in 2 cycles
The high-fat (HF) diet utilized the commercial Fullvit JP70 diet, finely ground and enhanced with heated liquid beef tallow This high-fat food regimen consisted of two variations, with Lot 3 (HF:A) featuring a 70% fat content and incorporating 50% carbohydrate sourced from gelatinized yam starch.
Lot 4 (HF:HR0): used a 70% fat diet with 70% carbohydrate source being 0-hour hydrolyzed yam starch retrograded in 2 cycles
Liquid beef tallow was incorporated into high-fat diets by first washing beef belly fat to eliminate impurities and odors, then chopping and cooking it until it transformed into a light yellow liquid This liquid fat was filtered to remove any residue and stored in a sealed jar in the freezer The diets were prepared according to the ratios outlined in Table 3.5, with the addition of a few grams of vanilla scent powder for flavor enhancement Diets ND:A and ND:HR0 were mixed with dry ingredients such as commercial feed, yam starch, protein, and CMC, combined with water to achieve the desired adhesion The mixture was then rolled out, cut into 1-2 cm bars, and dried for consumption.
HF diets were prepared by completely melting the beef fat and then adding the dry ingredients and mixing well Then, they are rolled thinly and frozen in the refrigerator.
Measurement method on in vivo experiments
3.9.1 Operations on experimental mice a) Operation of moving the mouse out of the cage
To safely handle a mouse, gently grasp the base or middle of its tail with your thumb and forefinger, and carefully remove it from the cage Next, transfer the mouse to a new cage, ensuring it is placed on a rough surface or a scale for proper weighing.
If the mouse is lifted by the tail, it may be seriously injured because the tail skin may peel off
Do not hang the mouse by its upper body or tail for a long time The mouse experiences high
24 levels of stress due to actions such as handling and trapping Therefore, wait for the mouse to calm down before applying any further restraint measures(Manzoor & Raza, 2013)
Figure 3 4 Operation of moving the mouse out of the cage (Mark Bowler, 2010) b) Blood sampling from the tail of the mouse
Mice over six weeks old are optimal for tail vein injections, as their blood vessels are sufficiently developed It is essential to thoroughly clean and disinfect the injection site before proceeding with the procedure (Hatakeyama et al., 2010).
Tail blood sampling in mice involves a straightforward procedure where a small section (1-2 mm) of the tail tip is cut to collect blood This method can be easily performed in any laboratory by using sharp scissors or a scalpel Blood can be obtained either through direct flow or by gently massaging the tail to 'milk' the blood into a capillary tube It is recommended to allow a recovery time of 2 hours after the initial cut before collecting the first sample, with subsequent samples taken by removing the scales and repeating the milking process This technique is ideal for obtaining small blood volumes, as larger samples may induce stress in the mouse, resulting in elevated catecholamine levels and increased endogenous glucose production Additionally, tail blood sampling is advantageous as it does not require catheterization, making it a less invasive option compared to arterial sampling.
The second method for blood sampling involves using a medical syringe needle to extract blood from the tail of the mouse Precise needle insertion is crucial for successful injection, making proper mouse handling techniques essential to the procedure Additionally, various restraining devices are available to help securely hold the mouse during the sampling process.
A practical method for handling mice involves using a 50ml centrifuge tube wrapped in black adhesive tape to minimize their stress A small hole is created at the tail end of the tube to accommodate the mouse's tail, which reveals three vessels: a central artery flanked by veins Arteries can be identified by the branching vessels leading to the veins For tail vein injections, a 28-gauge insulin needle bent at a 30-50° angle is recommended (Hatakeyama et al., 2010).
To ensure safe and effective injection in mice, the animal must be securely restrained, and its tail pulled straight back while monitoring its breathing and signs of anxiety Needles should be positioned nearly parallel to the vein surface for careful insertion, as a common mistake is penetrating too deeply due to the vein's proximity to the skin Once the needle tip is beneath the skin, it's essential to gently retract the syringe while continuing the insertion to confirm proper blood flow.
Figure 3 5 Mouse tail blood vessel diagram (Hatakeyama et al., 2010)
Figure 3 6 Procedure for blood sampling from the tail using needle
3.9.2 Determination of in vivo Glycemic index (iGI)
Figure 3 7 Diagram of in vivo experiment to investigate blood sugar index
In this study, mice were acclimatized on a normal diet until they reached a weight of 30±1 g Following a 12-hour fasting period, the mice were orally administered a sample suspension solution containing starch samples (HR0, HR4, HR10, HR18) or a glucose standard solution, both at a concentration of 7.5% (w/v) Blood samples were collected from the tail vein of each mouse before and at intervals of 15, 30, 60, 90, 120, and 180 minutes after consumption.
Calculate the area limited by the blood glucose concentration curve measured over 180 minutes using Origin 8.5.1 software and apply the corresponding GI calculation formula (where GIglucose0) (mg/dL) (Wolever T.M ,1986)
The study examines the glycemic index (GI) of yam starch, comparing the effects of gelatinized yam starch and 2-cycle retrograded hydrolyzed yam starch on blood glucose levels With a reference GI of 100 for glucose, the research highlights the area under the blood glucose concentration curve over approximately 180 minutes for both starch types, providing insights into their impact on glucose metabolism The findings indicate significant differences in the glycemic response between the two forms of yam starch, emphasizing the importance of starch processing methods in dietary glycemic control.
Table 3 4 Coding table of starch samples
The 0h hydrolyzed yam starch suspension sample was subjected to 2 cycles of retrogradation
The 4-h hydrolyzed yam starch suspension sample was retrograded in 2 cycles
The 10-h hydrolyzed yam starch suspension sample was retrograded in
The 18-h hydrolyzed yam starch suspension sample was retrograded in
3.9.3 Fasting plasma glucose (FPG) measurement method
Fasting plasma glucose (FPG) serves as a crucial diagnostic tool for diabetes mellitus and glucose tolerance, as noted by Sakaguchi et al (2016) In this study, mice undergo a 12-hour overnight fast with access to water, after which blood samples are collected from their tail veins at the beginning and end of the eight-week period to assess blood glucose levels.
3.9.4 Oral glucose tolerance test (OGTT)
In a controlled experiment, mice were fed various diets, including high-fat (HF), high-fat with additive (HF:A), high-fat with high resistance (HF:HR0), normal diet (ND), and normal diet with high resistance (ND:HR0) over a period of 8 weeks Following a 12-hour fasting period, the mice were administered a 7.5% glucose solution orally via an oral gavage tube Blood samples were collected from the tail vein at baseline (0 minutes) and subsequently at 15, 30, 60, 90, 120, and 180 minutes post-administration to monitor glucose levels (Ao & Jane, 2007).
Daily monitoring of mouse weights was conducted using a precise electronic scale (0.01 g) to effectively track their growth throughout the experiment The weekly weight gain (Δm, g) was calculated using a specific formula, ensuring accurate assessment of the mice's growth progress.
∆ 𝑚 = 𝑚 𝑛 − 𝑚 0 (3.7) where m n and m o are the average weights (g) of each group of mice at week n and week 0, respectively
Food Efficiency Ratio (FER) indicates the efficiency of energy absorption from food and its conversion into body mass in mice FER is calculated using the following formula:
𝑁 𝑛 × 100 (3.8) where 𝑀 0 is the initial weight of mice, 𝑀 𝑛 is the weight of mice at week n, and 𝑁 𝑛 is the total energy consumed by mice over n weeks
Cumulative energy intake (CI) in mice is determined by multiplying the total grams of food consumed over a specified number of weeks by the caloric value per gram of the experimental diet.
100 (3.9) where CI (kcal/individual/week) is the cumulative energy intake, n is the experimental week, F is the amount of food consumed by mice (g), and C is the calories per 100 g of food
3.9.6 Anatomical methods and determination of blood biochemical indicators
After 8 weeks of experimentation, mice were surgically operated on to collect liver, kidney, fat, and leg muscle tissues Prior to surgery, mice were anesthetized by spinal cord paralysis using cervical dislocation or ethyl ether anesthesia to minimize pain sensation This was done in accordance with the principles of 3Rs and bioethics To prevent cross-contamination into the blood and tissues of the mice, the operator wore gloves and sterilized the area
To immobilize the mouse and prevent movement, its tail was held firmly with the thumb and forefinger behind the skull This technique aimed to protect the heart and other vital organs from damage The abdomen was then sterilized using cotton soaked in 70% alcohol Following this, a careful incision was made from the abdomen to the chin, allowing access to the abdominal organs, and the ribs on both sides were subsequently cut and removed.
A 1.0 mL syringe pump was used to withdraw blood from the left ventricle of the heart with steady suction to prevent heart collapse (Parasuraman et al., 2010) The blood was then
Blood samples were collected in tubes with Heparin anticoagulant, ensuring each sample did not exceed 1.0 mL Multiple samples from each batch were pooled for analysis Following this, the body was dissected to extract and preserve liver, kidney, fat, and leg muscle tissues in 10% formalin solution, with careful handling to prevent tissue damage.
The General Clinic of Pham Ngoc Thach University of Medicine uses Beckman Coulter's
AU 400 machine to measure blood indexes The machine is located at 461 Su Van Hanh, Ward
RESULTS AND DISCUSSION
In vitro digestibility index of yam starch
Table 4 1 In vitro digestibility index of yam starch
Note: Values with different symbols in the same column indicate statistically significant differences (p HR18 > HR10 > HR4 > HR0 > N This suggests that higher hydrolysis rates lead to increased enzyme activity on starch granules, resulting in greater glucose production.
Gelatined raw starch (A) is a food rich in rapidly digestible starch (RDS), which is swiftly broken down by α-amylase, generating a readily available glucose stream (RAG) for absorption in the small intestine This process results in a rapid spike in blood sugar and insulin levels post-meal, making gelatinized raw starch (A) the starch sample with the highest glycemic index.
Modified starch samples (HR18, HR10, HR4, HR0) undergo a retrogradation process at low temperatures (4°C), which can be repeated to enhance resistant starch (RS) content, as noted by Jin Park et al (2004) Furthermore, Health Canada has classified RS as a dietary fiber, acknowledging its health benefits (Health Canada, 2012).
The European Food Safety Authority (EFSA, 2011) recognizes resistant starch (RS) for its health benefits, including its ability to reduce postprandial blood glucose responses (Zafar, 2018) Since RS does not release glucose in the gut, it has no impact on blood sugar levels (Copeland, 2024) Analysis of starch samples HR18, HR10, HR4, and HR0 reveals that they all possess low glycemic index values, specifically 49.6, 43.15, 39.67, and 35.27, respectively, indicating they are classified as low GI foods.
In vivo glycemic index of purple yam starch (iGI)
Figure 4.2 illustrates the glucose uptake levels in mice after administration of glucose and starch solutions The blood glucose concentration in the mice surged quickly, reaching its highest point at 30 minutes post-consumption of the glucose solution.
In a study examining various hydrolyzed starch samples, the peak blood glucose values were ranked as follows: Glu (193 mg/dL), A (170 mg/dL), HR18 (160.5 mg/dL), HR10 (152.5 mg/dL), HR4 (146.7 mg/dL), and HR0 (141.5 mg/dL) Following a 30-minute period, blood glucose levels gradually decreased, nearly returning to baseline after 180 minutes, with the HR0 starch group exhibiting the slowest decline in glucose concentration This aligns with the digestion index and estimated Glycemic Index (eGI), indicating that foods high in resistant starch contribute to a lower glycemic response by promoting a gradual increase in blood sugar levels Consequently, low-GI foods are recommended for diabetic patients due to their beneficial effects on blood glucose and insulin regulation (Koksel et al., 2024).
GLU A HR0 HR4 HR10 HR18
The study revealed a significant difference in the glycemic index (GI) of various starch samples, with the in vivo GI (iGI) being higher than the in vitro GI (eGI), attributed to physiological and dietary factors affecting digestion and glucose absorption The ranking of the iGI among mouse groups was Glu (100.0) > A (72.6) > HR18 (64.67) > HR10 (61.11) > HR4 (57.24) > HR0 (51.1) Notably, starch samples with higher resistant starch (RS) content exhibited lower GI indices, as RS slows digestion and glucose absorption, while rapidly digestible starch (RDS) increased the GI due to its quick digestion in the small intestine Among the tested starches, only HR0 had a blood sugar index below 55, classifying it as a low GI food and making it the optimal choice for further in vivo experiments, as it also showed increased SDS and RS indices, enhancing its nutritional value and potential to prevent metabolic diseases like obesity and diabetes.
Effect of starch on oral glucose tolerance (OGTT)
We used the glucose absorption test to evaluate the sensitivity of glucose concentration and the ability to absorb glucose in mice with different diets From figure 4.3, fasting blood sugar
The study found no significant differences in fasting blood sugar levels among standard diet groups, while high-fat diets exhibited notably higher blood sugar levels All diet groups maintained normal fasting blood sugar levels below 130 mg/dL, except for the high-fat diet without starch supplementation, which showed levels exceeding 150 mg/dL, indicating potential type 2 diabetes Following glucose absorption, blood sugar levels in mice rose, peaking at 30 minutes before gradually decreasing by the 180th minute The peak blood sugar levels were ranked as follows: ND:HR0 at 170.75 mg/dL, followed by ND:A at 183.0 mg/dL.
< ND (195.33 mg/dL) < HF:HR0 (209 mg/dL) < HF:A (220.6 mg/dL) < HF (240.75 mg/dL)
Obesity is linked to insulin resistance and impaired glucose absorption, as evidenced by the oral glucose tolerance test (OGTT) Research indicates that individuals on a high-fat diet (HF) exhibit the highest glucose sensitivity but struggle with glucose absorption and control Elevated levels of free fatty acids in the bloodstream contribute to insulin response defects, leading to insulin resistance and reduced glucose transport.
Supplementation with gelatinized raw starch and low-GI purple yam starch resulted in lower peak glucose levels at 30 minutes compared to the high-fat (HF) group, indicating a beneficial impact of carbohydrate supplementation on glucose uptake Notably, low-GI purple yam starch produced the lowest glucose peak in the HF group Resistant starch plays a crucial role in lowering blood fat levels by slowing digestion, which limits fat formation in the liver due to reduced glucose availability and insulin activation Furthermore, extensive research suggests that low-GI foods and diets can significantly lower the risk of diabetes and heart disease.
The area under the blood glucose curve (AUC) indicates the level of body glucose following food intake As illustrated in Figure 4.4, standard diet groups (ND, ND:A, ND:HR0) generally exhibit lower AUC values compared to the high-fat diet group While the standard diet groups demonstrate significant differences, these variations are not substantial Conversely, the high-fat diet groups display a distinct trend in their glucose response.
ND ND:A HF:A HF HF:HR0 ND:HR0
The study found that replacing carbohydrates with purple yam starch led to an increase in AUC, with the 70% low glycemic index (GI) starch (HF:HR0) resulting in the lowest glucose intake This was followed by the 50% gelatinized raw starch (HF:A) The groups were ranked by glucose intake in the following order: HF:HR0 < HF:A < HF.
Low glycemic index (GI) foods contribute to reduced insulin secretion, resulting in lower postprandial insulin levels This decrease in insulin production, while maintaining stable levels, can help minimize the buildup of free fatty acids, ultimately enhancing the body's metabolism and improving glucose uptake (Aller et al., 2011).
Fasting plasma glucose
After 8 weeks of experimentation, both glucose and insulin concentrations in serum showed a significant increase, particularly after 6 and 24 hours of fasting (Andrikopoulos et al., 2008) The fasting plasma glucose (FPG) levels of mouse groups indicated that the ND, ND:A, and ND:HR0 groups maintained normal FPG values below 130 mg/dL, while the high-fat diet group exhibited elevated FPG levels exceeding 130 mg/dL Specifically, the high-fat diet supplemented with gelatinized raw starch (HF:A) recorded an FPG level of 133.40 mg/dL, indicating prediabetes, while the HF group reached a concerning 162.80 mg/dL, signifying a doubling of FPG levels and corresponding to type 2 diabetes.
2 diabetes (T2D) Based on the FPG levels of the high-fat diet groups, we obtained the following order: HF (162.80 mg/dL) > HF:A (133.40 mg/dL) > HF:HR0 (111.2 mg/dL) e d f a b c
After 8 weeks of consumption, the high-fat diet groups HF and HF:A exhibited abnormal levels compared to other groups This indicates that supplementing purple yam starch with a low glycemic index (GI) in high-fat diets effectively reduces postprandial glucose response and lowers the risk of insulin resistance, helping to maintain normal blood glucose levels in individuals at risk of diabetes or obesity.
Figure 4 5 The fasting plasma glucose (FPG) levels of the experimental groups
Effect of starch on body weight and calories consumption
According to table 3.3, with 100g of food, both groups ND:A and ND:HR0 will provide
In a study comparing dietary impacts on mice, groups HF:A and HF:HR0 received a higher calorie intake of 575.33 kcal, while the ND groups were limited to only 375 kcal This significant difference in calorie consumption resulted in the ND groups exhibiting lower growth rates and reduced energy expenditure compared to their HF counterparts.
In the HF:HR0 starch-supplemented mouse group, a contrasting trend is observed, likely due to their diet, which is high in both fat and carbohydrates with elevated resistant starch (RS) content, leading to poor absorption In contrast, the raw starch-supplemented HF:A group demonstrates easier digestibility, resulting in a higher and more stable growth rate.
ND ND:A ND:HR0 HF HF:A HF:HR0
Figure 4 6 Body weight gain (BWG, Δm) of the experimental groups for 8 weeks
Figure 4 7 Cumulative energy intake (CI) of the experimental groups over 8 weeks
Figure 4 8 Cumulative food intake of experimental groups for 8 weeks
Figure 4 9 Cumulative FER value of groups trial for 8 weeks
According to figure 4.6 and appendix 8, a notable disparity in additional body weight gain exists between the high-fat (HF) and normal diet (ND) groups At the conclusion of week 8, the additional body weight gain for the mouse groups is ranked in ascending order as follows: NDHR0 < HF:HR0 < ND, with a significance level of p HF:A > HF:HR0) due to the high resistant starch content in HR0 starch, which promotes satiety and reduces appetite Significant disparities in energy consumption are evident between HF and ND groups, with HF groups consuming considerably more However, a high-fat diet may hinder nutrient absorption and transport in the gut, influencing weight regulation in mice Consequently, the FER index highlights a distinct difference between ND and HF groups.
Effect of starch on biochemical indices
Table 4 2 Biochemical indices of experimental mouse groups
Total cholesterol (TC) is a crucial metric for assessing cholesterol levels in the blood, encompassing low-density lipoprotein (LDL), high-density lipoprotein (HDL), and minor cholesterol types A TC range of 1.743-4.665 mmol/L is deemed normal for mouse health (Lea et al., 2017) As indicated in Table 4.2, all experimental mouse groups maintained TC levels within this normal range By the conclusion of week 8, the TC levels of the experimental groups were ranked significantly (p